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Past Issue in 2023
Volume: 13, Issue: 1
Cell Biology
Myonecrosis Induction by Intramuscular Injection of CTX
Skeletal muscle, one of the most abundant tissue in the body, is a highly regenerative tissue. Indeed, compared to other tissues that are not able to regenerate after injury, skeletal muscle can fully regenerate upon mechanically, chemically, and infection-induced trauma. Several injury models have been developed to thoroughly investigate the physiological mechanisms regulating skeletal muscle regeneration. This protocol describes how to induce muscle regeneration by taking advantage of a cardiotoxin (CTX)-induced muscle injury model. The overall steps include CTX injection of tibialis anterior (TA) muscles of BL6N mice, collection of regenerating muscles at different time points after CTX injury, and histological characterization of regenerating muscles. Our protocol, compared with others such as those for freeze-induced injury models, avoids laceration or infections of the muscles since it involves neither surgery nor suture. In addition, our protocol is highly reproducible, since it causes homogenous myonecrosis of the whole muscle, and further reduces animal pain and stress. Graphical abstract
Developmental Biology
Preparation of Caenorhabditis elegans for Scoring of Muscle-derived Exophers
Utilizingresources available from the mother's body to guarantee healthy offspring growth is the fundamental reproductive strategy. Recently, we showed that a class of the largest extracellular vesicles known as exophers, which are responsible for the removal of neurotoxic components from neurons (Melentijevic et al., 2017) and damaged mitochondria from cardiomyocytes (Nicolás-Ávila et al., 2020), are released by the Caenorhabditis elegans hermaphrodite body wall muscles (BWM), to support embryonic growth (Turek et al., 2021). Employing worms expressing fluorescent reporters in BWM cells, we found that exopher formation (exophergenesis) is sex-specific and fertility-dependent. Moreover, exophergenesis is regulated by the developing embryo in utero, and exophers serve as transporters for muscle-generated yolk proteins, which can be used to nourish the next generation. Given the specific regulation of muscular exophergenesis, and the fact that muscle-generated exophers are much larger than neuronal ones and have different targeting, their identification and quantification required a modified approach from that designed for neuronal-derived exophers (Arnold et al., 2020). Here, we present a methodology for assessing and quantifying muscle-derived exophers that can be easily extended to determine their function and regulation in various biological contexts. Graphical abstract
Immunology
Safety Profiling of Tumor-targeted T Cell–Bispecific Antibodies with Alveolus Lung- and Colon-on-Chip
Traditional drug safety assessments often fail to predict complications in humans, especially when the drug targets the immune system. Rodent-based preclinical animal models are often ill-suited for predicting immunotherapy-mediated adverse events in humans, in part because of the fundamental differences in immunological responses between species and the human relevant expression profile of the target antigen, if it is expected to be present in normal, healthy tissue. While human-relevant cell-based models of tissues and organs promise to bridge this gap, conventional in vitro two-dimensional models fail to provide the complexity required to model the biological mechanisms of immunotherapeutic effects. Also, like animal models, they fail to recapitulate physiologically relevant levels and patterns of organ-specific proteins, crucial for capturing pharmacology and safety liabilities. Organ-on-Chip models aim to overcome these limitations by combining micro-engineering with cultured primary human cells to recreate the complex multifactorial microenvironment and functions of native tissues and organs. In this protocol, we show the unprecedented capability of two human Organs-on-Chip models to evaluate the safety profile of T cell–bispecific antibodies (TCBs) targeting tumor antigens. These novel tools broaden the research options available for a mechanistic understanding of engineered therapeutic antibodies and for assessing safety in tissues susceptible to adverse events. Graphical abstract Figure 1. Graphical representation of the major steps in target-dependent T cell–bispecific antibodies engagement and immunomodulation, as performed in the Colon Intestine-Chip
Sample Preparation and Integrative Data Analysis of a Droplet-based Single-Cell ATAC-sequencing Using Murine Thymic Epithelial Cells
Accessible chromatin regions modulate gene expression by acting as cis-regulatory elements. Understanding the epigenetic landscape by mapping accessible regions of DNA is therefore imperative to decipher mechanisms of gene regulation under specific biological contexts of interest. The assay for transposase-accessible chromatin sequencing (ATAC-seq) has been widely used to detect accessible chromatin and the recent introduction of single-cell technology has increased resolution to the single-cell level. In a recent study, we used droplet-based, single-cell ATAC-seq technology (scATAC-seq) to reveal the epigenetic profile of the transit-amplifying subset of thymic epithelial cells (TECs), which was identified previously using single-cell RNA-sequencing technology (scRNA-seq). This protocol allows the preparation of nuclei from TECs in order to perform droplet-based scATAC-seq and its integrative analysis with scRNA-seq data obtained from the same cell population. Integrative analysis has the advantage of identifying cell types in scATAC-seq data based on cell cluster annotations in scRNA-seq analysis.
Microbiology
Sclerotinia sclerotiorum Protoplast Preparation and Transformation
Sclerotinia sclerotiorum causes white mold, leading to substantial losses on a wide variety of hosts around the world. Many genes encoding effector proteins play important roles in the pathogenesis of S. sclerotiorum. Therefore, establishment of a transformation system for the exploration of gene function is necessarily significant. Here, we introduce a modified protocol to acquire protoplasts for transformation and generate knockout strains, which completements the transformation system of S. sclerotiorum.
Molecular Biology
BONCAT-based Profiling of Nascent Small and Alternative Open Reading Frame-encoded Proteins
RIBO-seq and proteogenomics have revealed that mammalian genomes harbor thousands of unannotated small and alternative open reading frames (smORFs, <100 amino acids, and alt-ORFs, >100 amino acids, respectively). Several dozen mammalian smORF-encoded proteins (SEPs) and alt-ORF-encoded proteins (alt-proteins) have been shown to play important biological roles, while the overwhelming majority of smORFs and alt-ORFs remain uncharacterized, particularly at the molecular level. Functional proteomics has the potential to reveal key properties of unannotated SEPs and alt-proteins in high throughput, and an approach to identify SEPs and alt-proteins undergoing regulated synthesis should be of broad utility. Here, we introduce a chemoproteomic pipeline based on bio-orthogonal non-canonical amino acid tagging (BONCAT) (Dieterich et al., 2006) to profile nascent SEPs and alt-proteins in human cells. This approach is able to identify cellular stress-induced and cell-cycle regulated SEPs and alt-proteins in cells. Graphical abstract Schematic overview of BONCAT-based chemoproteomic profiling of nascent, unannotated small and alternative open reading frame-encoded proteins (SEPs and alt-proteins)
Staining and Scanning Protocol for Micro-Computed Tomography to Observe the Morphology of Soft Tissues in Ambrosia Beetles
Advances in imaging technology offer new opportunities in developmental biology. To observe the development of internal structures, microtome cross-sectioning followed by H&E staining on glass slides is a common procedure; however, this technique can be destructive, and artifacts can be introduced during the process. In this protocol, we describe a less invasive procedure with which we can stain insect samples and obtain reconstructed three-dimensional images using micro-computed tomography, or micro-CT (µCT). Specifically, we utilize the fungus-farming ambrosia beetle species Euwallacea validus to observe the morphology of mycangia, a critical internal organ with which beetles transport fungal symbionts. Not only this protocol is ideal to observe mycangia, our staining/scanning procedure can also be applied to observe other delicate tissues and small organs in arthropods. Graphical abstract
Neuroscience
Quantitative Analysis of Gene Expression in RNAscope-processed Brain Tissue
Molecular characterization of different cell types in rodent brains is a widely used and important approach in neuroscience. Fluorescent detection of transcripts using RNAscope (ACDBio) has quickly became a standard in situ hybridization (ISH) approach. Its sensitivity and specificity allow for the simultaneous detection of between three and forty-eight low abundance mRNAs in single cells (i.e., multiplexing or hiplexing), and, in contrast to other ISH techniques, it is performed in a shorter amount of time. Manual quantification of transcripts is a laborious and time-consuming task even for small portions of a larger tissue section. Herein, we present a protocol for creating high-quality images for quantification of RNAscope-labeled neurons in the rat brain. This protocol uses custom-made scripts within the open-source software QuPath to create an automated workflow for the careful optimization and validation of cell detection parameters. Moreover, we describe a method to derive mRNA signal thresholds using negative controls. This protocol and automated workflow may help scientists to reliably and reproducibly prepare and analyze rodent brain tissue for cell type characterization using RNAscope. Graphical abstract
Parasitoid Wasp Culturing and Assay to Study Parasitoid-induced Reproductive Modifications in Drosophila
In nature, parasitoid wasp infections are a major cause of insect mortality. Parasitoid wasps attack a vast range of insect species to lay their eggs. As a defense, insects evolved survival strategies to protect themselves from parasitoid infection. While a growing number of studies reported both host defensive tactics and parasitoid counter-offensives, we emphasize that this parasite–host relationship presents a unique ecological and evolutionary relevant model that is often challenging to replicate in a laboratory. Although maintaining parasitoid wasp cultures in the laboratory requires meticulous planning and can be labor intensive, a diverse set of wasp species that target many different insect types can be maintained in similar culture conditions. Here, we describe the protocol for culturing parasitoid wasp species on Drosophila larvae and pupae in laboratory conditions. We also detail an egg-laying assay to assess the reproductive modification of Drosophila females in response to parasitoid wasps. This behavioral study is relatively simple and easily adaptable to study environmental or genetic influences on egg-laying, a readout for female germline development. Neither the parasitoid culture conditions or the behavioral assay require special supplies or equipment, making them a powerful and versatile approach in research or teaching laboratory settings. Graphical abstract
Systems Biology
A Cartographic Tool to Predict Disease Risk-associated Pseudo-Dynamic Networks from Tissue-specific Gene Expression
Understanding how genes are differentially expressed across tissues is key to reveal the etiology of human diseases. Genes are never expressed in isolation, but rather co-expressed in a community; thus, they co-act through intricate but well-orchestrated networks. However, existing approaches cannot coalesce the full properties of gene–gene communication and interactions into networks. In particular, the unavailability of dynamic gene expression data might impair the application of existing network models to unleash the complexity of human diseases. To address this limitation, we developed a statistical pipeline named DRDNetPro to visualize and trace how genes dynamically interact with each other across diverse tissues, to ascertain health risk from static expression data. This protocol contains detailed tutorials designed to learn a series of networks, with the illustration example from the Genotype-Tissue Expression (GTEx) project. The proposed toolbox relies on the method developed in our published paper (Chen et al., 2022), coding all genes into bidirectional, signed, weighted, and feedback looped networks, which will provide profound genomic information enabling medical doctors to design precise medicine. Graphical abstract Flowchart illustrating the use of DRDNetPro. The left panel contains the summarized pipeline of DRDNetPro and the right panel contains one pseudo-illustrative example. See the Equipment and Procedure sections for detailed explanations.