Biology Methods and Protocols最新文献

{"title":"Engineered BSA nanoparticles: Synthesis, drug loading, and advanced characterization.","authors":"Hemlata, A Hariharan, Nandan Murali, Srabaita Roy, Soutik Betal, Saran Kumar, Shilpi Minocha","doi":"10.1093/biomethods/bpaf066","DOIUrl":"10.1093/biomethods/bpaf066","url":null,"abstract":"<p><p>Bovine serum albumin (BSA) nanoparticles have attracted a lot of interest as biocompatible and biodegradable carriers for a range of pharmacological and biological uses. BSA nanoparticles have several advantages over other types of nanoparticles, including their ability to increase the stability and solubility of encapsulated drugs, their non-toxicity, and their ease of surface modification. Cancer treatment, immunological modulation, enzyme immobilization, controlled release systems, bioimaging, and theranostics are some of its potential applications. This protocol offers a detailed and accessible methodology for the synthesis, drug encapsulation, and characterization of albumin nanoparticles, with particular emphasis on reproducibility and adaptability. The synthesis uses the desolvation process and crosslinking with the compound glutaraldehyde for stability. The crosslinking ratio, pH, and BSA content are important factors that can be adjusted to control size, surface charge, and dispersity. The methods used for characterization are described in detail, including dynamic light scattering for particle size and zeta potential, transmission and scanning electron microscopy for morphology, Fourier-transform infrared spectroscopy, and nanoparticle tracking analysis for stability assessment. The stability of the nanoparticles was evaluated under physiologically relevant ionic and pH conditions by dispersing them in phosphate-buffered saline, providing insight into their colloidal behavior in a simulated physiological environment. This technique facilitates the design of functionalized BSA nanoparticles for certain biomedical and therapeutic applications by acting as a fundamental reference for researchers. This work promotes innovation in nanoparticle-based technology and advances the field by standardizing preparation and characterization techniques.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"10 1","pages":"bpaf066"},"PeriodicalIF":1.3,"publicationDate":"2025-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12466926/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145186923","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 robust protocol for proteomic profiling of secreted proteins in conditioned culture medium.","authors":"Takayoshi Otsuka, Atsushi Hatano, Masaki Matsumoto, Hideaki Matsui","doi":"10.1093/biomethods/bpaf068","DOIUrl":"10.1093/biomethods/bpaf068","url":null,"abstract":"<p><p>Reliable secretome analysis is crucial for understanding cellular communication and developing therapeutic strategies. However, conventional protein quantification methods, such as the bicinchoninic acid (BCA) assay, can overestimate protein concentrations in concentrated culture media, leading to inconsistent protein loading and compromised quantitative accuracy in mass spectrometry-based proteomics. To address this methodological challenge, we developed an improved sample preparation method for secretome analysis. Our approach introduces a concentration rate-based normalization method that adjusts sample volumes according to the ultrafiltration concentration ratio, ensuring more consistent protein loading across samples. This method enabled reliable identification of 3468 secreted proteins with high reproducibility (<i>r</i> > 0.93) in a model system of nuclear DNA (nucDNA)-induced inflammation in HeLa cells. Secretome profiles were distinctly altered by nucDNA transfection, with 89 proteins showing significant differential release between control and nucDNA-transfected wild-type HeLa cells. Furthermore, we identified a subset of proteins, including chaperone and proteasome complexes, that were consistently released across all conditions, suggesting their potential utility as internal controls for secretome analysis. This study presents a practical solution to the methodological challenge in secretome analysis, enabling more reliable and reproducible secretome profiling. This improved methodology represents an important step toward establishing standardized protocols for secretome analysis, ultimately enhancing the quality and comparability of research in this rapidly growing field.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"10 1","pages":"bpaf068"},"PeriodicalIF":1.3,"publicationDate":"2025-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12461699/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145186973","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":"Integrating multiple microRNA functional similarity networks for improved disease-microRNA association prediction.","authors":"Duc-Hau Le","doi":"10.1093/biomethods/bpaf065","DOIUrl":"10.1093/biomethods/bpaf065","url":null,"abstract":"<p><p>MicroRNAs (miRNAs) play a critical role in disease mechanisms, making the identification of disease-associated miRNAs essential for precision medicine. We propose a novel computational method, multiplex-heterogeneous network for MiRNA-disease associations (MHMDA), which integrates multiple miRNA functional similarity networks and a disease similarity network into a multiplex-heterogeneous network. This approach employs a tailored random walk with restart algorithm to predict disease-miRNA associations, leveraging the complementary information from experimentally validated and predicted miRNA-target interactions, as well as disease phenotypic similarities. Evaluated on the human microRNA disease database and miR2Disease datasets using leave-one-out cross-validation and 5-fold cross-validation, MHMDA demonstrates superior performance, achieving area under the receiver operating characteristic curve values of 0.938 and 0.913 on human microRNA disease database and miR2Disease, respectively, and outperforming existing methods. The integration of multiplex networks enhances prediction accuracy by capturing diverse miRNA functional relationships, which directly contributes to the high area under the receiver operating characteristic curve and area under the precision-recall curve values observed. Additionally, MHMDA's stability across parameter variations and disease contexts underscores its robustness and potential for real-world applications in identifying novel disease-miRNA associations.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"10 1","pages":"bpaf065"},"PeriodicalIF":1.3,"publicationDate":"2025-09-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12410926/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145016322","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":"TockyLocus: quantitative analysis of flow cytometric fluorescent timer data in Nr4a3-Tocky and Foxp3-Tocky mice.","authors":"Masahiro Ono","doi":"10.1093/biomethods/bpaf060","DOIUrl":"10.1093/biomethods/bpaf060","url":null,"abstract":"<p><p>Fluorescent Timer proteins undergo a time-dependent shift from blue to red fluorescence after translation, providing a temporal record of transcriptional activity in Timer reporter systems. While Timer proteins are well suited for studying dynamic cellular processes such as T cell activation using the Timer-of-Cell-Kinetics-and-Activity (Tocky) framework, quantitative analysis of Timer-based flow cytometry data has yet to be fully standardized. In this study, we optimize quantitative analysis methods for the key parameter within the Tocky framework, Timer Angle, and introduce TockyLocus, an open-source <math><mi>R</mi></math> package that implements a five-category scheme based on biologically grounded angular intervals (designated as Tocky Loci). This approach is validated using both simulated and experimental datasets and enables downstream statistical testing and visualization of transcriptional dynamics in flow cytometry data. Using computational modelling of Timer protein kinetics, we define transcriptional dynamics in relation to key anchoring points in Timer Angle values at <math> <mrow> <mrow> <msup><mrow><mn>0</mn></mrow> <mo>°</mo></msup> </mrow> </mrow> </math> , <math> <mrow> <mrow> <msup> <mrow><mrow><mn>45</mn></mrow> </mrow> <mo>°</mo></msup> </mrow> </mrow> </math> , and <math> <mrow> <mrow> <msup> <mrow><mrow><mn>90</mn></mrow> </mrow> <mo>°</mo></msup> </mrow> </mrow> </math> . Comprehensive simulations with synthetic spike-in datasets further demonstrate the robustness of the five-locus approach, which captures the three key points and the intermediate regions between these points. Building on the TockyPrep preprocessing framework, we systematically evaluated categorization schemes ranging from three to seven loci on real-world datasets from Nr4a3-Tocky and Foxp3-Tocky mice. The five-locus model emerged as optimal, showing significant advantages in balancing biological interpretability and statistical robustness. Optimized algorithms implemented in the TockyLocus package now standardize quantitative analysis of Timer Angle data, enabling reproducible interpretation without reliance on arbitrary gating or complex assumptions. In summary, the five-locus categorization of Timer Angle data effectively links underlying biological dynamics to the percentage of cells in each Tocky Locus, providing a robust and interpretable framework for investigating transcriptional dynamics in immunology and related fields.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"10 1","pages":"bpaf060"},"PeriodicalIF":1.3,"publicationDate":"2025-08-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12464679/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145187003","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":"Constructing a norm for children's scientific drawing: Distribution features based on semantic similarity of large language models.","authors":"Yi Zhang, Fan Wei, Jingyi Li, Yan Wang, Yanyan Yu, Jianli Chen, Zipo Cai, Xinyu Liu, Wei Wang, Sensen Yao, Peng Wang, Zhong Wang","doi":"10.1093/biomethods/bpaf062","DOIUrl":"10.1093/biomethods/bpaf062","url":null,"abstract":"<p><p>The use of children's drawings to examining their conceptual understanding has been proven to be an effective method, but there are two major problems with previous research: (i) The content of the drawings heavily relies on the task, and the ecological validity of the conclusions is low. (ii) The interpretation of drawings relies too much on the subjective feelings of the researchers. To address this issue, this study uses the Large Language Model (LLM) to identify 1420 children's scientific drawings (covering nine scientific themes/concepts) and uses the word2vec algorithm to calculate their semantic similarity. The study explores whether there are consistent drawing representations for children on the same theme and attempts to establish a norm for children's scientific drawings, providing a baseline reference for follow-up children's drawing research. The results show that the representation of most drawings has consistency, manifested as most semantic similarity >0.8. At the same time, it was found that the consistency of the representation is independent of the accuracy (of LLM's recognition), indicating the existence of consistency bias. In the subsequent exploration of influencing factors, we used Kendall rank correlation coefficient to investigate the effects of \"sample size,\" \"abstract degree,\" and \"focus points\" on drawings and used word frequency statistics to explore whether children represented abstract themes/concepts by reproducing what was taught in class. It was found that accuracy (of LLM's recognition) is the most sensitive indicator, and data such as sample size and semantic similarity are related to it. The consistency between classroom experiments and teaching purpose is also an important factor, many students focus more on the experiments themselves rather than what they explain. In addition, most children tend to use examples they have seen in class to represent more abstract themes/concepts, indicating that they may need concrete examples to understand abstract things.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"10 1","pages":"bpaf062"},"PeriodicalIF":1.3,"publicationDate":"2025-08-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12380450/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144972610","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":"Fast and accurate quantification of double-strand breaks in microsatellites by digital PCR.","authors":"Cécile Palao, Adèle Kovacs, Maria Teresa Teixeira, Guy-Franck Richard","doi":"10.1093/biomethods/bpaf059","DOIUrl":"10.1093/biomethods/bpaf059","url":null,"abstract":"<p><p>DNA double-strand breaks (DSBs) represent critical events in genome integrity, arising from both endogenous cellular processes and exogenous factors. These breaks are implicated in various genomic aberrations and chromosomal rearrangements, leading to cancers and genetic disorders. Common and rare fragile sites, containing repetitive elements and non-B DNA structures, are particularly prone to breakage under replication stress, which play a pivotal role in cancer development and genetic diseases. Accurate quantification of DNA breaks in the context of repetitive sequences such as microsatellites or non-B DNA structures is technically challenging. We have been comparing four different methods to reliably quantify DSBs in repetitive DNA, namely Southern blot, DSB-PCR, real-time DSB-qPCR, and digital PCR (dPCR). We show here that dPCR offers enhanced sensitivity and specificity compared to other methods. This provides significant applications for future disease diagnosis, understanding molecular mechanisms generating chromosomal breakage and for the development of gene therapies for microsatellite expansion disorders.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"10 1","pages":"bpaf059"},"PeriodicalIF":1.3,"publicationDate":"2025-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12377901/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144972571","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":"Radiolabeling isolated mitochondria with Tc-99m: A first-in-field protocol and early feasibility findings.","authors":"Melanie Walker, Francisco Javier Miralles, Keiko Prijoles, Jacob S Kazmi, Jennifer Hough, David Lewis, Michael R Levitt, Yasemin Sancak","doi":"10.1093/biomethods/bpaf063","DOIUrl":"10.1093/biomethods/bpaf063","url":null,"abstract":"<p><p>Mitochondrial transplantation is a promising but still experimental strategy for treating ischemic and metabolic disorders. A key barrier to its advancement is the lack of scalable, non-invasive methods for tracking transplanted extracellular mitochondria <i>in vivo</i>. Technetium-99m (Tc-99m) radiopharmaceuticals, widely used in SPECT imaging, may offer a clinically compatible solution. Cryopreserved mitochondria derived from HEK-293 cells were incubated with Tc-99m sestamibi, tetrofosmin, pertechnetate, or control solutions. After brief incubation and washing, mitochondrial pellets were analyzed for retained radioactivity. ATP content was measured to assess metabolic function, and electron microscopy was used to evaluate ultrastructural integrity. Tc-99m sestamibi and tetrofosmin showed labeling efficiencies of 2.74% and 2.68%, respectively. Pertechnetate demonstrated minimal uptake (0.34%). Radiolabeled mitochondria retained ATP production comparable to controls. Electron microscopy showed preserved double membranes and cristae. Controls confirmed assay specificity and viability. To our knowledge, this is the first report of radiolabeling isolated mitochondria with clinically approved Tc-99m agents. This platform supports the development of SPECT-compatible protocols for visualizing viable transplanted mitochondria in recipient tissues.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"10 1","pages":"bpaf063"},"PeriodicalIF":1.3,"publicationDate":"2025-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12371404/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144972575","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":"An explainable AI approach for mapping multivariate regional brain age and clinical severity patterns in Alzheimer's disease.","authors":"Gauri Darekar, Taslim Murad, Hui-Yuan Miao, Deepa S Thakuri, Ganesh B Chand","doi":"10.1093/biomethods/bpaf051","DOIUrl":"10.1093/biomethods/bpaf051","url":null,"abstract":"<p><p>Age is a significant risk factor for mild cognitive impairment (MCI) and Alzheimer's disease (AD) and identifying brain age patterns is critical for comprehending the normal aging and MCI/AD processes. Prior studies have widely established the univariate relationships between brain regions and age, while multivariate associations remain largely unexplored. Herein, various artificial intelligence (AI) models were used to perform brain age prediction using an MRI dataset (<i>n</i> = 825). The optimal AI model was then integrated with the feature importance methods, namely Shapley additive explanations (SHAP), local interpretable model-agnostic explanations, and layer-wise relevance propagation, to identify the significant multivariate brain regions hierarchically involved in this prediction. Our results showed that the deep learning model (referred to as AgeNet) outperformed conventional machine learning models for brain age prediction, and that AgeNet integrated with SHAP (referred to as AgeNet-SHAP) identified all ground-truth perturbed regions as key predictors of brain age in semi-simulation, demonstrating the validity of our methodology. In the experimental dataset, when compared to cognitively normal (CN) participants, MCI exhibited moderate differences in brain regions, whereas AD showed highly robust and widely distributed regional differences. Individualized AgeNet-SHAP regional features further showed associations with clinical severity scores in the AD continuum. These results collectively facilitate data-driven explainable AI approaches for disease progression, diagnostics, prognostics, and personalized medicine efforts.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"10 1","pages":"bpaf051"},"PeriodicalIF":1.3,"publicationDate":"2025-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12377905/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144972608","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":"Application of dicentric chromosome assay for evaluation of radioprotective effect.","authors":"Marcela Milanová, Vojtěch Chmil, Aleš Tichý, Lenka Lecová","doi":"10.1093/biomethods/bpaf058","DOIUrl":"10.1093/biomethods/bpaf058","url":null,"abstract":"<p><p>The dicentric chromosome assay is a well-established biodosimetric method used to assess absorbed ionizing radiation doses by detecting dicentric chromosomal aberrations. Here, we present a detailed, reproducible protocol for applying the dicentric chromosome assay for <i>in vitro</i> evaluation of radioprotective agents, including novel piperazine derivatives compared with amifostine and its active metabolite WR-1065. The protocol covers all key steps-blood sample preparation, <i>in vitro</i> irradiation, lymphocyte culture, metaphase preparation, and scoring of dicentric chromosomes. It highlights critical stages that affect data quality and reproducibility. Integrating manual scoring with automated analysis using the Metafer system ensures accurate and efficient assessment. Thus, this protocol bridges the fields of biological dosimetry and preclinical screening of radioprotective agents, providing a reliable framework for emergency radiation dose estimation and the development of new radiation medical countermeasures.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"10 1","pages":"bpaf058"},"PeriodicalIF":1.3,"publicationDate":"2025-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12349920/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144849286","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 simple, cost-effective, method for creating electronic cigarette vapor condensate.","authors":"Jennifer M Piechowski, Brian Bagatto","doi":"10.1093/biomethods/bpaf055","DOIUrl":"10.1093/biomethods/bpaf055","url":null,"abstract":"<p><p>Methods to create electronic cigarette (e-cigarette) vapor condensate are needed for use in e-cigarette vapor exposure studies. There are currently several methods to produce condensate described in the literature, but they are often cost-prohibitive, complex, or potentially hazardous, thus limiting the true availability of these methods to many researchers in the field. Here, we developed a method to make e-cigarette vapor condensate utilizing a button-activated vaping device and inexpensive supplies such as a syringe, vinyl tubing of varying diameters, an assortment of fittings, a conical tube, and ordinary, hard-sided, ice packs. The method of condensate production described here produced a yield of 35 µL of condensate per 15 puffs of e-cigarette vapor. This method is cost-effective, easy to perform, and can be readily used by researchers at a wide variety of institutions.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"10 1","pages":"bpaf055"},"PeriodicalIF":1.3,"publicationDate":"2025-07-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12341675/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144838100","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}