Nature Protocols最新文献

Fabrication of optically transparent ultrasound transducers to integrate light and sound in multimodal biomedical systems. 在多模态生物医学系统中集成光声的光学透明超声换能器的制造。

IF 16 1区生物学

Nature Protocols Pub Date : 2026-05-08 DOI: 10.1038/s41596-026-01366-6

Donggyu Kim, Mingyu Ha, Seonghee Cho, Jaewoo Kim, Dasom Heo, Minsu Kim, Peeyush Malik, Jeongwoo Park, Chulhong Kim

{"title":"Fabrication of optically transparent ultrasound transducers to integrate light and sound in multimodal biomedical systems.","authors":"Donggyu Kim, Mingyu Ha, Seonghee Cho, Jaewoo Kim, Dasom Heo, Minsu Kim, Peeyush Malik, Jeongwoo Park, Chulhong Kim","doi":"10.1038/s41596-026-01366-6","DOIUrl":"https://doi.org/10.1038/s41596-026-01366-6","url":null,"abstract":"Optical and acoustic technologies are extensively utilized across sectors for imaging purposes and in theranostic applications. The integration of coaxially aligned optical and acoustic pathways results in synergistic systems that overcome the limitations of each approach and provide complementary advantages such as increased precision and efficiency, housed in a compact instrument. However, commercial ultrasound transducers are optically opaque and therefore necessitate oblique configurations that compromise system simplicity, spatial alignment and signal quality. The use of optically transparent materials for the acoustic and electrical components results in transparent ultrasound transducers (TUTs) that can be used in biomedical setups and do not require oblique pathways or complex alignment. Here we describe a comprehensive and customizable pipeline for TUT development, covering material selection, simulation, fabrication and performance characterization. The procedure includes the materials' selection best suited for various target applications, the design optimization via acoustic simulation and the fabrication steps required for surface processing, electrode deposition and the integration of the acoustic layers. We explain how to characterize the fabricated TUTs to ensure their optical and acoustic functionality and to confirm that their performance matches that predicted in the simulations. The entire procedure requires ~3 weeks for users with moderate experience in piezoelectric device fabrication, acoustic measurement and microfabrication techniques. This protocol could facilitate future developments in multimodal biomedical systems, particularly those requiring seamless and high-fidelity opto-ultrasound integration.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":16.0,"publicationDate":"2026-05-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147856803","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"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

Implementation of an adaptive-optics assisted isoSTED nanoscope. 一种自适应光学辅助定向纳米显微镜的实现。

IF 16 1区生物学

Nature Protocols Pub Date : 2026-05-08 DOI: 10.1038/s41596-026-01365-7

Yang Li, Dong-Ryoung Lee, Edward S Allgeyer, Lena K Schroeder, Shijie Tu, Joerg Bewersdorf, Xiang Hao

引用次数: 0

Receptor discovery for nanomaterial soft and hard coronas via a biosensor-based Fishing strategy. 通过基于生物传感器的捕鱼策略发现纳米软硬冠的受体。

IF 16 1区生物学

Nature Protocols Pub Date : 2026-05-07 DOI: 10.1038/s41596-026-01358-6

Didar Baimanov, Jing Wang, Chunying Chen, Liming Wang

{"title":"Receptor discovery for nanomaterial soft and hard coronas via a biosensor-based Fishing strategy.","authors":"Didar Baimanov, Jing Wang, Chunying Chen, Liming Wang","doi":"10.1038/s41596-026-01358-6","DOIUrl":"https://doi.org/10.1038/s41596-026-01358-6","url":null,"abstract":"Upon exposure to biological fluids, nanomaterials rapidly acquire dynamic layers of adsorbed proteins called the protein corona, redefining the biological identity of nanomaterials and governing their in vivo fate. While corona composition has been extensively profiled, two aspects remain poorly understood: dynamic evolution and the receptor-mediated mechanisms underlying its cellular recognition. Here we present a broadly applicable, real-time biosensor-based protocol. This workflow characterizes nanomaterial-protein interactions and identifies cell membrane receptors involved in corona recognition. The protocol distinguishes between soft corona, hard corona, and total corona layers. It can not only quantify dynamic and competitive interactions but also map receptor-corona and receptor-plasma protein interactions using a label-free biosensor-based platform. This method combines biolayer interferometry, or alternatively surface plasmon resonance, or magnetic isolation, with downstream proteomics. It enables functional dissection of the nano-bio interface and provides insights into how corona dynamics govern cellular uptake. The method is compatible with diverse nanomaterial types and biofluids, depending on the experimental scope. The protocol is designed for users with basic experience in areas such as nanotechnology, molecular interaction analysis, proteomic profiling, cell culture, and biofluid sample preparation. The entire process takes ~10 days to complete.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":16.0,"publicationDate":"2026-05-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147840382","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"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

A modular method for high-throughput measurement of ion channel currents in cardiac myocytes. 高通量测量心肌细胞离子通道电流的模块化方法。

IF 16 1区生物学

Nature Protocols Pub Date : 2026-05-07 DOI: 10.1038/s41596-026-01351-z

Fitzwilliam Seibertz, Izzatulo Sobitov, Marcus L Gerloff, Aiste Liutkute, Alexey Alekseev, Thomas Mager, Constanze Schmidt, Funsho E Fakuade, Niels Voigt

{"title":"A modular method for high-throughput measurement of ion channel currents in cardiac myocytes.","authors":"Fitzwilliam Seibertz, Izzatulo Sobitov, Marcus L Gerloff, Aiste Liutkute, Alexey Alekseev, Thomas Mager, Constanze Schmidt, Funsho E Fakuade, Niels Voigt","doi":"10.1038/s41596-026-01351-z","DOIUrl":"https://doi.org/10.1038/s41596-026-01351-z","url":null,"abstract":"The patch-clamp technique offers unparalleled insight into the electrical and biophysical behavior of excitable cells. However, it is a slow and low-throughput method that typically requires cells to be measured one by one. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are regularly subjected to this technique to unravel the molecular mechanisms of cardiac diseases. Their use in direct patient treatment and successful drug development has been limited due to the lack of applicable high-throughput patch-clamp methods suited to successful hiPSC-CM measurement. Here we present a protocol employing a patch-clamp robot that addresses these limitations by using planar patch-clamp technology. We outline how to collect and handle hiPSC-CM for these experiments, along with optimized patch-clamp protocols for direct functional measurement of major cardiac ion channels including Kir2.1, NaV1.5, CaV1.2, Kv11.1 and Kir3.1/3.4. We further explain how the liquid-handling properties of this setup allow multiple patch-clamp protocols to be combined in sequence while the cell remains in whole-cell configuration. This allows for over a hundred-fold increase in functional data acquisition. These procedures can be carried out within 1 d by both skilled and non-electrophysiologists; however, some experience in cell culture and handling is required. Overall, this protocol enhances fast and reliable functional characterization of hiPSCl-CM and may increase their applicability for rapid and safe drug development.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":16.0,"publicationDate":"2026-05-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147840432","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"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

Site-specific protein and peptide modification by redeploying an asparaginyl ligase for noncanonical reactions. 通过重新部署天冬酰胺连接酶进行非规范反应的位点特异性蛋白质和肽修饰。

IF 16 1区生物学

Nature Protocols Pub Date : 2026-04-29 DOI: 10.1038/s41596-026-01348-8

Simon J de Veer, Yan Zhou, Fabian B H Rehm, Thomas Durek, David J Craik

{"title":"Site-specific protein and peptide modification by redeploying an asparaginyl ligase for noncanonical reactions.","authors":"Simon J de Veer, Yan Zhou, Fabian B H Rehm, Thomas Durek, David J Craik","doi":"10.1038/s41596-026-01348-8","DOIUrl":"https://doi.org/10.1038/s41596-026-01348-8","url":null,"abstract":"The ability to precisely modify proteins and peptides is fundamental to studying their function and creating new variants or topologies with improved properties. Recent studies have transformed the scope of transpeptidases as versatile tools for site-specific modification of proteins and peptides. The engineered asparaginyl ligase OaAEP1 is an ultrafast transpeptidase that stands out owing to its ability to efficiently catalyze a diverse range of modifications that extend well beyond its natural function to generate backbone cyclic peptides in plants. In this Protocol Extension, we describe a framework for the design and application of noncanonical reactions catalyzed by OaAEP1 that provide access to engineered products with customized terminal or side-chain modifications. The reactions proceed cleanly under mild, nondenaturing conditions and can be applied to a broad array of substrates produced by chemical synthesis or recombinant expression, including folded proteins and peptides. After preparing the required substrates and reagents (~5 d) and expressing the recombinant enzyme in Escherichia coli (~3 d), OaAEP1-catalyzed reactions can be carried out in a matter of minutes to hours. We describe methods for installing non-native C-terminal modifications, including by conjugating commercially available nonpeptidic amines (reactive handles, carbohydrates and so on) or ligating a reversed (retro) substrate mimetic that enables production of genetically inaccessible C-to-C fusions. We also describe procedures for OaAEP1-catalyzed side-chain modification of proteins and peptides, which can be applied to generate side-chain-to-tail macrocyclic products, to label a specific side-chain amine with a dye or other reporter tag, or to produce defined protein-cyclic peptide fusions.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":16.0,"publicationDate":"2026-04-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147776788","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"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

Publisher Correction: Peptide-centric local stability assay (PELSA) for sensitive identification of ligand-targeting proteins and binding sites at proteome scale. 出版者更正：肽中心局部稳定性测定（PELSA）用于在蛋白质组尺度上对配体靶向蛋白和结合位点进行敏感鉴定。

IF 16 1区生物学

Nature Protocols Pub Date : 2026-04-28 DOI: 10.1038/s41596-026-01381-7

Keyun Wang, Kejia Li, Jiayang Yan, Lianji Xue, Yanni Ma, Haiyang Zhu, Ting Yu, Yan Wang, Mikhail Savitski, Mingliang Ye

引用次数: 0

Profiling large-scale protein occupancy on bacterial genomes using IPOD-HR. 利用IPOD-HR分析细菌基因组上的大规模蛋白质占用。

IF 16 1区生物学

Nature Protocols Pub Date : 2026-04-28 DOI: 10.1038/s41596-026-01357-7

Rebecca L Hurto, Jeremy W Schroeder, Julian Trouillon, Uwe Sauer, Lydia Freddolino

{"title":"Profiling large-scale protein occupancy on bacterial genomes using IPOD-HR.","authors":"Rebecca L Hurto, Jeremy W Schroeder, Julian Trouillon, Uwe Sauer, Lydia Freddolino","doi":"10.1038/s41596-026-01357-7","DOIUrl":"https://doi.org/10.1038/s41596-026-01357-7","url":null,"abstract":"Identifying genomic regions bound by individual proteins such as transcription factors is essential to understanding bacterial gene regulation; however, comprehensive understanding of the effect of protein occupancy on gene regulation would be prohibitively laborious and expensive to achieve using methods such as chromatin immunoprecipitation with sequencing (ChIP-seq) and ChIP with exonuclease treatment (ChIP-exo) for every protein and condition of interest. Here we describe a protocol for performing in vivo protein occupancy display-high resolution (IPOD-HR), a powerful method for genome-wide profiling of protein-bound DNA in prokaryotic systems. Although assay for transposase-accessible chromatin with sequencing (ATAC-seq) is the method of choice for assaying general protein occupancy in eukaryotic systems, bacterial nucleoid-associated proteins can affect ATAC-seq, rendering it unsuitable for use in bacteria. In contrast, IPOD-HR can be used to identify regions of bacterial genomes that are highly bound by proteins, regardless of the identity of the proteins bound, allowing the identification of condition- and genotype-dependent changes in protein occupancy associated with changes in gene regulation. The technique is coupled to RNA polymerase ChIP, followed by sequencing of the extracted samples and downstream analysis using open-source, automated software that we provide and actively maintain. Once cross-linked samples are obtained, the core DNA selection portion of the IPOD-HR protocol takes 3 calendar days to perform. The resulting DNA extracts are subjected to high-throughput sequencing, resulting in sequencing data that are analyzed, which typically requires a few additional days, depending on the number of samples and computing resources. The IPOD-HR experimental method requires familiarity with standard molecular biology techniques suitable for preparing Illumina sequencing inputs, and the computational post-processing pipeline requires basic knowledge of the Linux command line environment.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":16.0,"publicationDate":"2026-04-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147776833","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"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

Whole-mouse immunolabeling at cellular resolution for comprehensive 3D atlases. 全鼠免疫标记在细胞分辨率全面的三维地图集。

IF 16 1区生物学

Nature Protocols Pub Date : 2026-04-27 DOI: 10.1038/s41596-026-01363-9

Hongcheng Mai, Yuewei Wang, Yu Zhu, Yin Zhao, Ying Chen, Luciano Hoeher, Rami Al-Maskari, Jie Luo, Ali Erturk

{"title":"Whole-mouse immunolabeling at cellular resolution for comprehensive 3D atlases.","authors":"Hongcheng Mai, Yuewei Wang, Yu Zhu, Yin Zhao, Ying Chen, Luciano Hoeher, Rami Al-Maskari, Jie Luo, Ali Erturk","doi":"10.1038/s41596-026-01363-9","DOIUrl":"https://doi.org/10.1038/s41596-026-01363-9","url":null,"abstract":"Mapping complex biological systems and tracking disease progression at high resolution across the entire mammalian body has remained technically challenging. Here, to address this, we present wildDISCO (immunolabeling of wild-type mice and DISCO clearing), a comprehensive protocol for whole-body immunolabeling, optical clearing and imaging of mice at cellular resolution using standard IgG antibodies. This protocol optimizes tissue permeabilization using cyclodextrin as a potent enhancer of cholesterol extraction and membrane permeabilization, enabling deep antibody penetration across all organs. We detail procedures for sample preparation, tissue decolorization and decalcification, whole-body immunostaining, clearing, and subsequent 3D imaging, virtual reality visualization and whole-mouse atlas construction. The method allows comprehensive mapping of neuronal, vascular, lymphatic and immune systems, as well as systemic studies in disease models, including cancer and microbiome-host interaction studies. We anticipate that wildDISCO will serve as a broadly applicable platform for generating whole-body cellular atlases, enabling systems-level investigations of health and disease. Only standard immunohistochemistry facilities are required, but successful implementation may require initial technical training, particularly for researchers with limited prior experience in tissue clearing or 3D imaging workflows. From start to finish, the procedure takes 4 weeks.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":16.0,"publicationDate":"2026-04-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147776863","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"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

Preparation of C4'-modified nucleoside analogs. C4'修饰核苷类似物的制备。

IF 16 1区生物学

Nature Protocols Pub Date : 2026-04-27 DOI: 10.1038/s41596-026-01353-x

Thirupathi Nuligonda, Gautam Kumar, Jason W Wang, Lara K Mahal, Michael W Meanwell

{"title":"Preparation of C4'-modified nucleoside analogs.","authors":"Thirupathi Nuligonda, Gautam Kumar, Jason W Wang, Lara K Mahal, Michael W Meanwell","doi":"10.1038/s41596-026-01353-x","DOIUrl":"https://doi.org/10.1038/s41596-026-01353-x","url":null,"abstract":"C4'-modified nucleoside analogs continue to attract global attention for treating infectious diseases and as components in oligonucleotide therapeutics. Current preparations mostly employ lengthy semi-synthetic approaches that do not allow for the efficient exploration of the chemical space associated with this valuable nucleoside subclass. Here we describe the pilot-scale (250 mg) and process-scale (85 g) preparation of 4'-methyl-ribothymidine (4',5-dimethyluridine) using a de novo strategy. Both L- and D-nucleoside analogs are accessible, and 10 different C4' modifications and 20 different nucleobases can be used interchangeably to create new analogs. This protocol involves the use of an enantioselective proline-catalyzed aldol between 2,2-dimethoxyacetaldehyde and a dioxanone. A 1,2-addition into the aldol product installs the C4' modification. Subsequent cyclization via intramolecular trans-acetalization delivers the modified ribose core of the nucleoside analog. Peracetylation, followed by Vorbrüggen glycosylation, completes the route. The pilot- and process-scale protocols can be completed in ~5 and ~7 d, respectively, to deliver C4'-modified nucleoside analogs in good yields and excellent enantiopurity.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":16.0,"publicationDate":"2026-04-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147776773","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"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

Discovery of candidate therapeutic targets with Geneformer. 用Geneformer发现候选治疗靶点。

IF 16 1区生物学

Nature Protocols Pub Date : 2026-04-23 DOI: 10.1038/s41596-026-01364-8

Yujie Zhang, Madhavan S Venkatesh, Christina V Theodoris

{"title":"Discovery of candidate therapeutic targets with Geneformer.","authors":"Yujie Zhang, Madhavan S Venkatesh, Christina V Theodoris","doi":"10.1038/s41596-026-01364-8","DOIUrl":"https://doi.org/10.1038/s41596-026-01364-8","url":null,"abstract":"Mapping the connections between genes enables the identification of networks disrupted in disease. The approach, however, requires large amounts of data, making the discovery of therapeutic targets difficult in settings with limited data. We recently developed a foundational artificial intelligence model, Geneformer, pretrained on a large-scale corpus of single-cell transcriptomes (initially ~30 million, now >100 million) to enable context-aware predictions in network biology with limited data. Here, we cover the methodology for using Geneformer through a combination of zero-shot inference, fine-tuning and in silico perturbation. The procedure includes the tokenization of raw gene expression counts into rank value encodings aligned with the model's pretrained vocabulary. Separability of relevant phenotypes in the pretrained embedding space is first assessed with zero-shot embeddings. Fine-tuning is then performed either with a single task, for example, disease prediction within a specific cell type, or with multiple tasks to jointly learn cross-informative features, such as cell types and disease states. Performance is evaluated with confusion matrices, macro F1 scores and embedding analysis. Subsequently, in silico perturbation simulates gene repression or activation and quantifies the shift in cell state embeddings, prioritizing candidate targets by statistical and biological metrics. The approach also supports perturbation using a quantized model to enhance computational efficiency. Outputs include predictive models fine-tuned for context-specific cell state representations and rank-ordered predictions of perturbations to induce each target state. The full pipeline typically completes in under 2 days on a standard GPU workstation and requires only moderate Python experience.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":16.0,"publicationDate":"2026-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147776673","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"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