IF 4.3 3区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

Methods

Pub Date : 2026-01-08 DOI: 10.1016/j.ymeth.2026.01.004

Víctor Pola-Véliz , Sebastián B. Arredondo , Yennyfer Arancibia , Juan Ahumada , Sebastián Estay , Nicole Vidal , Paola A. Haeger , Marco Fuenzalida , Lorena Varela-Nallar , Fernando J. Bustos , Martin Montecino , Brigitte van Zundert

Memory formation activates a relatively sparse population of engram cells that store long-term memories. Changes in the epigenetic landscape and 3D chromatin architecture have been proposed as key candidate regulators of transcriptional waves that control gene expression in engram cells; however, isolating chromatin efficiently from engram cells has remained challenging. Double-transgenic Targeted Recombination in Active Populations (dTRAP) mice have enabled indelible EYFP labeling of hippocampal engram cells expressing the immediate-early gene (IEG) Arc when ArcCreER^T2 mice are crossed with R26R-STOP-floxed-EYFP mice and exposed to learning paradigms. A major limitation of dTRAP mice is that labeling of activated hippocampal Arc⁺ neurons with soluble EYFP compromises the efficiency of fluorescence-activated nuclear sorting (FANS) of engram nuclei, and hence isolation of chromatin. Here, we used viral-mediated delivery of GFP-KASH (AAV-PHP.eB-FLEX-EGFP-KASH) to ArcCreER^T2 mice -generating vkTRAP mice- to enable precise and robust endogenous perinuclear fluorescent tagging of activated hippocampal neurons following contextual fear conditioning (CFC). At 24 h post-CFC (24 h-CFC), vkTRAP mice exhibited a robust freezing behavior. Electrophysiological recordings in CA1 hippocampal slices showed occluded long-term potentiation (LTP). Efficient FANS-based isolation of hippocampal engram nuclei enabled chromatin immunoprecipitation (ChIP) assays (detecting H3K4me3, H3K9ac and H3K27ac) at promoters of immediate-early (Egr1) and plasticity-related (Dlg4/PSD95) genes. Expression peaks of both Egr1 and Dlg4/PSD95 transcripts during memory acquisition (1 h-CFC) and consolidation (24 h-CFC) were accompanied by active epigenetic histone mark profiles. We conclude that vkTRAP provides a robust model to study epigenomic regulation in engram cells.

记忆形成激活了相对稀少的印迹细胞，这些细胞储存了长期记忆。表观遗传景观和三维染色质结构的变化被认为是印迹细胞中控制基因表达的转录波的关键候选调节因子；然而，从印迹细胞中有效地分离染色质仍然具有挑战性。当ArcCreERT2小鼠与R26R- stop - floxedypfp小鼠杂交并暴露在学习范式中时，双转基因活性群体靶向重组（dTRAP）小鼠能够使表达立即早期基因（IEG） Arc的海马印迹细胞不可磨灭地标记EYFP。dTRAP小鼠的一个主要限制是，用可溶性EYFP标记激活的海马Arc+神经元会影响印迹核的荧光激活核分选（FANS）的效率，从而影响染色质的分离。在这里，我们使用病毒介导的GFP-KASH （AAV-PHP.eB-FLEX-EGFP-KASH）传递到ArcCreERT2小鼠-生成vkTRAP小鼠-以实现上下文恐惧条件反射（CFC）后激活的海马神经元的精确和强大的内源性核周荧光标记。在24 h（24 h- cfc）后，vkTRAP小鼠表现出强健的冻结行为。CA1海马切片电生理记录显示长时程增强（LTP）闭塞。海马印迹核的高效分离使染色质免疫沉淀（ChIP）测定（检测H3K4me3， H3K9ac和H3K27ac）在立即早期（Egr1）和可塑性相关（Dlg4/PSD95）基因启动子上。Egr1和Dlg4/PSD95转录本在记忆获取（1 h-CFC）和巩固（24 h-CFC）期间的表达高峰均伴有活跃的表观遗传组蛋白标记谱。我们得出结论，vkTRAP提供了一个强大的模型来研究印迹细胞的表观基因组调控。

{"title":"Viral-mediated fluorescent labelling of activated hippocampal memory engrams to study epigenetic dynamics associated with gene expression","authors":"Víctor Pola-Véliz , Sebastián B. Arredondo , Yennyfer Arancibia , Juan Ahumada , Sebastián Estay , Nicole Vidal , Paola A. Haeger , Marco Fuenzalida , Lorena Varela-Nallar , Fernando J. Bustos , Martin Montecino , Brigitte van Zundert","doi":"10.1016/j.ymeth.2026.01.004","DOIUrl":"10.1016/j.ymeth.2026.01.004","url":null,"abstract":"<div><div>Memory formation activates a relatively sparse population of engram cells that store long-term memories. Changes in the epigenetic landscape and 3D chromatin architecture have been proposed as key candidate regulators of transcriptional waves that control gene expression in engram cells; however, isolating chromatin efficiently from engram cells has remained challenging. Double-transgenic <em>T</em>argeted <em>R</em>ecombination in <em>A</em>ctive <em>P</em>opulations (dTRAP) mice have enabled indelible EYFP labeling of hippocampal engram cells expressing the immediate-early gene (IEG) <em>Arc</em> when ArcCreER<sup>T2</sup> mice are crossed with R26R-STOP-floxed-EYFP mice and exposed to learning paradigms. A major limitation of dTRAP mice is that labeling of activated hippocampal Arc<sup>+</sup> neurons with soluble EYFP compromises the efficiency of fluorescence-activated nuclear sorting (FANS) of engram nuclei, and hence isolation of chromatin. Here, we used viral-mediated delivery of GFP-KASH (AAV-PHP.eB-FLEX-EGFP-KASH) to ArcCreER<sup>T2</sup> mice -generating vkTRAP mice- to enable precise and robust endogenous perinuclear fluorescent tagging of activated hippocampal neurons following contextual fear conditioning (CFC). At 24 h post-CFC (24 h-CFC), vkTRAP mice exhibited a robust freezing behavior. Electrophysiological recordings in CA1 hippocampal slices showed occluded long-term potentiation (LTP). Efficient FANS-based isolation of hippocampal engram nuclei enabled chromatin immunoprecipitation (ChIP) assays (detecting H3K4me3, H3K9ac and H3K27ac) at promoters of immediate-early (<em>Egr1</em>) and plasticity-related (<em>Dlg4</em>/PSD95) genes. Expression peaks of both <em>Egr1</em> and <em>Dlg4/</em>PSD95 transcripts during memory acquisition (1 h-CFC) and consolidation (24 h-CFC) were accompanied by active epigenetic histone mark profiles. We conclude that vkTRAP provides a robust model to study epigenomic regulation in engram cells.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"247 ","pages":"Pages 85-94"},"PeriodicalIF":4.3,"publicationDate":"2026-01-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145942002","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

Rapid and improved surface passivation method for Single-Molecule experiments 快速改进的单分子表面钝化方法。

IF 4.3 3区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

Methods

Pub Date : 2026-01-07 DOI: 10.1016/j.ymeth.2026.01.003

Alyssa N. Gonneville , Alyssa E. Ward , Narisa Ria Naidoo , Francisco N. Barrera , Rajan Lamichhane

Single-molecule fluorescence experiments are a powerful tool for studying biomolecular interactions, including protein dynamics and oligomerization, protein–protein interactions, and protein-nucleic acid interactions. Biomolecules are commonly immobilized on the microscope surface to extend the observation time. However, non-specific interactions between biomolecules and the surface present a major challenge. The first critical step in these experiments is preparing the surface using polyethylene glycol (PEG) coated slides, which facilitate biomolecule immobilization while minimizing non-specific interactions. The surface treatment typically uses PEG-SVA (Succinimidyl Valerate) coated slides, and the protocol for the treatment is lengthy and time-consuming. To overcome this issue, we have developed a process that uses PEG-Silane to improve efficiency while maintaining reproducibility. Here, we present a one-step, rapid PEGylation methodology that can be completed in minutes rather than hours. We demonstrate its validity and feasibility through single-molecule fluorescence resonance energy transfer (smFRET) and single-molecule photobleaching experiments across various biological samples.

单分子荧光实验是研究生物分子相互作用的有力工具，包括蛋白质动力学和寡聚化、蛋白质-蛋白质相互作用和蛋白质-核酸相互作用。通常将生物分子固定在显微镜表面以延长观察时间。然而，生物分子与表面之间的非特异性相互作用是一个主要的挑战。这些实验的第一个关键步骤是使用聚乙二醇（PEG）涂层载玻片制备表面，这有助于生物分子的固定，同时最大限度地减少非特异性相互作用。表面处理通常使用PEG-SVA（琥珀酰戊酸酯）涂层载玻片，处理方案冗长且耗时。为了克服这个问题，我们开发了一种使用peg -硅烷的工艺，以提高效率，同时保持可重复性。在这里，我们提出了一个一步，快速聚乙二醇化方法，可以在几分钟内完成，而不是几个小时。我们通过不同生物样品的单分子荧光共振能量转移（smFRET）和单分子光漂白实验证明了其有效性和可行性。

{"title":"Rapid and improved surface passivation method for Single-Molecule experiments","authors":"Alyssa N. Gonneville , Alyssa E. Ward , Narisa Ria Naidoo , Francisco N. Barrera , Rajan Lamichhane","doi":"10.1016/j.ymeth.2026.01.003","DOIUrl":"10.1016/j.ymeth.2026.01.003","url":null,"abstract":"<div><div>Single-molecule fluorescence experiments are a powerful tool for studying biomolecular interactions, including protein dynamics and oligomerization, protein–protein interactions, and protein-nucleic acid interactions. Biomolecules are commonly immobilized on the microscope surface to extend the observation time. However, non-specific interactions between biomolecules and the surface present a major challenge. The first critical step in these experiments is preparing the surface using polyethylene glycol (PEG) coated slides, which facilitate biomolecule immobilization while minimizing non-specific interactions. The surface treatment typically uses PEG-SVA (Succinimidyl Valerate) coated slides, and the protocol for the treatment is lengthy and time-consuming. To overcome this issue, we have developed a process that uses PEG-Silane to improve efficiency while maintaining reproducibility. <em>Here</em>, we present a one-step, rapid PEGylation methodology that can be completed in minutes rather than hours. We demonstrate its validity and feasibility through single-molecule fluorescence resonance energy transfer (smFRET) and single-molecule photobleaching experiments across various biological samples.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"247 ","pages":"Pages 95-106"},"PeriodicalIF":4.3,"publicationDate":"2026-01-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145942009","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

Precision mapping of NF-κB-DNA binding: high-resolution insights via dynamic UV-laser footprinting NF-κB-DNA结合的精确定位：通过动态紫外激光足迹的高分辨率见解

IF 4.3 3区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

Methods

Pub Date : 2026-01-07 DOI: 10.1016/j.ymeth.2026.01.005

Imtiaz Nisar Lone , Gözdem Çavdar , Zahari Peshev , Seyit Kale , Dimitar Angelov

Sequence-specific binding is at the core of all DNA-templated processes, including the initiation of DNA replication, gene expression and DNA repair. Yet the kinetics and precision of these interactions remain difficult to capture at high resolution. Here, we present Dynamic UV Laser Footprinting (DULF), a novel technique that integrates UV laser footprinting with stopped-flow mixing to probe transcription factor (TF)-DNA interactions at millisecond temporal and single base-pair spatial resolution. Using NF-κB as a model TF, we demonstrate the feasibility of DULF in visualizing the spatiotemporal details of a sequence-specific TF-DNA interaction. The high temporal resolution of our data reveals that TF-DNA binding proceeds through a rapid recognition step and a subsequent slower stabilization phase. DULF also revealed that the p50 homodimer binds specifically to DNA outside the canonical binding site, emphasizing the role of flanking sequences in these interactions. All-atom molecular dynamics simulations confirmed that DNA sequence context, including flanking base pairs, modulates NF-κB binding stability and induces local structural changes such as bending, groove widening, and base unstacking. DULF offers a unique opportunity to study DNA-protein interactions at unprecedented resolution, providing insights into the mechanism of sequence-specific binding and stabilization of chromatin interactors.

序列特异性结合是所有DNA模板化过程的核心，包括DNA复制的起始、基因表达和DNA修复。然而，这些相互作用的动力学和精度仍然难以在高分辨率下捕获。在这里，我们提出了动态紫外激光足迹（DULF），这是一种将紫外激光足迹与停止流动混合相结合的新技术，可以在毫秒时间和单碱基对空间分辨率下探测转录因子(TF)-DNA相互作用。使用NF-κB作为模型TF，我们证明了DULF在可视化序列特异性TF- dna相互作用的时空细节方面的可行性。我们数据的高时间分辨率揭示了TF-DNA结合通过快速识别步骤和随后较慢的稳定阶段进行。DULF还揭示了p50同型二聚体特异性结合到典型结合位点外的DNA，强调了侧翼序列在这些相互作用中的作用。全原子分子动力学模拟证实，DNA序列背景（包括侧翼碱基对）调节NF-κB结合稳定性，并诱导局部结构变化，如弯曲、凹槽扩大和碱基解堆叠。DULF提供了一个独特的机会，以前所未有的分辨率研究dna -蛋白质相互作用，提供了对序列特异性结合和染色质相互作用稳定机制的见解。

{"title":"Precision mapping of NF-κB-DNA binding: high-resolution insights via dynamic UV-laser footprinting","authors":"Imtiaz Nisar Lone , Gözdem Çavdar , Zahari Peshev , Seyit Kale , Dimitar Angelov","doi":"10.1016/j.ymeth.2026.01.005","DOIUrl":"10.1016/j.ymeth.2026.01.005","url":null,"abstract":"<div><div>Sequence-specific binding is at the core of all DNA-templated processes, including the initiation of DNA replication, gene expression and DNA repair. Yet the kinetics and precision of these interactions remain difficult to capture at high resolution. <em>Here</em>, we present Dynamic UV Laser Footprinting (DULF), a novel technique that integrates UV laser footprinting with stopped-flow mixing to probe transcription factor (TF)-DNA interactions at millisecond temporal and single base-pair spatial resolution. Using NF-κB as a model TF, we demonstrate the feasibility of DULF in visualizing the spatiotemporal details of a sequence-specific TF-DNA interaction. The high temporal resolution of our data reveals that TF-DNA binding proceeds through a rapid recognition step and a subsequent slower stabilization phase. DULF also revealed that the p50 homodimer binds specifically to DNA outside the canonical binding site, emphasizing the role of flanking sequences in these interactions. All-atom molecular dynamics simulations confirmed that DNA sequence context, including flanking base pairs, modulates NF-κB binding stability and induces local structural changes such as bending, groove widening, and base unstacking. DULF offers a unique opportunity to study DNA-protein interactions at unprecedented resolution, providing insights into the mechanism of sequence-specific binding and stabilization of chromatin interactors.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"247 ","pages":"Pages 50-62"},"PeriodicalIF":4.3,"publicationDate":"2026-01-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145922632","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

ERAPID: an end‑to‑end RNA‑seq analysis pipeline for integrative candidate biomarker discovery with applications to neuropsychiatric disorders ERAPID：一个端到端RNA - seq分析管道，用于综合候选生物标志物的发现，并应用于神经精神疾病。

IF 4.3 3区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

Methods

Pub Date : 2026-01-05 DOI: 10.1016/j.ymeth.2026.01.001

Jihye Park , Seoyeon Youn , Kyuho Kang , Keunsoo Kang

RNA sequencing datasets in the gene expression omnibus (GEO) increasingly include NCBI-generated count matrices, enabling streamlined signature gene discovery. We present ERAPID, a computational framework that automatically processes this public data for robust differential expression analysis. The pipeline integrates metadata harmonization, surrogate variable analysis (SVA) to capture latent technical variation for batch correction, dual differential expression methods (DESeq2 and dream), gene set enrichment analysis (GSEA), and evidence-based gene prioritization via automated literature mining. ERAPID delivers interactive dashboards (volcano/MA plots, heatmaps, enrichment reports, searchable DEG tables) and supports an optional meta‑analysis step. Applied to a neuropsychiatric cohort (GSE80655), ERAPID completed analysis on a standard laptop in under an hour, recapitulating the reported association of EGR1 with schizophrenia. In an Alzheimer’s disease (AD) case study integrating four GEO datasets, ERAPID identified 17 DEGs consistently altered across all AD‑versus‑control comparisons (e.g., ADCYAP1, PPEF1, VGF, and CRH), with KEGG Alzheimer’s disease and oxidative phosphorylation pathways showing negative enrichment. Thus, ERAPID lowers the barrier to reusing public transcriptomes for signature gene discovery and biological interpretation.

基因表达综合（GEO）中的RNA测序数据集越来越多地包括ncbi生成的计数矩阵，从而简化了特征基因的发现。我们提出了ERAPID，这是一个计算框架，可以自动处理这些公共数据，用于稳健的差异表达分析。该管道集成了元数据协调、替代变量分析（SVA）以捕获潜在的技术变异以进行批量校正、双差异表达方法（DESeq2和dream）、基因集富集分析（GSEA）以及通过自动化文献挖掘进行循证基因优先级排序。ERAPID提供交互式仪表板（火山/MA图，热图，富集报告，可搜索的DEG表），并支持可选的元分析步骤。应用于神经精神病学队列（GSE80655）， ERAPID在一个小时内在一台标准笔记本电脑上完成了分析，概括了EGR1与精神分裂症的关联。在一项整合4个GEO数据集的阿尔茨海默病（AD）案例研究中，ERAPID鉴定出17个基因在所有AD与对照比较中一致改变（例如，ADCYAP1、PPEF1、VGF和CRH），其中KEGG阿尔茨海默病和氧化磷酸化途径显示负富集。因此，ERAPID降低了重复使用公共转录组进行特征基因发现和生物学解释的障碍。

{"title":"ERAPID: an end‑to‑end RNA‑seq analysis pipeline for integrative candidate biomarker discovery with applications to neuropsychiatric disorders","authors":"Jihye Park , Seoyeon Youn , Kyuho Kang , Keunsoo Kang","doi":"10.1016/j.ymeth.2026.01.001","DOIUrl":"10.1016/j.ymeth.2026.01.001","url":null,"abstract":"<div><div>RNA sequencing datasets in the gene expression omnibus (GEO) increasingly include NCBI-generated count matrices, enabling streamlined signature gene discovery. We present ERAPID, a computational framework that automatically processes this public data for robust differential expression analysis. The pipeline integrates metadata harmonization, surrogate variable analysis (SVA) to capture latent technical variation for batch correction, dual differential expression methods (DESeq2 and dream), gene set enrichment analysis (GSEA), and evidence-based gene prioritization via automated literature mining. ERAPID delivers interactive dashboards (volcano/MA plots, heatmaps, enrichment reports, searchable DEG tables) and supports an optional meta‑analysis step. Applied to a neuropsychiatric cohort (GSE80655), ERAPID completed analysis on a standard laptop in under an hour, recapitulating the reported association of <em>EGR1</em> with schizophrenia. In an Alzheimer’s disease (AD) case study integrating four GEO datasets, ERAPID identified 17 DEGs consistently altered across all AD‑versus‑control comparisons (e.g., <em>ADCYAP1</em>, <em>PPEF1</em>, <em>VGF</em>, and <em>CRH</em>), with KEGG Alzheimer’s disease and oxidative phosphorylation pathways showing negative enrichment. Thus, ERAPID lowers the barrier to reusing public transcriptomes for signature gene discovery and biological interpretation.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"247 ","pages":"Pages 34-40"},"PeriodicalIF":4.3,"publicationDate":"2026-01-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145916601","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

MetaChrome: an open-source, user-friendly tool for automated metaphase chromosome analysis MetaChrome：一个开源的，用户友好的工具，用于自动中期染色体分析。

IF 4.3 3区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

Methods

Pub Date : 2025-12-29 DOI: 10.1016/j.ymeth.2025.12.013

Md Abdul Kader Sagar , Yamini Dalal , Gianluca Pegoraro , Ganesan Arunkumar

DNA Fluorescence In Situ Hybridization (DNA FISH) is an essential technique to study chromosome biology and genetics, enabling precise visualization of specific genomic loci to study structural abnormalities, gene mapping, and chromosomal rearrangements. High-Throughput Imaging (HTI) can automate the analysis of DNA FISH chromosome images, but the accurate and automated segmentation of mitotic chromosomes and simultaneous colocalization of DNA FISH signals remains a challenge. While several commercial automated karyotyping tools partially solve these issues, open-source software that effectively combines robust chromosome segmentation with comprehensive colocalization analysis capabilities remains necessary. To address this unmet need, we developed MetaChrome, an open-source software platform built around a graphical user interface and explicitly designed for automated metaphase chromosome analysis. MetaChrome leverages fine-tuned deep learning models to automate metaphase chromosome segmentation, together with colocalization analysis of chromosome-specific FISH probes and immunofluorescent-labeled proteins. Importantly, MetaChrome achieves enhanced segmentation accuracy compared to traditional image processing methods by adopting a Cellpose segmentation model fine-tuned with manually annotated metaphase chromosome datasets. The fine-tuned model ensures the precise assignment of DNA FISH spots to individual chromosomes in an automated manner. This facilitates rapid identification of chromosomal abnormalities, reduces human error, and advances high-throughput chromosome analysis workflows, addressing a key bottleneck in chromosome biology research.

DNA荧光原位杂交（DNA FISH）是研究染色体生物学和遗传学的一项重要技术，能够精确地可视化特定的基因组位点，以研究结构异常、基因定位和染色体重排。高通量成像技术（HTI）可以实现DNA FISH染色体图像的自动化分析，但有丝分裂染色体的准确和自动化分割以及DNA FISH信号的同步共定位仍然是一个挑战。虽然一些商业自动化核型工具部分解决了这些问题，但有效结合强大的染色体分割和全面的共定位分析能力的开源软件仍然是必要的。为了解决这一未满足的需求，我们开发了MetaChrome，这是一个围绕图形用户界面构建的开源软件平台，明确设计用于中期染色体自动分析。MetaChrome利用微调的深度学习模型自动化中期染色体分割，以及染色体特异性FISH探针和免疫荧光标记蛋白的共定位分析。重要的是，与传统的图像处理方法相比，MetaChrome通过采用手动注释中期染色体数据集微调的Cellpose分割模型，实现了更高的分割精度。微调模型确保精确分配DNA FISH点到单个染色体在一个自动化的方式。这有助于快速识别染色体异常，减少人为错误，并推进高通量染色体分析工作流程，解决染色体生物学研究的关键瓶颈。

{"title":"MetaChrome: an open-source, user-friendly tool for automated metaphase chromosome analysis","authors":"Md Abdul Kader Sagar , Yamini Dalal , Gianluca Pegoraro , Ganesan Arunkumar","doi":"10.1016/j.ymeth.2025.12.013","DOIUrl":"10.1016/j.ymeth.2025.12.013","url":null,"abstract":"<div><div>DNA Fluorescence In Situ Hybridization (DNA FISH) is an essential technique to study chromosome biology and genetics, enabling precise visualization of specific genomic loci to study structural abnormalities, gene mapping, and chromosomal rearrangements. High-Throughput Imaging (HTI) can automate the analysis of DNA FISH chromosome images, but the accurate and automated segmentation of mitotic chromosomes and simultaneous colocalization of DNA FISH signals remains a challenge. While several commercial automated karyotyping tools partially solve these issues, open-source software that effectively combines robust chromosome segmentation with comprehensive colocalization analysis capabilities remains necessary. To address this unmet need, we developed MetaChrome, an open-source software platform built around a graphical user interface and explicitly designed for automated metaphase chromosome analysis. MetaChrome leverages fine-tuned deep learning models to automate metaphase chromosome segmentation, together with colocalization analysis of chromosome-specific FISH probes and immunofluorescent-labeled proteins. Importantly, MetaChrome achieves enhanced segmentation accuracy compared to traditional image processing methods by adopting a Cellpose segmentation model fine-tuned with manually annotated metaphase chromosome datasets. The fine-tuned model ensures the precise assignment of DNA FISH spots to individual chromosomes in an automated manner. This facilitates rapid identification of chromosomal abnormalities, reduces human error, and advances high-throughput chromosome analysis workflows, addressing a key bottleneck in chromosome biology research.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"247 ","pages":"Pages 12-24"},"PeriodicalIF":4.3,"publicationDate":"2025-12-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145877422","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

Korean longitudinal study on digitally optimized mental healthcare: a cohort profile 韩国对数字优化精神保健的纵向研究：队列概况。

IF 4.3 3区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

Methods

Pub Date : 2025-12-25 DOI: 10.1016/j.ymeth.2025.12.011

Ok Kim , Kyung-Hwa Choi , Tae Hui Kim , Sungmin Son , Jonghun Lee , Kyungmin Kim , Un Sun Chung , Eun-Jin Cheon , Ilju Lee , Hyunwoo Jung , Ho-Jang Kwon , Xue Han , Jonghyuk Choi , Jung Won Kim , Ah Lahm Shin , Jung Jae Lee

The Korean Longitudinal Study on Digitally Optimized Mental Healthcare is an innovative multicenter trial-ready cohort study. It aims to develop a digitally integrated mental healthcare platform that integrates robots, artificial intelligence, and local community services. A total of 3,100 participants, including 1,000 from the previous Chungnam Province cohort and 700 each from Chungnam, Gangwon, and Daegu, will be recruited between December 2024 and the end of 2027. Sociodemographic factors and physical health data are collected at enrollment using questionnaires. Every four months, formal tools are used to conduct psychiatric diagnoses and determine participants’ mental health condition, such as depressive symptoms, anxiety symptoms, suicidality, stress, insomnia, loneliness, quality of life, and disability. We assessed smartphone overdependence at regular intervals. Passive data on phone usage, life patterns, and health monitoring from a smart band are collected in real time. Weekly ecological momentary assessments monitor daily life and mood and detect risk situations early. A subset of 500 participants engages in a Living Lab, collecting multi-modal data through robots and sleep radar sensors while evaluating the usability of and satisfaction with these devices. This study will show how tailored digital applications and web-based platforms can facilitate personalized self-help interventions, enhance expert–user interaction, and promote active user engagement. This approach can potentially reduce stigma and improve public awareness of mental healthcare by shifting from a treatment-centered model to a community-based prevention framework.

韩国数字优化心理保健纵向研究是一项创新的多中心试验就绪队列研究。该项目旨在开发一个集成了机器人、人工智能和当地社区服务的数字化综合精神医疗平台。从2024年12月开始到2027年底为止，将从原忠南地区招募1000人，忠南、江原、大邱地区各招募700人，共招募3100人。社会人口因素和身体健康数据在登记时使用问卷收集。每四个月，使用正式工具进行精神诊断并确定参与者的心理健康状况，如抑郁症状、焦虑症状、自杀倾向、压力、失眠、孤独、生活质量和残疾。我们定期评估对智能手机的过度依赖。通过智能手环实时收集手机使用情况、生活模式和健康监测等被动数据。每周生态瞬间评估监测日常生活和情绪，并及早发现风险情况。500名参与者参与了一个生活实验室，通过机器人和睡眠雷达传感器收集多模态数据，同时评估这些设备的可用性和满意度。这项研究将展示量身定制的数字应用程序和基于网络的平台如何促进个性化自助干预，增强专家与用户的互动，并促进积极的用户参与。通过从以治疗为中心的模式转向以社区为基础的预防框架，这种方法有可能减少耻辱感，提高公众对精神卫生保健的认识。

{"title":"Korean longitudinal study on digitally optimized mental healthcare: a cohort profile","authors":"Ok Kim , Kyung-Hwa Choi , Tae Hui Kim , Sungmin Son , Jonghun Lee , Kyungmin Kim , Un Sun Chung , Eun-Jin Cheon , Ilju Lee , Hyunwoo Jung , Ho-Jang Kwon , Xue Han , Jonghyuk Choi , Jung Won Kim , Ah Lahm Shin , Jung Jae Lee","doi":"10.1016/j.ymeth.2025.12.011","DOIUrl":"10.1016/j.ymeth.2025.12.011","url":null,"abstract":"<div><div>The Korean Longitudinal Study on Digitally Optimized Mental Healthcare is an innovative multicenter trial-ready cohort study. It aims to develop a digitally integrated mental healthcare platform that integrates robots, artificial intelligence, and local community services. A total of 3,100 participants, including 1,000 from the previous Chungnam Province cohort and 700 each from Chungnam, Gangwon, and Daegu, will be recruited between December 2024 and the end of 2027. Sociodemographic factors and physical health data are collected at enrollment using questionnaires. Every four months, formal tools are used to conduct psychiatric diagnoses and determine participants’ mental health condition, such as depressive symptoms, anxiety symptoms, suicidality, stress, insomnia, loneliness, quality of life, and disability. We assessed smartphone overdependence at regular intervals. Passive data on phone usage, life patterns, and health monitoring from a smart band are collected in real time. Weekly ecological momentary assessments monitor daily life and mood and detect risk situations early. A subset of 500 participants engages in a Living Lab, collecting multi-modal data through robots and sleep radar sensors while evaluating the usability of and satisfaction with these devices. This study will show how tailored digital applications and web-based platforms can facilitate personalized self-help interventions, enhance expert–user interaction, and promote active user engagement. This approach can potentially reduce stigma and improve public awareness of mental healthcare by shifting from a treatment-centered model to a community-based prevention framework.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"247 ","pages":"Pages 41-49"},"PeriodicalIF":4.3,"publicationDate":"2025-12-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145846209","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

Cross-modal image generation with uncertainty quantification from echocardiogram to MRI 从超声心动图到MRI的不确定量化的交叉模态图像生成。

IF 4.3 3区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

Methods

Pub Date : 2025-12-23 DOI: 10.1016/j.ymeth.2025.12.012

Zakia Zinat Choudhury , Samiran Dey , Haoming Wang , Sean Coffey , Tapabrata Chakraborti , Brendan McCane

Medical imaging is fundamental to cardiovascular diagnostics, with modalities such as Transthoracic Echocardiography (TTE) and Cardiac Magnetic Resonance (CMR) offering complementary strengths. TTE provides real-time, non-invasive visualization of cardiac function but is often limited by operator dependency and incomplete views. In contrast, CMR delivers comprehensive, high-resolution structural assessments, although it comes with greater time and cost burdens. To address these limitations, this study explores cross-modal generative modeling techniques for synthesizing CMR-like images directly from TTE. We propose a novel architecture that combines a UNet backbone with a vision transformer, utilizing the UNet for feature extraction and the transformer for global attention to improve image synthesis quality. Quantitative and qualitative evaluations demonstrate the model’s ability to produce realistic and anatomically consistent CMR images, with strong potential to improve diagnostic accuracy and clinical decision-making across multiple image modalities.

医学成像是心血管诊断的基础，经胸超声心动图（TTE）和心脏磁共振（CMR）等方式提供了互补的优势。TTE提供实时、无创的心功能可视化，但常常受到操作者依赖性和视图不完整的限制。相比之下，CMR提供了全面、高分辨率的结构评估，尽管它需要更多的时间和成本负担。为了解决这些限制，本研究探索了直接从TTE合成cmr类图像的跨模态生成建模技术。我们提出了一种新的架构，将UNet骨干网与视觉转换器相结合，利用UNet进行特征提取，利用变压器进行全局关注，以提高图像合成质量。定量和定性评估表明，该模型能够产生真实的、解剖结构一致的CMR图像，具有提高多种图像模式诊断准确性和临床决策的强大潜力。

{"title":"Cross-modal image generation with uncertainty quantification from echocardiogram to MRI","authors":"Zakia Zinat Choudhury , Samiran Dey , Haoming Wang , Sean Coffey , Tapabrata Chakraborti , Brendan McCane","doi":"10.1016/j.ymeth.2025.12.012","DOIUrl":"10.1016/j.ymeth.2025.12.012","url":null,"abstract":"<div><div>Medical imaging is fundamental to cardiovascular diagnostics, with modalities such as Transthoracic Echocardiography (TTE) and Cardiac Magnetic Resonance (CMR) offering complementary strengths. TTE provides real-time, non-invasive visualization of cardiac function but is often limited by operator dependency and incomplete views. In contrast, CMR delivers comprehensive, high-resolution structural assessments, although it comes with greater time and cost burdens. To address these limitations, this study explores cross-modal generative modeling techniques for synthesizing CMR-like images directly from TTE. We propose a novel architecture that combines a UNet backbone with a vision transformer, utilizing the UNet for feature extraction and the transformer for global attention to improve image synthesis quality. Quantitative and qualitative evaluations demonstrate the model’s ability to produce realistic and anatomically consistent CMR images, with strong potential to improve diagnostic accuracy and clinical decision-making across multiple image modalities.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"247 ","pages":"Pages 1-11"},"PeriodicalIF":4.3,"publicationDate":"2025-12-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145831859","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

Gold nanoparticles for salivary-based sensors: A review 用于唾液传感器的金纳米颗粒：综述。

IF 4.3 3区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

Methods

Pub Date : 2025-12-20 DOI: 10.1016/j.ymeth.2025.12.008

Bahar Mosanaee, Saba Mazareei, Iliya Fattahiyan, Moein Ziyazadeh, Hamide Ehtesabi

Advancements in nanotechnology integrated with biomedical diagnostics have facilitated substantial progress in non-invasive health monitoring, with salivary biosensors utilizing gold nanoparticles (AuNPs) emerging as a novel approach for rapid, sensitive, and user-friendly point-of-care testing. This review article demonstrates how researchers utilize the distinctive physicochemical qualities of AuNPs, specifically their plasmonic responsiveness and bioconjugation ability, to improve diverse biosensing techniques. Rather than traditional blood-based diagnostics, AuNP-based devices utilize saliva as a molecular reservoir to monitor a variety of biomarkers such as hormones, metabolites, nucleic acids, or other analytes. The development of these biosensors extends beyond technological innovation to their incorporation into affordable, user-friendly devices that address clinical needs. Still, converting these biosensors into standardized health care diagnostic tools encounters similar challenges as other bioassays pertaining to biofluid-based complexity, burden of reproducibility, and regulatory approval. In addition to summarizing the extent of scientific and engineering accomplishments to date, this review highlights the opportunity for future implementation of AuNP-based biosensor tests in personalized medicine, advocating synergistic interdisciplinary approaches that integrate materials science, digital analytics, and device engineering to fully realize AuNP-based salivary diagnostics.

纳米技术与生物医学诊断相结合的进步促进了非侵入性健康监测的实质性进展，利用金纳米颗粒（AuNPs）的唾液生物传感器成为快速、敏感和用户友好的护理点检测的新方法。这篇综述文章展示了研究人员如何利用AuNPs独特的物理化学特性，特别是它们的等离子体响应性和生物偶联能力，来改进各种生物传感技术。与传统的基于血液的诊断不同，基于aunp的设备利用唾液作为分子库来监测各种生物标志物，如激素、代谢物、核酸或其他分析物。这些生物传感器的发展超越了技术创新，将其纳入可负担得起的、用户友好的设备中，以满足临床需求。然而，将这些生物传感器转化为标准化的医疗诊断工具，与其他生物检测方法一样，也面临着类似的挑战，包括基于生物流体的复杂性、可重复性负担和监管批准。除了总结迄今为止科学和工程成就的程度外，本综述还强调了未来在个性化医疗中实施基于aunp的生物传感器测试的机会，提倡综合材料科学、数字分析和设备工程的协同跨学科方法，以充分实现基于aunp的唾液诊断。

{"title":"Gold nanoparticles for salivary-based sensors: A review","authors":"Bahar Mosanaee, Saba Mazareei, Iliya Fattahiyan, Moein Ziyazadeh, Hamide Ehtesabi","doi":"10.1016/j.ymeth.2025.12.008","DOIUrl":"10.1016/j.ymeth.2025.12.008","url":null,"abstract":"<div><div>Advancements in nanotechnology integrated with biomedical diagnostics have facilitated substantial progress in non-invasive health monitoring, with salivary biosensors utilizing gold nanoparticles (AuNPs) emerging as a novel approach for rapid, sensitive, and user-friendly point-of-care testing. This review article demonstrates how researchers utilize the distinctive physicochemical qualities of AuNPs, specifically their plasmonic responsiveness and bioconjugation ability, to improve diverse biosensing techniques. Rather than traditional blood-based diagnostics, AuNP-based devices utilize saliva as a molecular reservoir to monitor a variety of biomarkers such as hormones, metabolites, nucleic acids, or other analytes. The development of these biosensors extends beyond technological innovation to their incorporation into affordable, user-friendly devices that address clinical needs. Still, converting these biosensors into standardized health care diagnostic tools encounters similar challenges as other bioassays pertaining to biofluid-based complexity, burden of reproducibility, and regulatory approval. In addition to summarizing the extent of scientific and engineering accomplishments to date, this review highlights the opportunity for future implementation of AuNP-based biosensor tests in personalized medicine, advocating synergistic interdisciplinary approaches that integrate materials science, digital analytics, and device engineering to fully realize AuNP-based salivary diagnostics.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"246 ","pages":"Pages 195-222"},"PeriodicalIF":4.3,"publicationDate":"2025-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145809162","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

Microplate assay for the quantification of catalase activity in biological samples 定量生物样品中过氧化氢酶活性的微孔板法。

IF 4.3 3区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

Methods

Pub Date : 2025-12-19 DOI: 10.1016/j.ymeth.2025.12.009

Unai Montejo , Gorane Beldarrain , Sheila Olza , Jon Ander Alart , Marc Chillida , Ignacio García-Alonso , Daniel Alonso-Alconada , Ana Alonso-Varona , Borja Herrero de la Parte

Catalase is a key antioxidant enzyme that protects cells from oxidative stress by decomposing hydrogen peroxide (H₂O₂). Traditional methods for quantifying catalase activity, such as the Aebi UV method and the dichromate assay, are limited by low sensitivity, interference from biological molecules and require large sample and reagent volumes. Here, we present an optimized, microplate assay based on the oxidation of cobalt(II) to cobalt(III) by H₂O₂, followed by complexation with ethylenediamine-tetraacetic acid (EDTA) to form a highly colored Co(III)-EDTA complex measurable at 570 nm. This method demonstrates superior sensitivity and dynamic range (10–0.039 U/mL) compared to previous colorimetric assays, while minimizing reaction volume and avoiding interference from common biological substances. The assay has been validated using standard bovine liver catalase, liver homogenates, serum and cell lysates, showing strong linearity and specificity. This approach offers a rapid, cost-effective, and robust alternative for catalase activity measurement in biomedical research.

过氧化氢酶是一种关键的抗氧化酶，通过分解过氧化氢（H2O2）来保护细胞免受氧化应激。传统的定量过氧化氢酶活性的方法，如Aebi紫外法和重铬酸盐法，受到灵敏度低、生物分子干扰和需要大量样品和试剂的限制。在这里，我们提出了一种优化的微孔板分析方法，该方法基于H2O2将钴（II）氧化为钴（III），然后与乙二胺-四乙酸（EDTA）络合形成高度着色的Co(III)-EDTA络合物，可在570 nm处测量。与以往的比色法相比，该方法具有更高的灵敏度和动态范围（10-0.039 U/mL），同时最大限度地减少了反应量，避免了常见生物物质的干扰。该方法已使用标准牛肝过氧化氢酶、肝脏匀浆、血清和细胞裂解液进行验证，显示出很强的线性和特异性。这种方法为生物医学研究中过氧化氢酶活性的测量提供了一种快速、经济、可靠的替代方法。

{"title":"Microplate assay for the quantification of catalase activity in biological samples","authors":"Unai Montejo , Gorane Beldarrain , Sheila Olza , Jon Ander Alart , Marc Chillida , Ignacio García-Alonso , Daniel Alonso-Alconada , Ana Alonso-Varona , Borja Herrero de la Parte","doi":"10.1016/j.ymeth.2025.12.009","DOIUrl":"10.1016/j.ymeth.2025.12.009","url":null,"abstract":"<div><div>Catalase is a key antioxidant enzyme that protects cells from oxidative stress by decomposing hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>). Traditional methods for quantifying catalase activity, such as the Aebi UV method and the dichromate assay, are limited by low sensitivity, interference from biological molecules and require large sample and reagent volumes. Here, we present an optimized, microplate assay based on the oxidation of cobalt(II) to cobalt(III) by H<sub>2</sub>O<sub>2</sub>, followed by complexation with ethylenediamine-tetraacetic acid (EDTA) to form a highly colored Co(III)-EDTA complex measurable at 570 nm. This method demonstrates superior sensitivity and dynamic range (10–0.039 U/mL) compared to previous colorimetric assays, while minimizing reaction volume and avoiding interference from common biological substances. The assay has been validated using standard bovine liver catalase, liver homogenates, serum and cell lysates, showing strong linearity and specificity. This approach offers a rapid, cost-effective, and robust alternative for catalase activity measurement in biomedical research.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"246 ","pages":"Pages 186-194"},"PeriodicalIF":4.3,"publicationDate":"2025-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145802874","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Establishment and application of a fully automated serum total free sulfhydryl assay based on 2,2′-DTDP 基于2,2′-DTDP的全自动血清总游离巯基测定方法的建立与应用。

IF 4.3 3区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

Methods

Pub Date : 2025-12-19 DOI: 10.1016/j.ymeth.2025.12.010

Jie Zeng , Zhiyu Shao , Siming Wang , Jun Dong , Weiyan Zhou , Haijian Zhao , Tianjiao Zhang , Jiangtao Zhang , Jing Wang , Hao Zheng , Wenxiang Chen , Chuanbao Zhang

Background

Serum free thiols (FTs) are sensitive biomarkers of oxidative stress, closely associated with aging and chronic disease risk. Existing DTNB-based detection methods suffer from product instability, affecting analytical reliability. A more stable and high-throughput method is needed.

Methods

We developed an automated FT assay using 2,2′-dithiodipyridine (2,2′-DTDP) on a clinical chemistry analyzer. Serum samples reacted with 2,2′-DTDP under optimized conditions, and absorbance at 340 nm was used to quantify FT levels. Method performance, stability, and associations with cardiovascular disease (CVD) risk factors were assessed in 341 healthy individuals.

Results

FTs reacted with 2,2′-DTDP to generate 2-TP, a stable product with maximum absorbance at 340 nm. The method demonstrated excellent linearity (0–1200 μmol/L), low imprecision (1.8 %–3.3 %), and reasonable recovery (≥80 %). Serum FTs were stable for at least one year at −70 °C, 12 h at 2–8 °C, and 2 h at room temperature. In the study population, FT levels were positively correlated with serum albumin, a major thiol carrier, and negatively correlated with age, triglyceride, glucose and creatinine, suggesting links to redox status and CVD risks. These associations support the potential of FTs as indicators of systemic oxidative balance and aging-related health decline.

Conclusion

This simple and robust 2,2′-DTDP-based method enables accurate, high-throughput measurement of serum FTs. It is well suited for exploring thiol antioxidant function and evaluating age-related and cardiovascular risk in clinical and research settings.

背景：血清游离硫醇（FTs）是氧化应激的敏感生物标志物，与衰老和慢性疾病风险密切相关。现有的基于dtnb的检测方法存在产品不稳定性，影响了分析的可靠性。需要一种更稳定和高通量的方法。方法：在临床化学分析仪上使用2,2′-二硫代二吡啶（2,2′-DTDP）建立了一种自动FT测定方法。血清样品在优化条件下与2,2'-DTDP反应，340 nm吸光度用于定量FT水平。方法在341名健康个体中评估了性能、稳定性以及与心血管疾病（CVD）危险因素的相关性。结果：ft与2,2′-DTDP反应生成2- tp，产物稳定，最大吸光度为340 nm。该方法线性良好（0 ~ 1200 μmol/L），不精密度低（1.8 % ~ 3.3 %），回收率合理（≥80 %）。血清FTs在-70 °C下稳定至少一年，在2-8 °C下稳定12 h，在室温下稳定2 h。在研究人群中，FT水平与血清白蛋白（主要硫醇载体）呈正相关，与年龄、甘油三酯、葡萄糖和肌酐呈负相关，提示与氧化还原状态和心血管疾病风险有关。这些关联支持FTs作为系统氧化平衡和衰老相关健康下降指标的潜力。结论：基于2,2'- ddpp的方法简单可靠，能够准确、高通量地测定血清FTs。它非常适合在临床和研究环境中探索硫醇抗氧化功能和评估年龄相关和心血管风险。

{"title":"Establishment and application of a fully automated serum total free sulfhydryl assay based on 2,2′-DTDP","authors":"Jie Zeng , Zhiyu Shao , Siming Wang , Jun Dong , Weiyan Zhou , Haijian Zhao , Tianjiao Zhang , Jiangtao Zhang , Jing Wang , Hao Zheng , Wenxiang Chen , Chuanbao Zhang","doi":"10.1016/j.ymeth.2025.12.010","DOIUrl":"10.1016/j.ymeth.2025.12.010","url":null,"abstract":"<div><h3>Background</h3><div>Serum free thiols (FTs) are sensitive biomarkers of oxidative stress, closely associated with aging and chronic disease risk. Existing DTNB-based detection methods suffer from product instability, affecting analytical reliability. A more stable and high-throughput method is needed.</div></div><div><h3>Methods</h3><div>We developed an automated FT assay using 2,2′-dithiodipyridine (2,2′-DTDP) on a clinical chemistry analyzer. Serum samples reacted with 2,2′-DTDP under optimized conditions, and absorbance at 340 nm was used to quantify FT levels. Method performance, stability, and associations with cardiovascular disease (CVD) risk factors were assessed in 341 healthy individuals.</div></div><div><h3>Results</h3><div>FTs reacted with 2,2′-DTDP to generate 2-TP, a stable product with maximum absorbance at 340 nm. The method demonstrated excellent linearity (0–1200 μmol/L), low imprecision (1.8 %–3.3 %), and reasonable recovery (≥80 %). Serum FTs were stable for at least one year at −70 °C, 12 h at 2–8 °C, and 2 h at room temperature. In the study population, FT levels were positively correlated with serum albumin, a major thiol carrier, and negatively correlated with age, triglyceride, glucose and creatinine, suggesting links to redox status and CVD risks. These associations support the potential of FTs as indicators of systemic oxidative balance and aging-related health decline.</div></div><div><h3>Conclusion</h3><div>This simple and robust 2,2′-DTDP-based method enables accurate, high-throughput measurement of serum FTs. It is well suited for exploring thiol antioxidant function and evaluating age-related and cardiovascular risk in clinical and research settings.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"246 ","pages":"Pages 236-242"},"PeriodicalIF":4.3,"publicationDate":"2025-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145802876","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

Methods最新文献

Background

Methods

Results

Conclusion