Nature Protocols最新文献_第5页

A workflow for statistical analysis and visualization of microbiome omics data using the R microeco package. 使用R microeco包的微生物组学数据的统计分析和可视化工作流程。

IF 16 1区生物学

Nature Protocols Pub Date : 2025-08-06 DOI: 10.1038/s41596-025-01239-4

Chi Liu, Felipe R P Mansoldo, Hankang Li, Alane Beatriz Vermelho, Raymond Jianxiong Zeng, Xiangzhen Li, Minjie Yao

{"title":"A workflow for statistical analysis and visualization of microbiome omics data using the R microeco package.","authors":"Chi Liu, Felipe R P Mansoldo, Hankang Li, Alane Beatriz Vermelho, Raymond Jianxiong Zeng, Xiangzhen Li, Minjie Yao","doi":"10.1038/s41596-025-01239-4","DOIUrl":"https://doi.org/10.1038/s41596-025-01239-4","url":null,"abstract":"The increasing complexity of experimental designs and the volume of data in the microbiome field, along with the diversification of omics data types, pose substantial challenges to statistical analysis and visualization. Here we present a step-by-step protocol based on the R microeco package ( https://github.com/ChiLiubio/microeco ) that details the statistical analysis and visualization of microbiome data. The omics data types shown consist of amplicon sequencing data, metagenomic sequencing data and nontargeted metabolomics data. The analysis of amplicon sequencing data specifically involves data preprocessing and normalization, core taxa, alpha diversity, beta diversity, differential abundance testing and machine learning. We consider various data analysis scenarios in each section to exhibit the comprehensiveness of the protocol. We emphasize that different normalized data produced by various methods are selected for subsequent analysis of each part based on the best analytical practices. Additionally, in the differential abundance test analysis, we adopt parametric community simulation to enable the performance evaluation of various testing approaches. For the analysis of metagenomic data, the focus is on how bioinformatic analysis data are read and preprocessed, which refers to the major usage differences from amplicon sequencing data. For metabolomics data, we mainly demonstrate the differential test, machine learning and association analysis with microbial abundances. To address some complex analyses, this protocol extensively combines different types of methods to build an analysis pipeline. This protocol is more comprehensive and scalable compared with alternative methods. The provided R codes can run in about 6 h on a laptop computer.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-08-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144794910","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

Mass spectrometry-based top-down proteomics for proteoform profiling of protein coronas. 基于质谱的自上而下蛋白质组学用于蛋白质冠状体的蛋白质形态分析。

IF 16 1区生物学

Nature Protocols Pub Date : 2025-08-05 DOI: 10.1038/s41596-025-01229-6

Seyed Amirhossein Sadeghi, Fei Fang, Reyhane Tabatabaeian Nimavard, Qianyi Wang, Guijie Zhu, Amir Ata Saei, Liangliang Sun, Morteza Mahmoudi

{"title":"Mass spectrometry-based top-down proteomics for proteoform profiling of protein coronas.","authors":"Seyed Amirhossein Sadeghi, Fei Fang, Reyhane Tabatabaeian Nimavard, Qianyi Wang, Guijie Zhu, Amir Ata Saei, Liangliang Sun, Morteza Mahmoudi","doi":"10.1038/s41596-025-01229-6","DOIUrl":"10.1038/s41596-025-01229-6","url":null,"abstract":"The protein corona is a layer of biomolecules-primarily proteins-that adsorbs to nanoparticle (NP) surfaces in biological fluids. If the purpose of the NP is therapeutic, this can have a profound effect on its biological activity and function in vivo. Protein corona formation can also be exploited for diagnostic purposes and to differentially enrich proteins for biomarker discovery. For all of these applications, it is useful to determine which proteins, and which specific proteoforms, bind to different types of NP. The traditional mass spectrometry (MS)-based bottom-up proteomics does not accurately identify specific proteoforms within the protein corona. This limitation impedes the nanomedicine field's ability to precisely predict the biological fate and pharmacokinetics of nanomedicines and their effectiveness in early-stage biomarker discovery and disease detection because many different proteoforms of the same gene could exist in the corona, and they have divergent biological functions. Here, we describe how to use capillary zone electrophoresis (CZE)-MS-based top-down proteomics to characterize the proteoform landscape of the protein corona. Our procedures detail the recovery of intact proteoforms from NP surfaces by using detergent-assisted proteoform elution and the measurement of these proteoforms by using CZE-tandem MS (MS/MS) and CZE-high-field asymmetric waveform ion mobility spectrometry (FAIMS)-MS/MS. The entire workflow is completed within 3-4 d. Using this protocol, hundreds of proteoforms from the protein corona of polystyrene NPs can be identified. Distinct protein corona proteoform profiles were observed from NPs with different physicochemical properties. The addition of FAIMS is beneficial for more in-depth proteoform characterization.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12407567/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144789601","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Electrocatalytic semi-hydrogenation of alkynes using water as the hydrogen source. 以水为氢源的炔的电催化半加氢反应。

IF 16 1区生物学

Nature Protocols Pub Date : 2025-08-04 DOI: 10.1038/s41596-025-01230-z

Ying Gao, Meng He, Yongmeng Wu, Bo-Hang Zhao, Cuibo Liu, Bin Zhang

{"title":"Electrocatalytic semi-hydrogenation of alkynes using water as the hydrogen source.","authors":"Ying Gao, Meng He, Yongmeng Wu, Bo-Hang Zhao, Cuibo Liu, Bin Zhang","doi":"10.1038/s41596-025-01230-z","DOIUrl":"10.1038/s41596-025-01230-z","url":null,"abstract":"The semi-hydrogenation of alkynes to alkenes, especially acetylene to ethylene, is an essential transformation that delivers raw materials and scaffolds for synthetic industries. Electrocatalytic hydrogenation, which is green and mild, provides an alternative strategy to the conventional hydrogenation process, which relies on high temperature, high pressure and flammable H2. This protocol describes an electrocatalytic semi-hydrogenation method to synthesize olefins with water as the hydrogen source under ambient temperature and pressure. Electrocatalytic semi-hydrogenation involves the adsorption and activation of alkynes and the cathodic generation of the active hydrogen (H*) intermediate from water dissociation, followed by the addition of H* to an adsorbed alkyne to yield an alkene. This process is generally assisted by Cu-based electrocatalysts (sulfur-modified Cu and Cu nanoparticles) and commercially available reaction vessels and is performed under a direct-current or constant potential power supply. Here we provide detailed procedures for catalyst design synthesis, alkene electrosynthesis and electrochemical in situ/ex situ spectroscopies for investigating reaction mechanisms. The semi-hydrogenation procedure can be performed within hours; it can also be flexibly adapted to synthetic procedures performed in batch or flow reactors and for various reaction times to meet the adjustable capacity requirements for fine or bulk chemicals. Compared with conventional approaches, the electrocatalytic semi-hydrogenation method eliminates the need for expensive and toxic hydrogenation reagents and conditions with elevated temperature and pressure. Our electrocatalytic semi-hydrogenation strategy has various advantages as a sustainable and alternative method to existing methods, including high alkene selectivity, operational simplicity, substrate universality and easily reproducible functional group compatibility.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-08-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144784793","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

Biomass-derived carbon dots for the initiation of conventional radical and ATRP-based photopolymerization processes. 生物质衍生的碳点用于引发传统的自由基和基于atrp的光聚合过程。

IF 16 1区生物学

Nature Protocols Pub Date : 2025-08-04 DOI: 10.1038/s41596-025-01210-3

Xiongfei Luo, Xue Liu, Hongda Guo, Ruiping Li, Min Wang, Xiaotong Li, Shujun Li, Shouxin Liu, Jian Li, Veronika Strehmel, Qunying Wang, Gorkem Yilmaz, Krzysztof Matyjaszewski, Bernd Strehmel, Zhijun Chen

{"title":"Biomass-derived carbon dots for the initiation of conventional radical and ATRP-based photopolymerization processes.","authors":"Xiongfei Luo, Xue Liu, Hongda Guo, Ruiping Li, Min Wang, Xiaotong Li, Shujun Li, Shouxin Liu, Jian Li, Veronika Strehmel, Qunying Wang, Gorkem Yilmaz, Krzysztof Matyjaszewski, Bernd Strehmel, Zhijun Chen","doi":"10.1038/s41596-025-01210-3","DOIUrl":"10.1038/s41596-025-01210-3","url":null,"abstract":"In recent years, there has been increasing interest in using carbon nanodots (CDs) as a component photoinitiator to initiate photopolymerization. These systems support conventional radical photopolymerization and light-mediated atom transfer radical polymerization (photo-ATRP), emphasizing single-component (Type I initiators) and multicomponent systems, which involve at least two reaction partners, specifically, the Type II CD initiator. The latter can function in both photoinduced conventional radical polymerization and photo-ATRP. CDs provide an important advantage by reducing toxicological concerns, as they are nontoxic to cells, and minimizing migration issues typically associated with molecular systems. Here we present two novel photopolymerization methods utilizing biomass-derived CDs as light-sensitive components. The first approach uses biobased furfural to create a Type I CD initiator for photoinduced uncontrolled radical polymerization, which initiates polymerization via homolytic bond cleavage of oxime ester groups attached to the CD surface. The second method employs sodium alginate to generate CDs capable of initiating photoinduced radical polymerization or activating alkyl halides in photo-ATRP processes. Key topics covered in these methods include (1) preparation and characterization of biomass-derived CDs; (2) experimental procedures for CD-assisted photo-induced conventional radical polymerization and photo-ATRP and (3) analysis of the resulting polymers. Preparing and characterizing the CDs takes ~4 d, while photochemical reactions can be conducted within 1 h, depending on requirements. Product separation and analysis take an additional 0.5 h. This protocol is designed for users with experience in polymer chemistry and CD handling.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-08-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144784792","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

Modeling heart rhythm using human engineered heart tissues. 利用人类工程心脏组织建模心律。

IF 16 1区生物学

Nature Protocols Pub Date : 2025-08-01 DOI: 10.1038/s41596-025-01217-w

Chengyi Tu, Arianne Caudal, Yu Liu, Sanjiv M Narayan, Joseph C Wu

{"title":"Modeling heart rhythm using human engineered heart tissues.","authors":"Chengyi Tu, Arianne Caudal, Yu Liu, Sanjiv M Narayan, Joseph C Wu","doi":"10.1038/s41596-025-01217-w","DOIUrl":"https://doi.org/10.1038/s41596-025-01217-w","url":null,"abstract":"Heart rate is both an indicator and modulator of cardiovascular health. Prolonged elevation in heart rate or irregular heart rhythm can trigger the onset of cardiac dysfunction, a condition termed 'tachycardia-induced cardiomyopathy'. While large animals have historically served as the primary model for studying this condition owing to their similar resting heart rates to humans, their use is limited by cost and throughput constraints. We recently developed the first engineered model of tachycardia-induced cardiomyopathy to overcome this technical bottleneck. Our model uses matured human engineered myocardium coupled with programmable electrical stimulation to emulate the pathophysiological changes in human heart rhythm. This in vitro model, capable of acutely and chronically modulating both beating rate and rhythm, recapitulated the clinical hallmarks of tachycardia-induced cardiomyopathy, and its utility was further validated via molecular comparisons against data from a canine model and human patients. Moreover, this model has improved the throughput and relevance to human genetics, enabling deep mechanistic explorations that were previously impossible. Here we present a comprehensive workflow detailing the fabrication and maturation of human engineered heart tissue, assembly of the electrical pacing system, functional analysis using open-source software and preparation for proteomic and transcriptomic analyses. This 5-week Protocol could be implemented by an experienced bench scientist with strong expertise in cell culture, ideally involving stem cell-derived cardiomyocytes. Given the broad implications of heart rhythm alterations in various cardiac conditions, this workflow can be employed with other biophysical and chemical cues to generate more complex and physiologically relevant cardiac models.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144765026","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

Comparative genomics approach to infer ancestral cell karyotypes and reconstruct the evolutionary trajectories of plant chromosomes. 比较基因组学方法推断祖先细胞核型和重建植物染色体的进化轨迹。

IF 16 1区生物学

Nature Protocols Pub Date : 2025-08-01 DOI: 10.1038/s41596-025-01173-5

Xiyin Wang, Bowen Song, Weiwei Liu, Yuelong Jia, Yingjie Li, Tao Wang, Minran Yang, Jiangli Wang, Fubo Hu, Huilong Qi, Huizhe Zhang, Xiaochang Xu, Zhenyi Wang, Yongchao Jin

{"title":"Comparative genomics approach to infer ancestral cell karyotypes and reconstruct the evolutionary trajectories of plant chromosomes.","authors":"Xiyin Wang, Bowen Song, Weiwei Liu, Yuelong Jia, Yingjie Li, Tao Wang, Minran Yang, Jiangli Wang, Fubo Hu, Huilong Qi, Huizhe Zhang, Xiaochang Xu, Zhenyi Wang, Yongchao Jin","doi":"10.1038/s41596-025-01173-5","DOIUrl":"https://doi.org/10.1038/s41596-025-01173-5","url":null,"abstract":"Chromosomes are key structural and functional components of heredity. Reconstruction of ancestral cell karyotypes (ACKs) and evolutionary trajectories of chromosomes (CETs) can reveal how structural and functional changes in chromosomes have occurred during evolution. The whole-genome duplication integrated analysis toolkit implements a telomere-centric model on the basis of the comparative analysis of gene collinearity within and between plant genomes to reconstruct the ACKs and CETs of many angiosperm plants whose genomes have been complicated by repeated polyploidization and subsequent repatterning. Here we summarize the steps for using whole-genome duplication integrated analysis to infer the gene collinearity within a genome or between genomes and to infer the repeated polyploidization specific to a plant or common to multiple plants or plant families. In addition, we provide an example with three grass genomes. We also describe procedures to infer ancestral chromosomes at key evolutionary nodes, to reconstruct CETs from the deep past to extant plants and to generate event-related hierarchical alignment of multiple genomes, which is realized by deciphering collinear genes produced by different polyploidization or speciation events. The Protocol guides users to infer ACKs and CETs in a plant taxon and between selected plants from different taxa, which is crucial to understand important sources of genetic innovation including chromosome evolution, genome complexity and origination and evolution of duplicated genes. This Protocol requires minimal bioinformatics knowledge, for example, retrieving data from public databases and running Python programs. Completing the protocol with the example data takes around 8 h.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144765025","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

Bioink design for organ-scale projection-based 3D bioprinting. 基于器官投影的3D生物打印的生物链接设计。

IF 16 1区生物学

Nature Protocols Pub Date : 2025-07-30 DOI: 10.1038/s41596-025-01221-0

Tianhong Qiao, Chaofan He, Pengcheng Xia, Guofeng Liu, Yuan Sun, Miao Sun, Yi Wang, Yiyu Cheng, Mengfei Yu, Yong He

{"title":"Bioink design for organ-scale projection-based 3D bioprinting.","authors":"Tianhong Qiao, Chaofan He, Pengcheng Xia, Guofeng Liu, Yuan Sun, Miao Sun, Yi Wang, Yiyu Cheng, Mengfei Yu, Yong He","doi":"10.1038/s41596-025-01221-0","DOIUrl":"https://doi.org/10.1038/s41596-025-01221-0","url":null,"abstract":"Projection-based three-dimensional bioprinting offers an approach for manufacturing biomimetic tissues with complex spatial structures and bioactivity, presenting potential for creating implantable organs or organoids to test drug response. Nevertheless, the extended printing times required for organ-scale manufacturing represents a challenge. Here we provide step-by-step instructions to manufacture organ-scale structures using bioinks while preserving high bioactivity. This approach incorporates Ficoll 400 to mitigate the heterogeneity of bioink with respect to refractive index and density, while 4-(2-aminoethyl)benzenesulfonyl fluoride and oil-sealing ensure the stability of the bioink components, thereby allowing extended printing times. This procedure also enables high-cell-viability printing via the calibration of the pH value of the bioink. This Protocol is appropriate for users with basic laboratory skills and fundamental knowledge in biotechnology to manufacture organ-scale structures for utilization in a wide variety of experimental designs. The approach is generalizable, as demonstrated by the successful printing of corpora cavernosa structures with a cell density of 10 million per milliliter, measuring 10 mm × 10 mm × 10 mm. After 7 d of culture, the cell viability was measured at 82.5%, highlighting the potential applicability in tissue engineering. All bioink preparation and printing steps are expected to take 5 h, while the development of printed structures requires 7 d of continuous culture.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-07-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144753876","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

Manufacturing of CRISPR-edited primary mouse CAR T cells for cancer immunotherapy. 用于癌症免疫治疗的crispr编辑原代小鼠CAR - T细胞的制造。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-07-25 DOI: 10.1038/s41596-025-01208-x

Puneeth Guruprasad, Ranjani Ramasubramanian, Siena Nason, Alberto Carturan, Shan Liu, Luca Paruzzo, Vladlena Hornet, Jacqueline Plesset, Ruchi P Patel, Vijay Bhoj, Gregory L Beatty, Marco Ruella

{"title":"Manufacturing of CRISPR-edited primary mouse CAR T cells for cancer immunotherapy.","authors":"Puneeth Guruprasad, Ranjani Ramasubramanian, Siena Nason, Alberto Carturan, Shan Liu, Luca Paruzzo, Vladlena Hornet, Jacqueline Plesset, Ruchi P Patel, Vijay Bhoj, Gregory L Beatty, Marco Ruella","doi":"10.1038/s41596-025-01208-x","DOIUrl":"https://doi.org/10.1038/s41596-025-01208-x","url":null,"abstract":"Editing chimeric antigen receptor (CAR) T cells by using CRISPR-Cas9 has become a routine strategy to improve their antitumor function or safety profile. Xenograft tumor models in immunodeficient mice are often used to evaluate the function of CRISPR-edited human CAR T cells. These models, however, lack functional immune systems and thus fail to recapitulate barriers such as the immunosuppressive tumor microenvironment (TME) that CAR T cells will encounter in patients. Thus, genetically modifying mouse CAR T cells for use in immune-intact models is an attractive approach to explore the impact of a given gene deletion on CAR T cells within a natural TME. Here, we describe a protocol to perform CRISPR-Cas9 editing in primary mouse T cells, thereby enabling studies of gene-edited CAR T within the TME and in the presence of a functional immune system. This protocol is integrated into a standard mouse CAR T manufacturing workflow, a process that typically spans ~5-6 days. The first stage of this protocol involves isolating mouse T cells, electroporating them with a ribonucleoprotein complex and activating them by using magnetic bead stimulation. The second stage involves transducing the CAR gene and expanding these cells, and the third stage focuses on validating knockout efficiency and the functionality of gene-edited mouse CAR T cells. This procedure requires a proficiency in aseptic cell culture techniques and a basic understanding of T cell biology. We anticipate that efficient and reliable genetic modification of mouse T cells will have wide-ranging applications for cancer immunotherapies and related fields.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144718212","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

Expansion of human hepatocytes and their application in three-dimensional culture and genetic manipulation. 人肝细胞的扩增及其在三维培养和基因操作中的应用。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-07-25 DOI: 10.1038/s41596-025-01211-2

Kun Zhang, Xiang Yuan, Shuang Lu, Yajing Shu, Chenhua Wang, Jin Cen, Baihua Wu, Lijian Hui

{"title":"Expansion of human hepatocytes and their application in three-dimensional culture and genetic manipulation.","authors":"Kun Zhang, Xiang Yuan, Shuang Lu, Yajing Shu, Chenhua Wang, Jin Cen, Baihua Wu, Lijian Hui","doi":"10.1038/s41596-025-01211-2","DOIUrl":"https://doi.org/10.1038/s41596-025-01211-2","url":null,"abstract":"Hepatocytes are one of the most important cell types in the liver, carrying out key functions. They are essential for hepatocyte-based therapy, disease modeling and drug development. However, the availability of primary human hepatocytes (PHHs) is limited by a shortage of donors. It is therefore of great value to expand PHHs in large quantities. Here we provide a detailed protocol for the large-scale expansion of PHHs (proliferating human hepatocytes, ProliHHs) derived from healthy donors and patients with inherited liver diseases, which can be rematured in a three-dimensional culture system. Moreover, we provide a protocol for the genetic manipulation of ProliHHs, including lentivirus transduction and CRISPR-Cas9-mediated knockout and knock-in. The protocol described here will help to realize the full potential of ProliHH-based therapy, organoid-based liver disease modeling and drug screening. The protocol to expand PHHs takes ~1-2 months, the protocol to establish the 3D-cultured ProliHHs takes ~8 d and the protocol to perform gene editing takes ~3 d. Personnel with basic scientific training can conduct these protocols.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144718211","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

Printed photonic crystal biochips for rapid and sensitive detection of biomarkers in various body fluids. 印刷光子晶体生物芯片，用于快速和灵敏地检测各种体液中的生物标志物。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-07-24 DOI: 10.1038/s41596-025-01215-y

Zewei Lian, Jimei Chi, Xu Yang, Lijun Cheng, Daixi Xie, Zhiyu Tan, Sisi Chen, Yang Yun, Yisilamu Yibulayimu, Wei Wu, Yanlin Song, Meng Su

{"title":"Printed photonic crystal biochips for rapid and sensitive detection of biomarkers in various body fluids.","authors":"Zewei Lian, Jimei Chi, Xu Yang, Lijun Cheng, Daixi Xie, Zhiyu Tan, Sisi Chen, Yang Yun, Yisilamu Yibulayimu, Wei Wu, Yanlin Song, Meng Su","doi":"10.1038/s41596-025-01215-y","DOIUrl":"https://doi.org/10.1038/s41596-025-01215-y","url":null,"abstract":"There is an urgent requirement to improve accessibility to diagnostic tools in remote areas. This requires assays that are easy to use, are cost-effective and produce rapid results. Important public health applications include early disease diagnosis, real-time monitoring, epidemic control and medical cost control. This protocol describes the fabrication of all-printed photonic crystal (PC) biochips for point-of-care testing of biomarkers. The photonic crystal material is prepared by the self-assembly of latex nanospheres that are printed onto a polyethylene terephthalate substrate. Photonic crystals composed of latex nanospheres of different sizes enhance the fluorescent signal emitted at different wavelengths, resulting in remarkably higher detection sensitivity. PC microarrays enable mass-printed preparation (up to 2,700 pieces can be printed per hour by one printer), and each microarray can be stored for a long time (>6 months) after heating. Biomarker specificity is achieved by the biofunctionalization of the nanospheres, for example, attaching capture antibodies. The detection involves the use of a fluorescently labeled detection antibody and a simple point-of-care detection device. This universal approach can be applied to the detection of many biomarkers, and the simultaneous detection of multiple biomarkers is also possible. Here we demonstrate describing how to prepare a chip that can be used to detect three inflammatory biomarkers in 10 min at low sample volumes at a cost of less than 3 CNY (~US$0.41) per PC codetection biochip. The biofunctionalization process including capture antibody coupling and blocking takes 3-4 h, and the detection process takes 20 min.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-07-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144708145","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