Methods最新文献

{"title":"Nanomaterial-based electrochemical sensors for phenolic antioxidants in foods and beverages: From design to device translation","authors":"Angga Hermawan ,&nbsp;Asmi Aris ,&nbsp;Munawar Khalil ,&nbsp;Ni Luh Wulan Septiani ,&nbsp;Teti Estiasih ,&nbsp;Hamidie Ronald Daniel Ray ,&nbsp;Miguel Palma ,&nbsp;Widiastuti Setyaningsih","doi":"10.1016/j.ymeth.2026.01.008","DOIUrl":"10.1016/j.ymeth.2026.01.008","url":null,"abstract":"<div><div>Nanomaterial-enabled electrochemical sensors are nearing the performance and practicality needed for routine, on-site monitoring of phenolic compounds in foods and beverages. Advances in nanomaterial dimensionality and hybrid architectures, from atomically doped nanoparticles and zero-dimensional clusters to two- and three-dimensional porous frameworks, have enhanced electron-transfer kinetics, expanded electroactive surface area, and enabled more selective surface chemistries. These gains align with progress in molecular recognition using enzymes, aptamers, molecularly imprinted polymers, and permselective antifouling coatings, as well as electrode-engineering strategies that translate nanoscale activity into reliable printed-electrodes. Although laboratory detection limits are often impressive (micromolar to low-nanomolar in controlled media), challenges remain in reproducibility, shelf life, and performance in complex matrices such as wine, olive oil, and fermented foods. Closing these gaps requires integrated solutions that unite printable, stable nanomaterial inks with simple on-cartridge sample conditioning, modular recognition layers, and robust on-board calibration and data-handling routines. To enable practical deployment, we propose a development pathway focused on scalable manufacturing and quality control of nanomaterial inks and electrodes, harmonized validation against chromatographic reference methods, durable antifouling and self-cleaning strategies, and an ecosystem approach that uses smartphone connectivity and cloud analytics to convert electrochemical signals into traceable, defensible decisions for industry, regulators, and consumers.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"247 ","pages":"Pages 132-160"},"PeriodicalIF":4.3,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146049740","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":"In vitro wound simulation: A high-throughput device for scratch assays","authors":"Jacob E. Labovitz ,&nbsp;Patrick Kulaga ,&nbsp;Eric M. DuBois,&nbsp;Kunyu Li,&nbsp;Timothy M. O’Shea","doi":"10.1016/j.ymeth.2025.12.007","DOIUrl":"10.1016/j.ymeth.2025.12.007","url":null,"abstract":"<div><div>Traumatic injury to the healthy central nervous system (CNS) causes mechanical tissue damage that results in localized cell death and blood–brain-barrier (BBB) disruption. CNS tissue damage stimulates a multicellular wound response to limit the extent of damage but fails to reestablish the normal function of injured tissue. There is strong interest in developing new strategies to augment regeneration after CNS injury. To enable therapy development, reliable assays to screen and identify molecular approaches to augment glial-based wound responses over fibrotic scarring are needed. Scratch assays, which involve mechanically removing cells from an <em>in vitro</em> culture, allow for the simulation of wounds with high throughput and tight control over applied treatments to mechanistically study cell migration and proliferation functions that are critical to effective repair. Current methods require researchers to individually scratch each well with a pipette tip, resulting in low throughput as well as inconsistent scratch widths, straightness, and efficacy within and between wells. Here, we describe the design of a quickly assembled (&lt;30 min), inexpensive (&lt;$110) scratch assay rig that readily creates uniform scratches that are straight (average tortuosity &lt; 1.1), have tunable widths (730–1100 µm), and fully remove damaged cells from the simulated wound region. Designed for a 24-well plate, the rig allows for high-throughput screening of varied experimental conditions or for testing many replicates. Application of the scratch assay device on an <em>in vitro</em> culture of neural progenitor cells (NPC) demonstrates the ability to detect differences in wound closure rate for three unique media conditions. These results support the implementation of this high-throughput scratch assay rig as a method to standardize and improve the efficiency of <em>in vitro</em> wound healing studies.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"246 ","pages":"Pages 223-235"},"PeriodicalIF":4.3,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145800327","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":"Investigation on structure–property relationships of MgO-SrO containing silicate-based bioactive glasses: An experimental and molecular dynamics simulation study","authors":"Amirhossein Moghanian ,&nbsp;Ramin Farmani ,&nbsp;Niloufar Kolivand ,&nbsp;Arman Tayebi ,&nbsp;Sirus Safaee","doi":"10.1016/j.ymeth.2025.12.001","DOIUrl":"10.1016/j.ymeth.2025.12.001","url":null,"abstract":"<div><div>Molecular dynamics (MD) simulations and experimental analysis were performed on MgO–SrO containing bioactive glasses (MSBGs) with the composition of 60SiO₂–(31–x)CaO–4P₂O₅–5MgO–xSrO (mol%) (x = 0, 1, 3, 5, 8, 10, 15, 20; M5S0–M5S20) to evaluate the structural properties, ion clustering, and dissolution behavior as a function of SrO content. Simulation employed the Buckingham potential for short-range interactions and Coulombic potentials for long-range forces. MSBGs had Si–O and P–O bond lengths of 1.609 Å and 1.491 Å, with O–Si–O and O–P–O bond angles centered at ∼109.3° and 109.4°, respectively, confirming tetrahedral SiO₄/PO₄ coordination. Across all compositions, Si–O–Si bonds dominated the majority of the distribution (88–89 %), with Si–O–P at 11–12 % and P–O–P negligible (∼0.3 %). Densities decreased from 2.913 g·cm⁻³ (M5S20) to 2.631 g·cm⁻³ (M5S0), reflecting network loosening with SrO substitution. Qⁿ distribution remained stable, with Q³/Q⁴ fractions of 38–43 % and 26–30 % for Si-based tetrahedra. R-factor analysis revealed optimal homogeneity for M5S5 (<span><math><mrow><msubsup><mi>R</mi><mrow><mi>S</mi><mi>i</mi><mo>/</mo><mi>P</mi></mrow><mrow><mi>S</mi><mi>r</mi></mrow></msubsup></mrow></math></span> = 0.838252), balancing reduced cation clustering and moderate network stability. ICP-AES showed M5S5 with a sustained release of Si⁴⁺, Mg²⁺, and Sr²⁺ over 24 h. Meanwhile, antibacterial study resulted in statistically significant increase in efficiency for M5S5 compared to M5S0 (***p &lt; 0.001). The combined computational and experimental findings identify M5S5 as the most promising candidate for biomedical applications requiring structural benefits, controlled ion release, and antibacterial efficiency.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"246 ","pages":"Pages 116-129"},"PeriodicalIF":4.3,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145712861","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":"Explainable machine learning-based prediction of psoriatic arthritis flares using heterogenous real-world data for personalised patient care","authors":"Pradip Moon ,&nbsp;Weizi Li ,&nbsp;Antoni Chan ,&nbsp;Bing Wang ,&nbsp;Eghosa Bazuaye","doi":"10.1016/j.ymeth.2025.10.010","DOIUrl":"10.1016/j.ymeth.2025.10.010","url":null,"abstract":"<div><div>Psoriatic arthritis (PsA) is a chronic inflammatory disease characterised by unpredictable flare-ups that are difficult to forecast, particularly in patients without an acute phase response. In this paper, we propose and apply an explainable, multimodal machine learning framework that jointly leverages structured temporal electronic patient records (EPRs) – sequential blood tests, disease activity scores, comorbidity burden, medications, and demographics – and unstructured clinical referral letters pre-processed with large language models ((LLMs, (Qwen-2.5 family)) to predict PsA flares. Gradient boosting models, Light Gradient Boosting Machine (LGBM) and eXtreme Gradient Boosting (XGBoost) were used to predict PsA flares, achieving the highest predictive performance 3 months before a clinic visit (accuracy = 92.8 %, AUROC = 0.94). Model performance gradually declined for longer timeframes (6 months: 78.2 %, AUROC = 0.80; 9 months: 76.6 %, AUROC = 0.78; 12 months: 72.2 %, AUROC = 0.75). LLMs applied to unstructured GP referral letters had limited standalone predictive value, but enhanced sensitivity and specificity when combined with the structured models in an ensemble approach. SHapley Additive exPlanations (SHAP) helped explain the prediction and demonstrated comorbidity count, disease scores, and immunosuppressive medications as the top predictors. Our results show that integrating both structured longitudinal data with unstructured clinical narratives using interpretable multimodal artificial intelligence can enable time-sensitive, personalised management of PsA flares and early clinical intervention.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"246 ","pages":"Pages 95-106"},"PeriodicalIF":4.3,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145585760","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":"Sensitive and specific detection of ctDNA using Copper-Free click chemistry and magnetic bead Technology","authors":"Reza Didarian ,&nbsp;Dilek Kanarya ,&nbsp;Sonya Sahin ,&nbsp;Canan Özyurt ,&nbsp;Serap Evran ,&nbsp;Dilek Odaci ,&nbsp;Nimet Yildirim-Tirgil","doi":"10.1016/j.ymeth.2025.11.010","DOIUrl":"10.1016/j.ymeth.2025.11.010","url":null,"abstract":"<div><div>Liquid biopsy, particularly the analysis of circulating tumor DNA (ctDNA), offers immense potential for non-invasive cancer diagnosis and monitoring. It provides a less invasive alternative to traditional tissue biopsies, enabling earlier detection and real-time assessment of disease progression. However, a significant hurdle in its widespread adoption is the extremely low concentration of ctDNA in biological samples, especially during the early stages of cancer, making sensitive and specific detection challenging. This work addresses the critical problem of developing a highly sensitive and specific method for low abundance ctDNA detection.</div><div>We developed a novel, highly sensitive, and specific method for ctDNA analysis, employing copper-free click chemistry (strain-promoted azide-alkyne cycloaddition, SPAAC) for enzyme-free amplification, coupled with magnetic bead-assisted fluorometric detection. This enzyme-free approach significantly enhanced specificity and reduced background noise. We meticulously optimized parameters, including primer length and annealing temperature, finding that 30-base primers and a 50 °C annealing temperature yielded optimal amplification efficiency. Our method successfully detected ctDNA at concentrations as low as 10 pM (15 bp primer). Agarose gel electrophoresis confirmed highly specific amplification with minimal non-specific products, and the assay demonstrated excellent allelic discrimination, accurately distinguishing single-nucleotide mutations. Importantly, the method proved robust in complex human serum samples, demonstrating its practical applicability.</div><div>This innovative, cost-effective, and enzyme-free platform overcomes many limitations of current ctDNA detection technologies. By enabling highly sensitive and specific detection of low abundance ctDNA, this methodology represents a significant leap forward for non-invasive cancer diagnostics, paving the way for earlier disease detection, improved treatment monitoring, and the broader implementation of personalized medicine.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"246 ","pages":"Pages 83-94"},"PeriodicalIF":4.3,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145659955","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":"Denaturation, rapid dilution refolding, and single-step purification of the core histones using desalting size-exclusion chromatography","authors":"Olufola O. Ige ,&nbsp;Thordur Hendrickson-Rebizant ,&nbsp;Wenxia Luo ,&nbsp;Marvellous Oyeyode ,&nbsp;Stefan Jacobson ,&nbsp;Fryda Malinalli Ortiz Bada ,&nbsp;Michael J. Rowley ,&nbsp;Rui Wen Liu ,&nbsp;James R Davie ,&nbsp;Adam Frankel ,&nbsp;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 &gt; 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}

{"title":"A smoothing method for DNA methylome analysis to enhance epigenomic signature detection in epigenome-wide association studies","authors":"Abderrahim Oussalah ,&nbsp;Loris Mousel ,&nbsp;David-Alexandre Trégouët ,&nbsp;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 × 10⁻²¹⁹) and was independently validated in a separate dataset, significantly distinguishing newborns from nonagenarians (<em>P</em> = 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 <em>P</em>-value threshold of 1 × 10⁻⁸. 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}

{"title":"Disease-related omics data analysis","authors":"Wei Peng ,&nbsp;Zhipeng Cai","doi":"10.1016/j.ymeth.2025.11.001","DOIUrl":"10.1016/j.ymeth.2025.11.001","url":null,"abstract":"","PeriodicalId":390,"journal":{"name":"Methods","volume":"246 ","pages":"Pages 10-11"},"PeriodicalIF":4.3,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145450491","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":"MDM2/p53-based live-cell quantitative FRET imaging for apoptosis drug discovery","authors":"Zhiyu Xiao,&nbsp;Lingmin Xie,&nbsp;Ziru Wu,&nbsp;Xinghong Cai,&nbsp;Zhengfei Zhuang,&nbsp;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_Dmax</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_Dmax</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_Dma</em>_x 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}

{"title":"Development and characterization of a membrane-permeant GRASP65-mimetic peptide that inhibits Golgi unlinking and cell cycle progression","authors":"Romina Ines Cervigni ,&nbsp;Raffaella Bonavita ,&nbsp;Maria Luisa Barretta ,&nbsp;Daniela Spano ,&nbsp;Inmaculada Ayala ,&nbsp;Fabiola Mascanzoni ,&nbsp;Roberta Iannitti ,&nbsp;Petra Henklein ,&nbsp;Alessandra Monti ,&nbsp;Maurizio Renna ,&nbsp;Nunzianna Doti ,&nbsp;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, 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.</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}