Nature Protocols最新文献

Mammalian development starts at fertilization and continually progresses until birth, except in cases in which an interruption is favorable to the embryo and the mother. Many mammals have the ability to pause development in case of suboptimal resources or routinely as part of their reproductive cycle-a phenomenon called 'embryonic diapause'. Diapause can be mimicked in vivo in mice via surgical removal of the ovaries or hormone injections. This procedure is laborious and invasive, ruling out its use across species. We have developed in vitro protocols through which mouse blastocysts, human blastoids and pluripotent stem cells from both species can be induced to enter a diapause-like dormant state via pharmacological inhibition of mTOR. Here, we describe in detail how embryos, blastoids and stem cells can be transitioned into and out of dormancy under different culture conditions. We further explain critical parameters to ensure success and propose experimental readouts. These in vitro embryonic dormancy setups can be used to uncover molecular mechanisms of dormancy, to test environmental or pharmacological effectors and to further innovate culture systems for species in which in vitro reproductive technologies are limited. We anticipate that researchers with ~1 year of embryo- and stem cell-handling experience should be able to achieve consistent results and evaluate outcomes. Altogether, inducing dormancy in vitro offers the possibility to slow down embryonic development for exploratory investigations of molecular mechanisms and eventually to expand the time window before implantation for clinical assays.

Elucidating the mechanical regulation of angiogenesis remains a challenge owing to the complexities of measuring cellular forces in this dynamic, multicellular and three-dimensional (3D) process. Current methods for force measurements typically involve traction force microscopy (TFM) applied to single cells or monolayers on 2D substrates or to individual cells within 3D extracellular matrix (ECM)-like gels. Here we present a protocol for mimicking and imaging dynamic early angiogenic sprouting into biomimetic matrices compatible with 3D TFM and for visualizing matrix degradation. Given that reliably acquiring sufficiently large 3D TFM datasets in multicellular systems is challenging, our protocol emphasizes best practices for higher-throughput data acquisition and for accurately imaging, analyzing and interpreting cell-ECM forces using our open-source TFMLAB software. As such, this assay provides a defined and reproducible system to study cellular forces and matrix degradation during angiogenesis in response to perturbations of cell-intrinsic signaling and mixed cell populations, as well as ECM cues. We further provide protocols for immunofluorescence analysis of angiogenic sprouts formed within the matrices and their retrieval from the hydrogel for downstream sequencing. Depending on the number of samples, sample preparation can take between 2 h and 4 h followed by a 15-17 h overnight wait time for angiogenic invasion. The 3D TFM data acquisition can take 2-6 h, while downstream processing of samples can take either 1 h (endothelial cell isolation) or up to 5 d (immunofluorescence). Notably, this workflow demands minimal prior expertise in programming, biophysics or molecular biology.

Practical lignin valorization strategies have the potential to enhance the profitability of lignocellulosic biorefineries. Leveraging the phenolic structure of lignin, commercially available lignins (for example, kraft, soda or biorefinery lignin) have been extensively studied as renewable substitutes for phenol in the synthesis of lignin-phenol-formaldehyde resin adhesives. However, the large-scale production and adoption of lignin-phenol-formaldehyde resin adhesives remain limited owing to challenges related to appearance, performance and cost. To address these limitations, this protocol outlines a comprehensive process for producing lignin adhesives with light colors, high adhesion properties and superior water and weather resistance. The procedure encompasses: (1) isolation of lignin from various types of biomass pretreatment liquors; (2) screening of high-quality lignins from the isolated lignins; and (3) performance assessment of lignin adhesives prepared directly from high-quality lignins without the need for chemical modification or additional processing. Additionally, this protocol provides a rapid and quantitative method for determining the condensation degree of lignin using a small amount of the isolated lignin sample (~50 mg) within a relatively short experimental time (5 h 20 min). This enables efficient quality evaluation and screening of high-quality lignins derived from different extraction methods and biomass sources. The entire process, from biomass to lignin adhesive fabrication, requires a total of 8 h 10 min and is designed for users with prior experience in biomass fractionation, chromatographic analysis and wood-based panel production.

Glioblastomas (GBMs) functionally integrate into diverse neuronal circuits within the central nervous system, which can promote tumor progression and affect neurons via neuron-to-glioma synapses. It remains challenging to identify and manipulate tumor-innervating neurons, which may remain localized or widely distributed throughout the brain. Building on GBM organoids (GBOs) derived from patient-resected surgical tissue, we present here detailed procedures for assessing interactions between tumors and neurons. We first discuss retrograde trans-monosynaptic tracing approaches to study the neuron-tumor connectome by using a rabies viral system in ex vivo human tissue and in xenogenic animal models. As a complementary approach, we then describe the use of anterograde transsynaptic tracing using herpes simplex virus in vivo and ex vivo to assess brain region-specific connectivity in GBMs. In addition, to facilitate the adaptability of these tracing methodologies in diverse systems, we provide procedures for the viral transduction into GBOs, the generation of assembloids comprising GBOs and human induced pluripotent stem cell-derived cortical organoids and the establishment of air-liquid interface cultures from surgical human brain tissue. Together, these techniques permit the flexible characterization and manipulation of tumor-neural circuits and can be easily adapted to other cancers with nervous system involvement. After the generation of GBOs and/or cortical organoids, transsynaptic tracing requires 12-35 d to complete ex vivo or in vivo. The procedure is suitable for users with expertise in human cell and organoid culture, viral production and transduction, rodent surgery and microscopy.

The construction of carbon‒nitrogen (C‒N) bonds is an essential transformation for synthesizing value-added organonitrogen compounds, including the raw materials for fertilizers, synthetic materials and pharmaceuticals. Electrocatalytic C‒N bond construction has emerged as an alternative strategy to traditional thermochemical routes, avoiding harsh and energy-intensive processes. This protocol describes an electrocatalytic C‒N bond construction strategy to synthesize organonitrogen from nitrogen oxides in water under ambient conditions, with a focus on several vital chemicals, such as urea, formamide, cyclohexanone oxime, amino acids and ¹⁵N-labeled amino acids. Here, we provide detailed procedures for catalyst and reaction device design, electrosynthesis, product quantification and measurements for investigating the reaction mechanisms. Four catalysts, namely, vacancy-rich ZnO, core-shell Cu@Zn, AgRu alloy and low-coordination Ag, are synthesized as cathode catalysts. Two types of electrolyzers, an H-type cell and a flow cell, are used for the electrocatalytic reaction. Characterization techniques such as electrochemical in situ Raman spectroscopy, in situ attenuated total reflectance-Fourier transform infrared spectroscopy, ex situ electron paramagnetic resonance and scanning flow cell-differential electrochemical mass spectrometry have been adopted to study the reaction mechanism. The synthesis amount of urea is at the micromole level, while the synthesis amounts of formamide, cyclohexanone oxime and amino acids are at the millimole level. The catalyst synthesis protocol requires 0.5-1.5 d, the electrosynthesis requires ≤11 h and the in situ characterization requires 0.5-1.5 h.

Cartilage is an essential component of the vertebrate skeleton, providing biomechanical support via its extracellular matrix composition. However, in many mammals, including humans and mice, numerous head, neck and chest cartilages produce little extracellular matrix and, instead, contain many large intracellular lipid vacuoles, which determine tissue size, shape and biomechanics. Such cartilages, termed lipocartilages, are made of individual cells called lipochondrocytes with distinct gene expression, lipid composition and metabolism. Lipochondrocytes significantly influence tissue-level physiology, regenerative potential and aging of skeletal elements. Here we provide a step-by-step protocol for the isolation of lipocartilage from mouse ear and the purification of its lipochondrocytes. We include instructions on how to microdissect ear lipocartilage for the purposes of lipid staining, wholemount imaging, morphometric analyses and biomechanical assays. Furthermore, we include a guide for the efficient dissociation of lipocartilages and the purification of individual lipochondrocytes by means of lipid-based buoyancy or cell sorting following fluorescent staining with neutral lipid dyes. With adequate dissection tools and sufficient practice, a researcher can cleanly isolate mouse ear lipocartilage within 20 min and purify lipochondrocytes within 4 h. Tissue biomechanics can be assayed by tensile testing within 30 min per sample. Although the protocol has only been validated in mice, it might be possible to adapt it for larger mammals, but modifications would probably be necessary, as lipocartilage is thicker. These guidelines will serve as a standard for future experiments on lipocartilage and have applications in the fields of developmental biology, bioengineering and metabolism.

Neural activity data can be associated with behavioral and physiological variables by analyzing their changes in the temporal domain. However, such relationships are often difficult to quantify and test, requiring advanced computational modeling approaches. Here, we provide a protocol for the statistical analysis of brain dynamics and for testing their associations with behavioral, physiological and other non-imaging variables. The protocol is based on an open-source Python package built on a generalization of the hidden Markov model (HMM)-the Gaussian-linear HMM-and supports multiple experimental modalities, including task-based and resting-state studies, often used to explore a wide range of questions in neuroscience and mental health. Our toolbox is available as both a Python library and a graphical interface, so it can be used by researchers with or without programming experience. Statistical inference is performed by using permutation-based methods and structured Monte Carlo resampling, and the framework can easily handle confounding variables, multiple testing corrections and hierarchical relationships within the data, among other features. The package includes tools developed to facilitate the intuitive visualization of statistical results, along with comprehensive documentation and step-by-step tutorials for data interpretation. Overall, the protocol covers the full workflow for the statistical analysis of functional neural data and their temporal dynamics.

Genomic loci positioning by sequencing (GPSeq) is a genome-wide method for mapping the radial organization of the genome in the nucleus of eukaryotic cells. GPSeq relies on in situ digestion of chromatin with a restriction enzyme that gradually diffuses inward from the nuclear periphery, followed by ligation of sequencing adapters to the digested restriction enzyme sites and library preparation for high-throughput sequencing. In parallel, ligation of labeled imaging adapters to the digested restriction enzyme recognition sites enables monitoring of the progression of radial digestion by fluorescence microscopy, providing an essential internal quality control before proceeding with sequencing. By comparing samples in which chromatin has been digested for increasing time intervals, a GPSeq score is calculated for every genomic bin into which the genome is arbitrarily divided, and genome-wide radial maps are generated with a resolution as high as 25 kb. These maps allow exploration of the radial distribution of (epi)genomic features, gene expression levels, mutational landscapes, and genomic profiles of DNA damage, when integrated with other omic data. Here, we present a detailed step-by-step protocol for performing GPSeq and preprocessing GPSeq data. The entire protocol requires ~2 weeks from the start of sample preparation to having ready-to-sequence libraries and intermediate levels of expertise in molecular biology, genomics and microscopy.

High-resolution mapping of active RNA polymerase II transcription initiation provides a dynamic view of gene expression and reveals the entire spectrum of RNA transcripts-from stable mRNAs to transient enhancer RNAs-which is critical for understanding gene regulation, deciphering transcriptional programs and defining regulatory element function. Here we present a detailed protocol for capped small RNA sequencing (csRNA-seq). Starting with total RNA, which can be readily isolated from fresh, frozen or fixed cells, tissues or patient samples, csRNA-seq selectively enriches for actively initiating 5'-capped RNA polymerase II transcripts. This approach captures both initiating stable protein-coding RNAs and non-coding RNAs, as well as rapidly degraded, transient transcripts such as enhancer or promoter divergent RNAs, providing a comprehensive snapshot of active cis-regulatory elements and facilitating the study of underlying regulatory mechanisms with high sensitivity. The protocol involves small RNA isolation, 5'-capped RNA enrichment and library generation, followed by sequencing. Key advantages of csRNA-seq over other nascent RNA-seq methods include (i) decoupling of sample collection and processing, (ii) broad compatibility with diverse eukaryotic sample types and organisms, (iii) high-resolution data defining active regulatory elements and their properties and (iv) scalability. Importantly, purified RNA is non-infectious and can be isolated from inactivated samples, including clinical or pathogenic specimens, allowing safe transport and analysis under standard laboratory conditions. This protocol empowers researchers with minimal experience in nascent transcriptomics to study gene regulation, cis-regulatory elements and transcription dynamics.