Nature Protocols最新文献

{"title":"An assay to quantify sexual commitment and stage conversion in the human malaria parasite Plasmodium falciparum","authors":"Kannan Venugopal, Eveline D. A. Borbon, Basil T. Thommen, Nicolas M. B. Brancucci, Lorena Coronado, Matthias Marti","doi":"10.1038/s41596-026-01378-2","DOIUrl":"10.1038/s41596-026-01378-2","url":null,"abstract":"Sexual commitment and stage conversion are two crucial steps in the blood stage cycle of the human malaria parasite, as they mark its initial decision and subsequent differentiation in preparation for transmission to the mosquito vector. Here we present a Protocol Update that allows tracking and quantification of sexual commitment (in the asexual replicative parent, schizont stage) and conversion (in the nonreplicative sexual progeny, gametocyte stage) rates in the human malaria parasite Plasmodium falciparum. For this purpose, we combine three critical tools we recently established and validated. First, we use a standardized minimal medium to efficiently and reproducibly increase sexual commitment. Second, we use Tubulin Tracker Deep Red to label developing gametocytes and quantify the sexual conversion rate in reporter and reporter-free lines. Third, we use a fluorescent transgenic reporter line for consecutive quantification of sexual commitment and stage conversion in the same experimental procedure. These tools enable multiple experimental readouts including flow cytometry, fixed and live cell microscopy (including high content imaging) and other applications such as fluorescence-activated cell sorting. We anticipate that the presented sexual commitment–conversion assay will enable novel screening approaches for the identification of transmission blocking antimalarials and critical knowledge gaps in the Plasmodium transmission cycle to be closed. The Protocol can be completed in 7 d from start to finish. This Protocol Update uses new transgenic reporter parasite lines and synchronized gametocyte production by shifting cultures to a defined medium. Tubulin Tracker Deep Red in combination with the reporter line enables the quantification of both sexual commitment and conversion in the same experiment.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"21 10","pages":"4431-4446"},"PeriodicalIF":18.4,"publicationDate":"2026-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148376285","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":"Author Correction: Direct inoculation of bioreactor-controlled stirred suspension culture with cryopreserved human pluripotent stem cells.","authors":"Kevin Cyrys, Felix Manstein, Wiebke Triebert, Nils Kriedemann, Carlos Alberto Hernandez-Bautista, Robert Zweigerdt","doi":"10.1038/s41596-026-01413-2","DOIUrl":"https://doi.org/10.1038/s41596-026-01413-2","url":null,"abstract":"","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":18.4,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148369340","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":"High-throughput measurements of protein domain functions using magnetic separation.","authors":"Abby R Thurm, Josh Tycko, Connor H Ludwig, Mark Ragheb, Nicole DelRosso, Gaelen T Hess, Michael C Bassik, Lacramioara Bintu","doi":"10.1038/s41596-026-01397-z","DOIUrl":"10.1038/s41596-026-01397-z","url":null,"abstract":"<p><p>High-throughput screening of protein domains enables the systematic discovery of protein sequences that encode specific cellular functions. Fluorescence-activated cell sorting-based assays have long been the standard readout for such screens but remain time- and resource-intensive, imposing practical limits on library size and coverage. Here we describe a scalable magnetic separation-based workflow that provides an alternative to fluorescence-activated cell sorting for screening large protein libraries in mammalian cells. We engineered a modular synthetic surface marker, consisting of a fusion between the fragment crystallizable (Fc) region of human immunoglobulin G and the transmembrane domain of platelet-derived growth factor receptor-β, that allows cells to be magnetically separated on the basis of surface reporter expression using Protein G-coated magnetic beads. The procedure covers pooled library cloning, lentiviral delivery, magnetic separation and sequencing-based quantification, enabling reproducible screening of more than 100,000 protein domain variants. The approach is suitable for the identification of functional protein domains capable of transcriptional and post-transcriptional RNA regulation and may lead to the selection of improved transmembrane domains for efficient protein surface display. The entire workflow, from library design to data analysis, can be completed in 4-6 weeks and requires skills in cell culture, molecular cloning and computational techniques. This scalable and accessible Protocol enables researchers to systematically measure protein domain functions across biological contexts, thus accelerating both biological discovery and protein engineering.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":18.4,"publicationDate":"2026-06-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148361697","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":"Inducing physiological polarity and performing gene editing using CRISPR-Cas9 in human trophoblast organoids.","authors":"Liheng Yang, Joshua Hatterschide, Rizban E Worota, Kaila Cooley, Carolyn B Coyne","doi":"10.1038/s41596-026-01402-5","DOIUrl":"https://doi.org/10.1038/s41596-026-01402-5","url":null,"abstract":"<p><p>Human trophoblast organoids (TOs) are three-dimensional ex vivo culture models that enable the study of placental development, physiology and pathology. A major limitation of TOs grown in Matrigel or other extracellular matrices is their apical-inward polarity, in which cytotrophoblasts (CTBs) line the outer surface and the multinucleated syncytiotrophoblast (STB) forms the interior layer, opposite to their orientation in vivo. Here we present a detailed protocol to reverse TO polarity, producing organoids that recapitulate the cellular orientation of human chorionic villi. Standard TOs with inward-facing STBs (STBⁱⁿ) undergo polarity reversal during suspension culture to generate outward-facing STBs (STB^out). In parallel, we describe a complementary protocol for CRISPR-Cas9-mediated gene editing in TOs and illustrate its application in generating CGA (hCG) knockout organoids, which disrupt placental hormone secretion, and MAVS knockouts, which impair antiviral signaling. The outward-facing STB protocol can be completed in ~2 weeks, whereas the establishment of stable gene-edited TO lines requires 2-3 months. Successful implementation requires experience in TO culture, lentiviral transduction and CRISPR-Cas9-based genome editing. Together, these protocols provide versatile and reproducible methods for modeling placental architecture and studying gene function in vitro, enabling functional interrogation of trophoblast biology within physiologically oriented organoids.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":18.4,"publicationDate":"2026-06-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148352524","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":"Photocatalytic low-temperature defluorination of PTFE.","authors":"Yi-Ming Chen, Shu-Hui Lei, Yu Liu, Hu Zhang, Jia-Le Fu, Zhi-Ao Tian, Yong-Ze Chen, Bo Yang, Bai-Chuan Ding, Yu-Fan Zou, Hao Zhang, Jian-Ping Qu, Yan-Biao Kang","doi":"10.1038/s41596-026-01390-6","DOIUrl":"https://doi.org/10.1038/s41596-026-01390-6","url":null,"abstract":"<p><p>Polytetrafluoroethylene (PTFE) is widely used owing to its exceptional chemical and thermal stability; however, these same properties render it highly persistent in the environment, contributing to long-term fluorinated waste. Conventional degradation methods, such as pyrolysis above 500 °C or treatment with alkali metals, are energy-intensive and operationally demanding, highlighting the need for mild and practical defluorination strategies for sustainable fluoropolymer recycling. Here we report a visible-light-driven photocatalytic protocol for PTFE defluorination under mild conditions. The method uses carbazole-based photocatalysts (CBZ6 and KQGZ), readily prepared in two to three steps from commercial precursors, and proceeds in standard glassware under purple light-emitting diode irradiation. Finely divided PTFE powder (~1-μm particle size, 20-mg scale) is treated with a photocatalyst, base and sacrificial reductant, with dispersion maintained by intermittent sonication. Critical parameters influencing reactivity include photocatalyst identity and loading, dispersion efficiency, base purity and physical form, light source configuration, temperature control and irradiation time. The reaction yields soluble fluoride salts and a defluorinated amorphous carbon, along with carbon-derived oxyanions. Fluoride ions are quantified by ¹⁹F NMR spectroscopy or ion chromatography and can be recovered as potassium fluoride by recrystallization. The amorphous carbon is characterized primarily by Raman spectroscopy, supported by X-ray photoelectron spectroscopy, solid-state NMR and IR spectroscopy. The protocol is straightforward and accessible to researchers with standard training in organic synthesis, including graduate students and experienced laboratory technicians. The full protocol, encompassing photocatalyst preparation, PTFE defluorination and product characterization, can be completed in 189 h (59 h hands-on time).</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":18.4,"publicationDate":"2026-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148331398","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":"Yeast nuclei-mediated precise delivery of synthetic megabase-scale human DNA into mammalian embryos.","authors":"Yue Liu, Jianting Zhou, Zichuan Liu, Ying-Jin Yuan","doi":"10.1038/s41596-026-01374-6","DOIUrl":"https://doi.org/10.1038/s41596-026-01374-6","url":null,"abstract":"<p><p>The cross-species delivery of megabase-scale synthetic DNA molecules, from microorganisms into mammalian cells, remains a major challenge for synthetic genomics. Recently, we developed nucleus isolation for chromosome extraction (NICE), a method that enables the isolation of yeast nuclei containing intact synthetic megabase-scale DNA with preserved chromatin structure. By leveraging the unique epigenomic features of Saccharomyces cerevisiae, which lacks cytosine methylation and repressive histone marks, synthetic DNA encapsulated within isolated yeast nuclei was successfully delivered into mouse early embryos, maintaining a naive state. This work established a unique platform for studying the establishment of de novo epigenetic modifications and their influence on transcriptional regulation over time. Here, we provide a detailed protocol for NICE, including the isolation of yeast nuclei and their subsequent delivery into mammalian embryos. The high-concentration and high-purity isolated nuclei can be stored at -80 °C for >6 months. Using microinjection, we achieved 100% delivery efficiency, reliably transferring isolated yeast nuclei into mouse embryos. The entire procedure, including pulsed-field gel electrophoresis verification, can be completed within ~5 d. When the isolated yeast nuclei are intended for cross-species delivery into embryos, prior familiarity with mammalian embryo microinjection techniques may be required. This protocol offers an efficient and reliable method for the delivery of large-scale genetic information, advancing the study of complex biological functions.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":18.4,"publicationDate":"2026-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148318133","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":"Facilitating structure-based drug discovery with an artificial intelligence-driven virtual screening platform.","authors":"Shukai Gu, Xujun Zhang, Mengwu Xiao, Yuntao Qian, Bo Liu, Hao Luo, Hongyan Du, Odin Zhang, Minjie Mou, Tingting Fu, Xiaorui Wang, Jingxuan Ge, Chao Shen, Feng Zhu, Xiaojun Yao, Huanxiang Liu, Tingjun Hou, Yu Kang","doi":"10.1038/s41596-026-01389-z","DOIUrl":"https://doi.org/10.1038/s41596-026-01389-z","url":null,"abstract":"<p><p>Structure-based virtual screening (VS) via molecular docking is a pivotal approach for hit identification. Many artificial intelligence (AI)-powered protein-ligand docking and scoring methods have demonstrated impressive speed and accuracy. Retrospective benchmarking studies using enrichment rate and computational efficiency on curated datasets have corroborated their potential for discovering bioactive compounds. However, determining which method suits a specific application and implementing it efficiently remains challenging. Here we present the Comprehensive VS Platform with AI Engine (CVSP-AIE) for drug discovery from compound libraries. It integrates three AI models: KarmaDock, a fast docking model that directly updates atomic coordinates; CarsiDock, an accurate docking model that predicts protein-ligand distances and reconstructs binding poses; and RTMScore, an accurate scoring model that learns residue-atom distance distributions for affinity prediction. Their hierarchical application enables dynamical balances in screening speed and accuracy. CVSP-AIE is available as an online web server ( https://cadd.zju.edu.cn/cvsp/ ) and a local software package. Users can efficiently initiate drug screening by uploading a protein and a known binder that defines the binding pocket. The following workflow involves (1) preprocessing, including protein structure repair and molecule standardization, (2) binding pose and affinity prediction powered by KarmaDock, CarsiDock and RTMScore and (3) postprocessing, comprising protein-ligand interaction calculation and visualization. It takes 30-45 min to hierarchically screen 100,000 compounds, and the output is a ranked list of molecules with predicted binding scores, intermolecular interaction profiles and interactive chemical space analysis. Users can also install locally the hierarchical screening module through command-line package for arbitrary-scale screening.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":18.4,"publicationDate":"2026-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148318154","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":"Multimodal imaging and quantification of lanthanide chelate-labeled micro- and nanoplastics in plants.","authors":"Chen Tu, Lianzhen Li, Jie Yang, Ruijie Li, Yudong Feng, Yongming Luo","doi":"10.1038/s41596-026-01392-4","DOIUrl":"https://doi.org/10.1038/s41596-026-01392-4","url":null,"abstract":"<p><p>Understanding the uptake and distribution of microplastics (MPs) and nanoplastics (NPs) in plants is crucial for assessing their environmental impact. This can be effectively achieved using labeled model particles. While conventional fluorophores are limited by plant autofluorescence, photobleaching, and poor quantitative resolution, in our work we incorporate stable lanthanide (europium) chelates, enabling long-lifetime luminescence detection that effectively eliminates this background interference. Here this protocol integrates time-gated fluorescence imaging microscopy, electron microscopy coupled with energy-dispersive X-ray spectroscopy, and inductively coupled plasma mass spectrometry. The solvent swelling labeling method is exemplified using MPs/NPs with different chemical compositions and shapes. For comparison, procedures using MPs/NPs labeled with conventional fluorescent dyes, such as Nile blue chloride (red fluorescence) and the amine-reactive probe 4-chloro-7-nitro-2,1,3-benzoxadiazole (for green fluorescence) are included. The workflow follows a tiered approach: time-gated fluorescence imaging microscopy rapidly localizes particles within tissues, electron microscopy coupled with energy-dispersive X-ray spectroscopy provides subcellular verification and elemental confirmation, and inductively coupled plasma mass spectrometry quantifies total particle accumulation with high sensitivity. This provides a robust framework for mechanistic studies of MPs/NPs uptake, translocation and partitioning from the subcellular to the whole-plant level. The protocol requires moderate expertise in microscopy and analytical chemistry and takes 2-4 months to complete, depending on the plant species (procedures for hydroponic and soil-grown wheat and lettuce are provided). It is designed specifically for hypothesis-driven research using well-defined, labeled model particles and is not intended for the environmental monitoring of unlabeled plastics in field samples.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":18.4,"publicationDate":"2026-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148318127","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":"Direct inoculation of bioreactor-controlled stirred suspension culture with cryopreserved human pluripotent stem cells.","authors":"Kevin Cyrys, Felix Manstein, Wiebke Triebert, Nils Kriedemann, Carlos Alberto Hernandez-Bautista, Robert Zweigerdt","doi":"10.1038/s41596-026-01398-y","DOIUrl":"10.1038/s41596-026-01398-y","url":null,"abstract":"<p><p>The routine mass applications of human pluripotent stem (hPS) cell-derived progenies for regenerative medicine or high-throughput drug screenings will depend on the standardized supply of high-quality cells via controlled, efficient bioprocesses. Recent suspension culture (three-dimensional, 3D) strategies for hPS cell production in stirred-tank bioreactors (STBR) support this development. However, bioprocess inoculation still depends on adherent (two-dimensional, 2D) preculture, which is labor intensive, resource demanding and poorly controlled and limits process automation. Here we describe the controlled in-process production and dissociation of 3D cultured hPS cell aggregates directly in STBRs, tackling these challenges. The resulting cells can be used for the generation of high-density cryostocks, subsequently enabling the direct inoculation of 3D cultures, thereby entirely omitting the need for 2D preculture and the associated limitations. A key feature of this Protocol is the nonenzymatic, EDTA-based hPS cell aggregate dissociation approach in STBRs, enabling the impeller-based mechanical control of the dissociation process. The resulting cell suspension can be used for process reinoculation and seed train-based upscaling, as well as for the cryopreservation of produced hPS cells, ideally via controlled-rate freezing. Together, the protocol enables the efficient and flexible hPS cell suspension culture over multiple passages, maintaining karyotype stability and pluripotency. This Protocol can be easily implemented by any cell culture-educated scientist without extensive bioprocess training. Cell thawing requires 1 h, the 2D preculture requires 9 days, 2D cell passaging requires 1 h, bioreactor preparation requires 2 days, (direct) bioreactor inoculation requires 1.5 h, 3D STBR cultivation requires 3-4 days and STBR-based aggregate dissociation requires 2 h.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":18.4,"publicationDate":"2026-06-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148302155","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":"Solid-phase synthesis of sterically hindered peptides via ribosome-mimicking molecular reactors.","authors":"Siyuan Wei, Xuchun Zhang, Yishan Guo, Fa Liu, Zhu-Jun Yao","doi":"10.1038/s41596-026-01383-5","DOIUrl":"https://doi.org/10.1038/s41596-026-01383-5","url":null,"abstract":"<p><p>The incorporation of N-methylated or α,α-disubstituted amino acids into peptides can markedly enhance their drug-like properties by improving proteolytic stability and membrane permeability. However, the solid-phase synthesis of such sterically hindered peptides remains a persistent challenge. Here we present a detailed protocol based on our recently developed ribosome-mimicking molecular reactor (RMMR) strategy, which substantially improves the synthetic efficiency of these difficult sequences via solid phase peptide synthesis (SPPS). This protocol provides a comprehensive, step-by-step procedure to facilitate a quick startup by the users, including the synthesis of Oxyma-C as the key activating unit precursor in the RMMR structure, the preparation of RMMR resin through modification of conventional SPPS resin and implementation of RMMR-SPPS in both manual and automated synthesizer formats. Peptides containing N-methylated and/or α,α-disubstituted amino acid(s) are synthesized efficiently using our strategy, with crude purities of up to 98% and respectable isolated yields, greatly surpassing existing methods. It is anticipated that this approach will facilitate broader adoption of RMMR-SPPS, addressing critical technical gaps and accelerating the discovery of orally bioavailable or intracellularly targeted macrocyclic peptide drug candidates.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":18.4,"publicationDate":"2026-06-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148272457","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}