Nature Protocols最新文献

Hydrogen peroxide (H₂O₂) is a natural product of aerobic metabolism. It acts as a signaling molecule and regulates fundamental cellular functions. However, it has remained difficult to measure intracellular H₂O₂ with high specificity and in a quantitative manner. Here, we present a detailed protocol for a chemogenetic method that enables the detection and quantitation of H₂O₂ in living cells by converting intracellular H₂O₂ into fluorescent or luminescent signals. This is achieved by expressing the engineered heme peroxidase APEX2 in cells and subcellular locations of interest and by providing an appropriate fluorogenic or luminogenic substrate from outside. This method differs fundamentally from previously developed genetically encoded H₂O₂ probes; those are reversible and measure the balance between probe thiol oxidation and reduction. By contrast, APEX2 turns over its substrate irreversibly and therefore directly measures endogenous H₂O₂ availability. Our detailed step-by-step protocol covers the generation of APEX2-expressing cell lines, the implementation of fluorescent and luminescent measurements and examples for application. Ectopic expression of APEX2 can be achieved in 3 days, while the actual measurements typically require 1-2 h. This protocol is intended for entry-level scientists.

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.

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.

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.

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.