Methods最新文献

{"title":"Non-invasive tools for analysis of plasma membrane protein topology in living cells","authors":"Daria Savenkova ,&nbsp;Leysan Bulatova ,&nbsp;Vera Skripova ,&nbsp;Ramziya Kiyamova ,&nbsp;Mikhail Bogdanov","doi":"10.1016/j.ymeth.2025.04.007","DOIUrl":"10.1016/j.ymeth.2025.04.007","url":null,"abstract":"<div><div>Membrane protein topology studies offer guidance to membrane protein structure, folding, and function, serving as a credible scaffold for designing site-directed mutagenesis and biochemical experiments, helping to identify functionally significant extracellular and intracellular regions, modeling three-dimensional structures, and building reliable mechanistic models. Membrane protein structure as a function of given lipid composition and physiological state of the cell is best probed in whole intact cells. A described simple and advanced immunofluorescence protocol applied to the transmembrane orientation of extramembrane domains permits a topology analysis of plasma membrane proteins in their native state in living unperturbed eucaryotic cells. The accessibility of native epitopes to corresponding antibodies is determined in intact and permeabilized cells to establish their extra- or intracellular or localization respectively. The ability of the given antibody to bind the epitope in intact live and permeabilized cells is then assessed routinely by intact and permeabilized cell immunofluorescent confocal microscopy or fluorescence flow cytometry parametric analyses in several hours. To ensure that the observed immunofluorescence is entirely a result of the binding of antibodies, cells are alive and the plasma membrane is intact, plasma membrane integrity is routinely monitored by co-incubating the cells with a cell membrane-impermeable probe, propidium iodide. Accordingly, plasma membrane side-specific immunostaining analysis was restricted to the propidium iodide-negative, non-permeabilized cell population. The strength of this technique is its simplicity since each native epitope is unique and there is no need to mutate any endogenous sites, introduce new epitopes, or engineer single, dual, or split colorimetric enzymatic reporters. Aside from its simplicity, the advantage of this approach is that the topology is documented in the context of full-length and fully biologically active membrane protein molecules, and topology mapping is carried out using whole live cells, thereby avoiding problems related to cell fixation or the conversion of cells into membrane vesicles with a uniform orientation. The protocol can be universally adapted to any cellular system to systematically map a uniform topology of target membrane protein.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"239 ","pages":"Pages 111-126"},"PeriodicalIF":4.2,"publicationDate":"2025-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143870670","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":"EffiCOVID-net: A highly efficient convolutional neural network for COVID-19 diagnosis using chest X-ray imaging","authors":"Sunil Kumar,&nbsp;Biswajit Bhowmik","doi":"10.1016/j.ymeth.2025.04.008","DOIUrl":"10.1016/j.ymeth.2025.04.008","url":null,"abstract":"<div><div>The global COVID-19 pandemic has drastically affected daily life, emphasizing the urgent need for early and accurate detection to provide adequate medical treatment, especially with limited antiviral options. Chest X-ray imaging has proven crucial for distinguishing COVID-19 from other respiratory conditions, providing an essential diagnostic tool. Deep learning (DL)-based models have proven highly effective in image diagnostics in recent years. Many of these models are computationally intensive and prone to overfitting, especially when trained on limited datasets. Additionally, conventional models often fail to capture multi-scale features, reducing diagnostic accuracy. This paper proposed a highly efficient convolutional neural network (CNN) called EffiCOVID-Net, incorporating diverse feature learning units. The proposed model consists of a bunch of EffiCOVID blocks that incorporate several layers of convolution containing (<span><math><mn>3</mn><mo>×</mo><mn>3</mn></math></span>) filters and recurrent connections to extract complex features while preserving spatial integrity. The performance of EffiCOVID-Net is rigorously evaluated using standard performance metrics on two publicly available COVID-19 chest X-ray datasets. Experimental results demonstrate that EffiCOVID-Net outperforms existing models, achieving 98.68% accuracy on the COVID-19 radiography dataset (<span><math><msub><mrow><mi>D</mi></mrow><mrow><mn>1</mn></mrow></msub></math></span>), 98.55% on the curated chest X-ray dataset (<span><math><msub><mrow><mi>D</mi></mrow><mrow><mn>2</mn></mrow></msub></math></span>), and 98.87% on the mixed dataset (<span><math><msub><mrow><mi>D</mi></mrow><mrow><mi>M</mi><mi>i</mi><mi>x</mi></mrow></msub></math></span>) in multi-class classification (COVID-19 vs. Normal vs. Pneumonia). For binary classification (COVID-19 vs. Normal), the model attains 99.06%, 99.78%, and 99.07% accuracy, respectively. Integrating Grad-CAM-based visualizations further enhances interpretability by highlighting critical regions influencing model predictions. EffiCOVID-Net's lightweight architecture ensures low computational overhead, making it suitable for deployment in resource-constrained clinical settings. A comparative analysis with existing methods highlights its superior accuracy, efficiency, and robustness performance. However, while the model enhances diagnostic workflows, it is best utilized as an assistive tool rather than a standalone diagnostic method.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"240 ","pages":"Pages 81-100"},"PeriodicalIF":4.2,"publicationDate":"2025-04-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143855970","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":"Methodologies for label free Raman microspectroscopic monitoring of viral replication processes in vitro","authors":"Iqra Chaudary ,&nbsp;Judit Barabas ,&nbsp;Ultan F. Power ,&nbsp;Luke O’Neill ,&nbsp;Hugh J Byrne ,&nbsp;Denise Denning","doi":"10.1016/j.ymeth.2025.04.010","DOIUrl":"10.1016/j.ymeth.2025.04.010","url":null,"abstract":"<div><div>This study demonstrates the use of Raman Spectroscopy, integrated with multivariate statistical analysis, to monitor the process of viral infection in cells, in-vitro, using the model example of Sendai Virus (SeV) infection in LLC-MK2 monkey kidney cells. A comprehensive methodology is described for determining a precise multiplicity of infection, 48 h post infection, tailored for analysis of viral-host interactions using Raman Microspectroscopy. SeV infected LLC-MK2 cells were fixed on a gold-coated glass slide for Raman spectroscopic analysis. 30-point spectra of uninfected control and 30-point spectra of SeV-infected cells were acquired, focusing randomly on the individual cells. Mean Raman spectra of the control and SeV-infected LLC-MK2 cells revealed spectral differences of peaks corresponding to nucleic acids (485 cm⁻¹, 785 cm⁻¹), lipids (1445 cm⁻¹) and proteins (1600 cm⁻¹, 1655 cm⁻¹). These changes in the relative intensities of Raman peaks indicate modifications in the biochemical content, potentially due to viral entry and replication inside the cells. Principal Components Analysis distinguished between control and SeV-infected LLC-MK2 cells, indicating significant biochemical alterations in response to the SeV infection. Partial Least Squares Discriminant Analysis can be employed to quantify the differentiation of the spectral datasets of the infected/noninfected cells, classifying them with 100 % sensitivity and specificity. The detailed methodology described in the study is potentially a powerful tool for tracking viral replication and detecting viral infections and has the potential to impact future research on host-virus interactions and viral diagnostics. Further research on these spectral differences can contribute to developing more efficient viral screening techniques and a better understanding of viral infections.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"240 ","pages":"Pages 73-80"},"PeriodicalIF":4.2,"publicationDate":"2025-04-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143855969","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 validation of novel RT-PCR assay for molecular diagnostic of viral variants using SARS-CoV-2 as a case study","authors":"Priyanshi Singh ,&nbsp;Gauri Misra ,&nbsp;Neelima Mishra ,&nbsp;Anupkumar Anvikar ,&nbsp;Varsha Potdar","doi":"10.1016/j.ymeth.2025.04.006","DOIUrl":"10.1016/j.ymeth.2025.04.006","url":null,"abstract":"<div><div>Emerging viruses have long posed significant challenges to global public health, frequently leading to widespread morbidity and mortality. The ongoing evolution of viruses driven by genetic mutations is critical in the emergence of these novel pathogens. Among the numerous viruses that have demonstrated this capability the SARS-CoV-2 responsible for the COVID-19 pandemic is the prime example of how viral mutations can profoundly impact disease dynamics, transmission, and control measures. In this study, we present the development of a multiplex RT-PCR assay, using allele-specific primer-probe tailored for molecular diagnostic of viral variants using SARS−CoV-2 as a case study. We conducted a comprehensive evaluation to validate the assay performance using a diverse panel of leftover clinical samples, including a few coded reference samples from external providers. This multiplex PCR typing method detects seven unique mutations of Omicron and two unique mutations of Delta strain with allele-specific primers and probe sets against the spike protein’s receptor-binding domain (RBD). The assay exhibits high analytical sensitivity, detecting about 1 x 10² copies/mL of SARS-CoV-2 RNA for each genetic variant tested, and possesses 100 % analytical specificity. Comparative analysis with existing commercial RT-PCR kits demonstrated better performance, particularly in detecting omicron and delta variants. This research highlights the translational potential of our approach in advancing diagnostic capabilities for emerging viral infections, enhancing public health responses to future outbreaks.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"240 ","pages":"Pages 54-62"},"PeriodicalIF":4.2,"publicationDate":"2025-04-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143839669","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":"Novel multitarget LAMP and PCR assays for the detection of Bordetella species","authors":"Maxim A. Koryukov ,&nbsp;Igor P. Oscorbin ,&nbsp;Maria A. Gordukova ,&nbsp;Irina E. Turina ,&nbsp;Alfiya I. Aminova ,&nbsp;Maxim L. Filipenko","doi":"10.1016/j.ymeth.2025.04.005","DOIUrl":"10.1016/j.ymeth.2025.04.005","url":null,"abstract":"<div><div>Whooping cough, or pertussis, is a highly contagious disease caused by several <em>Bordetella</em> species and continues to pose a significant global public health concern. The rising incidence of pertussis highlights the urgent need for effective public health strategies to address <em>Bordetella</em> infections. Rapid and species-specific diagnostic tools are essential for preventing <em>Bordetella</em> transmission and are vital components of anti-infective measures. This study aimed to develop novel loop-mediated isothermal amplification (LAMP) and quantitative PCR (qPCR) assays for the detection of four <em>Bordetella</em> species responsible for human respiratory tract infections: <em>B. pertussis, B. parapertussis, B. bronchiseptica</em>, and <em>B. holmesii</em>. The qPCR assay demonstrated a low limit of detection (LoD), reliably identifying up to 5 copies of target DNA per reaction. The LAMP assays were approximately three times faster than qPCR (30 min) but had higher LoDs. Notably, qLAMP had a limit of detection of 25 copies per reaction for all four <em>Bordetella</em> species. In contrast, vLAMP had a LoD of 25 copies per reaction for <em>B. pertussis</em> and <em>B. parapertussis</em>; and a LoD of 50 copies per reaction for <em>B. holmesii</em> and <em>B. bronchiseptica</em>. We validated the assays using nasal swab samples from patients with respiratory tract infections, analyzing a total of 651 samples with qPCR and 145 samples with LAMP. Both assays exhibited no cross-reactivity with common viral and bacterial respiratory pathogens. The concordance rate between qPCR and LAMP was 94.5%, underscoring the reliability of both methods for clinical application. These findings suggest that the developed qPCR and LAMP tests can be successfully integrated into clinical practice for the detection and management of <em>Bordetella</em> infections.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"240 ","pages":"Pages 63-72"},"PeriodicalIF":4.2,"publicationDate":"2025-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143839497","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":"Quantitative mass spectrometric analysis of C-terminal 36 amino acid peptides of alpha-1 antitrypsin in plasma using survey spectra","authors":"Harald Tammen ,&nbsp;Andreas Pich ,&nbsp;Rüdiger Hess ,&nbsp;Urszula Lechowicz ,&nbsp;Sabina Janciauskiene ,&nbsp;Joanna Chorostowska","doi":"10.1016/j.ymeth.2025.04.004","DOIUrl":"10.1016/j.ymeth.2025.04.004","url":null,"abstract":"<div><div>C-terminal peptides of alpha-1 antitrypsin (AAT) may serve as biomarkers for diseases such as sepsis, chronic obstructive pulmonary disease, liver disease, and autoimmune disorders. In this study, we present a robust and straightforward MS (mass spectrometry)-based method for quantifying AAT peptides 388–418 (C36) and its polymorphic variant (E400D, C36D) in plasma samples. Absolute quantification was accomplished using MALDI-MS reflectron spectra and ESI-MS MS1 scans, implemented in two independent laboratories. Two plasma preparation methods, methanol precipitation and ultrafiltration, were evaluated, with methanol precipitation yielding significantly higher recovery rates. The impact of freeze–thaw cycles on C36 levels was also assessed, revealing a significant increase in C36 levels after each cycle. Comparisons between MALDI-MS and ESI-MS showed strong concordance in C36 and C36D measurements. Furthermore, C36 and C36D levels correlated strongly with post-precipitation protein content across both MS methods. Normalizing C36 levels to protein content effectively mitigated variability. This method should be straightforward to implement in other laboratories, facilitating clinical studies to evaluate the diagnostic and prognostic significance of C36 peptides across various diseases.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"240 ","pages":"Pages 7-13"},"PeriodicalIF":4.2,"publicationDate":"2025-04-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143816565","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":"Immobilized animal liver microsomes: A versatile tool for efficient ester hydrolysis in chemo-enzymatic synthesis","authors":"Charline Monnier ,&nbsp;Rudolf Andrys ,&nbsp;Irene Castellino ,&nbsp;Veronika Mickova ,&nbsp;Annamaria Haleckova ,&nbsp;Jan Loskot ,&nbsp;Ondrej Benek ,&nbsp;Lucie Zemanova","doi":"10.1016/j.ymeth.2025.03.021","DOIUrl":"10.1016/j.ymeth.2025.03.021","url":null,"abstract":"<div><div>Animal liver microsomes are a rich source of carboxylesterases with potential for biocatalytic applications. However, their instability and difficulty in reuse limit their practical application. This study investigates the immobilization of animal liver microsomes from four species <em>Mus musculus</em> (house mouse), <em>Sus scrofa</em> (wild boar), <em>Dama dama</em> (fallow deer), and <em>Capreolus capreolus</em> (roe deer) on Perloza MG microparticles for enhanced stability and reusability. Immobilization significantly improved the stability and pH tolerance of the microsomes, particularly those from <em>D. dama</em>, maintaining esterase activity across a broad pH range (5–9) and enabling the reusability over ten consecutive cycles. The immobilized <em>D. dama</em> microsomes were successfully employed in a preparative-scale chemo-enzymatic synthesis of a cyclophilin D inhibitor, achieving a total reaction yield of 68% with 98% final product purity, demonstrating their potential for sustainable organic synthesis.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"240 ","pages":"Pages 35-46"},"PeriodicalIF":4.2,"publicationDate":"2025-04-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143825895","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 high-throughput and time-efficient Nanopore full-length 16S rRNA gene sequencing protocol for synthetic microbial communities","authors":"Xingjian Zhou,&nbsp;Karoline Faust","doi":"10.1016/j.ymeth.2025.04.003","DOIUrl":"10.1016/j.ymeth.2025.04.003","url":null,"abstract":"<div><div>Next-generation sequencing (NGS) has transitioned from primarily research-focused applications to a mature technology. However, resolving microbial community composition on the species level based on the 16S rRNA gene is impeded by several critical bottlenecks that limit the efficiency and scalability of analyses. Specifically, standard MiSeq sequencing suffers from read-length limitation; library preparation requires multiple labour-intensive steps from DNA isolation to amplification and barcoding; and prolonged turnaround times delay results. These challenges underscore the need for improved methods, which our study aims to address. Recent advances in Oxford Nanopore long-read sequencing technology (ONT), including a smaller and cheaper benchtop instrument and support for diverse sample types, have enabled faster sequencing in-house with reduced costs. To address the need for standardized, reproducible workflows, we present an optimized and state-of-the-art protocol for full-length 16S rRNA gene sequencing using the ONT MinION sequencing device. Furthermore, we quantified the reproducibility and accuracy of our protocol and compared it with previous MiSeq results. The results showed that the accuracy of our sequencing pipeline for synthetic communities is significantly higher than for MiSeq pipeline. In summary, our protocol elucidates the composition of synthetic microbial communities in an easy, fast and accurate manner while ensuring reproducible results.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"240 ","pages":"Pages 14-20"},"PeriodicalIF":4.2,"publicationDate":"2025-04-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143828114","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":"Transforming breast cancer diagnosis and treatment with large language Models: A comprehensive survey","authors":"Mohsen Ghorbian ,&nbsp;Mostafa Ghobaei-Arani ,&nbsp;Saied Ghorbian","doi":"10.1016/j.ymeth.2025.04.001","DOIUrl":"10.1016/j.ymeth.2025.04.001","url":null,"abstract":"<div><div>Breast cancer (BrCa), being one of the most prevalent forms of cancer in women, poses many challenges in the field of treatment and diagnosis due to its complex biological mechanisms. Early and accurate diagnosis plays a fundamental role in improving survival rates, but the limitations of existing imaging methods and clinical data interpretation often prevent optimal results. Large Language Models (LLMs), which are developed based on advanced architectures such as transformers, have brought about a significant revolution in data processing and medical decision-making. By analyzing a large volume of medical and clinical data, these models enable early diagnosis by identifying patterns in images and medical records and provide personalized treatment strategies by integrating genetic markers and clinical guidelines. Despite the transformative potential of these models, their use in BrCa management faces challenges such as data sensitivity, algorithm transparency, ethical considerations, and model compatibility with the details of medical applications that need to be addressed to achieve reliable results. This review systematically reviews the impact of LLMs on BrCa treatment and diagnosis. This study’s objectives include analyzing the role of LLM technology in diagnosing and treating this disease. The findings indicate that the application of LLMs has resulted in significant improvements in various aspects of BrCa management, such as a 35% increase in the Efficiency of Diagnosis and BrCa Treatment (EDBC), a 30% enhancement in the System’s Clinical Trust and Reliability (SCTR), and a 20% improvement in the quality of patient education and information (IPEI). Ultimately, this study demonstrates the importance of LLMs in advancing precision medicine for BrCa and paves the way for effective patient-centered care solutions.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"239 ","pages":"Pages 85-110"},"PeriodicalIF":4.2,"publicationDate":"2025-04-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143791482","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":"RNA aptamer-induced fluorescence enhancement for NADH monitoring in cellular environment","authors":"Mohammad Faysal Al Mazid ,&nbsp;Syafira Rizqi Eskasalam ,&nbsp;Jun-Seok Lee","doi":"10.1016/j.ymeth.2025.04.002","DOIUrl":"10.1016/j.ymeth.2025.04.002","url":null,"abstract":"<div><div>Cellular redox homeostasis is tightly regulated by the oxidation–reduction reactions of nicotinamide metabolites, including NAD(H) and NADP(H), which serve as essential cofactors in enzymatic processes related to energy metabolism. Monitoring intracellular NADH levels is therefore of significant interest. Most chemosensor designs to date rely on fluorescence turn-on mechanisms triggered by NADH oxidation, but these reaction-based sensors are inherently limited by NADH concentration and reaction kinetics. While NADH exhibits intrinsic fluorescence, its low quantum yield has led to the development of redox-sensitive substrates that emit fluorescence upon NADH oxidation. Here, we report an alternative fluorescence enhancement strategy based on an NADH-binding RNA aptamer. The interaction between NADH and a 49-base-pair RNA aptamer induces a 1.4-fold increase in fluorescence emission <em>in vitro</em> and an 1.8-fold increase in live-cell imaging. This fluorescence enhancement arises from aptamer-induced structural rigidity, analogous to the mechanism by which 4-(p-hydroxybenzylidene)-5-imidazolidinone (HBI) enhances fluorescence in green fluorescent protein. Using our aptamer-based assay, we established a live-cell fluorescence emission assay for real-time monitoring of cellular NADH dynamics.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"240 ","pages":""},"PeriodicalIF":4.2,"publicationDate":"2025-04-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143802131","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}