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Current Issue in 2025
Volume: 15, Issue: 21
Biochemistry
Lipid-Mediated Sequential Recruitment of Proteins Via Dual SLIPT and Dual SLIPTNVOC in Live Cells
Cellular phenomena such as signal integration and transmission are based on the correct spatiotemporal organization of biomolecules within the cell. Therefore, the targeted manipulation of such processes requires tools that can precisely induce the localizations and interactions of the key players relevant to these processes with high temporal resolution. Chemically induced dimerization (CID) techniques offer a powerful means to manipulate protein function with high temporal resolution and subcellular specificity, enabling direct control over cellular behavior. Here, we present the detailed synthesis and application of dual SLIPT and dual SLIPTNVOC, which expand the SLIPT (self-localizing ligand-induced protein translocation) platform by incorporating a dual-ligand CID system. Dual SLIPT and dual SLIPTNVOC independently sort into the inner leaflet of the plasma membrane via a lipid-like anchoring motif, where they present the two headgroup moieties trimethoprim (TMP) and HaloTag ligand (HTL), which recruit and dimerize any two iK6eDHFR- and HOB-tagged proteins of interest (POIs). Dual-SLIPTNVOC furthermore enables this protein dimerization of POIs at the inner leaflet of the plasma membrane in a pre-determined order and light-controlled manner. In this protocol, we detail the synthetic strategy to access dual SLIPT and dual SLIPTNVOC, while also providing the underlying rationale for key design and synthetic decisions, with the aim of offering a streamlined, accessible, and broadly implementable methodology. In addition to the detailed synthesis, we present representative applications and typical experimental outcomes and recommend strategies for data analysis to support effective use of the system. Notably, dual SLIPT and dual SLIPTNVOC represent the first CID systems to emulate endogenous lipidation-driven membrane targeting, while retaining hallmark advantages of CID technology—the precision over POI identity, recruitment sequence, high spatiotemporal control, and “plug-and-play” flexibility.
Cluster FLISA—A Method to Compare Protein Expression Efficiency Between Cell Lines and Subunit Clustering of Proteins
Nowadays, recombinant proteins are the focus of various research fields, and their use ranges from therapeutic investigations to cellular model systems for the development of therapeutic approaches. Cell systems used for the expression of recombinant proteins should be comparable in terms of yield and expression efficiency. In many research fields, it is desirable to obtain high protein concentrations. A method that combines an easy workflow with rapid results and affordable costs remains missing, and a standardized approach to determining protein concentration in transgenic cell lines is essential for more reliable data analysis. Our protocol demonstrates the cluster fluorescence-linked immunosorbent assay (FLISA), a technique that allows the exact quantification of comparable protein expression amounts. Moreover, it enables the detection of clustered or bound subunits of a protein without necessitating ultracentrifugation. In the present protocol, we demonstrate the utilization of two transgene cell lines, each expressing distinct recombinant proteins, to provide comparability of protein yields and detectable subunit clustering.
Bioinformatics and Computational Biology
In Silico Prediction and In Vitro Validation of Bacterial Interactions in the Plant Rhizosphere Using a Synthetic Bacterial Community
The rhizosphere, a 2–10 mm region surrounding the root surface, is colonized by numerous microorganisms, known as the rhizosphere microbiome. These microorganisms interact with each other, leading to emergent properties that affect plant fitness. Mapping these interactions is crucial to understanding microbial ecology in the rhizosphere and predicting and manipulating plant health. However, current methods do not capture the chemistry of the rhizosphere environment, and common plant–microbe interaction study setups do not map bacterial interactions in this niche. Additionally, studying bacterial interactions may require the creation of transgenic bacterial lines with markers for antibiotic resistance/fluorescent probes and even isotope labeling. Here, we describe a protocol for both in silico prediction and in vitro validation of bacterial interactions that closely recapitulate the major chemical constituents of the rhizosphere environment using a widely used Murashige & Skoog (MS)-based gnotobiotic plant growth system. We use the auto-fluorescent Pseudomonas, abundantly found in the rhizosphere, to estimate their interactions with other strains, thereby avoiding the need for the creation of transgenic bacterial strains. By combining artificial root exudate medium, plant cultivation medium, and a synthetic bacterial community (SynCom), we first simulate their interactions using genome-scale metabolic models (GSMMs) and then validate these interactions in vitro, using growth assays. We show that the GSMM-predicted interaction scores correlate moderately, yet significantly, with their in vitro validation. Given the complexity of interactions among rhizosphere microbiome members, this reproducible and efficient protocol will allow confident mapping of interactions of fluorescent Pseudomonas with other bacterial strains within the rhizosphere microbiome.
Computational Workflow for Genome-Wide DNA Methylation Profiling and Differential Methylation Analysis
DNA methylation is a crucial epigenetic modification that influences gene expression and plays a role in various biological processes. High-throughput sequencing techniques, such as bisulfite sequencing (BS-seq) and enzymatic methyl sequencing (EM-seq), enable genome-wide profiling of DNA methylation patterns with single-base resolution. In this protocol, we present a bioinformatics pipeline for analyzing genome-wide DNA methylation. We outline the step-by-step process of the essential analyses, including quality control using FASTQ for BS- and EM-seqs raw reads, read alignment with commonly used aligners such as Bowtie2 and BS-Seeker2, DNA methylation calling to generate CGmap files, identification of differentially methylated regions (DMRs) using tools including MethylC-analyzer and HOME, data visualization, and post-alignment analyses. Compared to existing workflows, this pipeline integrates multiple steps into a single protocol, lowering the technical barrier, improving reproducibility, and offering flexibility for both plant and animal methylome studies. To illustrate the application of BS-seq and EM-seq, we demonstrate a case study on analyzing a mutant in Arabidopsis thaliana with a mutation in the met1 gene, which encodes a DNA methyltransferase, and results in global CG hypomethylation and altered gene regulation. This example highlights the biological insights that can be gained through systematic methylome analysis. Our workflow is adaptable to any organism with a reference genome and provides a robust framework for uncovering methylation-associated regulatory mechanisms. All scripts and detailed instructions are provided in GitHub repository: https://github.com/PaoyangLab/Methylation_Analysis.
Biological Engineering
Production of Genetically Engineered Extracellular Vesicles for Targeted Protein Delivery
Extracellular vesicles (EVs) have emerged as promising carriers for the targeted delivery of therapeutic proteins to specific cells. Previously, we demonstrated that genetically engineered EVs can be used for targeted protein delivery. This protocol details the generation of mannose receptor (CD206)-targeted EVs using a modular plasmid system optimized for production in HEK293T cells. Three plasmids enable customizable EV budding, cargo loading, and surface modification for targeting to antigen-presenting cells (APCs). EVs are isolated via differential centrifugation and chromatography, characterized using transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA), and validated through functional uptake assays in primary human activated dendritic cells. Our approach combines flexibility in engineering required EVs with robust, reproducible isolation and characterization workflows. Its modularity allows easy adaptation to alternative targets or cargoes, which can be validated immediately through in vitro testing.
Cell Biology
Protocol for the Isolation and Analysis of Extracellular Vesicles From Peripheral Blood: Red Cell, Endothelial, and Platelet-Derived Extracellular Vesicles
This protocol describes the isolation and flow cytometric analysis of extracellular vesicles (EVs) derived from red blood cells, endothelial cells, and platelets in human peripheral blood. The protocol includes steps for preparing platelet-free plasma, fluorescent antibody staining, gating strategies, and technical controls. This protocol was developed within a study on EV release in snakebite-associated thrombotic microangiopathy; the protocol addresses challenges such as variable autofluorescence and heterogeneity in EV origin. It is flexible and can be adapted for alternative antibody panels targeting different cell populations or EV subtypes, including leukocyte-derived EVs.
Medicine
A Quantitative Spectrophotometric Assay Matched With Environmental Scanning Electron Microscopy to Measure Calcium Crystals in Human Osteoarthritic Synovial Fluid
In the field of osteoarthritis (OA), the identification of reliable diagnostic and prognostic biomarkers in patients with hip lesions such as femoroacetabular impingement (FAI) could have an immeasurable value. Calcium crystal detection in synovial fluids (SFs) is one tool currently available to diagnose patients with rheumatologic disorders. Crystals, such as monosodium urate (MSU) and calcium pyrophosphate (CPP), are identified qualitatively by compensated polarized light, whereas basic calcium phosphate (BCP) crystals are visualized under conventional light microscopy by Alizarin red S (ARS) staining. Here, we present an efficient and straightforward protocol to quantify calcium crystals by spectrophotometric analysis in human osteoarthritic SFs after staining with ARS. The type and size of the different crystal species are confirmed by environmental scanning electron microscopy (ESEM).
Microbiology
Optimized Protocol for the Collection, Cryopreservation, and In Vitro Cultivation of Human Gut Microbiota for Toxicomicrobiomics Applications
Xenobiotics, including environmental pollutants such as bisphenols, phthalates, and parabens, are widely present in food, cosmetics, packaging, and water. These compounds can reach the gastrointestinal tract and interact with the gut microbiota (GM), a complex microbial community that plays a key role in host immunity, metabolism, and barrier function. The GM engages in bidirectional communication with the host via the production of bioactive metabolites, including short-chain fatty acids, neurotransmitter precursors, and bile acid derivatives. Dysbiosis induced by xenobiotics can disrupt microbial metabolite production, impair gut barrier integrity, and contribute to the development of systemic disorders affecting distant organs such as the liver or brain. On the other hand, the GM can biotransform xenobiotics into metabolites with altered bioactivity or toxicity. In vitro models of the human GM offer a valuable tool to complement population-based and in vivo studies, enabling controlled investigation of causative effects and underlying mechanisms. Here, we present an optimized protocol for the collection, cryopreservation, and cultivation of human GM under strictly anaerobic conditions for toxicomicrobiomics applications. The method allows the assessment of xenobiotic–GM interactions in a cost-effective and ethically sustainable way. It is compatible with a wide range of downstream applications, including 16S rDNA sequencing, metabolomics, and endocrine activity assays. The protocol has been optimized to minimize oxygen exposure to less than 2 min, ensuring the viability of obligate anaerobes that dominate the gut ecosystem. This approach facilitates reproducible, mechanistic studies on the impact of environmental xenobiotics on human GM.
A Practical CRISPR-Based Method for Rapid Genome Editing in Caulobacter crescentus
The RNA-guided Cas enzyme specifically cuts chromosomes and introduces a targeted double-strand break, facilitating multiple kinds of genome editing, including gene deletion, insertion, and replacement. Caulobacter crescentus and its relatives, such as Agrobacterium fabrum and Sinorhizobium meliloti, have been widely studied for industrial, agricultural, and biomedical applications; however, their genetic manipulations are usually characterized as time-consuming and labor-intensive. C. crescentus and its relatives are known to be CRISPR/Cas-recalcitrant organisms due to intrinsic limitations of SpCas9 expression and possible CRISPR escapes. By fusing a reporting gene to the C terminus of SpCas9M and precisely manipulating the expression of SpCas9M, we developed a CRISPR/SpCas9M-reporting system and achieved efficient genome editing in C. crescentus and relatives. Here, we describe a protocol for rapid, marker-less, and convenient gene deletion by using the CRISPR/SpCas9M-reporting system in C. crescentus, as an example.
Molecular Biology
Library Preparation for Genome-Wide DNA Methylation Profiling
DNA methylation is a fundamental epigenetic mark with critical roles in epigenetic regulation, development, and genome stability across diverse organisms. Whole genome bisulfite sequencing (WGBS) enables single-base resolution mapping of cytosine methylation patterns and has become a standard method in epigenomics. This protocol provides a detailed, step-by-step workflow for WGBS library construction starting from genomic DNA. It includes steps of RNaseA treatment, DNA shearing, end-repair and A-tailing, adapter ligation, bisulfite conversion, library amplification, and quantification. Notably, the method uses self-prepared reagents and customizable index systems, avoiding the constraints of commercial library preparation kits. This flexibility supports cost-effective, scalable methylome profiling, suitable for diverse experimental designs, including high-throughput multiplexed sequencing.
Examining the Roles of m6A Sites in mRNA Using the Luciferase Gene Fused With Mutated RRACH Motifs
N6-methyladenosine (m6A) is the most abundant internal modification in mRNA and is regulated primarily by the balance between the METTL3 methylase complex and two demethylases, FTO (fat mass and obesity-associated protein) and ALKBH5 (α-ketoglutarate-dependent dioxygenase alkB homolog). Reflecting this prevalence, m6A participates in virtually every step of RNA metabolism, influencing a wide range of physiological and pathological processes. The first step in studying m6A is genome-wide mapping, typically performed by m6A-seq, which sequences RNA fragments immunoprecipitated with an m6A-specific antibody. This is followed by identification of RRACH motifs (R = A or G; H = A, C, or U) within these sequences, with m6A being located at the third nucleotide. The second step involves mutating the putative m6A sites to establish a causal link between the modification and downstream biological effects. Since the mapping step has been covered in several detailed protocols, this article focuses on the second step—mutagenesis of RRACH motifs and subsequent functional analysis of the mutations by ectopic expression. The 3′ untranslated region (UTR) of the mouse Runx2 gene is used as an example. The mutant and wild-type sequences are inserted into a luciferase reporter vector and transfected into 293FT cells to evaluate how loss of m6A affects luciferase protein levels. The same reporter plasmids are also used in an RNA stability assay with a transcription inhibitor. Although site-specific demethylation of endogenous mRNA would be preferable, it remains technically challenging despite many attempts. Thus, ectopic expression of the mutated target gene remains a widely used and practical alternative.
Neuroscience
Generation of 3D Human iPSC-Derived Multi-Cell Type Neurospheres for Studying Neuron, Astrocyte, and Microglia Crosstalk
Three-dimensional (3D) human brain tissue models derived from induced pluripotent stem cells (iPSCs) have transformed the study of neural development and disease in vitro. While cerebral organoids offer high structural complexity, their large size often leads to necrotic core formation, limiting reproducibility and challenging the integration of microglia. Here, we present a detailed, reproducible protocol for generating multi-cell type 3D neurospheres that incorporate neurons, astrocytes, and optionally microglia, all derived from the same iPSCs. While neurons and astrocytes differentiate spontaneously from neural precursor cells, generated by dual SMAD-inhibition (blocking BMP and TGF-b signaling), microglia are generated in parallel and can infiltrate the mature neurosphere tissue after plating neurospheres into 48-well plates. The system supports a range of downstream applications, including functional confocal live imaging of GCaMP6f after adeno-associated virus (AAV) transduction of neurospheres or immunofluorescence staining after fixation. Our approach has been successfully implemented across multiple laboratories, demonstrating its robustness and translational potential for studying neuron–glia interactions and modeling neurodegenerative processes.
Labeling Postsynaptic Densities for Super-Resolution Microscopy With Minimal Signal-Loss and Offset
Accurate labeling of excitatory postsynaptic sites remains a major challenge for high-resolution imaging due to the dense and sterically restricted environment of the postsynaptic density (PSD). Here, we present a protocol utilizing Sylites, 3 kDa synthetic peptide probes that bind with nanomolar affinity to key postsynaptic markers, PSD-95 and Gephyrin. eSylites (excitatory Sylites) specifically target the PDZ1 and PDZ2 domains of PSD-95, enabling precise and efficient labeling of excitatory postsynaptic density (ePSD). In contrast, iSylites (inhibitory Sylites) bind to the dimerizing E-domain of the Gephyrin C-terminus, allowing selective visualization of inhibitory postsynaptic density (iPSD). Their small size reduces linkage error and enhances accessibility compared to conventional antibodies, enabling clear separation of PSD-95 nanodomains in super-resolution microscopy. The protocol is compatible with co-labeling using standard antibodies and integrates seamlessly into multichannel immunocytochemistry workflows for primary neurons and brain tissue. This method enables robust, reproducible labeling of excitatory synapses with enhanced spatial resolution and can be readily adapted for expansion microscopy or live-cell applications.
Optogenetic Approach for Investigating Descending Control of Nociception in Ex Vivo Spinal Cord Preparation
Nociception is critically shaped by descending modulation of spinal circuits, yet its cellular and synaptic mechanisms remain poorly defined. Elucidating these mechanisms is technically challenging, as it requires simultaneous activation of primary afferents and descending fibers while monitoring the functioning of individual spinal neurons. Here, we present a method to investigate the influence of the rostral ventromedial medulla (RVM), a principal supraspinal structure mediating descending modulation, on the activity of spinal lamina I neurons. Our approach combines electrophysiological recordings in ex vivo intact spinal cord preparation with optogenetics, granting several advantages. First, ex vivo preparation spares rostrocaudal and mediolateral spinal architecture, preserving lamina I as well as primary afferent and descending inputs. Second, virally mediated channelrhodopsin-2 (ChR2) expression enables selective photostimulation of RVM-originating fibers. When coupled with patch-clamp recordings, this photostimulation allows identifying postsynaptic inputs from RVM to spinal neurons and revealing RVM-dependent presynaptic inhibition of primary afferent inputs. Overall, our approach is well-suited for investigating both pre- and postsynaptic mechanisms of descending modulation in physiological and pathological pain conditions.
Colocalizing Telomeres With PML or γH2AX Foci by IF-FISH in Mouse Brain Neurons
Telomere length maintenance is strongly linked to cellular aging, as telomeres progressively shorten with each cell division. This phenomenon is well-documented in mitotic, or dividing, cells. However, neurons are post-mitotic and do not undergo mitosis, meaning they lack the classical mechanisms through which telomere shortening occurs. Despite this, neurons retain telomeres that protect chromosomal ends. The role of telomeres in neurons has gained interest, particularly in the context of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), where aging is a major risk factor. This has sparked interest in investigating telomere maintenance mechanisms in post-mitotic neurons. Nevertheless, most existing telomere analysis techniques were developed for and optimized using mitotic cells, posing challenges for studying telomeres in non-dividing neuronal cells. Thus, this protocol adapts an already established technique, the combined immunofluorescence and telomere fluorescent in situ hybridization (IF-FISH) on mitotic cells to study the processes occurring at telomeres in cortical neurons of the mouse ALS transgenic model, TDP-43 rNLS. Specifically, it determines the occurrence of DNA damage and the alternative lengthening of telomeres (ALT) mechanism through simultaneous labeling of the DNA damage marker, γH2AX, or the ALT marker, promyelocytic leukemia (PML) protein, together with telomeres. Therefore, the protocol enables the visualization of DNA damage (γH2AX) or the ALT marker (PML) concurrently with telomeres. This technique can be successfully applied to brain tissue and enables the investigation of telomeres specifically in cortical neurons, rather than in bulk tissue, offering a significant advantage over Southern blot or qPCR-based techniques.
Plant Science
A Reliable In Planta Inoculation and Antifungal Screening Protocol for Rhizoctonia solani-Induced Sheath Blight in Rice
Sheath blight, caused by Rhizoctonia solani, is a major fungal disease of rice that leads to significant yield losses globally. Conventional inoculation methods often fail to achieve consistent and uniform infection, limiting their applicability in antifungal screening studies. This protocol describes a reliable in planta inoculation method for R. solani using mature sclerotia placed at the internodal region of tillering-stage rice seedlings. The procedure includes step-by-step instructions for seed germination, seedling preparation, pathogen culture, artificial inoculation, and post-infection application of antifungal treatments, including botanical compounds such as Ocimum gratissimum essential oil and thymol. Lesion development is monitored and quantified over time, and data are analyzed statistically to evaluate treatment efficacy. The protocol is optimized for reproducibility, scalability, and compatibility with sustainable disease management approaches. It provides a robust platform for evaluating antifungal agents in a biologically relevant and controlled environment.
Live-Cell Monitoring of Piecemeal Chloroplast Autophagy
When plants undergo senescence or experience carbon starvation, leaf cells degrade proteins in the chloroplasts on a massive scale via autophagy, an evolutionarily conserved process in which intracellular components are transported to the vacuole for degradation to facilitate nutrient recycling. Nonetheless, how portions of chloroplasts are released from the main chloroplast body and mobilized to the vacuole remains unclear. Here, we developed a method to observe the autophagic transport of chloroplast proteins in real time using confocal laser-scanning microscopy on transgenic plants expressing fluorescently labeled chloroplast components and autophagy-associated membranes. This protocol enabled us to track changes in chloroplast morphology during chloroplast-targeted autophagy on a timescale of seconds, and it could be adapted to monitor the dynamics of other intracellular processes in plant leaves.