Nature Protocols最新文献

Nanozymes are nanomaterials with enzyme-like catalytic properties. They are attractive reagents because they do not have the same limitations of natural enzymes (e.g., high cost, low stability and difficult storage). To test, optimize and compare nanozymes, it is important to establish fundamental principles and systematic standards to fully characterize their catalytic performance. Our 2018 protocol describes how to characterize the catalytic activity and kinetics of peroxidase nanozymes, the most widely used type of nanozyme. This approach was based on Michaelis-Menten enzyme kinetics and is now updated to take into account the unique physicochemical properties of nanomaterials that determine the catalytic kinetics of nanozymes. The updated procedure describes how to determine the number of active sites as well as other physicochemical properties such as surface area, shape and size. It also outlines how to calculate the hydroxyl adsorption energy from the crystal structure using the density functional theory method. The calculations now incorporate these measurements and computations to better characterize the catalytic kinetics of peroxidase nanozymes that have different shapes, sizes and compositions. This updated protocol better describes the catalytic performance of nanozymes and benefits the development of nanozyme research since further nanozyme development requires precise control of activity by engineering the electronic, geometric structure and atomic configuration of the catalytic sites of nanozymes. The characterization of the catalytic activity of peroxidase nanozymes and the evaluation of their kinetics can be performed in 4 h. The procedure is suitable for users with expertise in nano- and materials technology.

Single-cell proteomics by mass spectrometry (MS) allows the quantification of proteins with high specificity and sensitivity. To increase its throughput, we developed nano-proteomic sample preparation (nPOP), a method for parallel preparation of thousands of single cells in nanoliter-volume droplets deposited on glass slides. Here, we describe its protocol with emphasis on its flexibility to prepare samples for different multiplexed MS methods. An implementation using the plexDIA MS multiplexing method, which uses non-isobaric mass tags to barcode peptides from different samples for data-independent acquisition, demonstrates accurate quantification of ~3,000-3,700 proteins per human cell. A separate implementation with isobaric mass tags and prioritized data acquisition demonstrates analysis of 1,827 single cells at a rate of >1,000 single cells per day at a depth of 800-1,200 proteins per human cell. The protocol is implemented by using a cell-dispensing and liquid-handling robot-the CellenONE instrument-and uses readily available consumables, which should facilitate broad adoption. nPOP can be applied to all samples that can be processed to a single-cell suspension. It takes 1 or 2 d to prepare >3,000 single cells. We provide metrics and software (the QuantQC R package) for quality control and data exploration. QuantQC supports the robust scaling of nPOP to higher plex reagents for achieving reliable and scalable single-cell proteomics.

Covalent organic frameworks (COFs) are crystalline porous polymers constructed from organic building blocks into ordered two- or three-dimensional networks through dynamic covalent bonds. Attributed to their high porosity, well-defined structure, tailored functionality and excellent chemical stability, COFs have been considered ideal sorbents for various separation applications. The synthesis of COFs mainly employs the solvothermal method, which usually requires organic solvents in sealed Pyrex tubes, resulting in unscalable powdery products and environmental pollution that seriously limits their practical applications. Herein, our protocol focuses on an emerging synthesis method for COFs based on organic flux synthesis without adding solvents. The generality of this synthesis protocol has been applied in preparing various types of COFs, including olefin-linked, imide-linked, Schiff-based COFs on both gram and kilogram scales. Furthermore, organic flux synthesis avoids the disadvantages of solvothermal synthesis and enhances the crystallization and porosity of COFs. Typically, COF synthesis takes 3-5 d to complete, and subsequent washing procedures leading to pure COFs need 1 d. The procedure for kilogram-scale production of COFs with commercially available monomers is also provided. The resulting COFs are suitable for separation applications, particularly as adsorbent materials for industrial gas separation and water treatment applications. The protocol is suited for users with prior expertise in the synthesis of inorganic materials and porous organic materials.

Transcription factors (TFs) bind specific DNA sequences to regulate transcription. Apart from DNA sequences, local factors such as DNA accessibility and chromatin structure determine the affinity of a TF for any given locus. Including these factors when measuring TF-DNA affinities has proven difficult. To address this challenge, we recently developed a method called binding affinities in native chromatin by sequencing (BANC-seq). In BANC-seq, intact mammalian nuclei are incubated with a concentration range of epitope-tagged TF, followed by either chromatin immunoprecipitation or cleavage under target and release using nuclease with spike-in DNA. This allows determination of apparent dissociation constant (K_d^App) values, defined by the concentration of TF at which half-maximum binding occurs, across the genome. Here we present a detailed stepwise protocol for BANC-seq, including downstream data analysis. In principle, any molecular biologist should be able to perform a BANC-seq experiment in as little as 1.5 d (excluding analysis). However, preprocessing and analysis of the sequencing data does require some experience in command-line shell and R programming.

Human neural organoids represent promising models for studying neural function; however, organoids grown in vitro lack certain microenvironments and sensory inputs that are thought to be essential for maturation. The transplantation of patient-derived neural organoids into animal hosts helps overcome some of these limitations and offers an approach for neural organoid maturation and circuit integration. Here, we describe a method for transplanting human stem cell-derived cortical organoids (hCOs) into the somatosensory cortex of newborn rats. The differentiation of human induced pluripotent stem cells into hCOs occurs over 30-60 days, and the transplantation procedure itself requires ~0.5-1 hours per animal. The use of neonatal hosts provides a developmentally appropriate stage for circuit integration and allows the generation and experimental manipulation of a unit of human neural tissue within the cortex of a living animal host. After transplantation, animals can be maintained for hundreds of days, and transplanted hCO growth can be monitored by using brain magnetic resonance imaging. We describe the assessment of human neural circuit function in vivo by monitoring genetically encoded calcium responses and extracellular activity. To demonstrate human neuron-host functional integration, we also describe a procedure for engaging host neural circuits and for modulating animal behavior by using an optogenetic behavioral training paradigm. The transplanted human neurons can then undergo ex vivo characterization across modalities including dendritic morphology reconstruction, single-nucleus transcriptomics, optogenetic manipulation and electrophysiology. This approach may enable the discovery of cellular phenotypes from patient-derived cells and uncover mechanisms that contribute to human brain evolution from previously inaccessible developmental stages.

Network control theory (NCT) is a simple and powerful tool for studying how network topology informs and constrains the dynamics of a system. Compared to other structure-function coupling approaches, the strength of NCT lies in its capacity to predict the patterns of external control signals that may alter the dynamics of a system in a desired way. An interesting development for NCT in the neuroscience field is its application to study behavior and mental health symptoms. To date, NCT has been validated to study different aspects of the human structural connectome. NCT outputs can be monitored throughout developmental stages to study the effects of connectome topology on neural dynamics and, separately, to test the coherence of empirical datasets with brain function and stimulation. Here, we provide a comprehensive pipeline for applying NCT to structural connectomes by following two procedures. The main procedure focuses on computing the control energy associated with the transitions between specific neural activity states. The second procedure focuses on computing average controllability, which indexes nodes' general capacity to control the dynamics of the system. We provide recommendations for comparing NCT outputs against null network models, and we further support this approach with a Python-based software package called 'network control theory for python'. The procedures in this protocol are appropriate for users with a background in network neuroscience and experience in dynamical systems theory.

This paper introduces a comprehensive protocol leveraging open-access techniques to create small- to medium-scale 3D representations of the environment by using iPhone and iPad light detection and ranging (LiDAR). The protocol focuses on two capabilities of the iPhone LiDAR. The first capability is 3D modeling: iPhone LiDAR rapidly generates detailed indoor and outdoor 3D models, providing insights into object size, volume and geometry. The second capability is change detection: the 3D models created by the LiDAR sensor can be used for precise measurement of changes over time. Compared to other 3D topographic surveying methods, this method is rapid, high resolution, low cost and easy to use. The protocol outlines iPhone LiDAR scanning practices, model export and change detection. The expected results after executing the protocol are (i) a detailed 3D model of a small- to medium-sized object or area of interest and (ii) a distance point cloud revealing change between two point clouds of the same object or area between different times. The entire protocol can be conducted within 2 h by anyone with an iPhone with the LiDAR sensor and a computer. This protocol empowers scientists, students and community members conducting research with a cheap, easy-to-use method for addressing a range of questions and challenges, thus benefiting experts and the broader community.

Human early embryonic development is the cornerstone of a healthy baby. Abnormal early embryonic development may lead to developmental and pregnancy-related disorders. Accordingly, understanding the developmental events and mechanisms of human early embryonic development is very important. However, attempts to reveal these events and mechanisms are greatly hindered by the extreme inaccessibility of in vivo early human embryos. Fortunately, the emergence of in vitro culture (IVC) systems for mammalian embryos provides an alternative strategy. In recent years, different two-dimensional and three-dimensional IVC systems have been developed for human embryos. Ethical limitations restrict the IVC of human embryos beyond 14 days, which makes non-human primate embryos an ideal model for studying primate developmental events. Different culture systems have supported the development of monkey embryos to days postfertilization 14 and 25, respectively. The successful recapitulation of in vivo developmental events by these IVC embryos has greatly enriched our understanding of human early embryonic development, which undoubtedly helps us to develop possible strategies to predict or treat various gestation-related diseases and birth defects. In this protocol, we establish different two-dimensional and three-dimensional IVC systems for primate embryos, provide step-by-step culture procedures and notes, and summarize the advantages and limitations of different culture systems. Replicating this protocol requires a moderate level of experience in mammalian embryo IVC, and the embryo culture requires strict adherence to the procedures we have described.