IF 4.3 3区生物学

Methods Pub Date : 2025-12-01 Epub Date: 2025-09-06 DOI: 10.1016/j.ymeth.2025.09.001

Michaela Beranova , Petra Grossova , Jiri Demuth , Filip Kostelansky , Veronika Novakova , Petr Zimcik , Miroslav Miletin

{"title":"Strain promoted click labeling of oligonucleotides on solid-phase support","authors":"Michaela Beranova , Petra Grossova , Jiri Demuth , Filip Kostelansky , Veronika Novakova , Petr Zimcik , Miroslav Miletin","doi":"10.1016/j.ymeth.2025.09.001","DOIUrl":"10.1016/j.ymeth.2025.09.001","url":null,"abstract":"<div><div>Chemically modified oligonucleotides (ONs) are essential tools in molecular biology, diagnostics, and therapeutics. Strain-promoted azide–alkyne cycloaddition (SPAAC) offers an efficient and bioorthogonal method for ON functionalization. While SPAAC reactions on solid-phase support provide distinct advantages, particularly for the incorporation of lipophilic labels, factors influencing their efficiency remain poorly characterized. The interplay between the physicochemical properties of the modifying molecule, the nature of the solid support, and the labeling site within the ON chain has not been systematically evaluated. In this study, we systematically investigate how modifying molecule properties (size, polarity) and concentration, solid support type, labeling site within the ON chain, and reaction time influence efficiency of labeling. Our findings demonstrate that while polar modifying molecules react efficiently across all solid supports, lipophilic molecules can exhibit reduced reactivity on glass-based supports, particularly in positions close to the 3́-end of the oligonucleotide attached to the support. We further show that conjugation at the 5′-terminus consistently yields the highest efficiencies, with a gradual decline observed as the modification site approaches the 3′-end. A 1 mM concentration of labeling reagent was sufficient to achieve high yields on polystyrene support for all labels and on CPG 500 for the polar labels. The size of the modifying molecule had a lesser effect compared to other factors. The method also benefits from recovery and reusability of the unreacted label. These results enable the rational design of efficient ON labeling protocols on solid-phase support while minimizing reagent consumption, contributing to both cost-effectiveness and environmental sustainability.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"244 ","pages":"Pages 46-54"},"PeriodicalIF":4.3,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145022572","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}

引用次数: 0

Capturing G protein-coupled receptors into native lipid-bilayer nanodiscs using new diisobutylene/maleic acid (DIBMA) copolymers 利用新型二异丁烯/马来酸（DIBMA）共聚物将G蛋白偶联受体捕获到天然脂质双层纳米圆盘中。

IF 4.3 3区生物学

Methods Pub Date : 2025-12-01 Epub Date: 2025-09-04 DOI: 10.1016/j.ymeth.2025.08.013

Ci Chu , Carolyn Vargas , Maria Carolina Barbosa , Simon Sommerhage , Gerald N. Rechberger , David Pahovnik , Ema Žagar , Gunnar F. Schröder , Sandro Keller , Manuel Etzkorn

{"title":"Capturing G protein-coupled receptors into native lipid-bilayer nanodiscs using new diisobutylene/maleic acid (DIBMA) copolymers","authors":"Ci Chu , Carolyn Vargas , Maria Carolina Barbosa , Simon Sommerhage , Gerald N. Rechberger , David Pahovnik , Ema Žagar , Gunnar F. Schröder , Sandro Keller , Manuel Etzkorn","doi":"10.1016/j.ymeth.2025.08.013","DOIUrl":"10.1016/j.ymeth.2025.08.013","url":null,"abstract":"<div><div>Many membrane proteins, including G protein-coupled receptors (GPCRs), are susceptible to denaturation when extracted from their native membrane by detergents. Therefore, alternative methods have been developed, including amphiphilic copolymers that enable the direct extraction of functional membrane proteins along with their surrounding lipids. Among these amphiphilic copolymers, styrene/maleic acid (SMA) and diisobutylene/maleic acid (DIBMA) polymers have been extensively studied. Despite their many benefits, SMA and DIBMA polymers also have considerable drawbacks limiting their applications. Herein, we describe a series of new amphiphilic copolymers derived from DIBMA via partial amidation of the carboxylate pendant groups with various biocompatible amines. We characterize the new polymer’s nanodisc-forming properties and ability to extract the melanocortin 4 receptor (MC<sub>4</sub>R), a prototypical class A GPCR. While each new DIBMA variant displays features that may be favorable for selected applications, we identified a PEGylated DIBMA variant called mPEG<sub>4</sub>-DIBMA as particularly promising. In the tested system mPEG<sub>4</sub>-DIBMA abolishes unspecific interactions and outperforms other polymers by achieving higher extraction efficiencies of MC<sub>4</sub>R from Sf9 insect cell membranes. The new nanodisc-forming polymer combines two key advantages that are crucial for investigating GPCRs in a well-defined but still native lipid-bilayer environment, thus paving the way for manifold future applications.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"244 ","pages":"Pages 55-64"},"PeriodicalIF":4.3,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145008085","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}

引用次数: 0

LabImageXpert: imaging platform for mechanism-resolved detection of antimicrobial activity via β-galactosidase assay LabImageXpert：通过β-半乳糖苷酶测定进行机制分辨检测的成像平台。

IF 4.3 3区生物学

Methods Pub Date : 2025-12-01 Epub Date: 2025-10-12 DOI: 10.1016/j.ymeth.2025.10.004

Kusum Kharga , Deepak Kumar , Lokender Kumar

{"title":"LabImageXpert: imaging platform for mechanism-resolved detection of antimicrobial activity via β-galactosidase assay","authors":"Kusum Kharga , Deepak Kumar , Lokender Kumar","doi":"10.1016/j.ymeth.2025.10.004","DOIUrl":"10.1016/j.ymeth.2025.10.004","url":null,"abstract":"<div><div>Antimicrobial resistance (AMR) has emerged as a global crisis, responsible for millions of deaths annually. Traditionally used antimicrobial susceptibility testing (AST) methods, such as disk diffusion and broth dilution, suffer from limitations including prolonged incubation time, inconsistent results, and high instrumentation cost. To address these challenges, we present in this study our novel, cost-effective antimicrobial detection assay equipped with a highly sensitive quantification system, LabImageXpert. Our antimicrobial detection system leverages the ability of β-galactosidase to convert colorless X-gal to an intense insoluble blue colored product. Unlike traditional MIC assays, LabImageXpert enables image-based, mechanism-specific growth assessment. During our investigation, we tested compounds already known to possess antimicrobial properties against our antimicrobial assay and found that it could not only detect the antimicrobial activity but also reveal the mechanism of action behind their antimicrobial activity, i.e., bacteriostatic and bacteriolytic. Further, this system contains a high-resolution DSLR camera to image samples in a microtiter plate placed inside a special box. The captured images are processed using the open-source software. We performed an experiment exclusively to test its ability to quantify minute variations in bacterial growth over time. The results showed that it could detect minute deviations in average pixel intensity over time. LabImageXpert detected bacteriolytic activity of compounds within 3 h. Our findings suggest that the LabImageXpert provides a reproducible, scalable, and cost-effective alternative platform for antimicrobial research in teaching, research and clinical diagnostic labs.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"244 ","pages":"Pages 182-194"},"PeriodicalIF":4.3,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145290536","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}

引用次数: 0

IF-CRIB: A 3D-printable device to facilitate immunofluorescence experiments and its application in screening and characterizing cells expressing a degradable form of ERK2 IF-CRIB：一种3d打印设备，用于促进免疫荧光实验，并用于筛选和表征表达可降解形式ERK2的细胞。

IF 4.3 3区生物学

Methods Pub Date : 2025-11-01 Epub Date: 2025-08-09 DOI: 10.1016/j.ymeth.2025.08.004

Elsa Berliocchi , Cercina Onesto , Gilles Pagès , Philippe Lenormand , Roser Buscà

{"title":"IF-CRIB: A 3D-printable device to facilitate immunofluorescence experiments and its application in screening and characterizing cells expressing a degradable form of ERK2","authors":"Elsa Berliocchi , Cercina Onesto , Gilles Pagès , Philippe Lenormand , Roser Buscà","doi":"10.1016/j.ymeth.2025.08.004","DOIUrl":"10.1016/j.ymeth.2025.08.004","url":null,"abstract":"<div><div>Immunofluorescence-based detection of proteins in fixed cells is a powerful tool for research in cell and developmental biology. While a variety of immunofluorescence protocols exist, they can be time consuming or require expensive equipment which may not be accessible to all laboratories. A common challenge in these protocols is the numerous washing steps, particularly in experiments with numerous conditions. To address this, here we introduce the IF-CRIB device, a 3D-printable wash rack specifically designed for applications involving a high number of round coverslips with adherent cultured cells. We detail its design and the 3D printing process which can be easily used by any laboratory and we highlight that it facilitates the numerous washing steps. In addition, we present the IF-Express protocol, an optimized and effective method that enables fast and consistent immunofluorescence results. As an example of the utility of the IF-CRIB device and the IF-Express protocol, we describe their application in the screening and characterization of several NIH3T3 cell clones expressing a degradable form of ERK2 kinase (ERK2-dTAG) after treatment with the dTAG-13 compound. The generation of ERK2-dTAG clones involves a <em>knock-in</em> strategy. We provide a detailed methodology for clone selection, immunofluorescence screening, and characterization of ERK2-dTAG, including degradation kinetics, dose–response analysis, and nuclear translocation assays to assess ERK2-dTAG functionality. The IF-CRIB device and IF-Express protocol has been proven to be efficient for the obtention and characterization of ERK2dTAG-expressing clones thereby offering a powerful framework for studying ERK2 dynamics in cell biology and disease models.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"243 ","pages":"Pages 1-15"},"PeriodicalIF":4.3,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144820273","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}

引用次数: 0

Coffee – a ubiquitous substitute for uranyl acetate in staining of biological ultrathin sections for electron microscopy studies 咖啡-在电子显微镜研究的生物超薄切片染色中，醋酸铀酰的普遍替代品

IF 4.3 3区生物学

Methods Pub Date : 2025-11-01 Epub Date: 2025-08-21 DOI: 10.1016/j.ymeth.2025.08.009

Claudia Mayrhofer , Robert Zandonella , Willi Salvenmoser , Ilse Letofsky-Papst

{"title":"Coffee – a ubiquitous substitute for uranyl acetate in staining of biological ultrathin sections for electron microscopy studies","authors":"Claudia Mayrhofer , Robert Zandonella , Willi Salvenmoser , Ilse Letofsky-Papst","doi":"10.1016/j.ymeth.2025.08.009","DOIUrl":"10.1016/j.ymeth.2025.08.009","url":null,"abstract":"<div><div>The use of uranyl acetate, a staining agent successfully used for decades in electron microscopy of biological specimens, is now strictly regulated by law due to its toxicity and radioactivity. It is even banned in some laboratories. In the meantime, there are a number of substitutes on the market, none of which comes close to the very good staining results of uranyl acetate, or only partially, and some of which are also toxic. In this paper, two alternatives to uranyl acetate are presented, namely coffee, which is used in countless households, and pure chlorogenic acid, which is a component of coffee. We used the well-known zebrafish as biological test material, focusing on the mitochondrial membranes. The staining ability of coffee and chlorogenic acid compared with commercially available staining agents as well as uranyl acetate is assessed by the interference contrast between membranes and their environment. This work also describes how a subjective impression of good or bad contrast can be cast into an objective and comparable numerical value.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"243 ","pages":"Pages 76-82"},"PeriodicalIF":4.3,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144919785","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}

引用次数: 0

Exploiting Anisotropic Orientation in Nanoparticle-Aided Cryo-Electron Microscopy Sampling 利用纳米粒子辅助低温电子显微镜取样的各向异性取向

IF 4.3 3区生物学

Methods Pub Date : 2025-11-01 Epub Date: 2025-08-21 DOI: 10.1016/j.ymeth.2025.08.008

Yeeun Kim , Changin Kim , Yongsoo Yang , Hyotcherl Ihee

{"title":"Exploiting Anisotropic Orientation in Nanoparticle-Aided Cryo-Electron Microscopy Sampling","authors":"Yeeun Kim , Changin Kim , Yongsoo Yang , Hyotcherl Ihee","doi":"10.1016/j.ymeth.2025.08.008","DOIUrl":"10.1016/j.ymeth.2025.08.008","url":null,"abstract":"<div><div>Measuring the conformational distribution of small proteins is essential to understanding their role in biological systems. Multi-Tilt Nanoparticle-aided cryo-electron microscopy sampling (MT-NACS) was devised to measure the three-dimensional interparticle distance distribution (<em>P</em>(<em>d</em>)) of two gold nanoparticles (AuNPs) labeled on a protein by taking cryogenic electron microscopy (cryo-EM) images at multiple-tilt angles. However, tracking the same particles in a pseudo-tomographic manner during the multi-tilt cryo-EM experiments and data analysis requires extensive time and effort. Here, we report that proper incorporation of AuNP pair angle distribution allows reliable determination of the <em>P</em>(<em>d</em>) only using the cryo-EM images collected at a single tilt angle. The trends of structural changes in calmodulin (CaM) measured by single-tilt angle NACS (ST-NACS) and MT-NACS are consistent, as the tendencies of changes in the <em>P</em>(<em>d</em>) of AuNP-labeled CaM measured by both methods are similar. Our approach provides an efficient tool for measuring the conformational distribution and structural transition of small proteins.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"243 ","pages":"Pages 54-65"},"PeriodicalIF":4.3,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144907013","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}

引用次数: 0

Validation of combo ichroma as a reliable concentration-based alternative for AST and ALT measurement in liver disease monitoring 联合色度作为肝脏疾病监测中AST和ALT测量的可靠的基于浓度的替代方法的验证

IF 4.3 3区生物学

Methods Pub Date : 2025-11-01 Epub Date: 2025-08-27 DOI: 10.1016/j.ymeth.2025.08.011

Minsoo Kim , Su A Kim , Jeong Min Kim , Hee Young Kim , Ho Yeong Yoon , Sung Won Park , Daegyun Park , Ji Sook Han , Ki Tae Suk

{"title":"Validation of combo ichroma as a reliable concentration-based alternative for AST and ALT measurement in liver disease monitoring","authors":"Minsoo Kim , Su A Kim , Jeong Min Kim , Hee Young Kim , Ho Yeong Yoon , Sung Won Park , Daegyun Park , Ji Sook Han , Ki Tae Suk","doi":"10.1016/j.ymeth.2025.08.011","DOIUrl":"10.1016/j.ymeth.2025.08.011","url":null,"abstract":"<div><h3>Background</h3><div>Traditional assays for aspartate aminotransferase (AST) and alanine aminotransferase (ALT) measure enzymatic activity, which degrades during long-term frozen storage, threatening the accuracy of the assay. Instead, Combo ichroma (CI), a fluorescence immunoassay that quantifies AST and ALT concentrations, is a robust alternative for retrospective and point-of-care liver function testing, free from the influence of long-term storage.</div></div><div><h3>Methods</h3><div>Serum samples from 256 individuals (controls and patients with hepatitis, cirrhosis, or liver cancer) were collected and stored at −80 °C for an average of three years. AST and ALT were measured using CI, the 7180 clinical analyzer (HT), and Atellica CH 930 analyzer performed immediately after collection (HL). Correlations between AST, ALT, and AST/ALT ratios were analyzed. Random Forest Regression (RFR) models using CI or HT data were developed to predict HL-derived AST/ALT ratios.</div></div><div><h3>Results</h3><div>CI-AST showed strong correlation with HL-AST across all groups (R2 > 0.95), outperforming HT-AST, especially in controls (R2 = 0.58). CI-ALT moderately correlated with HL-ALT (R2 = 0.87), surpassing HT-ALT (R2 = 0.71). AST/ALT ratios varied across methods due to ALT variability, but RFR using CI data accurately predicted HL ratios (R2 = 0.85–0.91). Subgroup analysis confirmed CI’s superior concordance across etiologies.</div></div><div><h3>Conclusions</h3><div>CI enables activity-independent, reliable measurement of AST and ALT even after extended storage, outperforming enzymatic assays in precision and correlation. Its simplicity, and compatibility with machine learning models position CI as a promising tool for liver enzyme diagnostics in both clinical and resource-limited settings.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"243 ","pages":"Pages 66-75"},"PeriodicalIF":4.3,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144913714","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}

引用次数: 0

PCR-generated DNA templates enable efficient, rapid, and cost-effective mRNA synthesis pcr生成的DNA模板能够高效、快速、经济地合成mRNA。

IF 4.3 3区生物学

Methods Pub Date : 2025-11-01 Epub Date: 2025-08-16 DOI: 10.1016/j.ymeth.2025.08.007

Naveen Kumar , Nicholas C. Hazell , Jiani Bei , Tina Nguyen , Haitao Hu

{"title":"PCR-generated DNA templates enable efficient, rapid, and cost-effective mRNA synthesis","authors":"Naveen Kumar , Nicholas C. Hazell , Jiani Bei , Tina Nguyen , Haitao Hu","doi":"10.1016/j.ymeth.2025.08.007","DOIUrl":"10.1016/j.ymeth.2025.08.007","url":null,"abstract":"<div><div><em>In vitro</em> transcription (IVT) is a widely used technique for mRNA synthesis in both basic research and the development mRNA-based vaccines and therapies. The efficiency of IVT critically depends on the quality and integrity of the linear DNA templates. The conventional method for template DNA preparation involves plasmid propagation in bacteria followed by enzymatic linearization, which is labor-intensive and costly. Here, we describe a cell-free, PCR-based approach for generating high-quality, high-yield linear DNA templates. We extensively compared the PCR-based method with the conventional plasmid-based approach in terms of IVT efficiency, mRNA production, and the immunogenicity of the resulting mRNA-LNP (lipid nanoparticle) vaccines. Compared to the plasmid-derived DNA, the PCR-based method yielded higher amounts of both DNA templates and transcribed mRNA, while maintaining mRNA quality and integrity. Importantly, mRNA-LNP vaccines encoding the SARS-CoV-2 spike protein, generated from both methods, elicited robust and comparable immune responses in mice, with no significant differences observed between the two template methods. Our findings highlight the advantages of PCR-generated DNA templates as a rapid, efficient, and cost-effective alternative for mRNA synthesis, with broad applications in vaccine and therapeutic development.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"243 ","pages":"Pages 31-39"},"PeriodicalIF":4.3,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144870772","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}

引用次数: 0

Insights of Surface Enhancing Raman Spectroscopy for Biomedical Application 表面增强拉曼光谱在生物医学上的应用。

IF 4.3 3区生物学

Methods Pub Date : 2025-11-01 Epub Date: 2025-08-12 DOI: 10.1016/j.ymeth.2025.08.005

Neha Sharma , Anita Singh , Ratneshwar Kumar Ratnesh , Archana Adhana , Lalita Tyagi , Jay Singh

{"title":"Insights of Surface Enhancing Raman Spectroscopy for Biomedical Application","authors":"Neha Sharma , Anita Singh , Ratneshwar Kumar Ratnesh , Archana Adhana , Lalita Tyagi , Jay Singh","doi":"10.1016/j.ymeth.2025.08.005","DOIUrl":"10.1016/j.ymeth.2025.08.005","url":null,"abstract":"<div><div>Raman spectroscopy is a powerful, non-invasive analytical technique that enables rapid identification of molecules based on their unique spectral fingerprints. Its sensitivity has been significantly enhanced through the use of metal nanoparticles in Surface-Enhanced Raman Spectroscopy (SERS), where molecules adsorbed on rough metallic surfaces or colloids produce Raman signals amplified by several orders of magnitude. This enhancement has opened new possibilities for molecular detection, particularly in surface chemistry and biomedical diagnostics.</div><div>In clinical applications, timely and accurate diagnosis is critical, yet conventional bioanalytical methods often require multiple biochemical tests, leading to delays that are especially problematic in emergency settings. SERS provides a promising alternative, offering high sensitivity, specificity, and rapid analysis with minimal sample preparation.</div><div>This review explores the integration of Raman spectroscopy—especially SERS—for both <em>in vivo</em> and <em>ex vivo</em> biomedical diagnostics. It covers sample preparation techniques, spectral data interpretation, and the correlation of Raman signals with disease-specific biomarkers. Special focus is given to the application of Raman-based methods in diagnosing brain disorders, various cancers, drug abuse, and COVID-19. Finally, the article discusses future prospects and challenges to guide the continued advancement of biomedical Raman technologies.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"243 ","pages":"Pages 16-30"},"PeriodicalIF":4.3,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144854187","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}

引用次数: 0

Optimized CUT&Tag enables robust epigenome profiling in Schizosaccharomyces pombe 优化的CUT&Tag能够在裂糖菌pombe中实现稳健的表观基因组分析

IF 4.3 3区生物学

Methods Pub Date : 2025-11-01 Epub Date: 2025-08-22 DOI: 10.1016/j.ymeth.2025.08.010

Cheng-Zhi Huang , Kang-Di Zhou , Wei Ma

{"title":"Optimized CUT&Tag enables robust epigenome profiling in Schizosaccharomyces pombe","authors":"Cheng-Zhi Huang , Kang-Di Zhou , Wei Ma","doi":"10.1016/j.ymeth.2025.08.010","DOIUrl":"10.1016/j.ymeth.2025.08.010","url":null,"abstract":"<div><div>We optimized permeabilization for CUT&Tag in <em>S. pombe</em>, enabling robust H3K9me3 profiling using lightly fixed permeabilized sepheroplasts, overcoming limitations of ChIP-seq including crosslinking artifacts and high cell input. We established an optimized Cleavage Under Targets and Tagmentation (CUT&Tag) protocol for high-resolution epigenome profiling inusing Critical permeabilization refinements identified Lywallzyme as the optimal enzyme for spheroplast generation (>95 % efficiency in 60 min at 10 mg/mL), outperforming Zymolyase-20 T and combinatorial treatments. Systematic parameter optimization revealed concentration-dependent digestion kinetics and an inverse cell load-efficiency relationship (5 × 10<sup>5</sup> cells achieving > 90 % conversion in 50 min at 5 mg/mL). Validated through H3K9me3 mapping in wild-type and <!-->strains (10⁶ cells/replicate), this approach captured specific heterochromatic enrichment at centromeres/telomeres with complete signal ablation in mutants, while reduced spike-in DNA (0.2 pg) significantly enhanced signal-to-noise ratios. The protocol enables robust epigenomic analysis with minimal cell input and enhanced resolution.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"243 ","pages":"Pages 49-53"},"PeriodicalIF":4.3,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144895039","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}

引用次数: 0

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