Nature Protocols最新文献

{"title":"Absolute quantification of prokaryotes in the microbiome by 16S rRNA qPCR or ddPCR.","authors":"Boryana Doyle, Gabriella Z M Reynolds, Mai Dvorak, Dylan G Maghini, Aravind Natarajan, Ami S Bhatt","doi":"10.1038/s41596-025-01165-5","DOIUrl":"https://doi.org/10.1038/s41596-025-01165-5","url":null,"abstract":"<p><p>Measurements of prokaryotic absolute abundance can provide important insights into human gut microbiome biology and correct misinterpretations of relative abundance data. Despite the existence of several relatively well-established methods for making these measurements, most microbiome studies do not report absolute abundance. To enable researchers equipped with standard molecular biology capabilities to incorporate absolute quantification into their microbiome studies, we present a detailed, step-by-step protocol for rigorous and reproducible quantification of prokaryotic concentration in stool samples. We include methods for measuring stool sample moisture content, quantifying the concentration of the 16S rRNA prokaryotic marker gene by qPCR or digital droplet PCR (ddPCR) and analyzing the resulting data. We also highlight and provide strategies to overcome common pitfalls of the quantification method, such as 16S rRNA gene contamination. The final output of this approach is 16S rRNA copies per wet or dry gram of stool. In cases where samples have matched metagenomic sequencing information, data can be converted into absolute concentration of prokaryotes and taxon-specific absolute concentrations. To enable researchers to choose the appropriate method for their specific applications, we also compare and contrast our qPCR and ddPCR methods. In 4 days, ~80 samples can be taken from frozen stool to absolute concentration by using qPCR or ddPCR without the need for resequencing. Overall, this protocol provides a sensitive and straightforward way to measure the absolute concentration of prokaryotes in human gut microbiome samples stored with or without preservative.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-05-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144102220","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":"An injectable conductive hydrogel for closed-loop and robot-assisted rehabilitation via stretchable patch-type electrodes.","authors":"Subin Jin, Heewon Choi, Donghee Son, Mikyung Shin","doi":"10.1038/s41596-025-01184-2","DOIUrl":"https://doi.org/10.1038/s41596-025-01184-2","url":null,"abstract":"<p><p>Conventional therapies for severe musculoskeletal and neurological injuries require lengthy recovery periods, which may result in residual disabilities. As an innovative rehabilitation approach, the combination of soft conducting hydrogels as an injectable tissue prosthesis with self-healing, stretchable bioelectronic devices offers a promising solution to expedite tissue repair and enhance functional restoration. This class of tissue prostheses can help address the critical limitations of traditional materials and devices by providing a minimally invasive approach to filling tissue defects and reconstructing the electrophysiological environment. The integration of an injectable tissue prosthesis with exoskeleton robotics in closed-loop systems enables tailored rehabilitation interventions that optimize motor function efficiency. Here we provide the step-by-step instructions for the development and characterization of injectable tissue-interfacing conductive hydrogels and soft self-healing, stretchable bioelectronic devices. We also describe how to establish a fully integrated closed-loop rehabilitation system and show its efficacy in a rat model of volumetric muscle loss. Using this approach, we have achieved accelerated tissue regeneration and improved myofiber regeneration in rats, underscoring the potential of this approach to improve rehabilitation strategies for severe injuries. The protocol is suitable for users with experience in biomaterials, devices and animal handling and requires 30 d to complete.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-05-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144102226","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":"Tutorial: guidelines for the use of machine learning methods to mine genomes and proteomes for antibiotic discovery","authors":"Fangping Wan,&nbsp;Marcelo D. T. Torres,&nbsp;Changge Guan,&nbsp;Cesar de la Fuente-Nunez","doi":"10.1038/s41596-025-01144-w","DOIUrl":"10.1038/s41596-025-01144-w","url":null,"abstract":"Genomes and proteomes constitute a rich reservoir of molecular diversity. However, they have remained underexplored because of a lack of appropriate tools. In recent years, computational approaches have been developed to mine this unexplored biological information, or dark matter, accelerating the discovery of new antibiotic molecules. Such efforts have yielded a wide range of new molecules. These include peptides released via predicted proteolytic cleavage of larger proteins, termed ‘encrypted peptides’, which have been found to be widespread in nature. Molecules encoded by and translated from small open reading frames within genomic sequences have also been uncovered, further expanding the landscape of bioactive compounds. Here, we discuss computational approaches, including machine learning and artificial intelligence (AI) tools, which have been used to date to identify antimicrobial compounds, with a special emphasis on peptides. We also propose potential avenues for future exploration in this rapidly evolving field. Moreover, we provide an overview of the experimental methods commonly used to validate these computational predictions. We anticipate that efforts combining cutting-edge AI and experimental approaches for biological sequence mining will reveal new insights into host immunity and continue to accelerate discoveries in the fields of antibiotics and infectious diseases. In this Tutorial, we provide guidelines for the use of ML models to mine genomes and proteomes for the discovery of antimicrobial peptides, discussing the various stages of the ML workflow, recent advances in ML models and how to experimentally validate the computational predictions.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"20 10","pages":"2685-2697"},"PeriodicalIF":16.0,"publicationDate":"2025-05-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144079246","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":"Achieving transient and reversible optical transparency in live mice with tartrazine.","authors":"Carl H C Keck, Elizabeth Lea Schmidt, Su Zhao, Zhongyu Liu, Ling-Yi Zhang, Miao Cui, Xiaoyu Chen, Chonghe Wang, Han Cui, Mark L Brongersma, Guosong Hong","doi":"10.1038/s41596-025-01187-z","DOIUrl":"https://doi.org/10.1038/s41596-025-01187-z","url":null,"abstract":"<p><p>Optical imaging provides real-time visualization of tissues and cells at high spatial and temporal resolutions through techniques such as fluorescence microscopy, optical coherence tomography and photoacoustic imaging. However, overcoming light scattering, caused by mismatches in the refractive indices of tissue components such as water and lipids, still represents a major challenge, particularly when imaging through the thicker biological tissues of living animals. Despite advances in deep-tissue imaging, many optical methods struggle to achieve diffraction-limited resolution at depth or are unsuitable for use in live animals. Here we introduce a noninvasive approach to achieving transient and reversible optical transparency in live mice using absorbing dye molecules, using tartrazine as a representative example. Rooted in the fundamental physics of light-matter interactions, this approach enables reversible optical transparency in live animals and can be further applied ex vivo in freshly dissected tissues. In this Protocol, we detail the procedures for visualizing in vivo internal organs and muscle sarcomeres in the mouse abdomen and hindlimb through their respective transparency windows, showcasing a versatile approach for a variety of optical imaging applications in live animals. The entire protocol for an in vivo application can be implemented in just over 2 weeks by users with expertise in optical imaging and animal handling.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-05-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144034511","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":"Simple large-scale quantitative phenotyping and antimicrobial susceptibility testing with Q-PHAST.","authors":"Juan Carlos Nunez-Rodriguez, Miquel Àngel Schikora-Tamarit, Ewa Ksiezopolska, Toni Gabaldón","doi":"10.1038/s41596-025-01179-z","DOIUrl":"https://doi.org/10.1038/s41596-025-01179-z","url":null,"abstract":"<p><p>The characterization of antimicrobial susceptibility and other relevant phenotypes in large collections of microbial isolates is a common need across research and clinical microbiology laboratories. Robotization provides unprecedented throughput but involves costs that are prohibitive for the average laboratory. Here, using affordable materials and open-source software, we developed Q-PHAST (Quantitative PHenotyping and Antimicrobial Susceptibility Testing), a unique solution for cost-effective, large-scale phenotyping in a standard microbiology laboratory. Single colonies are grown in a deep 96-well master plate, from which diluted aliquots are used to generate 96 spots on different experimental plates containing solid medium with the substance and concentration of interest. These plates are incubated on inexpensive flatbed scanners that monitor the growth of each spot by obtaining images every 15 min. A simple, python-based software, which can be used via a graphical interface on various operating systems ( https://github.com/Gabaldonlab/Q-PHAST ), analyzes the images to infer growth, fitness (e.g., doubling rate) and susceptibility (e.g., minimum inhibitory concentration) measures. With <120 min of hands-on time per day for three consecutive days, ready-to-use results are obtained and presented in tables or graphs. This solution enables non-experts with limited resources to perform accurate quantitative phenotyping on hundreds of strains in parallel.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-05-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144028857","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":"Establishment of human expanded potential stem cell lines via preimplantation embryo cultivation and somatic cell reprogramming","authors":"Degong Ruan,&nbsp;Andy Chun Hang Chen,&nbsp;Timothy Theodore Ka Ki Tam,&nbsp;Wen Huang,&nbsp;Jilong Guo,&nbsp;Shao Xu,&nbsp;Hanzhang Ruan,&nbsp;Sze Wan Fong,&nbsp;Xueyan Liu,&nbsp;Xuefei Gao,&nbsp;William Shu Biu Yeung,&nbsp;Yin Lau Lee,&nbsp;Pentao Liu","doi":"10.1038/s41596-025-01168-2","DOIUrl":"10.1038/s41596-025-01168-2","url":null,"abstract":"We previously reported the derivation of expanded potential stem cells (EPSCs) by modulating signaling pathways involved in preimplantation embryogenesis. These cells exhibit expanded developmental potential into embryonic and extraembryonic lineages, and we have shown that human EPSCs (hEPSCs) possess trophoblast differentiation potency for generating human trophoblast stem cells. Here we report protocols for deriving stable hEPSC lines directly from morula or early blastocyst stages of human preimplantation embryos (hEPSC-em) and by reprogramming human dermal fibroblasts (human induced EPSCs) using six exogenous factors, as an extension to our previous protocols on deriving porcine EPSCs from preimplantation embryos and by reprogramming somatic cells. These hEPSC lines proliferate robustly over long-term passaging and are amenable to both simple indels and precision genome editing. We provide guidance for characterizing these newly established hEPSCs, including cell-cycle analysis, pluripotency validation and karyotyping. The hEPSCs form teratomas with embryonic and extraembryonic cell lineages and readily differentiate into human trophoblast stem cells in vitro. At the molecular level, hEPSCs have unique features such as high expression of core histone genes and low H3K27me3 levels resembling eight-cell/morula stage embryos. These properties make hEPSCs a valuable tool not only for studying early human development but also for potential applications in regenerative medicine. The protocols presented in this manuscript can be readily performed by postgraduate students or postdoctoral fellows and completed within around 2 months. This is a Protocol extension describing the establishment of human expanded potential stem cell lines from preimplantation embryos or by reprogramming somatic cells and their validation and characterization.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"20 10","pages":"2698-2734"},"PeriodicalIF":16.0,"publicationDate":"2025-04-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144044911","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":"Thioether-mediated protein ubiquitination in constructing affinity- and activity-based ubiquitinated protein probes.","authors":"Gregory A Davidson, Zeinab Moafian, Amanda R Sensi, Zhihao Zhuang","doi":"10.1038/s41596-025-01162-8","DOIUrl":"https://doi.org/10.1038/s41596-025-01162-8","url":null,"abstract":"<p><p>Protein ubiquitination, a critical regulatory mechanism and post-translational modification in eukaryotic cells, involves the formation of an isopeptide bond between ubiquitin (Ub) and targeted proteins. Despite extensive investigation into the roles played by protein ubiquitination in various cellular processes, many questions remain to be answered. A major challenge in the biochemical and biophysical characterization of protein ubiquitination, along with its associated pathways and protein players, lies in the generation of ubiquitinated proteins, either in mono- or poly-ubiquitinated forms. Enzymatic and chemical strategies have been reported to address this challenge; however, there are still unmet needs for the facile generation of ubiquitinated proteins in the quantity and homogeneity required to precisely decipher the role of various protein-specific ubiquitination events. In this protocol, we provide the ubiquitin research community with a chemical ubiquitination method enabled by an α-bromoketone-mediated ligation strategy. This method can be readily adapted to generate mono- and poly-ubiquitinated proteins of interest through a cysteine introduced to replace the target lysine, with the native cysteines mutated to serine. Using proliferating cell nuclear antigen (PCNA) as an example, we present herein a detailed protocol for generating di- and tri-Ub PCNA that contains a photo-activatable cross-linker for capturing potential reader proteins. The thioether-mediated protein ligation and purification typically takes 2-3 weeks. An important feature of our ubiquitination strategy is the ability to introduce a Michael-acceptor warhead to the linkage, allowing the generation of activity-based probes for deubiquitinases and ubiquitin-carrying enzymes such as HECT and RBR E3 ubiquitin ligases and E2 enzymes. As such, our method is highly versatile and can be readily adapted to investigate the readers and erasers of many proteins that undergo reversible ubiquitination.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-04-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144020115","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":"Single-nucleus total RNA sequencing of formalin-fixed paraffin-embedded samples using snRandom-seq","authors":"Ziye Xu,&nbsp;Yuexiao Lyu,&nbsp;Haide Chen,&nbsp;Yu Chen,&nbsp;Yongcheng Wang","doi":"10.1038/s41596-025-01170-8","DOIUrl":"10.1038/s41596-025-01170-8","url":null,"abstract":"Formalin-fixed paraffin-embedded (FFPE) samples represent a vast and valuable resource of patient material, often linked to extensive clinical history and follow-up data. However, achieving single-cell or single-nucleus RNA (sc/snRNA) profiling in these archived tissues remains challenging. To address this, we have developed snRandom-seq, a droplet- and random primer–based single-nucleus total RNA sequencing technology specifically designed for FFPE tissues. This method captures total RNAs by using random primers and demonstrates a low doublet rate (0.3%), increased RNA coverage and enhanced detection of non-coding and nascent RNAs compared to state-of-the-art high-throughput sc/snRNA-seq technologies. This protocol provides a comprehensive guide to isolating single nuclei from FFPE samples; performing in situ DNA blocking, reverse transcription and dA tailing reactions; barcoding single-nucleus droplets; and preparing sequencing libraries. The entire snRandom-seq process can be completed in 4 d. This platform serves as a powerful tool for snRNA-seq of clinical specimens, with broad applications in studying complex biological systems. This protocol describes single-nucleus total RNA-sequencing of formalin-fixed paraffin-embedded samples by using random primers to capture a broad spectrum of RNAs, including nascent and noncoding RNAs, for microfluidics-based droplet barcoding.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"20 10","pages":"2771-2802"},"PeriodicalIF":16.0,"publicationDate":"2025-04-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144008015","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":"Identification, assembly and characterization of tumor immunoglobulin transcripts from RNA sequencing data using IgSeqR.","authors":"Dean Bryant, Benjamin Sale, Giorgia Chiodin, Dylan Tatterton, Benjamin Stevens, Alyssa Adlaon, Erin Snook, James Batchelor, Alberto Orfao, Francesco Forconi","doi":"10.1038/s41596-025-01172-6","DOIUrl":"https://doi.org/10.1038/s41596-025-01172-6","url":null,"abstract":"<p><p>Immunoglobulin gene analysis provides fundamental insight into B cell receptor structure and function. In B cell tumors, it can provide information on the cell of origin and predict clinical outcomes. Its clinical value has been established in the two main types of chronic lymphocytic leukemia, which are distinguished by the expression of unmutated or mutated immunoglobulin heavy chain variable region (IGHV) genes, and is emerging in other B cell tumors. The traditional PCR and Sanger sequencing-based techniques for immunoglobulin gene analysis are labor-intensive and rely on attaining either a dominant sequence or a small number of subclonal sequences. Extraction of the expressed tumor immunoglobulin transcripts by using high-throughput RNA-sequencing (RNA-seq) can be faster, allow the collection of the tumor immunoglobulin sequence and match this with the rest of the RNA-seq data. Analytical tools are regularly sought to increase the accuracy, depth and speed of acquisition of the immunoglobulin transcript sequences and combine the immunoglobulin characteristics with other tumor features. We provide here a user-friendly protocol for the rapid (~1 h) de novo assembly, identification and accurate characterization of the full (leader to constant region) tumor immunoglobulin templated and non-templated transcript sequence from RNA-seq data ( https://github.com/ForconiLab/IgSeqR ). The derived amino acid sequences can be interrogated for their physicochemical characteristics and, in certain lymphomas, be used to predict tumor glycan types occupying acquired N-glycosylation sites. These features will then be available for association studies with the tumor transcriptome. The resulting information can also help refine diagnosis, prognosis and potential therapeutic targeting in the most common lymphomas.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144025240","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":"Empowering materials science with VASPKIT: a toolkit for enhanced simulation and analysis.","authors":"Wen-Tong Geng, Ya-Chao Liu, Nan Xu, Gang Tang, Yoshiyuki Kawazoe, Vei Wang","doi":"10.1038/s41596-025-01160-w","DOIUrl":"https://doi.org/10.1038/s41596-025-01160-w","url":null,"abstract":"<p><p>Driven by rapid advances in high-performance supercomputing, computational materials science has emerged as a powerful approach for exploring, designing, and predicting material properties at the atomic and molecular scales. Among the various computational tools developed in this field, the Vienna Ab initio Simulation Package (VASP) stands out as a widely adopted and highly versatile platform for performing first-principles density functional theory (DFT) calculations. VASP is widely used to explore electronic structures, phonon behavior, magnetic properties, thermodynamics and catalytic mechanisms across a diverse range of materials systems. Despite its robust capabilities, utilizing VASP requires expertise in setting up simulations and analyzing results, which can be time consuming and technically challenging. To address these barriers, VASPKIT was developed as a comprehensive toolkit to simplify the workflow for VASP users. VASPKIT streamlines both preprocessing and postprocessing tasks, enabling users to generate essential input files based on customizable parameters and automate computational workflows. The postprocessing features of VASPKIT allow for efficient analysis of electronic, mechanical, optical and catalytic properties, thereby substantially reducing the need for advanced programming expertise. This protocol provides a detailed guide to using VASPKIT, including practical examples to demonstrate its versatility and utility in conducting and analyzing DFT calculations. For instance, the computation of elastic constants, electronic band structures and density of states for a graphene system can typically be completed within half an hour, depending on the computational resources available. By offering step-by-step guidance, this protocol aims to further expand the accessibility and impact of VASPKIT in the field of computational materials science.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143972541","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}