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IF-CRIB: A 3D-printable device to facilitate immunofluorescence experiments and its application in screening and characterizing cells expressing a degradable form of ERK2 IF-CRIB：一种3d打印设备，用于促进免疫荧光实验，并用于筛选和表征表达可降解形式ERK2的细胞。
IF 4.3 3区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS
Methods
Pub Date : 2025-08-09 DOI: 10.1016/j.ymeth.2025.08.004
Elsa Berliocchi , Cercina Onesto , Gilles Pagès , Philippe Lenormand , Roser Buscà
Immunofluorescence-based detection of proteins in fixed cells is a powerful tool for research in cell and developmental biology. While a variety of immunofluorescence protocols exist, they can be time consuming or require expensive equipment which may not be accessible to all laboratories. A common challenge in these protocols is the numerous washing steps, particularly in experiments with numerous conditions. To address this, here we introduce the IF-CRIB device, a 3D-printable wash rack specifically designed for applications involving a high number of round coverslips with adherent cultured cells. We detail its design and the 3D printing process which can be easily used by any laboratory and we highlight that it facilitates the numerous washing steps. In addition, we present the IF-Express protocol, an optimized and effective method that enables fast and consistent immunofluorescence results. As an example of the utility of the IF-CRIB device and the IF-Express protocol, we describe their application in the screening and characterization of several NIH3T3 cell clones expressing a degradable form of ERK2 kinase (ERK2-dTAG) after treatment with the dTAG-13 compound. The generation of ERK2-dTAG clones involves a knock-in strategy. We provide a detailed methodology for clone selection, immunofluorescence screening, and characterization of ERK2-dTAG, including degradation kinetics, dose–response analysis, and nuclear translocation assays to assess ERK2-dTAG functionality. The IF-CRIB device and IF-Express protocol has been proven to be efficient for the obtention and characterization of ERK2dTAG-expressing clones thereby offering a powerful framework for studying ERK2 dynamics in cell biology and disease models.
基于免疫荧光的固定细胞蛋白检测是细胞和发育生物学研究的有力工具。虽然存在各种免疫荧光方案，但它们可能耗时或需要昂贵的设备，而这些设备可能不是所有实验室都能获得的。这些方案的一个共同挑战是大量的洗涤步骤，特别是在许多条件下的实验中。为了解决这个问题，我们在这里介绍IF-CRIB设备，这是一种3d打印洗涤架，专门设计用于涉及大量带有贴壁培养细胞的圆形盖子的应用。我们详细介绍了它的设计和3D打印过程，可以很容易地由任何实验室使用，我们强调，它有利于众多的洗涤步骤。此外，我们提出了IF-Express协议，这是一种优化和有效的方法，可实现快速和一致的免疫荧光结果。作为IF-CRIB设备和IF-Express协议的实用性的一个例子，我们描述了它们在筛选和表征几个NIH3T3细胞克隆中的应用，这些克隆在dTAG-13化合物处理后表达可降解形式的ERK2激酶（ERK2- dtag）。ERK2-dTAG克隆的产生涉及一种敲入策略。我们为ERK2-dTAG的克隆选择、免疫荧光筛选和表征提供了详细的方法，包括降解动力学、剂量反应分析和核易位分析，以评估ERK2-dTAG的功能。IF-CRIB设备和IF-Express协议已被证明是观察和表征表达erk2dtag的克隆的有效方法，从而为研究细胞生物学和疾病模型中的ERK2动力学提供了强大的框架。
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引用次数: 0
Identification of immunoregulatory peptides from gut microbiota modulating Epstein-Barr virus-induced gene 3 (EBI3) using a novel specific AlphaLISA™ assay 使用一种新的特异性AlphaLISA™检测方法鉴定肠道微生物群中调节eb病毒诱导基因3 （EBI3）的免疫调节肽
IF 4.3 3区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS
Methods
Pub Date : 2025-08-05 DOI: 10.1016/j.ymeth.2025.08.002
Sothea Touch , Maria Lucia Orsini Delgado , Parfait Evouna-Mengue , Lydie Surand , Souphalone Luangsay , Naeemunnisa Mohamed Anesary , Francesco Strozzi , Laurent Chêne , Antonietta Cultrone
Gut microbiota-derived compounds are pivotal in modulating host immunity by regulating the functions of various key innate and adaptive immune cells. Epstein-Barr virus-induced gene 3 (EBI3) serves as the beta subunit shared by the heterodimeric cytokines interleukin (IL)-27 and IL-35. Both these cytokines have been documented to inhibit the development of T helper 2 (Th2) and T helper 17 (Th17) cells, while enhancing the function of regulatory T cells (Tregs). EBI3, itself, has also been shown to regulate cell-mediated immune responses. Despite their critical roles in maintaining immune homeostasis, there is a significant lack of robust, high-throughput-compatible assays to evaluate the secretion of IL-27, IL-35, or EBI3.
In this study, we detail the development of a novel amplified luminescent proximity homogeneous assay (AlphaLISA™) to quantify EBI3 secretion by tolerogenic dendritic cells. We utilized this assay to screen a library of 9739 small proteins derived from the human gut microbiota to identify compounds that could stimulate EBI3 secretion.
Our findings revealed the immunoregulatory potential of VAC18, an unknown protein from Fusicatenibacter saccharivorans (Clostridium cluster XIVa) which significantly induces the secretion of both EBI3 and IL-27. This is the first study to demonstrate the effect of gut microbiota derived peptides on the balanced secretion of EBI3 and IL-27.
肠道微生物衍生的化合物通过调节各种关键先天和适应性免疫细胞的功能，在调节宿主免疫中起关键作用。Epstein-Barr病毒诱导的基因3 （EBI3）是异二聚体细胞因子白细胞介素(IL)-27和IL-35共享的β亚基。这两种细胞因子均可抑制辅助性T细胞2 （Th2）和辅助性T细胞17 （Th17）的发育，同时增强调节性T细胞（Tregs）的功能。EBI3本身也被证明可以调节细胞介导的免疫反应。尽管IL-27、IL-35或EBI3在维持免疫稳态中发挥着关键作用，但目前仍缺乏可靠的、高通量兼容的检测方法来评估其分泌。在这项研究中，我们详细介绍了一种新的放大发光接近均质测定（AlphaLISA™）的发展，以量化耐受性树突状细胞的EBI3分泌。我们利用该方法筛选了9739个来自人类肠道微生物群的小蛋白，以确定可以刺激EBI3分泌的化合物。我们的研究结果揭示了VAC18的免疫调节潜力，VAC18是一种来自糖化梭状杆菌（Clostridium cluster XIVa）的未知蛋白，可显著诱导EBI3和IL-27的分泌。这是首次证实肠道菌群衍生肽对EBI3和IL-27平衡分泌影响的研究。
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引用次数: 0
Recent advances in biosensors: structure, principles, classification, and application in bio-manufacturing 生物传感器的研究进展：结构、原理、分类及其在生物制造中的应用。
IF 4.3 3区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS
Methods
Pub Date : 2025-08-05 DOI: 10.1016/j.ymeth.2025.07.011
Weiguang Gao , Qi Wang , Weili Gong , Lan Zheng , Qingai Liu , Lihe Zhang , Yaohong Ma
Bio-manufacturing, as a frontier field of the new round of global scientific and technological revolution and industrial change, is becoming a core driving force for the future development of the bioeconomy. However, the bio-manufacturing process itself contains complex metabolic mechanisms and fine process control, which undoubtedly increases its technical difficulty. Especially at a time when the field of intelligent bio-manufacturing is developing rapidly, more stringent requirements have been placed on the ability to sense key biochemical information during the bio-manufacturing process. Biosensors based on biomolecular recognition provide powerful technical support for real time monitoring and precise control of key biochemical information that can rapidly sense the production process, and their applications have made remarkable progress. This article reviews the structure, classification, and working principle of biosensors and discusses their applications in bio-manufacturing, such as real-time monitoring of key biochemical parameters, intracellular and extracellular metabolite concentrations, and high-throughput screening for precise control and optimization of bioprocesses. The article also analyses the challenges facing biosensors, including the need for stability and reliability enhancements, and future directions. Researchers are developing new biometric components and sensor materials with advanced signal conversion techniques, while microelectronics and nanotechnology are driving the miniaturization and integration of sensors. These advances are expected to make biosensors more useful in microbial fermentation and biotechnology.
生物制造作为全球新一轮科技革命和产业变革的前沿领域，正在成为推动生物经济未来发展的核心动力。然而，生物制造过程本身包含复杂的代谢机制和精细的过程控制，这无疑增加了其技术难度。特别是在智能生物制造领域迅速发展的今天，对生物制造过程中关键生化信息的感知能力提出了更严格的要求。基于生物分子识别的生物传感器为实时监测和精确控制能够快速感知生产过程的关键生化信息提供了强有力的技术支持，其应用取得了显著进展。本文综述了生物传感器的结构、分类和工作原理，并讨论了它们在生物制造中的应用，如实时监测关键生化参数、细胞内和细胞外代谢物浓度、高通量筛选以精确控制和优化生物过程。文章还分析了生物传感器面临的挑战，包括对稳定性和可靠性增强的需求，以及未来的发展方向。研究人员正在开发具有先进信号转换技术的新型生物识别元件和传感器材料，而微电子和纳米技术正在推动传感器的小型化和集成化。这些进展有望使生物传感器在微生物发酵和生物技术方面发挥更大的作用。
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引用次数: 0
3DCellComposer − A versatile pipeline utilizing 2D cell segmentation methods for 3D cell segmentation 3DCellComposer -利用2D细胞分割方法进行3D细胞分割的多功能管道。
IF 4.3 3区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS
Methods
Pub Date : 2025-07-28 DOI: 10.1016/j.ymeth.2025.07.007
Haoran Chen , Ted Zhang, Matthew Ruffalo, Robert F. Murphy
Cell segmentation is crucial in bioimage informatics, as its accuracy directly impacts conclusions drawn from cellular analyses. While many approaches to 2D cell segmentation have been described, 3D cell segmentation has received much less attention. 3D segmentation faces significant challenges, including limited training data availability due to the difficulty of the task for human annotators, and inherent three-dimensional complexity. As a result, existing 3D cell segmentation methods often lack broad applicability across different imaging modalities. To address this, we developed a generalizable approach for using 2D cell segmentation methods to produce accurate 3D cell segmentations. We implemented this approach in 3DCellComposer, a versatile, open-source package that allows users to choose any existing 2D segmentation model appropriate for their tissue or cell type(s) without requiring any additional training. Importantly, we have enhanced our open source CellSegmentationEvaluator quality evaluation tool to support 3D images. It provides metrics that allow selection of the best approach for a given imaging source and modality, without the need for human annotations to assess performance. Using these metrics, we demonstrated that our approach produced high-quality 3D segmentations of multichannel tissue images. 3DCellComposer, when paired with well-trained 2D segmentation models, provides an important alternative to acquiring human-annotated 3D images for new sample types or imaging modalities and then training 3D segmentation models using them. It is expected to be of significant value for large scale projects such as the Human BioMolecular Atlas Program.
细胞分割在生物图像信息学中是至关重要的，因为它的准确性直接影响细胞分析得出的结论。虽然许多二维细胞分割的方法已经被描述，但三维细胞分割却很少受到关注。3D分割面临着巨大的挑战，包括由于人类注释者的任务困难而导致的训练数据可用性有限，以及固有的三维复杂性。因此，现有的三维细胞分割方法往往缺乏对不同成像模式的广泛适用性。为了解决这个问题，我们开发了一种可推广的方法，用于使用2D细胞分割方法来产生准确的3D细胞分割。我们在3DCellComposer中实现了这种方法，这是一个通用的开源软件包，允许用户选择适合其组织或细胞类型的任何现有2D分割模型，而无需任何额外的培训。重要的是，我们已经增强了我们的开源CellSegmentationEvaluator质量评估工具，以支持3D图像。它提供的指标允许为给定的成像源和模式选择最佳方法，而不需要人工注释来评估性能。使用这些指标，我们证明了我们的方法产生了高质量的多通道组织图像的3D分割。3DCellComposer与训练有素的2D分割模型配对时，为获取新的样本类型或成像模式的人工注释3D图像提供了重要的替代方案，然后使用它们训练3D分割模型。预计对人类生物分子图谱计划等大型项目具有重要价值。
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引用次数: 0
Bio-impedance spectroscopy-based classification of mental acuity in university students via machine-learning and deep-learning approaches 基于生物阻抗谱的大学生心理敏锐度机器学习和深度学习分类
IF 4.2 3区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS
Methods
Pub Date : 2025-07-24 DOI: 10.1016/j.ymeth.2025.07.009
Kusum Tara , Sadman Sakib , Md Hasibul Islam , Shadhon Chandra Mohonta , Md.Shamim Anower , Takenao Sugi
Mental acuity detection is crucial for identifying cognitive impairments linked to body composition imbalances and ensuring overall mental and physical fitness. This study introduces a deep-learning neural network (NN) model with MobileNetV2 deep learning architecture to classify mental acuity levels of university students—excellent, good, and average—using bioelectrical impedance spectroscopy (BIS)-based body composition and bio-impedance measurements. Body composition and bio-impedance features such as basal metabolic rate (BMR), body cell mass (BCM), total body water (TBW), bioelectrical impedance (BI), and phase angle (PA) were utilized as inputs for a feature-based random forest (RF) machine-learning model, achieving an accuracy of 88.26% and an F1-score of 84.89%. However, an image-based NN model with MobileNetV2 deep learning architecture, leveraging 2D impedance spectrum images, outperformed RF model, achieving exceptional accuracy of 98.39% and an F1-score of 97.83%. Additionally, the Nyquist diagram showed that excellent mental acuity had the smallest semicircle, average mental acuity had the largest, and good mental acuity level was intermediate. Similarly, feature analysis revealed that excellent mental acuity level corresponded to high BMR, BCM, TBW, and PA with low BI, while average mental acuity level had the opposite trend, while good mental acuity levels fell in between. The reliability and performance of the NN model in detecting mental acuity using 2D impedance spectrum analysis highlights its potential for this task. These results emphasize the value of deep-learning approaches in integrating BIS data for accurate mental acuity assessment and their broader implications for monitoring cognitive health.
精神敏锐度检测对于识别与身体成分失衡有关的认知障碍和确保整体身心健康至关重要。本研究采用基于生物电阻抗谱（BIS）的身体成分和生物阻抗测量，引入了一个具有MobileNetV2深度学习架构的深度学习神经网络（NN）模型，对大学生的心理敏锐度水平进行了优秀、良好和一般的分类。将基础代谢率（BMR）、体细胞质量（BCM）、总水量（TBW）、生物电阻抗（BI）和相位角（PA）等身体组成和生物阻抗特征作为基于特征的随机森林（RF）机器学习模型的输入，准确率为88.26%，f1得分为84.89%。然而，使用MobileNetV2深度学习架构的基于图像的神经网络模型，利用二维阻抗谱图像，优于RF模型，实现了98.39%的卓越准确率和97.83%的f1得分。此外，Nyquist图表显示，优秀的精神敏锐度的半圆最小，一般的精神敏锐度的半圆最大，良好的精神敏锐度的半圆为中间。同样，特征分析显示，优秀的精神敏锐度水平对应于高BMR、BCM、TBW和低BI的PA，而平均的精神敏锐度水平则相反，良好的精神敏锐度水平介于两者之间。神经网络模型在利用二维阻抗谱分析检测精神敏锐度方面的可靠性和性能突出了它在这项任务中的潜力。这些结果强调了深度学习方法在整合BIS数据以进行准确的精神敏锐度评估方面的价值，以及它们对监测认知健康的更广泛影响。
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引用次数: 0
A simple, accurate method for the measurement of lysosomal activity 测定溶酶体活性的一种简单、准确的方法。
IF 4.3 3区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS
Methods
Pub Date : 2025-07-24 DOI: 10.1016/j.ymeth.2025.07.008
Yoshito Iwai , Yuriko Furuya , Yuji Oguro , Keiji Yamamoto
Lysosomes are responsible for the degradation of intra- and extracellular components and are thus essential for the quality control of proteins and organelles. Lysosomal dysfunction leads to lysosomal storage diseases, and it is therefore important to identify which types of stress cause functional abnormalities. Lysosomal function is generally evaluated by measuring the enzyme activity of lysosomes with fluorescent dyes. However, fluorescence microscopy can lead to different outcomes due to variations in the field of view, the analysis software used, and the parameter settings. We therefore developed a method that uses only a microplate reader and DQ Green BSA, a dye that emits fluorescence upon lysosomal degradation, to ascertain lysosomal activity. HEK293 cells were treated with DQ Green BSA with or without bafilomycin A1 and lysates extracted using cell lysis buffer. Fluorescence intensities and protein concentrations in the cell lysates were then measured using a microplate reader and the bicinchoninic acid method, respectively, and the fluorescence intensity divided by the protein concentration. Results indicated a significant lysosome inhibitor-induced dose-dependent decrease in the lysosomal activity. The Z’-factor of 0.77 obtained using the proposed method is a significant improvement over the − 0.06 obtained using the conventional method. The versatility of the method was evaluated with different cell types, cell lysis buffers, inhibitors, and protease substrates. These results suggest that the method works regardless of the cells or reagents used, and indicates the relative simplicity and accuracy of the proposed method compared to the currently utilized method.
溶酶体负责细胞内和细胞外成分的降解，因此对蛋白质和细胞器的质量控制至关重要。溶酶体功能障碍导致溶酶体贮积病，因此确定哪些类型的应激导致功能异常是很重要的。溶酶体的功能一般是通过荧光染料测定溶酶体的酶活性来评价的。然而，由于视野、使用的分析软件和参数设置的变化，荧光显微镜可以导致不同的结果。因此，我们开发了一种方法，仅使用微孔板读取器和DQ Green BSA（一种在溶酶体降解时发出荧光的染料）来确定溶酶体的活性。用含或不含巴菲霉素A1的DQ Green BSA和放射免疫沉淀缓冲液提取的裂解物处理HEK293细胞。然后分别使用微孔板读取器和比辛醌酸法测量细胞裂解液中的荧光强度和蛋白质浓度，并将荧光强度除以蛋白质浓度。结果显示溶酶体抑制剂诱导溶酶体活性呈剂量依赖性降低。该方法得到的Z′因子为0.77，比传统方法得到的 - 0.06有显著提高。用不同的细胞类型、细胞裂解缓冲液、抑制剂和蛋白酶底物对该方法的通用性进行了评估，结果表明，无论使用何种细胞或试剂，该方法都有效，表明与目前使用的方法相比，所提出的方法相对简单和准确。
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引用次数: 0
Interpretable multimodal learning for tumor protein-metal binding: Progress, challenges, and perspectives 肿瘤蛋白-金属结合的可解释多模式学习：进展、挑战和前景。
IF 4.3 3区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS
Methods
Pub Date : 2025-07-21 DOI: 10.1016/j.ymeth.2025.07.004
Xiaokun Liu , Sayedmohammadreza Rastegari , Yijun Huang , Sxe Chang Cheong , Weikang Liu , Wenjie Zhao , Qihao Tian , Hongming Wang , Yingjie Guo , Shuo Zhou , Sina Tabakhi , Xianyuan Liu , Zheqing Zhu , Wei Sang , Haiping Lu
In cancer therapeutics, protein-metal binding mechanisms critically govern the pharmacokinetics and targeting efficacy of drugs, thereby fundamentally shaping the rational design of anticancer metallodrugs. While conventional laboratory methods used to study such mechanisms are often costly, low throughput, and limited in capturing dynamic biological processes, machine learning (ML) has emerged as a promising alternative. Despite increasing efforts to develop protein-metal binding datasets and ML algorithms, the application of ML in tumor protein-metal binding remains limited. Key challenges include a shortage of high-quality, tumor-specific datasets, insufficient consideration of multiple data modalities, and the complexity of interpreting results due to the “black box” nature of complex ML models. This paper summarizes recent progress and ongoing challenges in using ML to predict tumor protein-metal binding, focusing on data, modeling, and interpretability. We present multimodal protein-metal binding datasets and outline strategies for acquiring, curating, and preprocessing them for training ML models. Moreover, we explore the complementary value provided by different data modalities and examine methods for their integration. We also review approaches for improving model interpretability to support more trustworthy decisions in cancer research. Finally, we offer our perspective on research opportunities and propose strategies to address the scarcity of tumor protein data and the limited number of predictive models for tumor protein-metal binding. We also highlight two promising directions for effective metal-based drug design: integrating protein-protein interaction data to provide structural insights into metal-binding events and predicting structural changes in tumor proteins after metal binding.
在癌症治疗中，蛋白质-金属结合机制对药物的药代动力学和靶向疗效起着至关重要的作用，从而从根本上决定了抗癌金属药物的合理设计。虽然用于研究此类机制的传统实验室方法通常成本高，吞吐量低，并且在捕获动态生物过程方面受到限制，但机器学习（ML）已成为一种有前途的替代方法。尽管越来越多的人致力于开发蛋白质-金属结合数据集和ML算法，但ML在肿瘤蛋白质-金属结合中的应用仍然有限。主要挑战包括缺乏高质量的肿瘤特异性数据集，对多种数据模式的考虑不足，以及由于复杂ML模型的“黑箱”性质而导致解释结果的复杂性。本文总结了利用机器学习预测肿瘤蛋白-金属结合的最新进展和面临的挑战，重点是数据、建模和可解释性。我们提出了多模态蛋白质-金属结合数据集，并概述了获取、管理和预处理这些数据集用于训练ML模型的策略。此外，我们还探讨了不同数据模式提供的互补价值，并研究了它们整合的方法。我们还回顾了提高模型可解释性的方法，以支持癌症研究中更可靠的决策。最后，我们提出了我们对研究机会的看法，并提出了解决肿瘤蛋白质数据稀缺和肿瘤蛋白质-金属结合预测模型数量有限的策略。我们还强调了有效的基于金属的药物设计的两个有希望的方向：整合蛋白质-蛋白质相互作用数据以提供金属结合事件的结构见解和预测金属结合后肿瘤蛋白的结构变化。
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引用次数: 0
Circularization enhances RNA aptamer binding and Stability: Evidence from in-cell NMR 环状化增强RNA适体结合和稳定性：来自细胞内核磁共振的证据
IF 4.2 3区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS
Methods
Pub Date : 2025-07-16 DOI: 10.1016/j.ymeth.2025.07.006
Omar Eladl
RNA aptamers are emerging as promising molecular probes due to their high specificity and low immunogenicity. However, their clinical potential is often limited by instability under physiological conditions, primarily due to exonucleolytic degradation and structural flexibility. To address these challenges in a model system, we designed a circular RNA aptamer targeting the HIV-1 Tat protein.
Enzymatic circularization of the aptamer was performed using T4 RNA ligase, and circularization was confirmed by mobility shift assays and RNase R digestion. Binding affinity was assessed via filter dot blot assay, while structural stability was evaluated using 1D imino proton NMR and CLEANEX-PM experiments. Intracellular stability was monitored using in-cell NMR following transfection into HeLa cells.
In our study, the circular aptamer showed approximately tenfold higher binding affinity compared to its linear counterpart, as determined by filter binding assay. NMR analysis indicated improved structural rigidity, preservation of native base pairing, and reduced solvent exchange. Notably, in-cell NMR revealed that the circular aptamer remained detectable up to 18 h post-transfection, whereas the linear aptamer degraded within a few hours.
In this system, circularization substantially improved the binding performance and intracellular persistence of the RNA aptamer. These findings demonstrate the feasibility of applying RNA circularization to enhance aptamer functionality in living cells and lay the groundwork for future therapeutic exploration.
RNA适体因其高特异性和低免疫原性而成为一种很有前途的分子探针。然而，它们的临床潜力往往受到生理条件下不稳定性的限制，主要是由于核外溶降解和结构灵活性。为了在模型系统中解决这些挑战，我们设计了一个针对HIV-1 Tat蛋白的环状RNA适体。利用T4 RNA连接酶对适体进行酶环化，并通过迁移率转移实验和RNase R酶切证实了环化。结合亲和性通过滤点印迹法评估，结构稳定性通过1D亚质子核磁共振和CLEANEX-PM实验评估。转染到HeLa细胞后，使用细胞内核磁共振监测细胞内稳定性。在我们的研究中，圆形适配体的结合亲和力比其线性适配体高约10倍，这是通过过滤结合实验确定的。核磁共振分析表明，它能提高结构刚性，保持天然碱基对，减少溶剂交换。值得注意的是，细胞内核磁共振显示，圆形适配体在转染后18小时内仍然可检测到，而线性适配体在几小时内降解。在这个系统中，环状化大大提高了RNA适配体的结合性能和细胞内持久性。这些发现证明了应用RNA循环增强活细胞适体功能的可行性，并为未来的治疗探索奠定了基础。
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引用次数: 0
Ultrasensitive detection of MMP-2 via T7 RNA polymerase and CRISPR/Cas13a-Enhanced electrochemiluminescence biosensor for COPD diagnosis 通过T7 RNA聚合酶和CRISPR/ cas13a增强的电化学发光生物传感器超灵敏检测MMP-2在COPD诊断中的应用
IF 4.2 3区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS
Methods
Pub Date : 2025-07-15 DOI: 10.1016/j.ymeth.2025.07.005
Wensi Li , Zhichao Tang , Xiang Zhu , Beijuan Wang , Xiuqin Zhang , Ze Ji , Suhang Cheng
In this work, an electrochemiluminescence (ECL) biosensor integrating T7 RNA polymerase amplification and CRISPR/Cas13a-mediated signal enhancement was developed for the ultrasensitive detection of matrix metalloproteinase-2 (MMP-2), a key biomarker associated with chronic inflammatory diseases such as COPD. A peptide nucleic acid (PNA) probe was designed to respond specifically to MMP-2 cleavage, enabling the release of DNA templates for subsequent T7 RNA polymerase-driven transcription amplification. The generated RNA triggers the collateral cleavage activity of CRISPR/Cas13a, resulting in a significant amplification of the ECL signal. The biosensor’s surface was constructed using a AuNPs/Ti3C2Tx/Ru(II)-PEI nanocomposite, which enhanced signal transduction and stability. Under optimized conditions, the proposed biosensor achieved a detection limit as low as 62.05 fM, demonstrating superior sensitivity compared to conventional methods, as summarized in Table 1. The platform also exhibited excellent specificity and anti-interference capability, ensuring reliable detection of MMP-2 in complex biological samples. This study provides a simple yet highly efficient strategy for enzymatic biomarker detection, offering great potential for clinical applications in early disease diagnosis and monitoring.
在这项工作中，开发了一种集成T7 RNA聚合酶扩增和CRISPR/ cas13a介导的信号增强的电化学发光（ECL）生物传感器，用于超灵敏检测基质金属蛋白酶-2 (MMP-2)，这是与慢性炎症性疾病（如COPD）相关的关键生物标志物。设计了一种肽核酸（PNA）探针，专门响应MMP-2的切割，从而释放DNA模板，用于随后的T7 RNA聚合酶驱动的转录扩增。生成的RNA触发CRISPR/Cas13a的侧支裂解活性，导致ECL信号显著扩增。生物传感器表面采用AuNPs/Ti3C2Tx/Ru(II)-PEI纳米复合材料构建，增强了信号转导和稳定性。在优化条件下，所提出的生物传感器的检测限低至62.05 fM，与传统方法相比具有更高的灵敏度，如表1所示。该平台还具有出色的特异性和抗干扰能力，确保了复杂生物样品中MMP-2的可靠检测。本研究提供了一种简单而高效的酶促生物标志物检测策略，在疾病早期诊断和监测方面具有很大的临床应用潜力。
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引用次数: 0
A photoactivatable chemical lipidomics approach for local sphingolipid metabolic analysis 用于局部鞘脂代谢分析的光活化化学脂组学方法
IF 4.2 3区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS
Methods
Pub Date : 2025-07-08 DOI: 10.1016/j.ymeth.2025.07.002
Xiaoxue Wang , Zhiyi Zhang , Shuwei Tian , Suihan Feng
In eukaryotic cells, lipid metabolism is tightly regulated depending on the subcellular localization, which is essential for maintaining lipid homeostasis. However, understanding compartmentalized lipid metabolism remains challenging due to limited availability of suitable techniques. In this study, we present a chemical lipidomics approach that combines photoactivatable probes with high resolution mass spectrometry and stable-isotope labelling to analyze lipid dynamics at subcellular resolution. We applied this method to analyze the metabolism of 1-deoxysphingolipid (DoxSL), a non-canonical lipid species linked to various metabolic diseases and neuropathy, whose metabolism remains largely unexplored. Using the photoactivatable probes, we selectively delivered 1-deoxysphinganine, a key DoxSL intermediate, to mitochondria upon photo-illumination and subsequently analyzed its local metabolic products over time. Our data show that most 1-deoxysphinganine delivered to mitochondria is rapidly converted into 1-deoxyceramides, while only a small fraction forms oxidized products. Further lipidomic analysis revealed that 1-deoxyceramides are transported to the extracellular space and that DoxSL is also present in mouse and human serum samples. In summary, we developed novel probes to track lipid dynamics with high spatiotemporal resolution in a non-invasive manner and provided new insights into sphingolipid metabolism.
在真核细胞中，脂质代谢受到亚细胞定位的严格调控，这对于维持脂质稳态至关重要。然而，由于可用的合适技术有限，理解区隔化脂质代谢仍然具有挑战性。在这项研究中，我们提出了一种化学脂质组学方法，将光激活探针与高分辨率质谱和稳定同位素标记相结合，在亚细胞分辨率下分析脂质动力学。我们应用这种方法分析了1-脱氧鞘脂（DoxSL）的代谢，这是一种与各种代谢疾病和神经病变有关的非典型脂质物种，其代谢在很大程度上仍未被探索。使用光激活探针，我们在光照下选择性地将1-脱氧鞘氨氨酸（一种关键的DoxSL中间体）递送到线粒体，随后分析了其随时间变化的局部代谢产物。我们的数据表明，大多数传递到线粒体的1-脱氧鞘氨酸迅速转化为1-脱氧神经酰胺，而只有一小部分形成氧化产物。进一步的脂质组学分析表明，1-脱氧神经酰胺被运输到细胞外空间，DoxSL也存在于小鼠和人血清样本中。总之，我们开发了一种新颖的探针，以非侵入性的方式高时空分辨率跟踪脂质动力学，并为鞘脂代谢提供了新的见解。
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