Methods最新文献

{"title":"Development and interpretation of a pathomics-based model for the prediction of immune therapy response in colorectal cancer.","authors":"Yan Luo, Qiang Tian, Lei Xu, Dongmei Zeng, Hua Zhang, Tianyu Zeng, Hua Tang, Chao Wang, Yihua Chen","doi":"10.1016/j.ymeth.2025.05.012","DOIUrl":"https://doi.org/10.1016/j.ymeth.2025.05.012","url":null,"abstract":"<p><p>Colorectal cancer (CRC) is the third most common malignancy and the second leading cause of cancer-related deaths worldwide, with a 5-year survival rate below 20 %. Immunotherapy, particularly immune checkpoint blockade (ICB)-based therapies, has become an important approach for CRC treatment. However, only specific patient subsets demonstrate significant clinical benefits. Although the TIDE algorithm can predict immunotherapy responses, the reliance on transcriptome sequencing data limits its clinical applicability. Recent advances in artificial intelligence and computational pathology provide new avenues for medical image analysis.In this study, we classified TCGA-CRC samples into immunotherapy responder and non-responder groups using the TIDE algorithm. Further, a pathomics model based on convolutional neural networks was constructed to directly predict immunotherapy responses from histopathological images. Single-cell analysis revealed that fibroblasts may induce immunotherapy resistance in CRC through collagen-CD44 and ITGA1 + ITGB1 signaling axes. The developed pathomics model demonstrated excellent classification performance in the test set, with an AUC of 0.88 at the patch level and 0.85 at the patient level. Moreover, key pathomics features were identified through SHAP analysis. This innovative predictive tool provides a novel method for clinical decision-making in CRC immunotherapy, with potential to optimize treatment strategies and advance precision medicine.</p>","PeriodicalId":390,"journal":{"name":"Methods","volume":" ","pages":""},"PeriodicalIF":4.2,"publicationDate":"2025-05-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144207316","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":"Metabolic impacts of long-chain fatty acids on cardiomyocyte maturation in neonatal mammalian hearts","authors":"Seong-Eung Cha ,&nbsp;Mi Nam Lee ,&nbsp;Eung-Sam Kim","doi":"10.1016/j.ymeth.2025.05.010","DOIUrl":"10.1016/j.ymeth.2025.05.010","url":null,"abstract":"<div><div>Cardiomyocytes are essential models for cardiac disease modeling, drug development, and regenerative therapies. Specifically, human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as widely used cellular models with high reproducibility. However, cardiomyocytes generated <em>in vitro</em> tend to remain immature and insufficient in replicating the electrophysiological and mechanical functions of adult cardiomyocytes, limiting the clinical and experimental applications of these models. Thus, various biochemical and biophysical strategies have been explored to promote the maturation of cardiomyocytes, to address these limitations, and more accurately mimic the characteristics of mature cardiomyocytes. This review summarizes recent studies on multiple methodologies employed to induce cardiomyocyte maturation, with a particular emphasis on the role of long-chain fatty acids (LCFAs). The evidence summarized in this review is derived from studies utilizing cardiomyocytes from neonatal mice or rats and hiPSC-CMs. Meanwhile, immature cardiomyocytes have been demonstrated to predominantly rely on glycolysis, transitioning to oxidative phosphorylation through maturation, which enhances electrical stability, contractility, and structural organization. LCFAs play a key role in the cardiomyocyte maturation process by serving as key metabolic factors that generate ATP through mitochondrial β-oxidation, thereby improving metabolic efficiency. Additionally, LCFAs are involved in activating cytoskeletal components and signaling pathways integral to cardiomyocyte contractility. Importantly, studies suggest that when multiple biochemical and biophysical stimuli are simultaneously applied, various aspects of cardiomyocyte maturation are synergistically accelerated. Therefore, future studies focusing on the coordinated application of these regulatory factors are expected to enhance the maturation process, ultimately contributing to the generation of mature cardiomyocytes suitable for regenerative medicine and other advanced applications.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"241 ","pages":"Pages 114-127"},"PeriodicalIF":4.2,"publicationDate":"2025-05-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144184526","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 the Azu-Eos as a fluorescence-free staining method for enhanced bright-field microscopy in the comet assay","authors":"Penyo Ivanov,&nbsp;Dessislava Staneva,&nbsp;Milena Georgieva,&nbsp;George Miloshev","doi":"10.1016/j.ymeth.2025.05.011","DOIUrl":"10.1016/j.ymeth.2025.05.011","url":null,"abstract":"<div><div>The comet assay is a valuable technique for assessing cellular DNA damage due to intrinsic and environmental factors, particularly in studies of genotoxicity and mutagenesis. However, its broader application is limited by the need for fluorescent staining, the requirement for expensive fluorescent microscopes, and the inability to preserve and re-examine slides over time. We developed the Azure B-Eosin Y (Azu-Eos) staining method to visualise the comets under a standard bright-field microscope to overcome these limitations. The newly developed DNA-staining technique eliminates the need for fluorescence and allows unlimited slide storage and re-examination without quality deterioration. We optimised various factors to ensure the reliability and quality of bright-field stained comets, achieving results comparable to and even better than those obtained with traditional fluorescent dyes. This cost-effective, sensitive, and fluorescence-free method has broad potential applications in medicine, ecology, and environmental monitoring, enabling fast and on-site genotoxicity testing.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"241 ","pages":"Pages 103-113"},"PeriodicalIF":4.2,"publicationDate":"2025-05-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144179830","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":"Surface-enhanced Raman scattering (SERS) technology: Emerging applications in cancer imaging and precision medicine","authors":"Biqing Chen,&nbsp;Jiayin Gao,&nbsp;Haizhu Sun,&nbsp;Zhi Chen,&nbsp;Xiaohong Qiu","doi":"10.1016/j.ymeth.2025.05.009","DOIUrl":"10.1016/j.ymeth.2025.05.009","url":null,"abstract":"<div><div>Recent advancements in Surface-enhanced Raman Scattering (SERS) bioprobes have substantially enhanced their bioimaging capabilities for disease theranostics. This review systematically analyzes three categories of engineered SERS probes: noble metal nanostructures, metal oxide hybrids, and multifunctional composite materials, emphasizing their optimized designs for targeted tumor detection and image-guided surgery. Key developments include improved in vitro biosensing platforms for rapid tumor screening and advanced in vivo probes enabling real-time intraoperative imaging with molecular specificity. The integration of SERS with complementary modalities (fluorescence, photoacoustic, MRI) is critically examined as a strategy to overcome individual technical limitations and achieve multiscale tissue characterization. Technical progress in spatial resolution enhancement, multiplex biomarker detection, and biocompatibility optimization is quantitatively highlighted. Current challenges in signal consistency across biological systems and scalable probe manufacturing are discussed, proposing standardized evaluation frameworks as essential for clinical translation. This work establishes SERS as a multimodal imaging cornerstone for precision oncology, particularly in tumor margin delineation and metastatic lesion identification.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"241 ","pages":"Pages 67-93"},"PeriodicalIF":4.2,"publicationDate":"2025-05-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144131959","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":"Enhancing RNA Delivery: Practical insights into NeoLNPTM transfection reagent","authors":"Xiaobao Chen ,&nbsp;Yan Wu ,&nbsp;Li Liu ,&nbsp;Wei Wang","doi":"10.1016/j.ymeth.2025.05.007","DOIUrl":"10.1016/j.ymeth.2025.05.007","url":null,"abstract":"<div><div>The rapid advancement of RNA-based therapeutics, particularly in the wake of COVID-19 vaccine success, has prompted significant research into optimizing RNA delivery mechanisms. This study evaluates the NeoLNP^TM RNA Transfection Kit developed by Scindy Pharmaceutical, which utilizes lipid nanoparticles (LNPs) for efficient RNA encapsulation and delivery. We systematically investigate various parameters affecting transfection efficiency, including RNA concentration, RNA/LNP volume ratios, mixing techniques, LNP stability, and culture media. Our results demonstrate that the optimal RNA concentration for transfection efficiency is around 40–60 ng/µL, with a 1:0.75–1:1 RNA-to-LNP ratio yielding the highest protein expression. Additionally, we find that gentle mixing techniques outperform harsher methods, and the stability of LNP-RNA complexes significantly influences transfection outcomes. This research provides practical guidelines for enhancing RNA transfection efficiency, paving the way for more effective RNA therapeutics.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"241 ","pages":"Pages 59-66"},"PeriodicalIF":4.2,"publicationDate":"2025-05-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144116469","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":"Quorum sensing: An essential factor in culturing the human promyelocytic leukemia cell line HL-60 and its neutrophil-related functions","authors":"Danfeng Li ,&nbsp;Tian Yang ,&nbsp;Siyuan Ma ,&nbsp;Xinwei Lyu ,&nbsp;Cheng Hu ,&nbsp;Jiayin Yan ,&nbsp;Lihong Guo ,&nbsp;Jiali Tan","doi":"10.1016/j.ymeth.2025.05.008","DOIUrl":"10.1016/j.ymeth.2025.05.008","url":null,"abstract":"<div><div>HL-60 cells are frequently employed as a standard <em>in vitro</em> model for neutrophil research and extensively utilized. However, the cultivation of HL-60 cells presents a recurring challenge. Historically, cell culture density has been ignored in the consistency of culture conditions. Here, we optimized the culture protocol and explored the impact of culture density on HL-60 cells. Additionally, we investigated the differentiated rate and antibacterial potential of differentiated HL-60 (dHL-60) neutrophils across varying cell density cultures. The findings revealed a positive correlation between cell proliferation activity and cell density, suggesting that increased density facilitates enhanced cell proliferation. Furthermore, as the density of the cell culture increased, there was a concomitant rise in the differentiation rate of HL-60 cells into neutrophils upon stimulation. Importantly, this elevated density also led to significantly higher levels of mitochondrial reactive oxygen species (ROS) production and bacterial phagocytosis. Further investigation revealed that small extracellular vesicles (sEVs) are crucial communicator in quorum sensing within HL-60 cells. Supplementation of HL60-derived sEVs (hEVs) in low-density cell populations resulted in a restoration of cell proliferation, in dose-dependent tendency. Conversely, the inhibition of EV-secretion in HL-60 cells restrains cell growth and proliferation. Overall, our study not only optimized the HL-60 cell culture protocol but also elucidated the critical role of culture density in enhancing HL-60 cell proliferation and antibacterial activity. This finding offers a noteworthy consideration for <em>in vitro</em> experiments of HL-60 cells and suggests the involvement of a quorum sensing mechanism within the neutrophil microenvironment.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"241 ","pages":"Pages 94-102"},"PeriodicalIF":4.2,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144126136","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":"The influence of structured light scanning probe configuration on the 3D scanning accuracy of the maxillofacial region in a smiling state: An in vitro study","authors":"Miaosheng Guan ,&nbsp;Xiaoyu Wang ,&nbsp;Xue Li ,&nbsp;Yaqi Wang ,&nbsp;Kun Yan ,&nbsp;Ran Huo ,&nbsp;Tao Song ,&nbsp;Lin Liu ,&nbsp;Hongbo Li","doi":"10.1016/j.ymeth.2025.05.006","DOIUrl":"10.1016/j.ymeth.2025.05.006","url":null,"abstract":"<div><div>Currently, single-unit, dual-unit, and triple-unit structured light 3D scanning technologies have become the predominant facial scanning methods. However, the impact of different unit strategies on facial scanning accuracy remains unclear. A standardized 3D facial model in a smiling state was established. Key point reference coordinates and 3D data were obtained using a coordinate measurement instrument and an industrial-grade laser 3D scanner. Three structured light scanning techniques (single-, dual-, triple-unit) were utilized to capture the 3D information of the model. Linear distance deviations and 3D surface deviations (trueness and precision) of the three scanning strategies were compared. The triple-unit scanning strategy exhibited the lowest deviation among 20 trueness indicators and 22 precision indicators for linear distance measurements (P &lt; 0.05). Furthermore, the accuracy of the triple-unit strategy (trueness: 0.1607 ± 0.0201 mm, precision: 0.0161 ± 0.0112 mm) for overall facial scanning was significantly lower than that of the single-unit and dual-unit strategies, particularly in critical regions for oral and maxillofacial aesthetic analysis, such as the orbital, nasal, and perioral regions. The triple-unit structured light scanning strategy significantly enhances the accuracy of facial 3D scanning, particularly when acquiring 3D facial information from the midline and perioral regions. This in vitro study demonstrates that the triple-unit structured light 3D scanning strategy effectively improves the accuracy of facial scanning, especially in the oral-maxillofacial aesthetic regions. This approach provides a foundation and support for both preoperative planning and postoperative evaluation of aesthetic restoration.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"241 ","pages":"Pages 43-50"},"PeriodicalIF":4.2,"publicationDate":"2025-05-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144071803","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":"Multi-filter based signed heterogeneous graph convolutional networks for predicting activating/inhibiting drug-target interactions","authors":"Ming Chen ,&nbsp;Haike Li ,&nbsp;Yunhan Pan ,&nbsp;Yinglong Dai ,&nbsp;Xiujuan Lei ,&nbsp;Yi Pan","doi":"10.1016/j.ymeth.2025.05.005","DOIUrl":"10.1016/j.ymeth.2025.05.005","url":null,"abstract":"<div><div>The prediction of mechanisms within drug-target interactions (DTIs) can boost the drug discovery process, which has traditionally relied on time-consuming and expensive laboratory experiments. Despite much more attention has been paid to predicting DTIs, but few studies focused on their activating/inhibiting mechanisms. In this work, we model DTIs on signed heterogeneous networks, through categorizing activating/inhibiting DTIs into signed links, and accordingly introducing the coherence/incoherence between drugs on a common target to construct signed drug-drug links. We propose a multi-filter based signed heterogeneous graph convolutional network (MFSHGCN) for drugs and targets embedding, via employing dual filters on both the signed drug-drug sub-graph and the signed DTI sub-graph to converge the spectral information from positive and negative edges. We further put forward an end-to-end framework to predict activation and inhibition within DTIs. The comparison results demonstrate the introduction of coherence/incoherence of drug pairs and the design of our multi-filter system can effectively improve the prediction metrics, even without relying on rich node information and interactions from drug pairs or target pairs. Case studies on breast cancer and lung cancer confirm the model's feasibility.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"241 ","pages":"Pages 51-58"},"PeriodicalIF":4.2,"publicationDate":"2025-05-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144092458","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":"An NGS-based approach for precise and footprint-free CRISPR-based gene editing in human stem cells","authors":"Bert Vandendriessche ,&nbsp;Jolien Huyghebaert ,&nbsp;Kirsten Van Rossem ,&nbsp;Tycho Canter Cremers ,&nbsp;Kevin De Man ,&nbsp;Ewa Sieliwonczyk ,&nbsp;Hanne Boen ,&nbsp;Dogan Akdeniz ,&nbsp;Laura Rabaut ,&nbsp;Jolien Schippers ,&nbsp;Peter Ponsaerts ,&nbsp;R. Frank Kooy ,&nbsp;Bart Loeys ,&nbsp;Dorien Schepers ,&nbsp;Maaike Alaerts","doi":"10.1016/j.ymeth.2025.05.004","DOIUrl":"10.1016/j.ymeth.2025.05.004","url":null,"abstract":"<div><div>Precise gene editing with conventional CRISPR/Cas9 is often constrained by low <em>knock-in</em> (KI) efficiencies (≈ 2–20 %) in human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs). This limitation typically necessitates labour-intensive manual isolation and genotyping of hundreds of colonies to identify correctly edited cells. Fluorescence- or antibiotic-based enrichment methods facilitate the identification process but can compromise cell viability and genomic integrity. Here, we present a footprint-free editing strategy that combines low-density seeding with next-generation sequencing (NGS) to rapidly identify cell populations containing precisely modified clones. By optimising the transfection workflow and adhering to CRISPR/Cas9 KI design principles, we achieved high average editing efficiencies of 64 % in hiPSCs (introducing a Brugada syndrome-associated variant) and 51 % in hESCs (introducing a neurodevelopmental disorder (NDD)-associated variant). Furthermore, under suboptimal CRISPR design conditions, this approach successfully identified hESC clones carrying a second NDD-associated variant, despite average KI efficiencies below 1 %. Importantly, genomic integrity was preserved throughout subcloning rounds, as confirmed by Sanger sequencing and single nucleotide polymorphism (SNP) array analysis. Hence, this NGS-based enrichment strategy reliably identifies desired KI clones under both optimal and challenging conditions, reducing the need for extensive colony screening and offering an effective alternative to fluorescence- and antibiotic-based selection methods.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"241 ","pages":"Pages 33-42"},"PeriodicalIF":4.2,"publicationDate":"2025-05-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143947684","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":"Robust temporal knowledge inference via pathway snapshots with liquid neural network","authors":"Peifu Han ,&nbsp;Jianmin Wang ,&nbsp;Dayan Liu ,&nbsp;Lin Liu ,&nbsp;Tao Song","doi":"10.1016/j.ymeth.2025.05.003","DOIUrl":"10.1016/j.ymeth.2025.05.003","url":null,"abstract":"<div><div>Static graphs play a pivotal role in modeling and analyzing biological and biomedical data. However, many real-world scenarios—such as disease progression and drug pharmacokinetic processes—exhibit dynamic behaviors. Consequently, static graph methods often struggle to robustly address new environments characterized by complex and previously unseen relationship changes. Here, we propose a method for constructing temporal knowledge inference agents tailored to disease pathways, enabling effective relation reasoning beyond their training environment under complex shifts. To achieve this, we developed an imitation learning framework using liquid neural networks, a class of continuous-time neural models inspired by the brain function that are causal and adaptable to changing conditions. Our findings indicate that liquid agents can distill the essential tasks from knowledge graph inputs while accounting temporal evolution, thereby enabling the transfer of temporal skills to novel time nodes. Compared to state-of-the-art deep reinforcement learning agents, experiments demonstrate that temporal robustness in decision-making emerges uniquely in liquid networks.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"241 ","pages":"Pages 24-32"},"PeriodicalIF":4.2,"publicationDate":"2025-05-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143942678","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}