Methods最新文献_第4页

Novel approaches to biomarker discover 生物标志物发现的新方法

IF 4.2 3区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

Methods

Pub Date : 2025-02-01 DOI: 10.1016/j.ymeth.2025.01.005

Brian K. McFarlin PhD (Special Issue Editor)

引用次数: 0

Validation of plasmonic-based biosensors for rapid and in depth characterization of monoclonal antibodies directed against rabbit haemorrhagic and foot-and-mouth disease viruses in biological samples 验证基于等离子体的生物传感器在生物样品中快速和深入地表征针对兔出血性和口蹄疫病毒的单克隆抗体。

IF 4.2 3区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

Methods

Pub Date : 2025-02-01 DOI: 10.1016/j.ymeth.2024.12.003

Chiara Urbinati , Giulia Pezzoni , Patrizia Cavadini , Vittoria Di Giovanni , Lorenzo Capucci , Marco Rusnati

ELISA and RT-PCR represent the standard tools for the sensitive identification of viruses in biological samples, but they lack the capacity to finely characterize the binding of viruses or viral antigens to monoclonal antibodies (MAbs). Biosensing technologies are gaining increasing importance as powerful MAb characterization tools in the field of virology. Surface plasmon resonance (SPR) is an optical biosensing technology already used for the in depth characterization of MAbs of diagnostic and therapeutic value. Rabbit haemorrhagic disease virus (RHDV) and foot-and-mouth disease virus (FMDV) are top veterinary issues for which the development of novel methods aimed at the characterization of antiviral MAbs represents a priority with important livestock healthcare and economic implications. With these premises in mind, here we prepared a series of SPR biosensors by immobilizing RHDV2 or its 6S subunit by different strategies that were then used to characterize the binding capacity of a panel of anti-RHDV2 MAbs. From the comparison of the results obtained, the biosensor composed of intact RHDV2 captured with catcher-MAb covalently immobilized to the surface showed the best analytical performances. To evaluate the versatility of the biosensor, the same strategy was then adopted using FMVD in cell extracts. The results obtained are discussed in view of the exploitation of SPR in the rapid and resilient fine characterization of antiviral MAbs for diagnostic or therapeutic purposes in the field of animal virology.

ELISA和RT-PCR是生物样品中病毒敏感鉴定的标准工具，但它们缺乏精细表征病毒或病毒抗原与单克隆抗体（mab）结合的能力。生物传感技术作为一种强大的单克隆抗体鉴定工具，在病毒学领域正变得越来越重要。表面等离子体共振（SPR）是一种光学生物传感技术，已被用于深入表征具有诊断和治疗价值的单克隆抗体。兔出血性疾病病毒（RHDV）和口蹄疫病毒（FMDV）是兽医面临的首要问题，因此开发针对抗病毒单克隆抗体特征的新方法是具有重要牲畜保健和经济意义的优先事项。考虑到这些前提，在这里，我们通过不同的策略固定RHDV2或其6S亚基制备了一系列SPR生物传感器，然后用于表征抗RHDV2单克隆抗体的结合能力。结果表明，以捕集剂-单抗共价固定在表面捕获的完整RHDV2组成的生物传感器的分析性能最好。为了评估生物传感器的通用性，然后在细胞提取物中使用FMVD采用相同的策略。本文讨论了SPR在动物病毒学诊断和治疗领域中对抗病毒单克隆抗体的快速和弹性精细鉴定中的应用。

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引用次数: 0

CHiTA: A scarless high-throughput pipeline for characterization of ribozymes 一种无疤痕的高通量管道用于核酶的表征。

IF 4.2 3区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

Methods

Pub Date : 2025-02-01 DOI: 10.1016/j.ymeth.2024.12.007

Lauren N. McKinley , Philip C. Bevilacqua

Small self-cleaving ribozymes are catalytic RNAs that cleave their phosphodiester backbone rapidly and site-specifically, without the assistance of proteins. Their catalytic properties make them ideal targets for applications in RNA pharmaceuticals and bioengineering. Consequently, computational pipelines that predict or design thousands of self-cleaving ribozyme candidates have been developed. Traditional experimental techniques for verifying the activity of these putative ribozymes, however, are low-throughput and time intensive. High-throughput (HT) pipelines that employ next-generation sequencing (NGS) analyze the activity of these thousands of ribozymes simultaneously. Until recently, the application of these HT pipelines has been limited to studying all single and double mutants of a select representative ribozyme. Unfortunately, this prevents the exploration of candidates having different lengths, circular permutations, and auxiliary stem-loops. Moreover, pipelines that analyze ribozymes en masse often include transcription of non-native flanking sequences that preclude accurate assessment of the intrinsic rate of ribozyme self-cleavage. To overcome these limitations, we developed a HT pipeline, “Cleavage High-Throughput Assay (CHiTA)”, which employs NGS and massively parallel oligonucleotide synthesis (MPOS) to characterize ribozyme activity for thousands of candidates in a scarless fashion. Herein, we describe detailed strategies and protocols to implement CHiTA to measure the activity of putative ribozymes from a wide range of ribozyme classes.

小的自切割核酶是一种催化rna，它可以在没有蛋白质的帮助下快速和特异地切割磷酸二酯骨架。它们的催化特性使其成为RNA制药和生物工程应用的理想靶标。因此，预测或设计数千种自裂核酶候选物的计算管道已经开发出来。然而，用于验证这些假定的核酶活性的传统实验技术是低通量和时间密集型的。采用下一代测序（NGS）的高通量（HT）管道同时分析这数千种核酶的活性。直到最近，这些HT管道的应用仅限于研究选定的代表性核酶的所有单突变体和双突变体。不幸的是，这阻碍了对具有不同长度、圆形排列和辅助茎环的候选体的探索。此外，大量分析核酶的管道通常包括非天然侧翼序列的转录，这妨碍了对核酶自裂内在速率的准确评估。为了克服这些限制，我们开发了一种HT管道，“切割高通量测定（CHiTA）”，该管道采用NGS和大规模平行寡核苷酸合成（MPOS）以无疤的方式表征数千种候选核酶的活性。在此，我们描述了实施CHiTA的详细策略和方案，以测量来自广泛核酶类的假定核酶的活性。

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引用次数: 0

SV-JIM, detailed pairwise structural variant calling using long-reads and genome assemblies SV-JIM，详细的两两结构变异调用使用长读和基因组组装。

IF 4.2 3区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

Methods

Pub Date : 2025-02-01 DOI: 10.1016/j.ymeth.2024.12.015

Clarence Todd , Lingling Jin , Ian McQuillan

This paper proposes a detailed process for SV calling that permits a data-driven assessment of multiple SV callers that uses both genome assemblies and long-reads. The process is implemented as a software pipeline named Structural Variant − Jaccard Index Measure, or SVJIM, using the Snakemake [20] workflow management system. Like most state-of-the-art SV callers, SV-JIM detects the presence of variations between pairs of genomes, but it streamlines the numerous SV calling stages into a single process for user convenience and evaluates the multiple SV sets produced using the Jaccard index measure to identify those with the highest consistency among the included SV callers. SV-JIM then produces aggregated SV results based on how many callers supported the reported SVs. For validation, SV-JIM was assessed through three case studies on the Homo sapiens genome and two plant genomes – Brassica nigra and Arabidopsis thaliana. Executing SV-JIM identified a significant amount of inter-caller variance which varied by tens of thousands of results on the larger Brassica nigra and Homo sapiens genomes. Further, aggregating the SV sets helped simplify better retention of the less frequently occurring SV types by requiring a level of minimum support rather than from a specific SV caller combination. Finally, these case studies identified a potential for inflated precision reporting that can occur during evaluation. SV-JIM is available publicly under MIT license at https://github.com/USask-BINFO/SV-JIM.

本文提出了一个详细的SV调用过程，该过程允许使用基因组组装和长读段对多个SV调用者进行数据驱动评估。该过程使用Snakemake[20]工作流管理系统，作为一个名为结构变体- Jaccard索引度量（SVJIM）的软件管道来实现。像大多数最先进的SV呼叫者一样，SV- jim检测基因组对之间的差异，但为了方便用户，它将众多SV调用阶段简化为一个过程，并使用Jaccard指数测量评估产生的多个SV集，以识别在所包括的SV呼叫者中一致性最高的那些。然后，SV- jim根据支持报告的SV的调用者的数量生成聚合的SV结果。为了验证SV-JIM的有效性，我们对三个智人基因组和两个植物基因组（芸苔和拟南芥）进行了案例研究。执行SV-JIM识别出大量的呼叫者之间的差异，这些差异在较大的芸芥和智人基因组上有成千上万的结果。此外，通过要求一定程度的最小支持，而不是特定的SV调用者组合，聚合SV集有助于更好地简化对出现频率较低的SV类型的保留。最后，这些案例研究确定了在评估期间可能发生的夸大精度报告的可能性。SV-JIM在MIT许可下可在https://github.com/USask-BINFO/SV-JIM上公开获得。

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引用次数: 0

Nanotechnological approaches to improve corticosteroids ocular therapy 改进皮质类固醇眼部疗法的纳米技术方法。

IF 4.2 3区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

Methods

Pub Date : 2025-02-01 DOI: 10.1016/j.ymeth.2024.12.008

María Lina Formica, Juan Matías Pernochi Scerbo, Hamoudi Ghassan Awde Alfonso, Pablo Tomás Palmieri, Julieta Ribotta, Santiago Daniel Palma

The administration of corticosteroids is the first-line treatment of the clinical conditions with ocular inflammation. Nonetheless, ocular physiological mechanisms, anatomical barriers and corticosteroid properties prevent it from reaching the target site. Thus, frequent topical administered doses or ocular injections are required, leading to a higher risk of adverse events and poor patient compliance.

Designing novel drug delivery systems based on nanotechnological tools is a useful approach to overcome disadvantages associated with the ocular delivery of corticosteroids. Nanoparticle-based drug delivery systems represent an alternative to the current dosage forms for the ocular administration of corticosteroids, since due to their particle size and the properties of their materials, they can increase their solubility, improve ocular permeability, control their release and increase bioavailability after their ocular administration. In this way, lipid and polymer-based nanoparticles have been the main strategies developed, giving rise to novel patent applications to protect these innovative drug delivery systems as a product, its preparation or administration method. Additionally, it should be noted that at least 10 clinical trials are being carried out to evaluate the ocular application of different pharmaceutical formulations based on corticosteroid-loaded nanoparticles.

Through a comprehensive and extensive analysis, this review highlights the impact of nanotechnology applications in ocular inflammation therapy with corticosteroids.

使用皮质类固醇是治疗眼部炎症临床症状的首选方法。然而，眼部生理机制、解剖屏障和皮质类固醇的特性阻碍了皮质类固醇到达目标部位。因此，需要频繁地局部给药或眼部注射，导致不良反应风险较高，患者依从性较差。设计基于纳米技术工具的新型给药系统是克服皮质类固醇眼部给药相关缺点的有效方法。以纳米颗粒为基础的给药系统是眼部给药皮质类固醇现有剂型的替代品，因为由于其粒度和材料的特性，它们可以在眼部给药后增加溶解度、改善眼部渗透性、控制释放和增加生物利用度。因此，以脂质和聚合物为基础的纳米粒子已成为开发的主要策略，并产生了新的专利申请，以保护这些创新的给药系统，包括产品、其制备或给药方法。此外，值得注意的是，目前至少有 10 项临床试验正在进行，以评估基于皮质类固醇纳米颗粒的不同药物制剂在眼部的应用。通过全面而广泛的分析，本综述强调了纳米技术应用在皮质类固醇眼部炎症治疗中的影响。

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引用次数: 0

Inferring multi-slice spatially resolved gene expression from H&E-stained histology images with STMCL 从h&e染色组织学图像推断STMCL的多层空间分辨基因表达。

IF 4.2 3区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

Methods

Pub Date : 2025-02-01 DOI: 10.1016/j.ymeth.2024.11.016

Zhiceng Shi , Fangfang Zhu , Wenwen Min

Spatial transcriptomics has significantly advanced the measurement of spatial gene expression in the field of biology. However, the high cost of ST limits its application in large-scale studies. Using deep learning to predict spatial gene expression from H&E-stained histology images offers a more cost-effective alternative, but existing methods fail to fully leverage the multimodal information provided by Spatial transcriptomics and pathology images. In response, this paper proposes STMCL, a novel multimodal contrastive learning framework. STMCL integrates multimodal information, including histology images, gene expression features of spots, and their locations, to accurately infer spatial gene expression profiles. We tested four different types of multi-slice spatial transcriptomics datasets generated by the 10X Genomics platform. The results indicate that STMCL has advantages over baseline methods in predicting spatial gene expression profiles. Furthermore, STMCL is capable of capturing cancer-specific highly expressed genes and preserving gene expression patterns while maintaining the original spatial structure of gene expression. Our code is available at https://github.com/wenwenmin/STMCL.

空间转录组学极大地促进了生物学领域中空间基因表达的测量。然而，ST的高成本限制了其在大规模研究中的应用。利用深度学习从h&e染色的组织学图像中预测空间基因表达提供了一种更具成本效益的替代方法，但现有方法无法充分利用空间转录组学和病理图像提供的多模态信息。为此，本文提出了一种新的多模态对比学习框架——STMCL。STMCL集成了组织图像、斑点基因表达特征及其位置等多模态信息，准确推断出空间基因表达谱。我们测试了由10X Genomics平台生成的四种不同类型的多层空间转录组学数据集。结果表明，STMCL在预测空间基因表达谱方面优于基线方法。此外，STMCL能够捕获癌症特异性高表达基因，在保持基因表达原始空间结构的同时保留基因表达模式。我们的代码可在https://github.com/wenwenmin/STMCL上获得。

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引用次数: 0

Microplate fluorescence quenching for high throughput screening of affinity constants – Serum albumins and zearalenones case study 用于高通量筛选亲和常数的微孔板荧光淬灭技术--血清白蛋白和玉米赤霉烯酮案例研究。

IF 4.2 3区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

Methods

Pub Date : 2025-02-01 DOI: 10.1016/j.ymeth.2024.12.011

Heiðar Már Aðalsteinsson , Lavínia Quartin Pinto , Tatiana Q. Aguiar , José A. Teixeira , Luís Abrunhosa

Measurements of changes in fluorescence signal is one of the most commonly applied methods for studying protein-ligand affinities. These measurements are generally carried out using cuvettes in spectrofluorometers, which can only measure one sample at a time. This makes screening procedures for multiple ligands and proteins extremely laborious, as each protein must be measured with multiple ligand concentrations, and usually in triplicate. Moreover, multiple equations exist to extract the affinity constants and other information from the data, and their underlying assumptions are often disregarded. In this study, the affinities of human, bovine and rat serum albumins for the mycotoxin zearalenone and five of its common derivatives were measured in 96-well microplates, allowing quick measurements of multiple samples using less reagent amounts. In comparison to measurements using a cuvette in a spectrofluorometer, the microplate method was shown to reproduce the affinity constants accurately. The results were discussed in terms of common pitfalls regarding experimental setup and available equations to analyze protein-ligand binding in fluorescence quenching assays. The commonly used Stern-Volmer equation was discussed in detail and the results used to show how inaccurate it is when a fluorescent protein-ligand complex is formed, and when other underlying approximations are ignored.

测量荧光信号的变化是研究蛋白质与配体亲和关系最常用的方法之一。这些测量通常使用荧光分光计中的比色皿进行，一次只能测量一个样品。这使得多种配体和蛋白质的筛选过程非常费力，因为每种蛋白质必须用多种配体浓度进行测量，而且通常是三份。此外，存在多个方程来从数据中提取亲和常数和其他信息，而它们的基本假设往往被忽视。在本研究中，人、牛和大鼠血清白蛋白对真菌毒素玉米赤霉烯酮及其五种常见衍生物的亲和力在96孔微孔板上进行了测量，允许使用较少的试剂量快速测量多个样品。与在荧光分光计中使用比色皿进行测量相比，微孔板方法可以准确地再现亲和常数。结果讨论了关于实验设置和可用的方程来分析荧光猝灭测定中的蛋白质配体结合的常见陷阱。详细讨论了常用的斯特恩-沃尔默方程，并使用结果来显示当荧光蛋白-配体复合物形成时，以及当其他潜在的近似被忽略时，它是多么不准确。

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引用次数: 0

Retraction notice to “A methodological framework for rigorous systematic reviews: Tailoring comprehensive analyses to clinicians and healthcare professionals” [Methods 225 (2024) 38–43]

IF 4.2 3区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

Methods

Pub Date : 2025-02-01 DOI: 10.1016/j.ymeth.2025.01.007

Stefano Mancin , Marco Sguanci , Giuliano Anastasi , Lea Godino , Alessio Lo Cascio , Emanuela Morenghi , Michela Piredda , Maria Grazia De Marinis

引用次数: 0

BCDB: A dual-branch network based on transformer for predicting transcription factor binding sites BCDB：基于 Transformer 的双分支网络，用于预测转录因子结合位点。

IF 4.2 3区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

Methods

Pub Date : 2025-02-01 DOI: 10.1016/j.ymeth.2024.12.006

Jia He , Yupeng Zhang , Yuhang Liu , Zhigan Zhou , Tianhao Li , Yongqing Zhang , Boqia Xie

Transcription factor binding sites (TFBSs) are critical in regulating gene expression. Precisely locating TFBSs can reveal the mechanisms of action of different transcription factors in gene transcription. Various deep learning methods have been proposed to predict TFBS; however, these models often need help demonstrating ideal performance under limited data conditions. Furthermore, these models typically have complex structures, which makes their decision-making processes difficult to transparentize. Addressing these issues, we have developed a framework named BCDB. This framework integrates multi-scale DNA information and employs a dual-branch output strategy. Integrating DNABERT, convolutional neural networks (CNN), and multi-head attention mechanisms enhances the feature extraction capabilities, significantly improving the accuracy of predictions. This innovative method aims to balance the extraction of global and local information, enhancing predictive performance while utilizing attention mechanisms to provide an intuitive way to explain the model's predictions, thus strengthening the overall interpretability of the model. Prediction results on 165 ChIP-seq datasets show that BCDB significantly outperforms other existing deep learning methods in terms of performance. Additionally, since the BCDB model utilizes transfer learning methods, it can transfer knowledge learned from many unlabeled data to specific cell line prediction tasks, allowing our model to achieve cross-cell line TFBS prediction. The source code for BCDB is available on https://github.com/ZhangLab312/BCDB.

转录因子结合位点（TFBSs）是调控基因表达的关键。精确定位TFBSs可以揭示不同转录因子在基因转录中的作用机制。人们提出了各种深度学习方法来预测TFBS；然而，这些模型通常需要在有限的数据条件下证明理想的性能。此外，这些模型通常具有复杂的结构，这使得它们的决策过程难以透明。为了解决这些问题，我们开发了一个名为BCDB的框架。该框架集成了多尺度DNA信息，并采用双分支输出策略。结合DNABERT、卷积神经网络（CNN）和多头注意机制，增强了特征提取能力，显著提高了预测的准确性。这种创新的方法旨在平衡全局和局部信息的提取，提高预测性能，同时利用注意机制提供一种直观的方式来解释模型的预测，从而增强模型的整体可解释性。165个ChIP-seq数据集的预测结果表明，BCDB在性能方面明显优于其他现有的深度学习方法。此外，由于BCDB模型使用迁移学习方法，它可以将从许多未标记数据中学习到的知识转移到特定的细胞系预测任务中，从而使我们的模型实现跨细胞系TFBS预测。BCDB的源代码可在https://github.com/ZhangLab312/BCDB上获得。

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引用次数: 0

Optimized biochemical method for human Polyphosphate quantification 优化人多磷酸盐定量的生化方法。

IF 4.2 3区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

Methods

Pub Date : 2025-02-01 DOI: 10.1016/j.ymeth.2025.01.001

Blanca Lázaro , Ana Sarrias , Francisco J. Tadeo , Joan Marc Martínez-Láinez , Ainhoa Fernández , Eva Quandt , Blanca Depares , Tobias Dürr-Mayer , Henning Jessen , Javier Jiménez , Josep Clotet , Samuel Bru

Polyphosphate (polyP), a biopolymer composed of phosphates, impacts a wide range of biological functions and pathological conditions in all organisms. However, polyP’s intricate physiology and structure in human cells have remained elusive, largely due to the lack of a reliable quantification method including its extraction. In this study, we assess critical points in the whole process: extraction, purification, and quantification polyP from human cell lines. We developed a highly efficient method that extracts between 3 and 100 times more polyP than previously achieved. Supported by Nuclear Magnetic Resonance (NMR), our approach confirms that mammalian polyP is primarily a linear unbranched polymer. We applied the optimized method to commonly used human cell lines, uncovering important variations of intracellular polyP that correlate with the expression levels of specific polyP converting enzymes. This study underscores the importance of employing several techniques for polyP characterization in parallel and provides a valuable and standardized tool for further exploration in this field.

聚磷酸盐（polyP）是一种由磷酸盐组成的生物聚合物，在所有生物体中影响着广泛的生物功能和病理状况。然而，由于缺乏可靠的定量方法，包括其提取，息肉蛋白在人类细胞中复杂的生理和结构仍然是难以捉摸的。在这项研究中，我们评估了整个过程的关键点：从人细胞系中提取、纯化和定量息肉。我们开发了一种高效的方法，提取的息肉比以前多3到100倍。在核磁共振（NMR）的支持下，我们的方法证实了哺乳动物息肉主要是一种线性无支化聚合物。我们将优化的方法应用于常用的人类细胞系，发现细胞内息肉蛋白的重要变化与特定息肉蛋白转化酶的表达水平相关。本研究强调了采用多种技术并行表征息肉的重要性，并为该领域的进一步探索提供了有价值的标准化工具。

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引用次数: 0