Methods最新文献

{"title":"ZEMs: Zebrafish embedding molds for high-throughput imaging of zebrafish embryos and larvae.","authors":"Yugyeong Sim, Eunbeom Lee, Jinyoung Jeong","doi":"10.1016/j.ymeth.2025.10.001","DOIUrl":"https://doi.org/10.1016/j.ymeth.2025.10.001","url":null,"abstract":"<p><p>Zebrafish imaging is a powerful tool for observing physiological responses in real time, from the whole organism to the organ, tissue, and cellular levels. It enables researchers to derive biological meaning by observing morphological and histological changes, cell migration, and more. To analyze such dynamic phenomena, the acquisition of high-quality and consistent images is essential. However, it remains challenging to acquire standardized images at specific regions of interest in zebrafish. In this study, we developed a customized imaging platform, the zebrafish embedding mold (ZEM), designed to facilitate imaging of zebrafish embryos and larvae. Three types of molds were fabricated to accommodate different developmental stages and imaging orientations. The ZEM provided stable positioning of embryos (0-2 days post-fertilization, dpf) and larvae (3-7 dpf), enabling improved imaging of developmental stages, morphological changes, and fluorescence signals. Using this platform, we successfully analyzed the biodistribution and accumulation patterns of fluorescent polystyrene nanoplastics, as well as morphological alteration induced by exposure to the environmental pollutant benzo[a]pyrene. The ZEM ensured consistent specimen orientation in lateral, dorsal and ventral view, enabling quantitative image-based analysis and reliable toxicological assessment. This platform has the potential to be utilized for image-based screening and mechanistic studies, supporting multi-time point observations, reproducible image acquisition, and statistical analysis using the zebrafish model.</p>","PeriodicalId":390,"journal":{"name":"Methods","volume":" ","pages":""},"PeriodicalIF":4.3,"publicationDate":"2025-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145231032","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":"Comparative analysis of macroscopic and microscopic optical absorbance in hemagglutination assay.","authors":"Dong-Gyu Jeon, Chung-Young Lee, Chang-Hee Cho, Gang Ho Lee, Yongmin Chang, Sung-Wook Nam","doi":"10.1016/j.ymeth.2025.09.011","DOIUrl":"https://doi.org/10.1016/j.ymeth.2025.09.011","url":null,"abstract":"<p><p>We report a comparative study of macroscopic and microscopic optical absorbance in hemagglutination (HA) assay. Red blood cells (RBC) have unique optical absorbance properties with characteristic peaks including Soret, Qv, and Qo. In addition, RBC absorbs the light by showing dark contrast in bright field image in optical microscopy, implying that RBC tends to increase the local optical density (OD). By systematic analysis of macroscopic and microscopic measurements of ODs and UV-Visible (UV-Vis) spectroscopy, we developed a phenomenological model of RBC agglutination and non-agglutination. The chemical reaction in antigen-antibody reaction of RBCs is a catastrophic event such that the networking of RBC clumps is initiated at a critical RBC concentration. We analyzed the dependence of the RBC concentration on OD. At the critical concentration of RBCs, the ODs are dropped or saturated for RBC agglutination, on the other hand, the ODs keep increasing as the increase of RBC concentration for RBC non-agglutination. By the analysis of UV-Vis spectroscopy for HA assay, we provide an optimum wavelength as 480 nm to 520 nm, away from RBC characteristic peaks. For further validation, we demonstrated the OD-based HA assay for the detection of H1N1 influenza A virus. Our investigation described here provides an insight into how to utilize physical properties of RBCs for novel HA assay platform.</p>","PeriodicalId":390,"journal":{"name":"Methods","volume":" ","pages":""},"PeriodicalIF":4.3,"publicationDate":"2025-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145231083","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":"UHPLC-Orbitrap-MS/MS metabolite profiling combined with HPLC-PDA targeted chemical fingerprint reveals the geographic-environment (G × E) effect on chemical constituents of Bilvamula (Aegle marmelos root).","authors":"Ramdas, Mausam Singh, Amrat Pal Singh, Neerja Tiwari, Karuna Shanker","doi":"10.1016/j.ymeth.2025.09.010","DOIUrl":"https://doi.org/10.1016/j.ymeth.2025.09.010","url":null,"abstract":"<p><p>Aegle marmelos (AM) (Rutaceae family) holds significant economic value due to its uses in food, traditional medicines, and timber. Studies have confirmed that its extracts and phytochemicals possess various pharmacological actions, including anti-obesity, diuretic, anti-inflammatory, and chemopreventive. However, challenges remain due to the limited information on its secondary metabolites and the lack of validated methodologies. Natural variability in the raw material further complicates the assessment of therapeutic efficacy. Hence, we have conducted the untargeted metabolite profiling of A. marmelos root (AMR) using UHPLC-Orbitrap-MS/MS analysis, identifying 69 phytochemicals of diverse classes. Additionally, we have isolated and characterised 09 key phytochemicals to evaluate the chemical variability in AMR from 15 geographical locations. HPLC-PDA method, compliant with ICH Q2 (R2) guidelines, was developed and validated to quantitate 02 alkaloids (skimmianine, O-Methyl tembamide) and 07 coumarins [umbelliferone, xanthotoxol, marmin, and 7-(6-Hydroxy-7-methoxy-3,7-dimethyl-(2E)-2-octenyloxy) coumarin, 7-(3,7-Dimethyl-6-oxo-(2E)-2-octenyloxy) coumarin, marmelosin, and auraptene]. Chemometric analysis has distinguished 15 AMR ecotypes, and hierarchical cluster analysis resulted in AM-S4 as a distinctive ecotype, which was verified by principal component analysis with 96 % data variance. Partial least square-discriminate analysis predicted a relationship between targeted metabolites and ecotypes. The metabolites associated with a discriminatory pattern of AMR ecotypes were identified by variable importance for projection (VIP) score. Unlike previous reports, the present method fulfils the ISO: 17025-2017 requirement by evaluating the measurement of uncertainty (MU) to ensure the accuracy and traceability of the results. Overall, present study summarises comprehensive metabolite profiling, multi-components classification of AMR to define genetic-environment (G × E) effect, and quality evaluation of AMR derived medicinal product.</p>","PeriodicalId":390,"journal":{"name":"Methods","volume":" ","pages":""},"PeriodicalIF":4.3,"publicationDate":"2025-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145228392","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 newly developed ferroptosis-related gene signature for forecasting prognosis in uveal melanoma.","authors":"Yifei Zhang, Junfang Li, Yu Zhang, Yi Qu","doi":"10.1016/j.ymeth.2025.08.001","DOIUrl":"10.1016/j.ymeth.2025.08.001","url":null,"abstract":"<p><strong>Background: </strong>Uveal melanoma is the most common primary intraocular malignancy in adults, characterized by significant inter-patient heterogeneity and poor long-term prognosis. Ferroptosis, an iron-dependent form of regulated cell death, has emerged as a promising avenue for cancer therapy, yet its role in UVM remains insufficiently explored.</p><p><strong>Methods: </strong>We systematically screened ferroptosis-related genes (FRGs) associated with UVM prognosis by integrating univariate Cox regression, LASSO regression, and Random Forest algorithms. A prognostic signature was constructed and validated using data from The Cancer Genome Atlas and Gene Expression Omnibus cohorts. We further evaluated the model's association with clinicopathological features, tumor immune microenvironment, and drug sensitivity. In vitro experiments were conducted to validate the functional role of SIRT3, the most pivotal gene in the signature.</p><p><strong>Results: </strong>A robust FRG-based prognostic model comprising SIRT3, RRM2, and PANX2 was developed and successfully stratified patients into high- and low-risk groups with significantly different survival outcomes. Multivariate Cox regression confirmed the risk score as an independent prognostic factor. High-risk patients exhibited immunosuppressive microenvironmental features, including elevated regulatory T cells and macrophage infiltration, as well as lower predicted response to immune checkpoint blockade. Drug sensitivity analysis identified ten compounds with lower predicted IC50 values in the high-risk group, suggesting potential therapeutic relevance. Functional assays demonstrated that SIRT3 knockdown promoted UVM cell proliferation and migration, while attenuating susceptibility to ferroptosis, highlighting its tumor-suppressive role.</p><p><strong>Conclusion: </strong>This study presents a novel FRG signature with strong prognostic and immunological implications in UVM. The model offers a promising tool for risk stratification and may assist in guiding ferroptosis- and immunotherapy-based precision treatment. Our findings also suggest that SIRT3 acts as a ferroptosis sensitizer in UVM, representing a potential therapeutic target warranting further investigation.</p>","PeriodicalId":390,"journal":{"name":"Methods","volume":" ","pages":"187-199"},"PeriodicalIF":4.3,"publicationDate":"2025-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144787991","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":"Development of capillary electrophoresis method to measure albumin thiol oxidation in dystrophic humans and animal models of Duchenne muscular dystrophy.","authors":"Angelo Patrick R Bautista, Jessica R Terrill, Marisa N Duong, Gabriella Angelica, Irene Tsioutsias, Christopher P James, Aude Lafoux, Corinne Huchet, Peter G Arthur","doi":"10.1016/j.ymeth.2025.07.010","DOIUrl":"10.1016/j.ymeth.2025.07.010","url":null,"abstract":"<p><p>Inflammatory responses evident in many diseases involve the generation of oxidants which can cause oxidant-induced post-translational modifications to proteins. Albumin, the most abundant plasma protein, contains a free thiol group which is susceptible to oxidative modification. We propose that albumin thiol oxidation (AlbOx) could be a useful biomarker to monitor changes in inflammatory activity and oxidative stress. To measure AlbOx in humans and animal models, we developed a fast, sensitive, simple, and reproducible capillary electrophoresis method (CE-AlbOx). This method can analyse total, reversible, and irreversible oxidation of albumin. The method only requires a small volume of sample (<10 μL blood), has an intra/interday variation of <2 %, and has a total run time of 17 min. We validated the usefulness of AlbOx as a biomarker of chronic inflammation by analysing samples from patients with, and animal models of, Duchenne muscular dystrophy (DMD), a disease associated with chronic inflammation. The main findings in this study are (1) dystrophic humans and animals have higher oxidised albumin compared to healthy controls, (2) mouse albumin has two reactive cysteine groups, and (3) our method is the first to quantify the different oxidation states of mouse albumin. In conclusion, we have developed a new method to measure albumin oxidation in humans and animals using capillary electrophoresis. The simple methodology of the CE-AlbOx method makes it advantageous to current methods and can be readily used as a biomarker of inflammation and oxidative stress in both humans and animal models.</p>","PeriodicalId":390,"journal":{"name":"Methods","volume":" ","pages":"159-168"},"PeriodicalIF":4.3,"publicationDate":"2025-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144768179","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":"DEF-DSVM: A deep ensemble feature learning and deepSVM approach for multifaceted analysis and diagnosis of Alzheimer's disease from EEG signals.","authors":"Shabnam Hesari, Hamidreza Ghaffari, Khosro Rezaee","doi":"10.1016/j.ymeth.2025.08.003","DOIUrl":"10.1016/j.ymeth.2025.08.003","url":null,"abstract":"<p><p>Early detection of Alzheimer's disease (AD) and its precursor, mild cognitive impairment (MCI), is paramount for timely intervention and effective disease management. This study introduces a novel computer-aided diagnostic model that leverages electroencephalogram (EEG) data to precisely identify and classify AD and MCI. A comprehensive preprocessing pipeline is employed, incorporating discrete wavelet transform (DWT) for EEG signal decomposition into relevant subbands and subsequent signal windowing to address non-stationarity. Spectrograms derived from these preprocessed signals serve as input for a deep ensemble feature learning and deep support vector machine (DEF-DSVM) architecture. The DEF-DSVM model significantly enhances the accuracy of diagnosing both MCI and AD, achieving an impressive 98.17% accuracy rate that surpasses contemporary state-of-the-art methods. Beyond diagnostic precision, the model effectively identifies specific EEG subbands-namely alpha, theta, and delta-instrumental in elucidating the pathophysiology of AD and MCI. The structure's generalizability and robustness are validated using the Figshare dataset, encompassing, AD, MCI, and control classes. To ensure a rigorous assessment of the model's performance, the Leave-One-Subject-Out (LOSO) cross-validation procedure is employed in lieu of the traditional K-fold approach, mitigating the risk of overoptimistic performance estimates and providing a more accurate reflection of the model's ability to generalize to novel, unseen subjects. Further evaluation of the method's generalizability through its application to an EEG dataset related to attention deficit hyperactivity disorder (ADHD) highlights its broader clinical utility across various neurodegenerative disorders. These findings establish the DEF-DSVM model as a reliable and potent tool for the early diagnosis and monitoring of AD and MCI, offering substantial accuracy gains and demonstrating its potential for widespread application across different neurological conditions.</p>","PeriodicalId":390,"journal":{"name":"Methods","volume":" ","pages":"169-186"},"PeriodicalIF":4.3,"publicationDate":"2025-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144793172","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":"Modern membrane biophysics approaches.","authors":"Francisco N Barrera","doi":"10.1016/j.ymeth.2025.09.009","DOIUrl":"https://doi.org/10.1016/j.ymeth.2025.09.009","url":null,"abstract":"","PeriodicalId":390,"journal":{"name":"Methods","volume":" ","pages":""},"PeriodicalIF":4.3,"publicationDate":"2025-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145184424","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":"Integrated isotyping and CDR identification of mouse monoclonal antibodies using multiplex RT-PCR","authors":"Junmo Hwang ,&nbsp;Eunbi Kim ,&nbsp;Jina Kim ,&nbsp;Sujin Shin ,&nbsp;Hyun-Ho Lim","doi":"10.1016/j.ymeth.2025.09.008","DOIUrl":"10.1016/j.ymeth.2025.09.008","url":null,"abstract":"<div><div>The complementarity-determining regions (CDRs) of monoclonal antibodies are essential for antigen recognition and antibody engineering. Accurate determination of CDR sequences typically requires cDNA synthesis from hybridoma-derived mRNA followed by sequencing of the variable regions. However, murine monoclonal antibodies are composed of diverse heavy and light chain isotypes, necessitating prior isotype determination to select appropriate primers for cDNA synthesis. Conventional workflows rely on immunoassays for isotype identification, which adds time and complexity. Here, we developed a streamlined, isotype-independent workflow for the molecular characterization of mouse monoclonal antibodies. A multiplex set of reverse transcription primers (Multiplex-RT) incorporating a universal adaptor sequence was designed to enable cDNA synthesis across major murine isotypes without prior isotype knowledge. Variable regions were subsequently amplified by isotype-specific PCR (Iso-PCR), allowing identification of antibody isotypes, IgG subclasses, and CDR sequences in a single workflow. We applied this method to characterize a murine antibody targeting the astrocytic membrane protein MLC1 and engineered a human-mouse chimeric antibody by grafting murine CDRs onto a human IgG1 backbone. The chimeric antibody retained antigen-binding activity, as demonstrated by immunoprecipitation and immunoblotting. This workflow provides a rapid and reliable strategy for sequencing and isotyping mouse monoclonal antibodies and facilitates downstream applications in antibody discovery, recombinant production, and engineering.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"244 ","pages":"Pages 134-142"},"PeriodicalIF":4.3,"publicationDate":"2025-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145155811","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":"Analytical capability of Raman spectroscopy to detect biochemical changes in red blood cell membrane disorders","authors":"Panchanil Sarmah ,&nbsp;Ruchee Khanna ,&nbsp;Aseefhali Bankapur","doi":"10.1016/j.ymeth.2025.09.007","DOIUrl":"10.1016/j.ymeth.2025.09.007","url":null,"abstract":"<div><div>Detection of RBC membrane disorders with currently available modalities, such as osmotic fragility test (OFT), EMA binding test, ektacytometry, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and next-generation sequencing (NGS), has been challenging as they either lack biochemical inferences or are complex in nature. Raman spectroscopy, a highly analytical method known to produce molecular fingerprints, has proven potential in extracting biochemical information from single individual cells. Recent advancements in membrane-targeted Raman measurements using excitation spots of donut and line intensity profiles can transform lab-on-chip Raman-activated cell sorting methods into a potential technique for RBC membrane disorder diagnosis. We, therefore, conjecture that Raman spectroscopy can be a strong contender as a diagnostic modality in RBC membranopathies. In this comprehensive review<strong>,</strong> we have attempted to encompass the disorder-specific molecular defects, present diagnostic modalities, and their limitations, and explored the translational possibility of Raman spectroscopy as a diagnostic tool for membranopathies.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"244 ","pages":"Pages 118-133"},"PeriodicalIF":4.3,"publicationDate":"2025-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145124154","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":"Advancing antisense oligonucleotide delivery through click chemistry based chemical conjugation with designed short non-cationic peptides for Duchenne muscular dystrophy","authors":"Surojit Ghosh ,&nbsp;Mohammad Umar Arshi ,&nbsp;Satyajit Ghosh ,&nbsp;Moumita Jash ,&nbsp;Nabanita Mukherjee ,&nbsp;Aniket Jana ,&nbsp;Arun Shastry ,&nbsp;Deepika Karanth ,&nbsp;Khushbu Nishad ,&nbsp;Nirmal Kumar Rana ,&nbsp;Sudipta Bhattacharyya ,&nbsp;Surajit Ghosh","doi":"10.1016/j.ymeth.2025.09.005","DOIUrl":"10.1016/j.ymeth.2025.09.005","url":null,"abstract":"<div><div>Duchenne muscular dystrophy (DMD) is a fatal X-linked neuromuscular disease caused by frame shift mutations in the gene encoding dystrophin. 2́-O-methyl phosphorothioate (2′-OMePS) serves as an antisense RNA platform clinically used in DMD patients to facilitate exon skipping and production of an internally truncated, yet functional dystrophin protein. Effective delivery and uptake of antisense oligonucleotides (ASOs) by target cells are crucial for their efficacy. Peptide-conjugated ASOs offer a promising next-generation platform, where a cell-penetrating peptide (CPP) is linked to the 2′-OMePS backbone to enhance cellular uptake. Herein, we designed and synthesized a new non-cationic short CPP sequence that can be efficiently conjugated with the negatively charged 2′-OMePS ASO backbone using click chemistry. Conjugation of the lead peptide ETWWK to 2′-OMePS ASO resulted in significant cellular internalization with precise nuclear localization of the ASO cargo. Cellular uptake was assessed in C2C12 and human DMD patient-derived myoblast cells via fluorescence microscopy and flow cytometry. Additionally, the synthesized ETWWK-ASO conjugate exhibits a significant 1.94 fold upregulation of dystrophin protein in the clinically relevant DMD patient-derived cell line. Our findings suggest that the identified peptide holds promise for facilitating ASO delivery at the site of splicing. This study highlights the efficient conjugation of CPPs to negatively charged 2′-OMePS ASO through tailored conjugation strategies, and will eventually be a therapeutic avenue for future ASO-based DMD treatments.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"244 ","pages":"Pages 92-107"},"PeriodicalIF":4.3,"publicationDate":"2025-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145079293","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}