Nature Protocols最新文献_第5页

Absolute quantification of prokaryotes in the microbiome by 16S rRNA qPCR or ddPCR. 用16S rRNA qPCR或ddPCR对微生物组中的原核生物进行绝对定量。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-05-19 DOI: 10.1038/s41596-025-01165-5

Boryana Doyle, Gabriella Z M Reynolds, Mai Dvorak, Dylan G Maghini, Aravind Natarajan, Ami S Bhatt

{"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":"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.","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}

引用次数: 0

An injectable conductive hydrogel for closed-loop and robot-assisted rehabilitation via stretchable patch-type electrodes. 一种可注射的导电水凝胶，通过可拉伸的贴片型电极用于闭环和机器人辅助康复。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-05-19 DOI: 10.1038/s41596-025-01184-2

Subin Jin, Heewon Choi, Donghee Son, Mikyung Shin

{"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":"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.","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}

引用次数: 0

Tutorial: guidelines for the use of machine learning methods to mine genomes and proteomes for antibiotic discovery. 教程：使用机器学习方法挖掘基因组和蛋白质组以发现抗生素的指南。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-05-14 DOI: 10.1038/s41596-025-01144-w

Fangping Wan, Marcelo D T Torres, Changge Guan, Cesar de la Fuente-Nunez

{"title":"Tutorial: guidelines for the use of machine learning methods to mine genomes and proteomes for antibiotic discovery.","authors":"Fangping Wan, Marcelo D T Torres, Changge Guan, Cesar de la Fuente-Nunez","doi":"10.1038/s41596-025-01144-w","DOIUrl":"https://doi.org/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.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"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}

引用次数: 0

Achieving transient and reversible optical transparency in live mice with tartrazine. 酒黄石在活小鼠体内实现瞬时可逆的光学透明。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-05-13 DOI: 10.1038/s41596-025-01187-z

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

{"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":"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.","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}

引用次数: 0

Simple large-scale quantitative phenotyping and antimicrobial susceptibility testing with Q-PHAST. Q-PHAST简易大规模定量表型及药敏试验。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-05-12 DOI: 10.1038/s41596-025-01179-z

Juan Carlos Nunez-Rodriguez, Miquel Àngel Schikora-Tamarit, Ewa Ksiezopolska, Toni Gabaldón

{"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":"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.","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}

引用次数: 0

Versatile synthesis of uniform mesoporous superparticles from stable monomicelle units. 利用稳定的单微粒单元多用途合成均匀的介孔超微粒。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-05-01 Epub Date: 2024-11-13 DOI: 10.1038/s41596-024-01073-0

Zaiwang Zhao, Pengfei Zhang, Yujuan Zhao, Lipeng Wang, Jie Zhang, Fanxing Bu, Wanhai Zhou, Ruizheng Zhao, Xingmiao Zhang, Zirui Lv, Yupu Liu, Yuan Xia, Wei Zhang, Tiancong Zhao, Dongliang Chao, Wei Li, Dongyuan Zhao

{"title":"Versatile synthesis of uniform mesoporous superparticles from stable monomicelle units.","authors":"Zaiwang Zhao, Pengfei Zhang, Yujuan Zhao, Lipeng Wang, Jie Zhang, Fanxing Bu, Wanhai Zhou, Ruizheng Zhao, Xingmiao Zhang, Zirui Lv, Yupu Liu, Yuan Xia, Wei Zhang, Tiancong Zhao, Dongliang Chao, Wei Li, Dongyuan Zhao","doi":"10.1038/s41596-024-01073-0","DOIUrl":"10.1038/s41596-024-01073-0","url":null,"abstract":"Superstructures with architectural complexity and unique functionalities are promising for a variety of practical applications in many fields, including mechanics, sensing, photonics, catalysis, drug delivery and energy storage/conversion. In the past five years, a number of attempts have been made to build superparticles based on amphiphilic polymeric micelle units, but most have failed owing to their inherent poor stability. Determining how to stabilize micelles and control their superassembly is critical to obtaining the desired mesoporous superparticles. Here we provide a detailed procedure for the preparation of ultrastable polymeric monomicelle building units, the creation of a library of ultrasmall organic-inorganic nanohybrids, the modular superassembly of monomicelles into hierarchical superstructures and creation of novel multilevel mesoporous superstructures. The protocol enables precise control of the number of monomicelle units and the derived mesopores for superparticles. We show that ultrafine nanohybrids display enhanced mechanical antipressure performance compared with pristine polymeric micelles, and describe the functional characterization of mesoporous superstructures that exhibit excellent oxygen reduction reactivity. Except for the time (4.5 d) needed for the preparation of the triblock polystyrene-block-poly(4-vinylpyridine)-block-poly(ethylene oxide) PS-PVP-PEO or the polystyrene-block-poly(acrylic acid)-block-poly(ethylene oxide) (PS-PAA-PEO) copolymer, the synthesis of the ultrastable monomicelle, ultrafine organic-inorganic nanohybrids, hierarchical superstructures and mesoporous superparticles require ~6, 30, 8 and 24 h, respectively. The time needed for all characterizations and applications are 18 and 10 h, respectively.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":"1310-1351"},"PeriodicalIF":13.1,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142623899","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

Using the Chemotion repository to deposit and access FAIR research data for chemistry experiments. 使用 Chemotion 存储库存放和访问化学实验的 FAIR 研究数据。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-05-01 DOI: 10.1038/s41596-024-01074-z

Pei-Chi Huang, Chia-Lin Lin, Pierre Tremouilhac, Nicole Jung, Stefan Bräse

引用次数: 0

Enhancing consistency in brain imaging research for population neuroimaging. 加强脑成像研究的一致性，促进群体神经成像。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-05-01 DOI: 10.1038/s41596-024-01117-5

Ryan L Muetzel

引用次数: 0

Preparing submicrometer crystals for electron diffraction. 制备亚微米电子衍射晶体。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-05-01 DOI: 10.1038/s41596-024-01098-5

Alaa Shaikhqasem, Milton T Stubbs

引用次数: 0

Direct and quantitative analysis of tRNA acylation using intact tRNA liquid chromatography-mass spectrometry. 使用完整tRNA液相色谱-质谱法直接定量分析tRNA酰化。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-05-01 Epub Date: 2025-01-06 DOI: 10.1038/s41596-024-01086-9

Riley Fricke, Isaac Knudson, Cameron Verdayne Swenson, Sarah Smaga, Alanna Schepartz

{"title":"Direct and quantitative analysis of tRNA acylation using intact tRNA liquid chromatography-mass spectrometry.","authors":"Riley Fricke, Isaac Knudson, Cameron Verdayne Swenson, Sarah Smaga, Alanna Schepartz","doi":"10.1038/s41596-024-01086-9","DOIUrl":"https://doi.org/10.1038/s41596-024-01086-9","url":null,"abstract":"Aminoacyl-tRNA synthetases (aaRSs) provide an essential functional link between an mRNA sequence and the protein it encodes. aaRS enzymes catalyze a two-step chemical reaction that acylates specific tRNAs with a cognate α-amino acid. In addition to their role in translation, acylated tRNAs contribute to non-ribosomal natural product biosynthesis and are implicated in multiple human diseases. In synthetic biology, the acylation of tRNAs with a non-canonical α-amino acid or, more recently, a non-α-amino acid monomer is a critical first step in the incorporation of these monomers into proteins, where they can be used for fundamental and applied science. These endeavors all demand an understanding of aaRS activity and specificity. Here, we describe a liquid chromatography-mass spectrometry assay that directly monitors aaRS activity by detecting the intact acyl-tRNA product. After a simple tRNA acylation reaction workup, acyl- and non-acyl-tRNA molecules are resolved by using ion-pairing reverse-phase chromatography, and their exact masses are determined by using high-resolution time-of-flight mass spectrometry. Our assay is fast and simple, quantifies reaction yields as low as 0.23% and can also be used on tRNAs acylated with flexizyme to detect products that are undetectable by using standard techniques. The protocol requires basic expertise in molecular biology, liquid chromatography-mass spectrometry and RNase-free techniques. This protocol takes ≥5 h to complete, depending on the number of samples.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"20 5","pages":"1246-1274"},"PeriodicalIF":13.1,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143993949","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