Nature Protocols最新文献

The advancement of research on human cerebellar development and diseases has been hindered by the lack of a cell-based system that mirrors the cellular diversity and functional characteristics of the human cerebellum. Here, we describe our protocol for a human pluripotent stem cell-derived human cerebellar organoid (hCerO) model, which successfully replicates the cellular diversity of the fetal cerebellum along with some of its distinct cytoarchitectural features. Our approach involves the patterning of human pluripotent stem cells, resulting in the generation of both cerebellar excitatory and inhibitory progenitor populations-specifically, the rhombic lip and ventricular zone progenitors, respectively. This patterning strategy leads to the reproducible differentiation of the major neurons of the cerebellum such as granule cells and Purkinje cells within just one month of culture. hCerOs serve as platforms for molecular, cellular and functional assays, including single-cell transcriptomics, immunohistochemistry and investigations into calcium dynamics and electrophysiological properties. Remarkably, the cultivation of hCerOs for up to 8 months enables the healthy survival and maturation of Purkinje cells, which exhibit molecular and electrophysiological features akin to their in vivo counterparts. Overall, our protocol generates and allows for the long-term culture of all major cell types within the cerebellum. Consequently, this significant advancement provides the developmental neurobiology field with a robust platform for exploring both cerebellar development and diseases within an all-human system. This protocol can be easily implemented by a technician with cell culture experience and takes 1-2 months to complete with an option for extended maturation over the course of several months.

Structure determines function. The discovery of the DNA double-helix structure revealed how genetic information is stored and copied. In the mammalian cell nucleus, up to two meters of DNA is compacted by histones to form nucleosome/DNA particle chains that form euchromatin and heterochromatin domains, chromosome territories and mitotic chromosomes upon cell division. A critical question is what are the structures, interactions and 3D organization of DNA as chromatin in the nucleus and how do they determine DNA replication timing, gene expression and ultimately cell fate. To visualize genomic DNA across these different length scales in the nucleus, we developed ChromEMT, a method that selectively enhances the electron density and contrast of DNA and interacting nucleosome particles, which enables nucleosome chains, chromatin domains, chromatin ultrastructure and 3D organization to be imaged and reconstructed by using multi-tilt electron microscopy tomography (EMT). ChromEMT exploits a membrane-permeable, fluorescent DNA-binding dye, DRAQ5, which upon excitation drives the photo-oxidation and precipitation of diaminobenzidine polymers on the surface of DNA/nucleosome particles that are visible in the electron microscope when stained with osmium. Here, we describe a detailed protocol for ChromEMT, including DRAQ5 staining, photo-oxidation, sample preparation and multi-tilt EMT that can be applied broadly to reconstruct genomic DNA structure and 3D interactions in cells and tissues and different kingdoms of life. The entire procedure takes ~9 days and requires expertise in electron microscopy sample sectioning and acquisition of multi-tilt EMT data sets.

Chiral plasmonic nanostructures are in high demand because of their unique optical properties, which are applicable to polarization control, chiral sensing and biomedical applications. An easy and scalable synthesis method for these nanostructures may facilitate their development further. We have reported the synthesis for 432-symmetric chiral plasmonic nanoparticles by using a seed-mediated colloidal method facilitated by a chiral amino acid and peptides. Among those, 432 helicoid III nanoparticles particularly exhibited well-defined chiral morphologies and exceptional chiroptic properties, evidenced by a Kuhn's dissymmetry factor (g-factor) of 0.2, making them valuable for various applications. Here, we detail the synthesis stages, including the synthesis of seed nanoparticles, the verification of each stage outcome and the calibration of synthesis conditions. We further illustrate the troubleshooting section and video-document the stages to facilitate the reliable reproduction of 432 helicoid III nanoparticles. The procedure requires 8 h to complete and can be carried out by users with expertise in chemistry or materials science.

Protein-protein interactions underpin nearly all biological processes, and understanding the molecular mechanisms that govern these interactions is crucial for the progress of biomedical sciences. The emergence of artificial intelligence-driven computational tools can help reshape the methods of structural biology; however, model data often require empirical validation. The large scale of predictive modeling data will therefore benefit from optimized methodologies for the high-throughput biochemical characterization of protein-protein interactions. Biolayer interferometry is one of very few approaches that can determine the rate of biomolecular interactions, called kinetics, and, of the commonly available kinetic measurement techniques, it is the most suitable for high-throughput experimental designs. Here we provide step-by-step instructions on how to perform kinetics experiments using biolayer interferometry. We further describe the basis and execution of competition and epitope binning experiments, which are particularly useful for antibody and nanobody screening applications. The procedure requires 3 h to complete and is suitable for users with minimal experience with biochemical techniques.

RNAs play critical roles in most biological processes. Although the three-dimensional (3D) structures of RNAs primarily determine their functions, it remains challenging to experimentally determine these 3D structures due to their conformational heterogeneity and intrinsic dynamics. Cryogenic electron microscopy (cryo-EM) has recently played an emerging role in resolving dynamic conformational changes and understanding structure-function relationships of RNAs including ribozymes, riboswitches and bacterial and viral noncoding RNAs. A variety of methods and pipelines have been developed to facilitate cryo-EM structure determination of challenging RNA targets with small molecular weights at subnanometer to near-atomic resolutions. While a wide range of conditions have been used to prepare RNAs for cryo-EM analysis, correlations between the variables in these conditions and cryo-EM visualizations and reconstructions remain underexplored, which continue to hinder optimizations of RNA samples for high-resolution cryo-EM structure determination. Here we present a protocol that describes rigorous screenings and iterative optimizations of RNA preparation conditions that facilitate cryo-EM structure determination, supplemented by cryo-EM data processing pipelines that resolve RNA dynamics and conformational changes and RNA modeling algorithms that generate atomic coordinates based on moderate- to high-resolution cryo-EM density maps. The current protocol is designed for users with basic skills and experience in RNA biochemistry, cryo-EM and RNA modeling. The expected time to carry out this protocol may range from 3 days to more than 3 weeks, depending on the many variables described in the protocol. For particularly challenging RNA targets, this protocol could also serve as a starting point for further optimizations.

Glycosaminoglycans (GAGs) are linear, unbranched heteropolysaccharides whose structural complexity determines their function. Accurate quantification of GAGs in biofluids at high throughput is relevant for numerous biomedical applications. However, because of the structural variability of GAGs in biofluids, existing protocols require complex pre-analytical procedures, have limited throughput and lack accuracy. Here, we describe the extraction and quantification of GAGs by using ultra-high-performance liquid chromatography coupled with triple-quadrupole mass spectrometry (UHPLC-MS/MS). Designed for 96-well plates, this method enables the processing of up to 82 study samples per plate, with the remaining 14 wells used for calibrators and controls. Key steps include the enzymatic depolymerization of GAGs, their derivatization with 2-aminoacridone and their quantification via UHPLC-MS/MS. Each plate can be analyzed in a single UHPLC-MS/MS run, offering the quantitative and scalable analysis of 17 disaccharides from chondroitin sulfate, heparan sulfate and hyaluronic acid, with a level of precision and reproducibility sufficient for their use as biomarkers. The procedure from sample thawing to initiating the UHPLC-MS/MS run can be completed in ~1.5 d plus 15 min of MS runtime per sample, and it is structured to fit within ordinary working shifts, thus making it a valuable tool for clinical laboratories seeking high-throughput analysis of GAGs. The protocol requires expertise in UHPLC-MS/MS.

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.