Nature Protocols最新文献

Hydrogels, as 3D cross-linked hydrophilic networks that exhibit favorable flexibility, cargo loading and release abilities and structure and function designability, are desirable for diverse biomedical applications. For in vivo implementation, however, hydrogels often suffer from swelling-weakened mechanical strength, uncontrollable cargo release and complex composition, inevitably hindering further translation. Despite different reported synthetic approaches, the development of a facile yet universal method capable of fabricating hydrogels with dynamically adjustable structure and function remains difficult. Recently, inspired by biological tissues, we have developed a versatile biological membrane hybridization strategy to generate structurally and functionally programmable hydrogels. Specifically, biological membranes are used as a cross-linker to form a cross-linked network through a supramolecular-covalent cascade reaction route. This protocol demonstrates the construction of two biological membrane-hybridized hydrogels, including liposome-hybridized muscle-mimicking hydrogels with swelling-strengthening mechanical behavior and extracellular vesicle-hybridized skin-mimicking hydrogels with enhanced mechanical strength, lubricity, antibacterial activity and immunoactivity. We describe the detailed preparation procedures and characterize the structures and functions of the obtained hydrogels. We also expand the applicability of this biological membrane hybridization strategy to further tune the structure and function of the biomimetic hydrogels by incorporating a second network. This protocol provides a robust preparative platform to develop dual structure- and function-tunable hydrogels for different biomedical applications. Excluding the synthesis of reactive group-functionalized biological membranes, the fabrication of muscle-mimicking hydrogels takes ~3 d, while the construction of skin-mimicking hydrogels takes ~1 d. The implementation of the protocol requires expertise in polymer modification, hydrogel preparation, nanoscale vesicles, surface functionalization and cell culture.

Extracellular vesicles are a heterogeneous group of membrane-bound vesicles involved in cell-cell communication, formed at the plasma membrane (ectosomes) or by endocytosis (exosomes). Most exosome studies so far have focused on in vitro systems or exosomes derived from bodily fluids, while tissue-derived exosomes remain underexplored. Here we present a protocol using cell-type-specific promoter-driven reporter constructs for the targeted labeling and subsequent isolation of exosomes from specific cell types in vivo from mouse tissues. The differentiation between exosomes and ectosomes remains challenging due to limitations of current isolation techniques that are primarily based on size, density or surface markers. To address this issue, our approach leverages genetic engineering to mark exosomes specifically, enabling their precise identification and isolation from a complex biological pool of heterogenous extracellular vesicles. The isolated cell-type-specific exosomes are characterized by electron microscopy, nanoparticle tracking analysis, antibody exosome array assay and other established techniques. The labeling and isolation of exosomes spans 2-3 days and is designed to be accessible to researchers with fundamental laboratory competencies. This protocol facilitates the study of exosome-mediated cellular communication by enabling the isolation of cell-type-specific exosomes from either individual cell types or multiple cell types in combination. Most experiments within the protocol have used murine wound-edge skin tissue, but the protocol can, in principle, also be applied to other tissues to isolate exosomes, with a few modifications as required. This methodology opens new avenues for exploring the functional roles of cell-type-specific exosomes in intercellular communication.

Despite their limited cargo capacity (<5 kb), adeno-associated viral (AAV) vectors remain the gold standard for in vivo delivery of therapeutic genes. Dual AAV vectors have emerged as a valuable tool for delivering large therapeutic genes and CRISPR tools to overcome this limitation. Here we provide a detailed protocol for the design, production and evaluation of dual AAV vectors. We offer guidelines for selecting a suitable dual AAV strategy, designing and cloning the genes to be delivered, and conducting in vitro evaluations of expression efficiency. In addition, we detail the production of dual AAVs and their assessment in human cellular models, such as induced pluripotent stem cell-derived retinal organoids. Finally, we outline the administration of dual AAVs via different routes in mice and the assessment of transgene-derived RNA and protein expression in various tissues. Overall, the instructions in this Protocol will aid in the efficient in vivo delivery of large DNA fragments using dual AAVs. This Protocol is adaptable to a wide range of model organisms as well as to human organoid cultures and, depending on the application, can be completed in 15-44 weeks.

Structural biology is fundamental to understanding the molecular basis of biological processes. While machine learning-based protein structure prediction has advanced considerably, experimentally determined structures remain indispensable for guiding structure-function analyses and for improving predictive modeling. However, experimental studies of protein complexes continue to pose challenges, particularly due to the necessity of high protein concentrations and purity for downstream analyses such as cryogenic electron microscopy. Transient transformation of Nicotiana benthamiana has emerged as a promising expression system for recombinant protein production, offering advantages such as low operating costs, rapid cultivation, short experimental turnaround and scalability compared with other established platforms such as insect or human cell culture systems. Here we present a versatile protocol leveraging N. benthamiana for the purification and structural analysis of protein complexes of diverse origin and composition, exemplified by six oligomeric complexes ranging from ~140 to ~660 kDa, originating from plant, vertebrate, fungal and bacterial species. In most cases, purification only requires a single epitope tag, simplifying workflows and reducing complications that come with multitag and sequential affinity purifications. The protocol enables rapid application, allowing protein sample production in fewer than 7 days. Critical parameters influencing expression and purification efficiency include codon alteration, epitope tag selection and detergent supplementation.

Metabolism is a fundamental process that shapes the pharmacological and toxicological profiles of drugs, making metabolite identification and analysis critical in drug development and biological research. Global Natural Products Social Networking (GNPS) is a community-driven infrastructure for mass spectrometry data analysis, storage and knowledge dissemination. GNPS2 is an improved version of the platform offering higher processing speeds, improved data analysis tools and a more intuitive user interface. Molecular networking based on tandem mass spectrometry spectral alignments, combined with other tools in the GNPS2 analysis environment, enables the discovery of candidate drug metabolites without prior knowledge, even from complex biological matrices. This protocol represents an extension of a previously established protocol for fundamental molecular networking in GNPS, with a specific focus on metabolism studies. This article uses the example of the drug sildenafil to identify candidate metabolites obtained from liquid chromatography-quadrupole time-of-flight mass spectrometry analysis of liver microsomal fractions and mice plasma to guide the reader through a step-by-step process consisting of five GNPS2-based analytical workflows. It demonstrates how the tools in GNPS2 can be used not only to identify candidate drug metabolites from in vitro studies but also to evaluate the translational relevance of these in vitro findings to humans by using reverse metabolomics. We provide a step-by-step analytical approach based on published studies to showcase how GNPS2 can be effectively applied in drug metabolism studies.

Scanning probe microscopy (SPM) is a powerful technique for mapping nanoscale surface properties through tip-sample interactions. Thermal scanning-probe lithography (tSPL) is an advanced SPM variant that uses a silicon tip on a heated cantilever to sculpt and measure the topography of polymer films with nanometer precision. The surfaces produced by tSPL-smooth topographic landscapes-allow mathematically defined contours to be fabricated on the nanoscale, enabling sophisticated functionalities for photonic, electronic, chemical and biological technologies. Evaluating the physical effects of a landscape requires fitting arbitrary mathematical functions to SPM datasets; however, this capability does not exist in standard analysis programs. Here, we provide an open-source software package (FunFit) to fit analytical functions to SPM data and develop a fabrication and characterization protocol based on this analysis. We demonstrate the benefit of this approach by patterning periodic and quasi-periodic landscapes in a polymer resist with tSPL, which we transfer to hexagonal boron nitride (hBN) flakes with high fidelity via reactive ion etching. The topographic landscapes in polymers and hBN are measured with tSPL and atomic force microscopy, respectively. Within the FunFit program, the datasets are corrected for artifacts, fit with analytical functions and compared, providing critical feedback on the fabrication procedure. This approach can improve analysis, reproducibility and process development for a broad range of SPM experiments. The protocol can be performed within a working day by a trained graduate student or researcher, where fabrication and characterization take a few hours, and software analysis takes a few minutes.

The versatility of lanthanide-doped near-infrared (NIR, 700-1,700 nm) luminescent nanoparticles makes them valuable tools in various scientific and technological fields, from bioimaging to information security. However, the luminescence intensity of typical lanthanide-doped nanoparticles is significantly influenced by the efficiency of the sensitizer. The introduction of transition metal ions (such as Cr³⁺, Mn²⁺ and Ni²⁺) can greatly enrich the library of lanthanide NIR luminescence nanoparticles. We have reported a new crystalline nanoparticle, Na₃CrF₆, for high-brightness NIR emission from lanthanide activators (such as Er³⁺, Tm³⁺, Yb³⁺ or Nd³⁺). As an emerging luminescent material, a straightforward and scalable synthesis approach for these nanostructures holds promise for their broader application. Here we have refined and standardized the steps for transition metal-sensitized lanthanide luminescent nanoparticles, thereby establishing a library of advanced luminescent materials for researchers engaged in luminescent materials. The Protocol enables the precise preparation of chromium-, manganese- and nickel-trifluoroacetate, the synthesis of three types of transition metal-sensitized lanthanide nanoparticle and the fabrication of chromium-sensitized lanthanide homogeneous and heterogeneous nanostructure. Moreover, we provide verification protocols for each step's output and guidelines for adjusting synthesis conditions. To aid in the reproducible synthesis of these nanoparticles, we also include a troubleshooting guide of the various stages. The estimated duration for synthesizing transition metal trifluoroacetate, transition metal-sensitized lanthanide nanoparticles and core-shell transition metal-sensitized lanthanide nanoparticles are ~70, 30 and 30 h, respectively. These procedures can be carried out by users with expertise in chemistry or materials science.

Tandem repeats (TRs) are highly variable loci in the human genome that are linked to various human phenotypes. Accurate and reliable genotyping of TRs is important in understanding population TR variation dynamics and their effects in TR-trait association studies. In this protocol, we describe how to generate high-quality consensus TR genotypes for population genomics studies. In particular, we detail steps to: (i) perform TR genotyping from short-read whole-genome sequencing data by using the HipSTR, GangSTR, adVNTR and ExpansionHunter tools, (ii) perform quality control checks on TR genotypes by using TRTools and (iii) integrate TR genotypes from different tools by using EnsembleTR. We further discuss how to visualize and investigate TR variation patterns to identify population-specific expansions and perform TR-trait association analyses. We demonstrate the utility of these steps by analyzing a small dataset from the 1000 Genomes Project. In addition, we recapitulate a previously identified association between TR length and gene expression in the African population and provide a generalized discussion on TR analysis and its relevance to identifying complex traits. The expected time for installing the necessary software for each section is ~10 min. The expected run time on the user's desired dataset can vary from hours to days depending on factors such as the size of the data, input parameters and the capacity of the computing infrastructure.

Nephron progenitor cells (NPCs) have a central role in kidney organogenesis: they self-renew and differentiate into nephrons, the functional units of the kidney. Human pluripotent stem cells (hPSCs) can transiently produce induced nephron progenitor-like cells (iNPCs), which then differentiate into nephron organoids. Here, we describe a protocol to purify and expand the hPSC-derived iNPCs in a regular monolayer culture format with an optimized iNPC culture medium. Under this culture condition, iNPCs are programmed to a state with their transcriptome much closer to primary human NPCs than the transient hPSC-derived iNPCs. By following this protocol, iNPC lines can be derived from any hPSC lines, exhibiting a stable cell proliferation rate and retaining NPC marker gene expression over long-term culture. We also describe a protocol to generate nephron organoids from the iNPC lines. These iNPC-derived nephron organoids show minimal off-target cell types compared to hPSC-derived kidney organoids, with enhanced podocyte maturity. This protocol consists of a modified 10-d protocol to generate iNPCs from hPSCs, an iNPC expansion phase with a unique chemically defined iNPC expansion medium called 'hNPSR-v2' and a stepwise 21-d differentiation protocol to generate nephron organoids from iNPCs on an air-liquid interface. Experience in culturing and differentiating hPSCs is required to conduct this protocol, which can be executed within 1.5-2 months.