Pub Date : 2026-02-01Epub Date: 2025-12-11DOI: 10.1016/j.ymeth.2025.12.003
Olufola O. Ige , Thordur Hendrickson-Rebizant , Wenxia Luo , Marvellous Oyeyode , Stefan Jacobson , Fryda Malinalli Ortiz Bada , Michael J. Rowley , Rui Wen Liu , James R Davie , Adam Frankel , Ted M. Lakowski
Nucleosomes, composed of DNA and histone octamers, regulate gene expression through histone modifications such as lysine acetylation and methylation. These modifications are added by writers, removed by erasers, and interpreted by readers to control gene expression, chromatin structure, and essential cellular processes such as differentiation and development. Accurate Post Translational modifications analysis requires high-purity histones due to the sensitivity of epigenetic assays. Recombinant histones are expressed in E. coli as inclusion bodies, requiring denaturation, refolding, and purification. These traditional purification methods involve complicated and lengthy protocols taking days and potentially exposing the histones to oxidation and proteolytic degradation. We developed a rapid method for refolding histones from inclusion bodies in a one-step purification using a desalting column achieving > 90 % purity. This method is compared to our previous High Performance Liquid Chromatography (HPLC)-based protocol. Our single-step desalting purification reduces purification time from multiple days to one day, lowers operational cost, and eliminates the need for reverse-phase HPLC, making high-purity histone production accessible to laboratories without specialized chromatography infrastructure.
{"title":"Denaturation, rapid dilution refolding, and single-step purification of the core histones using desalting size-exclusion chromatography","authors":"Olufola O. Ige , Thordur Hendrickson-Rebizant , Wenxia Luo , Marvellous Oyeyode , Stefan Jacobson , Fryda Malinalli Ortiz Bada , Michael J. Rowley , Rui Wen Liu , James R Davie , Adam Frankel , Ted M. Lakowski","doi":"10.1016/j.ymeth.2025.12.003","DOIUrl":"10.1016/j.ymeth.2025.12.003","url":null,"abstract":"<div><div>Nucleosomes, composed of DNA and histone octamers, regulate gene expression through histone modifications such as lysine acetylation and methylation. These modifications are added by writers, removed by erasers, and interpreted by readers to control gene expression, chromatin structure, and essential cellular processes such as differentiation and development. Accurate Post Translational modifications analysis requires high-purity histones due to the sensitivity of epigenetic assays. Recombinant histones are expressed in <em>E. coli</em> as inclusion bodies, requiring denaturation, refolding, and purification. These traditional purification methods involve complicated and lengthy protocols taking days and potentially exposing the histones to oxidation and proteolytic degradation. We developed a rapid method for refolding histones from inclusion bodies in a one-step purification using a desalting column achieving > 90 % purity. This method is compared to our previous High Performance Liquid Chromatography (HPLC)-based protocol. Our single-step desalting purification reduces purification time from multiple days to one day, lowers operational cost, and eliminates the need for reverse-phase HPLC, making high-purity histone production accessible to laboratories without specialized chromatography infrastructure.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"246 ","pages":"Pages 142-150"},"PeriodicalIF":4.3,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145740425","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}
Epigenome‐wide association studies (EWAS) are instrumental for mapping DNA methylation changes in human traits and diseases but often suffer from low statistical power and false positives, especially in small cohorts. We developed an EWAS smoothing method that exploits co‐methylation of adjacent CpG probes within CpG islands via a sliding‐window average and generalized it using Savitzky-Golay filtering. We applied the smoothing approach—with window widths of 1–3 CpGs and, for generalization, Savitzky-Golay filters of varying polynomial orders and window sizes—across five distinct EWAS settings. Performance was quantified by signal‐to‐noise ratio (SNR), noise‐variance reduction, variance ratio (VR), Bayes factors, and sample‐size sensitivity. In the MMACHC epimutation dataset, a 5‐CpG window (width, w = 2) increased SNR by 90 %, reduced noise variance by 80 %, and elevated VR by 176 % at the target CpG island, with no genome‐wide false positives. For MLH1, smoothing preserved the top association and suppressed background signals. In the aging EWAS, a “Polyepigenetic CpG aging score” was derived following smoothing. This score correlated strongly with chronological age in the discovery cohort (Spearman’s ρ = 0.89; P = 3.0 × 10−219) and was independently validated in a separate dataset, significantly distinguishing newborns from nonagenarians (P = 3.4 × 10−8). Savitzky-Golay filtering of order 0 with a 5‐CpG window yielded optimal SNR across bootstrap iterations, supporting this configuration as a robust choice for methylation array smoothing. As an extension of the Savitzky-Golay-based smoothing framework, reanalysis of a liver cancer dataset identified five top loci surpassing a smoothed P-value threshold of 1 × 10−8. Among these, MIR10A within the HOXB3 locus was the only previously reported functionally relevant site. In conclusion, the smoothing method improves EWAS performance by enhancing SNR, enabling detection of meaningful associations even in small cohorts, and offers a valuable tool for reanalyzing existing Infinium methylation array datasets to uncover previously undetected epigenomic signatures.
{"title":"A smoothing method for DNA methylome analysis to enhance epigenomic signature detection in epigenome-wide association studies","authors":"Abderrahim Oussalah , Loris Mousel , David-Alexandre Trégouët , Jean-Louis Guéant","doi":"10.1016/j.ymeth.2025.11.005","DOIUrl":"10.1016/j.ymeth.2025.11.005","url":null,"abstract":"<div><div>Epigenome‐wide association studies (EWAS) are instrumental for mapping DNA methylation changes in human traits and diseases but often suffer from low statistical power and false positives, especially in small cohorts. We developed an EWAS smoothing method that exploits co‐methylation of adjacent CpG probes within CpG islands via a sliding‐window average and generalized it using Savitzky-Golay filtering. We applied the smoothing approach—with window widths of 1–3 CpGs and, for generalization, Savitzky-Golay filters of varying polynomial orders and window sizes—across five distinct EWAS settings. Performance was quantified by signal‐to‐noise ratio (SNR), noise‐variance reduction, variance ratio (VR), Bayes factors, and sample‐size sensitivity. In the <em>MMACHC</em> epimutation dataset, a 5‐CpG window (width, <em>w</em> = 2) increased SNR by 90 %, reduced noise variance by 80 %, and elevated VR by 176 % at the target CpG island, with no genome‐wide false positives. For <em>MLH1</em>, smoothing preserved the top association and suppressed background signals. In the aging EWAS, a “Polyepigenetic CpG aging score” was derived following smoothing. This score correlated strongly with chronological age in the discovery cohort (Spearman’s <em>ρ</em> = 0.89; <em>P</em> = 3.0 × 10<sup>−219</sup>) and was independently validated in a separate dataset, significantly distinguishing newborns from nonagenarians (<em>P</em> = 3.4 × 10<sup>−8</sup>). Savitzky-Golay filtering of order 0 with a 5‐CpG window yielded optimal SNR across bootstrap iterations, supporting this configuration as a robust choice for methylation array smoothing. As an extension of the Savitzky-Golay-based smoothing framework, reanalysis of a liver cancer dataset identified five top loci surpassing a smoothed <em>P</em>-value threshold of 1 × 10<sup>−8</sup>. Among these, <em>MIR10A</em> within the <em>HOXB3</em> locus was the only previously reported functionally relevant site. In conclusion, the smoothing method improves EWAS performance by enhancing SNR, enabling detection of meaningful associations even in small cohorts, and offers a valuable tool for reanalyzing existing Infinium methylation array datasets to uncover previously undetected epigenomic signatures.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"246 ","pages":"Pages 34-47"},"PeriodicalIF":4.3,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145581598","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}
Targeting the interaction between P53 and MDM2 to re-activate P53 to induce apoptosis is an important strategy for cancer treatment. In this study, based on the unique advantages of in situ visualization, dynamic imaging, and quantitative analysis of living cell FRET imaging, a method for screening apoptotic drugs targeting p53-MDM2 interaction was developed. A stable model of Nutlin-3-induced apoptosis was established in MCF-7 cells, which was verified by reducing mitochondrial membrane potential and increasing the proportion of nuclear chromatin condensation (from 9.16 % to 50.55 %). Biochemical methods such as WB analysis found that after activating P53, BAX expression was up-regulated through a Puma-independent pathway, which promoted BAX oligomerization. Live-cell quantitative FRET imaging found that the maximum donor center FRET efficiency (EDmax) of CFP-p53 and YFP-MDM2 decreased from 0.50 to 0.22 after Nutlin-3 treatment, and the co-localization coefficient decreased significantly from 83 % to 22 %, confirmed that Nutlin-3 directly disrupted the interaction between P53/MDM2, promoting P53 nuclear translocation and apoptosis. This indicated that Nutlin-3 was a direct inhibitor of the P53/MDM2 interaction. Apoptosis drug screening was performed in MCF-7 cells, and we found that the EDmax was 0.29 and 0.31 for the cells treated with DOX and RSV, respectively, and 0.48 for the IKE-treated cells and 0.43 for the SOR-treated cells, indicating that DOX and RSV, but not IKE and SOR, were potential P53/MDM2-dependent apoptotic drugs. In addition, Nutlin-3 treatment decreased the EDmax value in p53 wild-type U2OS cells from 0.43 to 0.20. In summary, our method can identify p53-MDM2 interaction inhibitors in living cells, providing a quantitative in vivo supplement for traditional target-based drug discovery.
{"title":"MDM2/p53-based live-cell quantitative FRET imaging for apoptosis drug discovery","authors":"Zhiyu Xiao, Lingmin Xie, Ziru Wu, Xinghong Cai, Zhengfei Zhuang, Tongsheng Chen","doi":"10.1016/j.ymeth.2025.12.002","DOIUrl":"10.1016/j.ymeth.2025.12.002","url":null,"abstract":"<div><div>Targeting the interaction between P53 and MDM2 to re-activate P53 to induce apoptosis is an important strategy for cancer treatment. In this study, based on the unique advantages of in situ visualization, dynamic imaging, and quantitative analysis of living cell FRET imaging, a method for screening apoptotic drugs targeting p53-MDM2 interaction was developed. A stable model of Nutlin-3-induced apoptosis was established in MCF-7 cells, which was verified by reducing mitochondrial membrane potential and increasing the proportion of nuclear chromatin condensation (from 9.16 % to 50.55 %). Biochemical methods such as WB analysis found that after activating P53, BAX expression was up-regulated through a Puma-independent pathway, which promoted BAX oligomerization. Live-cell quantitative FRET imaging found that the maximum donor center FRET efficiency (<em>E<sub>Dmax</sub></em>) of CFP-p53 and YFP-MDM2 decreased from 0.50 to 0.22 after Nutlin-3 treatment, and the co-localization coefficient decreased significantly from 83 % to 22 %, confirmed that Nutlin-3 directly disrupted the interaction between P53/MDM2, promoting P53 nuclear translocation and apoptosis. This indicated that Nutlin-3 was a direct inhibitor of the P53/MDM2 interaction. Apoptosis drug screening was performed in MCF-7 cells, and we found that the <em>E<sub>Dmax</sub></em> was 0.29 and 0.31 for the cells treated with DOX and RSV, respectively, and 0.48 for the IKE-treated cells and 0.43 for the SOR-treated cells, indicating that DOX and RSV, but not IKE and SOR, were potential P53/MDM2-dependent apoptotic drugs. In addition, Nutlin-3 treatment decreased the <em>E<sub>Dma</sub></em><sub>x</sub> value in p53 wild-type U2OS cells from 0.43 to 0.20. In summary, our method can identify p53-MDM2 interaction inhibitors in living cells, providing a quantitative in vivo supplement for traditional target-based drug discovery.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"246 ","pages":"Pages 107-115"},"PeriodicalIF":4.3,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145695665","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}
The Golgi complex is central to cellular homeostasis and serves as a key processing and sorting hub for protein trafficking. In many cell types, the Golgi complex is organized as interconnected stacks of cisternae, forming a structure known as the Golgi ribbon. This ribbon undergoes dynamic remodelling during physiological processes, such as cell division, and under pathological conditions, including cancer and neurodegeneration. A critical step in the unlinking of the Golgi ribbon involves the phosphorylation of the stacking protein GRASP65, which leads to the separation of the ribbon into individual stacks, a process necessary for the G2/M transition of the cell cycle. However, existing tools for selectively manipulating the GRASP65 role in ribbon organization are limited by non-specific effects or technical challenges.
Here, we present the development and characterization of a membrane-permeable peptide, R8-GRASP65-S277, derived from GRASP65 and containing the phosphorylation site Ser277, which is essential for Golgi unlinking. This peptide effectively inhibited Golgi unlinking and mitotic entry in several cell lines, including cancer models. In contrast, a control peptide with a non-phosphorylatable alanine substitution (R8-GRASP65-S277A) showed no such effect, confirming the specificity of the tool. Furthermore, the R8-GRASP65-S277 peptide reversed Golgi unlinking induced by the chemotherapeutic agent doxorubicin, demonstrating its utility in studying stress-induced Golgi disassembly.
These findings establish the R8-GRASP65-S277 peptide as a specific, potent, and scalable tool for probing the molecular mechanisms of Golgi unlinking, its regulation of cell cycle progression, and its potential contributions to pathological states.
{"title":"Development and characterization of a membrane-permeant GRASP65-mimetic peptide that inhibits Golgi unlinking and cell cycle progression","authors":"Romina Ines Cervigni , Raffaella Bonavita , Maria Luisa Barretta , Daniela Spano , Inmaculada Ayala , Fabiola Mascanzoni , Roberta Iannitti , Petra Henklein , Alessandra Monti , Maurizio Renna , Nunzianna Doti , Antonino Colanzi","doi":"10.1016/j.ymeth.2025.11.008","DOIUrl":"10.1016/j.ymeth.2025.11.008","url":null,"abstract":"<div><div>The Golgi complex is central to cellular homeostasis and serves as a key processing and sorting hub for protein trafficking. In many cell types, the Golgi complex is organized as interconnected stacks of cisternae, forming a structure known as the Golgi ribbon. This ribbon undergoes dynamic remodelling during physiological processes, such as cell division, and under pathological conditions, including cancer and neurodegeneration. A critical step in the unlinking of the Golgi ribbon involves the phosphorylation of the stacking protein GRASP65, which leads to the separation of the ribbon into individual stacks, a process necessary for the G2/M transition of the cell cycle. However, existing tools for selectively manipulating the GRASP65 role in ribbon organization are limited by non-specific effects or technical challenges.</div><div>Here, we present the development and characterization of a membrane-permeable peptide, R<sub>8</sub>-GRASP65-S277, derived from GRASP65 and containing the phosphorylation site Ser277, which is essential for Golgi unlinking. This peptide effectively inhibited Golgi unlinking and mitotic entry in several cell lines, including cancer models. In contrast, a control peptide with a non-phosphorylatable alanine substitution (R<sub>8</sub>-GRASP65-S277A) showed no such effect, confirming the specificity of the tool. Furthermore, the R<sub>8</sub>-GRASP65-S277 peptide reversed Golgi unlinking induced by the chemotherapeutic agent doxorubicin, demonstrating its utility in studying stress-induced Golgi disassembly.</div><div>These findings establish the R8-GRASP65-S277 peptide as a specific, potent, and scalable tool for probing the molecular mechanisms of Golgi unlinking, its regulation of cell cycle progression, and its potential contributions to pathological states.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"246 ","pages":"Pages 130-141"},"PeriodicalIF":4.3,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145666546","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}
Exome sequencing (ES) has transformed genomic research and clinical diagnostics by enabling precise identification of disease-associated variants within protein-coding regions, which, while representing a minority of the genome, include many well-characterized pathogenic mutations. This review provides a comprehensive overview of ES methodology, data analysis pipelines, clinical relevance, and ethical considerations. We describe the ES workflow from DNA extraction and library preparation to target enrichment, sequencing to ES data analysis. We have also evaluated major capture technologies and sequencing platforms, including short-read and emerging long-read systems. Furthermore, we discuss computational analysis tools such as GATK, FreeBayes, DeepVariant, and Platypus, and strategies to improve accuracy through rigorous quality control, coverage optimization, and orthogonal validation. Beyond rare disease and cancer genomics, ES has expanded into pharmacogenomics, population-scale studies, and integrative multi-omics frameworks that combine transcriptomic and proteomic data to enhance functional interpretation. We highlight actionable examples such as CYP2C19 variants influencing clopidogrel metabolism, illustrating ES’s growing role in personalized medicine. Challenges (including variant interpretation complexity, false positives, and data standardization) are critically discussed. The review also addresses ethical, legal, and social dimensions of ES, including informed consent, data privacy, incidental findings, and adherence to ACMG, HIPAA, and GDPR. Finally, we outline future directions emphasizing machine learning–based variant prioritization, single-cell sequencing integration, and scalable bioinformatics infrastructures to enhance accuracy and clinical translation. Collectively, these developments position ES as a pivotal tool bridging genomic discovery, disease diagnostics, and precision healthcare in the era of personalized medicine.
{"title":"From sample to clinical insight: a review of exome sequencing in disease diagnostics","authors":"Gowrang Kasaba Manjunath , Rohit Kumar Verma , Abhijit Berua , Shweta Mahalingam , Tikam Chand Dakal , Abhishek Kumar","doi":"10.1016/j.ymeth.2025.11.007","DOIUrl":"10.1016/j.ymeth.2025.11.007","url":null,"abstract":"<div><div>Exome sequencing (ES) has transformed genomic research and clinical diagnostics by enabling precise identification of disease-associated variants within protein-coding regions, which, while representing a minority of the genome, include many well-characterized pathogenic mutations. This review provides a comprehensive overview of ES methodology, data analysis pipelines, clinical relevance, and ethical considerations. We describe the ES workflow from DNA extraction and library preparation to target enrichment, sequencing to ES data analysis. We have also evaluated major capture technologies and sequencing platforms, including short-read and emerging long-read systems. Furthermore, we discuss computational analysis tools such as GATK, FreeBayes, DeepVariant, and Platypus, and strategies to improve accuracy through rigorous quality control, coverage optimization, and orthogonal validation. Beyond rare disease and cancer genomics, ES has expanded into pharmacogenomics, population-scale studies, and integrative multi-omics frameworks that combine transcriptomic and proteomic data to enhance functional interpretation. We highlight actionable examples such as CYP2C19 variants influencing clopidogrel metabolism, illustrating ES’s growing role in personalized medicine. Challenges (including variant interpretation complexity, false positives, and data standardization) are critically discussed. The review also addresses ethical, legal, and social dimensions of ES, including informed consent, data privacy, incidental findings, and adherence to ACMG, HIPAA, and GDPR. Finally, we outline future directions emphasizing machine learning–based variant prioritization, single-cell sequencing integration, and scalable bioinformatics infrastructures to enhance accuracy and clinical translation. Collectively, these developments position ES as a pivotal tool bridging genomic discovery, disease diagnostics, and precision healthcare in the era of personalized medicine.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"246 ","pages":"Pages 12-33"},"PeriodicalIF":4.3,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145572733","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}
Pub Date : 2026-02-01Epub Date: 2025-12-19DOI: 10.1016/j.ymeth.2025.12.009
Unai Montejo , Gorane Beldarrain , Sheila Olza , Jon Ander Alart , Marc Chillida , Ignacio García-Alonso , Daniel Alonso-Alconada , Ana Alonso-Varona , Borja Herrero de la Parte
Catalase is a key antioxidant enzyme that protects cells from oxidative stress by decomposing hydrogen peroxide (H2O2). Traditional methods for quantifying catalase activity, such as the Aebi UV method and the dichromate assay, are limited by low sensitivity, interference from biological molecules and require large sample and reagent volumes. Here, we present an optimized, microplate assay based on the oxidation of cobalt(II) to cobalt(III) by H2O2, followed by complexation with ethylenediamine-tetraacetic acid (EDTA) to form a highly colored Co(III)-EDTA complex measurable at 570 nm. This method demonstrates superior sensitivity and dynamic range (10–0.039 U/mL) compared to previous colorimetric assays, while minimizing reaction volume and avoiding interference from common biological substances. The assay has been validated using standard bovine liver catalase, liver homogenates, serum and cell lysates, showing strong linearity and specificity. This approach offers a rapid, cost-effective, and robust alternative for catalase activity measurement in biomedical research.
{"title":"Microplate assay for the quantification of catalase activity in biological samples","authors":"Unai Montejo , Gorane Beldarrain , Sheila Olza , Jon Ander Alart , Marc Chillida , Ignacio García-Alonso , Daniel Alonso-Alconada , Ana Alonso-Varona , Borja Herrero de la Parte","doi":"10.1016/j.ymeth.2025.12.009","DOIUrl":"10.1016/j.ymeth.2025.12.009","url":null,"abstract":"<div><div>Catalase is a key antioxidant enzyme that protects cells from oxidative stress by decomposing hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>). Traditional methods for quantifying catalase activity, such as the Aebi UV method and the dichromate assay, are limited by low sensitivity, interference from biological molecules and require large sample and reagent volumes. Here, we present an optimized, microplate assay based on the oxidation of cobalt(II) to cobalt(III) by H<sub>2</sub>O<sub>2</sub>, followed by complexation with ethylenediamine-tetraacetic acid (EDTA) to form a highly colored Co(III)-EDTA complex measurable at 570 nm. This method demonstrates superior sensitivity and dynamic range (10–0.039 U/mL) compared to previous colorimetric assays, while minimizing reaction volume and avoiding interference from common biological substances. The assay has been validated using standard bovine liver catalase, liver homogenates, serum and cell lysates, showing strong linearity and specificity. This approach offers a rapid, cost-effective, and robust alternative for catalase activity measurement in biomedical research.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"246 ","pages":"Pages 186-194"},"PeriodicalIF":4.3,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145802874","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}
Pub Date : 2026-02-01Epub Date: 2025-11-18DOI: 10.1016/j.ymeth.2025.10.011
Archana Mathur , Abbas Mufaddal Dudhiyawala , Sudeepa Roy Dey , Snehanshu Saha
The innovations in classifying breast cancer into malignant and benign categories and further categorizing it into molecular subtypes have reshaped healthcare services, enabling accurate diagnosis of these complex conditions. Identification of molecular subtypes of breast cancer is one of the most important treatment challenges, as these subtypes can have an enormous effect on the prognosis and treatment approaches. Data integration from various modalities, such as transcriptomics, imaging, and genomics, has been crucial in leveraging new opportunities to increase classification accuracy and improve individualized treatment plans. These heterogeneous data sources are examined by applying deep learning algorithms, which provide further insights into the complex patterns that traditional approaches often overlook. In this paper, we explore the various modalities researchers use to investigate breast cancer and the intriguing fusion techniques employed to combine these modalities. We also review the most recent models (traditional, machine learning, and deep learning), emphasizing their improvements over traditional classification methods and the molecular subtype categorization of breast cancer. Furthermore, the emphasis of this review is to examine techniques to process the entire image of the breast tissue slide, which is challenging, particularly due to its size. We explore recent advances in multiple instance learning tasks and the use of attention-based transformers and similar architectures for annotating the WSI slides before using them for cancer classification. We additionally discuss the interpretability tools—attention maps, saliency maps and model explainability— in the context of transformers. In a nutshell, we aim to provide an in-depth look at the revolutionary capabilities of deep learning models in precision oncology and guide future research paths in this crucial field by synthesizing existing studies.
{"title":"Toward accurate breast cancer classification: A review of multi-modal machine learning approaches","authors":"Archana Mathur , Abbas Mufaddal Dudhiyawala , Sudeepa Roy Dey , Snehanshu Saha","doi":"10.1016/j.ymeth.2025.10.011","DOIUrl":"10.1016/j.ymeth.2025.10.011","url":null,"abstract":"<div><div>The innovations in classifying breast cancer into malignant and benign categories and further categorizing it into molecular subtypes have reshaped healthcare services, enabling accurate diagnosis of these complex conditions. Identification of molecular subtypes of breast cancer is one of the most important treatment challenges, as these subtypes can have an enormous effect on the prognosis and treatment approaches. Data integration from various modalities, such as transcriptomics, imaging, and genomics, has been crucial in leveraging new opportunities to increase classification accuracy and improve individualized treatment plans. These heterogeneous data sources are examined by applying deep learning algorithms, which provide further insights into the complex patterns that traditional approaches often overlook. In this paper, we explore the various modalities researchers use to investigate breast cancer and the intriguing fusion techniques employed to combine these modalities. We also review the most recent models (traditional, machine learning, and deep learning), emphasizing their improvements over traditional classification methods and the molecular subtype categorization of breast cancer. Furthermore, the emphasis of this review is to examine techniques to process the entire image of the breast tissue slide, which is challenging, particularly due to its size. We explore recent advances in multiple instance learning tasks and the use of attention-based transformers and similar architectures for annotating the WSI slides before using them for cancer classification. We additionally discuss the interpretability tools—attention maps, saliency maps and model explainability— in the context of transformers. In a nutshell, we aim to provide an in-depth look at the revolutionary capabilities of deep learning models in precision oncology and guide future research paths in this crucial field by synthesizing existing studies.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"246 ","pages":"Pages 48-61"},"PeriodicalIF":4.3,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145562223","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}
Pub Date : 2026-02-01Epub Date: 2025-12-15DOI: 10.1016/j.ymeth.2025.12.005
Jing Yi Lai , Kae Yi Tan , Choo Hock Tan , Yee Siew Choong , Theam Soon Lim
Monoclonal antibodies are commonly expressed using mammalian cell lines due to their required post-translational modifications. However, this process can become costly and requires investment in specialized equipment and facilities. The use of eukaryotic cell Leishmania tarentolae offers a cost-effective alternative while still allowing N-linked glycosylation to occur. The focus of this study explores the use of L. tarentolae as a platform for expressing a monoclonal IgG antibody against the long-chain neurotoxin (LNTX) from the venom of the Monocled Cobra, Naja kaouthia. The monoclonal antibody was isolated from a human naïve phage display library and expressed as single-chain fragment variable (scFv) in Escherichia coli with good specificity against the LNTX of Naja kaouthia. The scFv was then converted to the scFv-Fc format and full-length IgG for expression in L. tarentolae. The IgG expression was achieved using a 2A peptide-based bicistronic vector system with good expression yield. The expressed IgG demonstrated homogeneity in size and minimal degradation. Therefore, L. tarentolae can be considered a possible alternative platform for the expression of human scFv-Fc and IgG antibodies.
单克隆抗体通常在哺乳动物细胞系中表达，因为它们需要翻译后修饰。然而，这个过程可能会变得昂贵，需要投资专门的设备和设施。使用真核细胞利什曼绦虫提供了一种具有成本效益的替代方案，同时仍然允许n -链糖基化发生。本研究的重点是探索利用L. tarentolae作为表达单克隆IgG抗体的平台，抗来自单眼眼镜蛇Naja kaouthia毒液的长链神经毒素（LNTX）。该单克隆抗体从人naïve噬菌体展示文库中分离得到，在大肠杆菌中以单链片段变量（single-chain fragment variable, scFv）的形式表达，具有较好的特异性。然后将scFv转化为scFv- fc格式和全长IgG，在猪乳杆菌中表达。IgG的表达采用基于2A肽的双链载体系统，表达量高。表达的IgG大小均匀，降解程度最低。因此，可以将链乳杆菌视为表达人scFv-Fc和IgG抗体的可能替代平台。
{"title":"Application of a 2A-peptide system for polycistronic human IgG production in Leishmania tarentolae","authors":"Jing Yi Lai , Kae Yi Tan , Choo Hock Tan , Yee Siew Choong , Theam Soon Lim","doi":"10.1016/j.ymeth.2025.12.005","DOIUrl":"10.1016/j.ymeth.2025.12.005","url":null,"abstract":"<div><div>Monoclonal antibodies are commonly expressed using mammalian cell lines due to their required post-translational modifications. However, this process can become costly and requires investment in specialized equipment and facilities. The use of eukaryotic cell <em>Leishmania tarentolae</em> offers a cost-effective alternative while still allowing N-linked glycosylation to occur. The focus of this study explores the use of <em>L. tarentolae</em> as a platform for expressing a monoclonal IgG antibody against the long-chain neurotoxin (LNTX) from the venom of the Monocled Cobra, <em>Naja kaouthia</em>. The monoclonal antibody was isolated from a human naïve phage display library and expressed as single-chain fragment variable (scFv) in <em>Escherichia coli</em> with good specificity against the LNTX of <em>Naja kaouthia</em>. The scFv was then converted to the scFv-Fc format and full-length IgG for expression in <em>L. tarentolae</em>. The IgG expression was achieved using a 2A peptide-based bicistronic vector system with good expression yield. The expressed IgG demonstrated homogeneity in size and minimal degradation. Therefore, <em>L. tarentolae</em> can be considered a possible alternative platform for the expression of human scFv-Fc and IgG antibodies.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"246 ","pages":"Pages 174-185"},"PeriodicalIF":4.3,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145773170","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}
Pub Date : 2026-02-01Epub Date: 2025-11-28DOI: 10.1016/j.ymeth.2025.11.009
Tejas S. Patil , Deepvardhan P. Chaudhari , Utkarsh U. Bhamare , Mahesh B. Palkar , Mahendra R. Mahajan , Sopan N. Nangare
Millions of women worldwide suffer from a variety of health conditions, including vaginal bacterial and yeast infections, sexually transmitted infections (STIs), urinary tract infections, pelvic inflammatory disorders, and hormonal abnormalities. Despite major advances in biomedical research, traditional intravaginal drug administration systems such as gels, creams, and suppositories frequently encounter issues such as fast drug clearance, leakage, and uneven mucosal retention, reducing therapeutic effectiveness. Microneedles, a painless and less invasive drug delivery technology, represent a viable alternative to standard formulations because they allow for accurate, controlled, and localized drug administration. Therefore, the present review focuses on possibility of microneedle-based techniques for intravaginal medication administration. In brief, it covers the need for breakthroughs in vaginal drug administration. It provides an overview of several types of microneedles, including solid, hollow, dissolving, coated, and hydrogel-forming, and their manufacturing procedures. Then, it delves into their use in intravaginal drug administration, highlighting their capacity to improve drug penetration and retention. Finally, it discusses future problems, prospective advancements, and the larger implications of microneedle technology in vaginal therapy. Microneedle-based intravaginal medication delivery is a huge step forward in targeted vaginal infection treatment. Notably, microneedles easily cross the cervicovaginal mucus barrier, increasing drug absorption at the target region while being minimally invasive. Future studies should focus on improving microneedle formulations, assessing long-term safety, and investigating their potential for wider clinical applications.
{"title":"Fabrication of advanced microneedle-based targeted intravaginal drug delivery devices: therapeutic opportunities and translational challenges","authors":"Tejas S. Patil , Deepvardhan P. Chaudhari , Utkarsh U. Bhamare , Mahesh B. Palkar , Mahendra R. Mahajan , Sopan N. Nangare","doi":"10.1016/j.ymeth.2025.11.009","DOIUrl":"10.1016/j.ymeth.2025.11.009","url":null,"abstract":"<div><div>Millions of women worldwide suffer from a variety of health conditions, including vaginal bacterial and yeast infections, sexually transmitted infections (STIs), urinary tract infections, pelvic inflammatory disorders, and hormonal abnormalities. Despite major advances in biomedical research, traditional intravaginal drug administration systems such as gels, creams, and suppositories frequently encounter issues such as fast drug clearance, leakage, and uneven mucosal retention, reducing therapeutic effectiveness. Microneedles, a painless and less invasive drug delivery technology, represent a viable alternative to standard formulations because they allow for accurate, controlled, and localized drug administration. Therefore, the present review focuses on possibility of microneedle-based techniques for intravaginal medication administration. In brief, it covers the need for breakthroughs in vaginal drug administration. It provides an overview of several types of microneedles, including solid, hollow, dissolving, coated, and hydrogel-forming, and their manufacturing procedures. Then, it delves into their use in intravaginal drug administration, highlighting their capacity to improve drug penetration and retention. Finally, it discusses future problems, prospective advancements, and the larger implications of microneedle technology in vaginal therapy. Microneedle-based intravaginal medication delivery is a huge step forward in targeted vaginal infection treatment. Notably, microneedles easily cross the cervicovaginal mucus barrier, increasing drug absorption at the target region while being minimally invasive. Future studies should focus on improving microneedle formulations, assessing long-term safety, and investigating their potential for wider clinical applications.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"246 ","pages":"Pages 62-82"},"PeriodicalIF":4.3,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145647037","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}
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