Molecular Biology

The quality of cellular products used in biological research can impact the accuracy of results. Epstein–Barr virus (EBV) is a latent virus that spreads extensively worldwide, and cell lines used in experiments may carry EBV and pose an infection risk. The presence of EBV in a single cell line can contaminate other cell lines used in the same laboratory, affecting experimental results. Existing tests to detect EBV can be divided into three categories: nucleic acid assays, serological assays, and in situ hybridization assays. However, most methods are time-consuming, expensive, and not conducive to high-volume clinical screening. Therefore, a simple system that allows for the rapid detection of EBV in multiple contexts, including both cell culture and tissue samples, remains necessary. In our research, we developed EBV detection systems: (1) a polymerase chain reaction (PCR)-based detection system, (2) a recombinase polymerase amplification (RPA)-based detection system, and (3) a combined RPA-lateral flow assay (LFA) detection system. The minimum EBV detection limits were 1 × 10³ copy numbers for the RPA-based and RPA-LFA systems and 1 × 10⁴ copy numbers for the PCR-based system. Both the PCR and RPA detection systems were applied to 192 cell lines, and the results were consistent with those of the assays specified in industry standards. A total of 10 EBV-positive cell lines were identified. The combined RPA-LFA system is simple to operate, allowing for rapid result visualization. This system can be implemented in laboratories and cell banks as part of a daily quality control strategy to ensure cell quality and experimental safety and may represent a potential new technique for the rapid detection of EBV in clinical samples.

Two aconitase isoforms are present in mammalian cells: the mitochondrial aconitase (ACO2) that catalyzes the reversible isomerization of citrate to isocitrate in the citric acid cycle, and the bifunctional cytosolic enzyme (ACO1), which also plays a role as an RNA-binding protein in the regulation of intracellular iron metabolism. Aconitase activities in the different subcellular compartments can be selectively inactivated by different genetic defects, iron depletion, and oxidative or nitrative stress. Aconitase contains a [4Fe-4S]²⁺ cluster that is essential for substrate coordination and catalysis. Many Fe-S clusters are sensitive to oxidative stress, nitrative stress, and reduced iron availability, which forms the basis of redox- and iron-mediated regulation of intermediary metabolism via aconitase and other Fe-S cluster-containing metabolic enzymes, such as succinate dehydrogenase. As such, ACO1 and ACO2 activities can serve as compartment-specific surrogate markers of oxygen levels, reactive oxygen species (ROS), reactive nitrogen species (RNS), iron bioavailability, and the status of intermediary and iron metabolism. Here, we provide a protocol describing a non-denaturing polyacrylamide gel electrophoresis (PAGE)-based procedure that has been successfully used to monitor ACO1 and ACO2 aconitase activities simultaneously in human and mouse cells and tissues.

Gene expression analysis is a fundamental technique to elucidate the regulatory mechanisms of genes of interest or to reveal the patterns of plant response to environmental stimuli. Traditionally, gene expression analyses have required RNA extraction, followed by cDNA synthesis and qPCR analyses. However, this conventional method is costly and time-consuming, limiting the amount of data collected. The protocol outlined in this study, which utilizes a chemiluminescence system, offers a cost-effective and rapid method for assessing the expression of Arabidopsis (Arabidopsis thaliana) genes, exemplified by analyzing the nitrate-inducible expression of a major nitrate transporter gene, nitrate transporter 2.1 (NRT2.1). A reporter construct, containing the NRT2.1 promoter fused to the firefly luciferase gene, was introduced into wild-type and mutant Arabidopsis plants. Seeds obtained from the transgenic lines were grown for 3 days in 96-well microplates containing a nitrate-free nutrient solution. After 3 days, the nutrient solution was replaced with a fresh batch, which was supplemented with luciferin potassium. One hour later, nitrate was added at various concentrations, and the temporal expression pattern of NRT2.1 was analyzed by monitoring the chemiluminescence signals. This method allowed for the cost-effective, quantitative, and high-throughput analysis of NRT2.1 expression over time under the effects of various nutrient conditions and genetic backgrounds.

The COVID-19 pandemic led to the rapid development of antibody-based therapeutics and vaccines targeting the SARS-CoV-2 spike protein. Several antibodies have been instrumental in protecting vulnerable populations, but their utility was limited by the emergence of spike variants with diminished susceptibility to antibody binding and neutralization. Moreover, these spike variants exhibited reduced neutralization by polyclonal antibodies in vaccinated individuals. Accordingly, the characterization of antibody binding to spike variants is critical to define antibody potency and understand the impact of amino acid changes. A key challenge in this effort is poor spike stability, with most current methods assessing antibody binding using individual domains instead of the intact spike or variants with stabilizing amino acid changes in the ectodomain (e.g., 2P or HexaPro). The use of non-native spike may not accurately predict antibody binding if changes lie within the epitope or alter epitope accessibility by altering spike dynamics. Here, we present methods to characterize antibody affinity for and activity against unmodified SARS-CoV-2 spike protein variants displayed on a mammalian cell membrane that recapitulates the native spike environment on infected cells. These include a flow cytometry–based method to determine the effective antibody binding affinity (K_D) and an antibody-dependent cellular cytotoxicity (ADCC) assay to assess Fc-mediated activities. These methods can readily evaluate antibody activity across a panel of spike variants and contribute to our understanding of spike/antibody co-evolution.

Gene-edited human pluripotent stem cells provide attractive model systems to functionally interrogate the role of specific genetic variants in relevant cell types. However, the need to isolate and screen edited clones often remains a bottleneck, in particular when recombination rates are sub-optimal. Here, we present a protocol for flexible gene editing combining Cas9 ribonucleoprotein with donor templates delivered by adeno-associated virus (AAV) vectors to yield high rates of homologous recombination. To streamline the workflow, we designed a modular system for one-step assembly of targeting vectors based on Golden Gate cloning and developed a rapid protocol for small-scale isolation of AAV virions of serotype DJ. High homology-directed repair (HDR) rates in human pluripotent stem cells (hPSCs), ~70% in ACTB and ~30% in LMNB1, were achieved using this approach, also with short (300 bp) homology arms. The modular design of donor templates is flexible and allows for the generation of conditional and/or complex alleles. This protocol thus provides a flexible and efficient strategy workflow to rapidly generate gene-edited hPSC lines.

Genome-wide gene expression analysis is a commonly used method to quantitatively examine the transcriptional signature of any tissue or cell state. Standard bulk cell RNA sequencing (RNA-seq) quantifies RNAs in the cells of the tissue type of interest through massive parallel sequencing of cDNA synthesized from the cellular RNA. The subsequent analysis of global RNA expression and normalization of RNA expression levels between two or more samples generally assumes that cells from all samples produce equivalent amounts of RNA per cell. This assumption may be invalid in cells where MYC or MYCN expression levels are markedly different and thus, overall mRNA expression per cell is altered. Here, we describe an approach for RNA-seq analysis of MYCN-amplified neuroblastoma cells during treatment with retinoic acid, which causes dramatic downregulation of MYCN expression and induces growth arrest and differentiation of the cells. Our procedure employs spiked-in RNA standards added in ratio to the number of cells in each sample prior to RNA extraction. In the analysis of differential gene expression, the expression level of each gene is standardized to the spiked-in RNA standard to accurately assess gene expression levels per cell in conditions of high and low MYCN expression. Our protocol thus provides a step-by-step experimental approach for normalizing RNA-seq expression data on a per-cell-number basis, allowing accurate assessment of differential gene expression in cells expressing markedly different levels of MYC or MYCN.

Enzyme-catalyzed proximity labeling is a potent technique for the discernment of subtle molecular interactions and subcellular localization, furnishing contextual insights into the protein of interest within cells. Although ascorbate peroxidase2 (APEX2) has proven effective in this approach when overexpressed, its compatibility with endogenous proteins remains untested. We improved this technique for studying native protein–protein interactions in live Drosophila ovary tissue. Through CRISPR/Cas9 genome editing, APEX2 was fused with the endogenous dysfusion gene. By pre-treating the tissue with Triton X-100 to enhance biotin-phenol penetration, we successfully identified multiple proteins interacting with the native Dysfusion proteins that reside on the inner nuclear membrane. Our protocol offers a comprehensive workflow for delineating the interactome networks of ovarian components in Drosophila, aiding future studies on endogenous protein–protein interactions in various tissues of other animals.

Regulation of gene expression in response to various biological processes, including extracellular stimulation and environmental adaptation, requires nascent mRNA synthesis and translation. Simultaneous analysis of the coordinated regulation of dynamic mRNA synthesis and translation using the same experiment remains a major challenge in the field. Here, we describe a step-by-step protocol for the simultaneous measurement of transcription of nascent mRNA and its translation at the gene level during the acute unfolded protein response (UPR) in HEK293 cells by combining 4-thiouridine metabolic mRNA labeling with translational ribosome affinity purification (TRAP) using a monoclonal antibody against evolutionarily conserved ribosomal P-stalk proteins (P-TRAP). Since P-TRAP captures full-length RNAs bound to ribosomes, it is compatible with 3' mRNA-seq, which analyzes the uridine-rich 3' UTRs of polyadenylated RNAs, allowing robust quantification of T>C conversions. Our nascent P-TRAP (nP-TRAP) method, in which P-TRAP is combined with metabolic mRNA labeling, can serve as a simple and powerful tool to analyze the coordinated regulation of transcription and translation of individual genes in cultured cells.

Single-cell transcriptomic analyses have emerged as very powerful tools to query the gene expression changes at the single-cell level in physiological and pathological conditions. The quality of the analysis is heavily dependent on tissue digestion protocols, with the goal of preserving thousands of single live cells to submit to the subsequent processing steps and analysis. Multiple digestion protocols that use different enzymes to digest the tissues have been described. Harsh digestion can damage certain cell types, but this might be required to digest especially fibrotic tissue as in our experimental condition. In this paper, we summarize a collagenase type I digestion protocol for preparing the single-cell suspension from fibrovascular tissues surgically removed from patients with proliferative diabetic retinopathy (PDR) for single-cell RNA sequencing (scRNA-Seq) analyses. We also provide a detailed description of the data analysis that we implemented in a previously published study.

Candida albicans is the most common human fungal pathogen, able to reside in a broad range of niches within the human body. Even though C. albicans systemic infection is associated with high mortality, the fungus has historically received relatively little attention, resulting in a lack of optimized molecular and fluorescent tools. Over the last decade, some extra focus has been put on the optimization of fluorescent proteins (FPs) of C. albicans. However, as the FPs are GFP-type, they require an aerobic environment and a relatively long period to fully mature. Recently, we have shown the application of a novel type of fluorogen-based FP, with an improved version of fluorescence activating and absorption shifting tag (iFAST), in C. albicans. Due to the dynamic relation between iFAST and its fluorogens, the system has the advantage of being reversible in terms of fluorescence. Furthermore, the combination of iFAST with different fluorogens results in different spectral and cellular properties, allowing customization of the system.