Methods in molecular biology最新文献_第7页

Comprehensive Assessment of Neutrophil Function in Autoimmune Diseases. 自身免疫性疾病中性粒细胞功能的综合评价。

Methods in molecular biology Pub Date : 2026-01-01 DOI: 10.1007/978-1-0716-4997-8_7

Quiterie Labaye, Daouda Abba Moussa, Mar Naranjo-Gomez

引用次数: 0

Identification and Characterization of Human Autoreactive T Cells for Autoimmune Research. 用于自身免疫研究的人类自身反应性T细胞的鉴定和表征

Methods in molecular biology Pub Date : 2026-01-01 DOI: 10.1007/978-1-0716-4997-8_1

Lenka Súkeníková, Daniela Latorre

引用次数: 0

Manual Immunofluorescence Staining for Dystrophin and Key Dystrophin-Associated Proteins. 肌营养不良蛋白和关键肌营养不良蛋白相关蛋白的手工免疫荧光染色。

Methods in molecular biology Pub Date : 2026-01-01 DOI: 10.1007/978-1-0716-4811-7_4

Hui Meng, Emily M Ott, Mariah J Prom, Margaret J Beatka, Tatyana A Vetter, Michael W Lawlor

{"title":"Manual Immunofluorescence Staining for Dystrophin and Key Dystrophin-Associated Proteins.","authors":"Hui Meng, Emily M Ott, Mariah J Prom, Margaret J Beatka, Tatyana A Vetter, Michael W Lawlor","doi":"10.1007/978-1-0716-4811-7_4","DOIUrl":"10.1007/978-1-0716-4811-7_4","url":null,"abstract":"The detection of dystrophin and the proteins of the dystrophin-associated protein complex (DAPC) by immunostaining allows for the detection of the presence and localization of these proteins in slide-mounted sections of skeletal muscle or heart tissue. This technique has been used historically for the diagnosis and characterization of muscle disorders including Duchenne muscular dystrophy and many of the limb girdle and congenital muscular dystrophies. The extensive usefulness of this technique has allowed the development of numerous antibodies that can be used in immunostaining assays. As an increasing number of research and translational science studies have recently focused on the restoration of dystrophin as a therapeutic strategy, high-quality immunostaining techniques and analysis remain sought-after scientific practices. This chapter describes useful procedures to perform the immunofluorescence staining for dystrophin and several other useful DAPC proteins, while also providing guidance based on our clinical diagnostic and translational science research experience from working in the muscular dystrophy field.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2975 ","pages":"47-65"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145200149","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Preparation, Sectioning, and Histochemical Staining of Muscle Tissue. 肌肉组织的制备、切片和组织化学染色。

Methods in molecular biology Pub Date : 2026-01-01 DOI: 10.1007/978-1-0716-4811-7_3

Emma C Frair, Stefan Nicolau

引用次数: 0

Quantifying Arbuscular Mycorrhizal Fungal Colonization via Anthocyanin Pigmentation in Medicago truncatula Roots. 通过花青素色素沉积测定丛枝菌根真菌定植。

Methods in molecular biology Pub Date : 2026-01-01 DOI: 10.1007/978-1-0716-4981-7_18

Anil Kumar, Fuyu Li, Qiuju Li

{"title":"Quantifying Arbuscular Mycorrhizal Fungal Colonization via Anthocyanin Pigmentation in Medicago truncatula Roots.","authors":"Anil Kumar, Fuyu Li, Qiuju Li","doi":"10.1007/978-1-0716-4981-7_18","DOIUrl":"10.1007/978-1-0716-4981-7_18","url":null,"abstract":"Plant responses to environmental stimuli are often shaped by a history of previous interactions, forming the foundation for stress memory and adaptive plasticity. Arbuscular mycorrhizal (AM) fungi establish a mutualistic relationship with most land plants, enhancing nutrient uptake and stress resilience, and are increasingly recognized as biological agents contributing to plant stress memory. However, quantifying AM colonization, especially in large-scale or time-course experiments investigating priming or memory effects, remains a technical bottleneck. Conventional staining methods are time-consuming, destructive, and incompatible with live imaging. This chapter presents a robust, nondestructive, and quantitative protocol to assess AM colonization in Medicago truncatula roots using a visible anthocyanin pigmentation marker. The method employs a synthetic construct expressing the R2R3 MYB transcription factor MtLAP1, driven by the AM-inducible Kunitz Protease Inhibitor 106 (KPI106) promoter, enabling visualization of arbuscule-containing root cells through purple/red pigmentation. The protocol encompasses Agrobacterium rhizogenes-mediated hairy root transformation, standardized mycorrhization assays, and anthocyanin pigment extraction and quantification. Anthocyanin accumulation correlates strongly with conventional staining-based colonization estimates, and the system enables early detection, live imaging, and high-throughput screening of mutants with altered AM phenotypes. This method offers a powerful tool for dissecting the functional role of mycorrhizal symbiosis in plant stress memory and is especially suited for forward genetic screens, stress priming experiments, and live-tracking of root-fungus interactions over time.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2988 ","pages":"195-205"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145232931","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Thermomemory Assay of Arabidopsis Plants. 拟南芥植物的温度记忆测定。

Methods in molecular biology Pub Date : 2026-01-01 DOI: 10.1007/978-1-0716-4981-7_1

Justyna J Olas, Philip A Wigge

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Characterization of Bioactive Saponins from Sea Cucumbers. 海参中生物活性皂苷的研究。

Methods in molecular biology Pub Date : 2026-01-01 DOI: 10.1007/978-1-0716-5384-5_10

Karuna Bhaskaran, Rajesh Chandra Misra, Anne Osbourn, Ramesha Thimmappa

{"title":"Characterization of Bioactive Saponins from Sea Cucumbers.","authors":"Karuna Bhaskaran, Rajesh Chandra Misra, Anne Osbourn, Ramesha Thimmappa","doi":"10.1007/978-1-0716-5384-5_10","DOIUrl":"https://doi.org/10.1007/978-1-0716-5384-5_10","url":null,"abstract":"Sea cucumbers are highly valued for their bioactive constituents, particularly triterpenoid saponins, which exhibit diverse pharmacological properties. The complex amphiphilic nature of saponins poses significant challenges in their extraction and purification. Their structural diversity and amphiphilicity demand carefully optimized protocols to maximize yield and purity. Moreover, the variability of saponin profiles between different sea cucumber species and tissues necessitates reproducible methods that can be scaled from small laboratory samples to larger quantities suitable for industrial applications. This chapter describes comprehensive, reproducible protocols for both small- and large-scale extraction of saponins from sea cucumber tissues, followed by chromatographic separation and bioactivity-guided fractionation. Subsequently, analytical techniques such as thin-layer chromatography (TLC), yeast growth inhibition assays, and liquid chromatography-mass spectrometry (LC-MS) are detailed. These detailed extraction, purification, and analytical protocols facilitate downstream applications such as structural characterization, quantification, and functional studies of saponins.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"3054 ","pages":"133-142"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148369119","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Synteny Analysis and Visualization of Gene Clusters. 基因簇的合成分析与可视化。

Methods in molecular biology Pub Date : 2026-01-01 DOI: 10.1007/978-1-0716-5384-5_6

Chanchan Liu, Dishuai Li, Qinan Wu

引用次数: 0

Methods for Functional Validation of Terpenoid Metabolic Clusters in Nicotiana benthamiana and Aspergillus oryzae. 本烟和米曲霉萜类代谢簇的功能验证方法。

Methods in molecular biology Pub Date : 2026-01-01 DOI: 10.1007/978-1-0716-5384-5_9

Zheyong Xue, Chang Li, Xue Yin, Jiaojiao Lu, Yanhong Guo

{"title":"Methods for Functional Validation of Terpenoid Metabolic Clusters in Nicotiana benthamiana and Aspergillus oryzae.","authors":"Zheyong Xue, Chang Li, Xue Yin, Jiaojiao Lu, Yanhong Guo","doi":"10.1007/978-1-0716-5384-5_9","DOIUrl":"https://doi.org/10.1007/978-1-0716-5384-5_9","url":null,"abstract":"Terpenoids represent one of the most structurally diverse classes of natural products, with vast potential applications in medicine, food, and agriculture. However, exploiting this potential has historically been challenging due to their low abundance in natural sources and the economic inefficiency of synthetic chemistry approaches for such complex structures. Heterologous biosynthesis offers a promising solution to these limitations. To date, multiple heterologous hosts have been developed for terpenoid production. Among these, Nicotiana benthamiana has emerged as a prominent platform for reconstituting plant-derived natural product pathways. Its compatibility with Agrobacterium-mediated transient expression, a scalable and flexible method, enables rapid integration and functional analysis of genes from diverse plant species. Conversely, Aspergillus oryzae has gained attention as a preferred host for fungal terpenoid pathways, owing to its highly efficient fungal expression system. Herein we present methodologies for functionally validating terpenoid biosynthetic clusters in N. benthamiana and A. oryzae.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"3054 ","pages":"123-132"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148369061","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Molecular Lego in the Green Factory: Engineering Plant Metabolism with Golden Gate Assembly. 绿色工厂中的分子乐高：用金门组装来设计植物代谢。

Methods in molecular biology Pub Date : 2026-01-01 DOI: 10.1007/978-1-0716-5384-5_8

Rajesh Chandra Misra, Ramesha Thimmappa, Anne Osbourn

{"title":"Molecular Lego in the Green Factory: Engineering Plant Metabolism with Golden Gate Assembly.","authors":"Rajesh Chandra Misra, Ramesha Thimmappa, Anne Osbourn","doi":"10.1007/978-1-0716-5384-5_8","DOIUrl":"https://doi.org/10.1007/978-1-0716-5384-5_8","url":null,"abstract":"Plant specialized metabolites encompass a diverse array of bioactive compounds with significant pharmaceutical, nutraceutical, and industrial relevance, and they play vital roles in plant defense, signaling, and ecological interactions. However, their complex biosynthetic pathways and low natural yields present a major bottleneck for commercial exploitation. Synthetic biology provides new tools for the rational design and engineering of metabolic pathways to enhance specialized metabolite biosynthesis in plants as well as in microbes such as E. coli and yeast. Here, we report a synthetic biology framework utilizing Golden Gate assembly (GGA) for the modular construction and optimization of plant metabolic pathways. Golden Gate enables scarless, directional assembly of multigene constructs with high efficiency, ideal for refactoring biosynthetic gene clusters and for the systematic design of complex metabolic networks. Golden Gate assembly can efficiently join up to 10-20 fragments simultaneously in a single reaction, a significant improvement over traditional molecular biology cloning methods. We developed and validated a standardized module of promoters, untranslated regions (UTRs), coding sequences, and regulatory elements compatible with plant transformation systems. Using this platform, we successfully reconstructed the biosynthetic pathway for plant specialized metabolites by transient expression in Nicotiana benthamiana, demonstrating significant improvements in construct stability. Our results highlight the potential of Golden Gate as a cornerstone technology for plant metabolic engineering, offering a scalable and versatile strategy to enhance the production of valuable specialized metabolites and set the stage for custom metabolic rewiring aimed at sustainable production of high-value compounds.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"3054 ","pages":"105-121"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148369069","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0