Nature Protocols最新文献

{"title":"Microfluidic gradients create a stem cell model of the human central nervous system.","authors":"Peter Serles, Giorgia Quadrato","doi":"10.1038/s41596-025-01269-y","DOIUrl":"https://doi.org/10.1038/s41596-025-01269-y","url":null,"abstract":"","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145251975","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}

{"title":"Measurement of electrochemical brain activity with fast-scan cyclic voltammetry during functional magnetic resonance imaging.","authors":"Tatiana A Shnitko, Lindsay R Walton, Tong-Yu Rainey Peng, Sung-Ho Lee, Tzu-Hao Harry Chao, Matthew D Verber, R Mark Wightman, Yen-Yu Ian Shih","doi":"10.1038/s41596-025-01250-9","DOIUrl":"https://doi.org/10.1038/s41596-025-01250-9","url":null,"abstract":"<p><p>One of the challenges associated with functional magnetic resonance imaging (MRI) studies is integrating and causally linking complementary functional information, often obtained using different modalities. Achieving this integration requires synchronizing the spatiotemporal multimodal datasets without mutual interference. Here we present a protocol for integrating electrochemical measurements with functional MRI, enabling the simultaneous assessment of neurochemical dynamics and brain-wide activity. This Protocol addresses challenges such as artifact interference and hardware incompatibility by providing magnetic resonance-compatible electrode designs, synchronized data acquisition settings and detailed in vitro and in vivo procedures. Using dopamine as an example, the protocol demonstrates how to measure neurochemical signals with fast-scan cyclic voltammetry (FSCV) in a flow-cell setup or in vivo in rats during MRI scanning. These procedures are adaptable to various analytes measurable by FSCV or other electrochemical techniques, such as amperometry and aptamer-based sensing. By offering step-by-step guidance, this Protocol facilitates studies of neurovascular coupling with the neurochemical basis of large-scale brain networks in health and disease and could be adapted in clinical settings. The procedure requires expertise in MRI, FSCV and stereotaxic surgeries and can be completed in 7 days.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145251980","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}

{"title":"Generation of spatially patterned human neural tube-like structures using microfluidic gradient devices.","authors":"Xufeng Xue, Omar M Rahman, Shiyu Sun, Jeyoon Bok, Aoife Tang, Jianping Fu","doi":"10.1038/s41596-025-01266-1","DOIUrl":"https://doi.org/10.1038/s41596-025-01266-1","url":null,"abstract":"<p><p>The functional complexity and anatomical organization of the nervous system are established during regional patterning of its embryonic precursor-the neural tube. Human pluripotent stem (hPS) cell-based models have emerged as valuable complements to animal models for studying neural development. Here we present the design and implementation of a microfluidic gradient device for modeling human neural tube formation and regional patterning with hPS cells. The microfluidic device enables the formation of tubular or spherical colonies of hPS cells at prescribed locations within microfluidic channels, allowing the cell colonies to form lumenal structures while being exposed to well-controlled chemical gradients for rostral-caudal and/or dorsal-ventral patterning, resulting in the formation of a microfluidic neural tube-like structure (μNTLS) or a forebrain-like structure (μFBLS). The μNTLS recapitulates important hallmarks of early human neural development, including well-defined lumenal morphologies, spatially organized regional marker expression, emergence of secondary signaling centers and the development of neural crest cells. The dorsal-ventral patterned μFBLS further recapitulates spatially segregated dorsal and ventral regions, as well as the layered segregation of early neurons from neural progenitors, mimicking human forebrain pallium and subpallium development. Both the μNTLS and μFBLS are compatible with long-term culture, live imaging, immunofluorescence staining and single-cell sequencing, serving as robust systems for studying human neurodevelopment and disease. This protocol can be implemented by a researcher with polydimethylsiloxane soft lithography and cell culture experience and takes ~8-41 d to complete, depending on the types of neural structure to model and their developmental stages, with an option for prolonged culture to promote neuronal maturation.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145251985","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}

{"title":"Investigating non-proliferative cell states with inducible CRISPR screens.","authors":"Jeroen M Bugter, Roland Rad","doi":"10.1038/s41596-025-01252-7","DOIUrl":"https://doi.org/10.1038/s41596-025-01252-7","url":null,"abstract":"","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145251993","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}

{"title":"Inducible CRISPR-Cas9 screening platform to interrogate non-proliferative cellular states.","authors":"Gabriele Casagrande Raffi, Hendrik J Kuiken, Cor Lieftink, Rene Bernards, Roderick L Beijersbergen, Liqin Wang","doi":"10.1038/s41596-025-01251-8","DOIUrl":"https://doi.org/10.1038/s41596-025-01251-8","url":null,"abstract":"<p><p>CRISPR screens have revolutionized the study of diverse biological processes, particularly in cancer research. Both pooled and arrayed CRISPR screens have facilitated the identification of essential genes for cell survival and proliferation, drivers of drug resistance and synthetic lethal interactions. However, applying loss-of-function CRISPR screening to non-proliferative states remains challenging, largely because of slower editing and the poor sensitivity of identifying guide RNAs that 'drop out' in a population of non-dividing cells. Here, we present a detailed protocol to accomplish this, using an inducible Cas9 system that offers precise temporal control over Cas9 expression. This inducible system allows gene editing to occur only after the non-proliferative state is fully established. We describe the complete procedure for generating an inducible Cas9-expressing model and for measuring editing efficiency by using flow cytometry. In addition, we discuss how to optimize key parameters for performing successful CRISPR screens in various non-proliferative states. We describe a detailed workflow for performing a screen in senescent cells to identify senolytic targets. This protocol is accessible to researchers with experience in molecular biology techniques and can be completed in 8-12 weeks, from the generation of an inducible Cas9 cell line clone to the analysis of a CRISPR screen for hit identification. These techniques can be applied by researchers across different fields, including stem cell differentiation, immune cell development, aging and cancer research.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145251936","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}

{"title":"Addendum: Visualizing plant salt stress with a NaCl-responsive fluorescent probe.","authors":"Xiaoxie Ma, Xiaoyan Zeng, Yurou Huang, Sheng Hua Liu, Jun Yin, Guang-Fu Yang","doi":"10.1038/s41596-025-01280-3","DOIUrl":"https://doi.org/10.1038/s41596-025-01280-3","url":null,"abstract":"","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-10-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145239233","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}

{"title":"Report Approval for Transcranial Electrical Stimulation (RATES): expert recommendation based on a Delphi consensus study.","authors":"Vahid Nejati, Zahra Vaziri, Andrea Antal, Daria Antonenko, Roozbeh Behroozmand, Sven Bestmann, Jerome Brunelin, Andre R Brunoni, Sandra Carvalho, Nick J Davis, Peter G Enticott, Andreas J Fallgatter, Roberta Ferrucci, Paul B Fitzgerald, Masashi Hamada, Roy H Hamilton, Kate E Hoy, Shapour Jaberzadeh, Asif Jamil, Roi Cohen Kadosh, Bart Krekelberg, Steven Laureys, Leonor J Romero Lauro, Colleen K Loo, Donel Martin, Giovanni Martinotti, Marine Mondino, Antonio Oliviero, Maria Concetta Pellicciari, Christian Plewnia, Gorana Pobric, Rudi De Raedt, Lais B Razza, Lorenzo Rocchi, Mohammad Ali Salehinejad, Azin Sarraj Khorrami, Martin Schecklmann, Hartwig Roman Siebner, Stephan F Taylor, Marie-Anne Vanderhasselt, Sven Vanneste, Carmelo M Vicario, Adam J Woods, Ulf Ziemann, Michael A Nitsche","doi":"10.1038/s41596-025-01259-0","DOIUrl":"https://doi.org/10.1038/s41596-025-01259-0","url":null,"abstract":"<p><p>Transcranial electrical stimulation (tES) has gained substantial momentum as a research and therapeutic tool; however, it suffers from challenges related to reproducibility and quality assessment due to the absence of standardized reporting practices. Here we aim to develop a comprehensive and consensus-based checklist for conducting and reporting tES studies to enhance the quality of research and reports. In this Consensus Statement, we used a Delphi approach conducted across three rounds and involving 38 experts to identify crucial elements required to report in tES studies. This consensus-driven approach included the evaluation of the interquartile deviation (>1.00), the percentage of positive responses (above 60%) and mean importance ratings (<3), hence ensuring the creation of a robust and well-balanced checklist. These metrics were utilized to assess both the consensus reached and importance ratings for each item. Consensus was reached, leading to the retention of 66 out of the initial 70 items. These items were categorized into five groups: participants (12 items), stimulation device (9 items), electrodes (12 items), current (12 items) and procedure (25 items). We then distilled a shorter version of the checklist, which includes the 26 items deemed essential. The Report Approval for Transcranial Electrical Stimulation (RATES) checklist is relevant to those carrying out and assessing tES studies, as it provides a structured framework for researchers to consider and report. For reviewers, it can serve as a tool to assess completeness, comprehensiveness and transparency of reports. In addition, the RATES checklist aims to promote a deeper understanding of tES and facilitates comparisons between studies within the field. Overall, the RATES checklist provides a shared reference point that may improve research quality, foster harmonization in reporting and, ultimately, enhance the interpretability and reproducibility of findings in both research and clinical contexts.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145225641","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}

{"title":"Iron-catalyzed stereoselective glycosylation for 1,2-cis-aminoglycoside assembly.","authors":"Zixiang Jiang, Dakang Zhang, Pinzhi Wang, Le Yin, Hao Xu","doi":"10.1038/s41596-025-01263-4","DOIUrl":"https://doi.org/10.1038/s41596-025-01263-4","url":null,"abstract":"<p><p>Complex carbohydrates are essential to life processes, but it is challenging to isolate these molecules from natural sources in high homogeneity. Therefore, complex-glycan synthesis becomes critical to improving our understanding of their important functions. Due to their complexity, synthesis is still difficult for nonexperts. One of the key challenges is to search for general solutions for highly 1,2-cis-selective glycosylation, which will directly assemble 1,2-cis-2-aminoglycosides that are incorporated in numerous biologically important complex glycans and glycoconjugates. Here we describe an iron-catalyzed, chemical glycosylation method for rapid assembly of 1,2-cis-aminoglycosidic linkages. The iron catalyst is commercially available, and the bench-stable supporting ligand and amination reagents are easily prepared from abundant, readily available starting materials. This catalytic, exclusively 1,2-cis-selective glycosylation is effective for a broad range of glycosyl donors and acceptors, and it can be operated in a continuous fashion and scaled up to the multigram scale. The reactivity of this glycosylation is tunable for both electron-rich and electron-deficient substrates by modulating amination reagents. The glycosylation proceeds through a unique mechanism in which the iron catalyst activates a glycosyl acceptor and an oxidant when it facilitates the cooperative atom transfer of both moieties to a glycosyl donor in an exclusively cis-selective manner. This glycosylation protocol takes several hours to operate. It complements the existing 1,2-cis-selective glycosylation methods and effectively addresses the challenge of achieving both generality and high stereoselectivity in the 1,2-cis-selective aminoglycosylation.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145225516","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}

{"title":"Enzymatic X-ray absorption spectroelectrochemistry.","authors":"Rafael N P Colombo, Graziela C Sedenho, Itamar T Neckel, Frank N Crespilho","doi":"10.1038/s41596-025-01254-5","DOIUrl":"https://doi.org/10.1038/s41596-025-01254-5","url":null,"abstract":"<p><p>Understanding the redox properties and catalytic behavior of proteins is critical for harnessing their functions in biocatalysis and to promote efficient bio-inspired catalysts design. Enzymatic X-ray absorption spectroelectrochemistry (XA-SEC) combines the insights of X-ray absorption spectroscopy with the precision of electrochemical methods to elucidate enzymes' redox properties and catalytic behavior. Here we describe how to perform enzymatic XA-SEC experiments. The procedure begins with the preparation of the carbon-based working electrode to enhance enzyme immobilization. We exemplify with the efficient immobilization of bilirubin oxidase from Myrothecium verrucaria on the electrode surface, utilizing nanomaterials to enhance biomaterial loading and electron-transfer at the enzyme-electrode interface. Next, we guide researchers through setting up a standard three-electrode electrochemical cell, ensuring proper electrical connections and electrolyte preparation. Our Protocol details the Cu K-edge X-ray absorption spectroscopy measurement procedure at the synchrotron light sources, with in situ electrochemical control. Real-time redox processes are monitored through direct electron transfer analysis, providing valuable thermodynamic and kinetic information. It is important to determine the stability and activity of the analyzed protein under X-ray beam exposure; our approach typically results in stable electrochemical and spectroscopic signals for long experimental runs, showcasing the enzyme's robust performance and efficient protein immobilization. The method's ability to correlate XA-SEC data with direct electron transfer and substrate-biding analysis provides a powerful tool for advancing our understanding of enzymatic electrocatalysis and opens new avenues for developing sustainable bioelectrochemical technologies.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145213104","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}

{"title":"Fabrication and characterization of optoelectronic in-sensor computing devices.","authors":"Guang Zeng, Sijie Ma, Tianqing Wan, Hongye Chen, Jiewei Chen, Yang Chai","doi":"10.1038/s41596-025-01262-5","DOIUrl":"https://doi.org/10.1038/s41596-025-01262-5","url":null,"abstract":"<p><p>Bioinspired in-sensor computing devices can process information at sensory terminals by leveraging physical principles, thereby reducing latency and energy consumption during computation while simultaneously enhancing the efficiency of data processing and real-time analysis. Optoelectronic devices exhibit in-sensor computing functions, such as feature enhancement and data compression, by tuning the defect states of the semiconductor channels and thereby modulating the photoresponsivity and time constants of the sensors. These functionalities are critically dependent on precise fabrication and testing protocols. Here we present a detailed procedure for fabricating and characterizing in-sensor computing devices based on nanoscale semiconductor thin films. We explain how to test such optoelectronic devices, including the testing of visual adaptation and motion perception responses. When using semiconductor materials obtained from commercial suppliers, this procedure is time efficient and results in highly reproducible device performance. Nevertheless, all device fabrication and testing steps are generalizable and can be extended to other semiconductor thin films grown using different methods. The procedure is intended for researchers experienced in cleanroom operations and microfabrication techniques and can be completed in ~14 d. The use of bioinspired optoelectronic devices enables the development of a framework for advancing in-sensor computing technologies.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145213235","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}