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Past Issue in 2025
Volume: 15, Issue: 3
Biochemistry
Determination of Dissociation Constants for the Interaction of Myosin-5a with its Cargo Protein Using Microscale Thermophoresis (MST)
Myosin-5a (Myo5a) is an actin-dependent molecular motor that recognizes a diverse range of cargo proteins through its tail domain, playing a crucial role in the transport and localization of various organelles within the cell. We have identified a new interaction between Myo5a and its cargo protein melanophilin (Mlph), i.e., the interaction between the middle tail domain of Myo5a (Myo5a-MTD) and the actin-binding domain of Mlph (Mlph-ABD), by GST pulldown assay. We then intend to obtain the dissociation constant between Myo5a-MTD and Mlph-ABD using isothermal titration calorimetry (ITC) or microscale thermophoresis (MST), both of which are two commonly used methods for determining quantitative data on protein interactions. The advantages of MST over ITC include less protein usage, shorter operation time, and higher sensitivity. In this protocol, we present a method for using MST to determine the dissociation constants of Myo5a-MTD and Mlph-ABD, which were purified through overexpression in bacteria using affinity chromatography. The dissociation constant values obtained directly reflect the binding strength between these two proteins and provide a foundation for the isolation and purification of the complex in the future.
Cell-Sonar, an Easy and Low-cost Method to Track a Target Protein by Expression Changes of Specific Protein Markers
Different research methods aim to clarify the intracellular trafficking of target proteins or unknown pathways. Currently, existing methods are mostly complex and expensive, requiring expert knowledge. Detailed microscopy for protein co-localization detection or omic technologies, which provide holistic network data, are elaborate, mostly complex, and expensive to apply. Our protocol illustrates a method to track a target protein by detecting expression changes of user-selected marker proteins that directly or indirectly interact with the target. Modulation of protein expression indicates interactions between the target and marker protein. Even without co-localization analysis, the results of the protein expression change are the first insights into the target's fate. Moreover, the use of the cell-sonar is straightforward and affordable, and the results are rapidly available. Furthermore, this method could also be used to determine if and how pathways are affected by compounds added to the cells. In conclusion, our method is adaptable to a wide range of proteins, easy to apply, inexpensive, and expandable with substances that affect proteins.
Bioinformatics and Computational Biology
Optimal Dual RNA-Seq Mapping for Accurate Pathogen Detection in Complex Eukaryotic Hosts
Dual RNA-Seq technology has significantly advanced the study of biological interactions between two organisms by allowing parallel transcriptomic analysis. Existing analysis methods employ various combinations of open-source bioinformatics tools to process dual RNA-Seq data. Upon reviewing these methods, we intend to explore crucial criteria for selecting standard tools and methods, especially focusing on critical steps such as trimming and mapping reads to the reference genome. In order to validate the different combinatorial approaches, we performed benchmarking using top-ranking tools and a publicly available dual RNA-Seq Sequence Read Archive (SRA) dataset. An important observation while evaluating the mapping approach is that when the adapter trimmed reads are first mapped to the pathogen genome, more reads align to the pathogen genome than the unmapped reads derived from the traditional host-first mapping approach. This mapping method prevents the misalignment of pathogen reads to the host genome due to their shorter length. In this way, the pathogenic read information found at lesser proportions in a complex eukaryotic dataset is precisely obtained. This protocol presents a comprehensive comparison of these possible approaches, resulting in a robust unified standard methodology.
Model Architecture Analysis and Implementation of TENET for Cell–Cell Interaction Network Reconstruction Using Spatial Transcriptomics Data
Cellular communication relies on the intricate interplay of signaling molecules, which come together to form the cell–cell interaction (CCI) network that orchestrates tissue behavior. Researchers have shown that shallow neural networks can effectively reconstruct the CCI from the abundant molecular data captured in spatial transcriptomics (ST). However, in scenarios characterized by sparse connections and excessive noise within the CCI, shallow networks are often susceptible to inaccuracies, leading to suboptimal reconstruction outcomes. To achieve a more comprehensive and precise CCI reconstruction, we propose a novel method called triple-enhancement-based graph neural network (TENET). The TENET framework has been implemented and evaluated on both real and synthetic ST datasets. This protocol primarily introduces our network architecture and its implementation.
Biological Engineering
Rapid Sampling of Large Quantities of Interstitial Fluid from Human Skin Using Microneedles and a Vacuum-assisted Skin Patch
Interstitial fluid (ISF) is a promising diagnostic sample due to its extensive biomolecular content while being safer and less invasive to collect than blood. However, existing ISF sampling methods are time-consuming, require specialized equipment, and yield small amounts of fluid (
Biophysics
High-resolution Cryo-EM Structure Determination of a-Synuclein—A Prototypical Amyloid Fibril
The physiological role of a-synuclein (a-syn), an intrinsically disordered presynaptic neuronal protein, is believed to impact the release of neurotransmitters through interactions with the SNARE complex. However, under certain cellular conditions that are not well understood, a-syn will self-assemble into β-sheet-rich fibrils that accumulate and form insoluble neuronal inclusions. Studies of patient-derived brain tissues have concluded that these inclusions are associated with Parkinson’s disease, the second most common neurodegenerative disorder, and other synuclein-related diseases called synucleinopathies. In addition, repetitions of specific mutations to the SNCA gene, the gene that encodes a-syn, result in an increased disposition for synucleinopathies. The latest advances in cryo-EM structure determination and real-space helical reconstruction methods have resulted in over 60 in vitro structures of a-syn fibrils solved to date, with a handful of these reaching a resolution below 2.5 Å. Here, we provide a protocol for a-syn protein expression, purification, and fibrilization. We detail how sample quality is assessed by negative stain transmission electron microscopy (NS-TEM) analysis and followed by sample vitrification using the Vitrobot Mark IV vitrification robot. We provide a detailed step-by-step protocol for high-resolution cryo-EM structure determination of a-syn fibrils using RELION and a series of specialized helical reconstruction tools that can be run within RELION. Finally, we detail how ChimeraX, Coot, and Phenix are used to build and refine a molecular model into the high-resolution cryo-EM map. This workflow resulted in a 2.04 Å structure of a-syn fibrils with excellent resolution of residues 36–97 and an additional island of density for residues 15–22 that had not been previously reported. This workflow should serve as a starting point for individuals new to the neurodegeneration and structural biology fields. Together, this procedure lays the foundation for advanced structural studies of a-syn and other amyloid fibrils.
Cancer Biology
Real-time IncuCyte® Assay for the Dynamic Assessment of Live and Dead Cells in 2D Cultures
Cell viability and cytotoxicity assays are commonly used to investigate protein function and to evaluate drug efficacy in cancer and other disease models. Cytotoxicity is the measure of dead or damaged cells and is often quantified using assays based on cellular characteristics such as membrane integrity or mitochondrial metabolism. However, these assays are typically limited to endpoint analysis and lack emulation of physiological conditions. The IncuCyte Live and Dead Cell assay described here leverages common cell permeability methodologies but uses fluorescence microscopy channels to image both live and dead cells over time and phase microscopy channels to measure confluency. Cytotox green reagent is a cell membrane–impermeable dye that can only be taken up by cells with poor cell membrane integrity. NucLight rapid red dye is a cell membrane–permeable nuclear dye that can be taken up by all cells. Based on dye uptake and fluorescence intensity, the IncuCyte software can be used to analyze images for live and dead cell detection and quantification. Phase microscopy is used to determine confluency and can be further quantified using the IncuCyte software. We provide an application of this assay, using it to calculate IC50 and EC50 values for the assessment of drug efficacy.
Profiling the Secretome of Glioblastoma Cells Under Histone Deacetylase Inhibition Using Mass Spectrometry
Glioblastoma (GBM) is the most aggressive brain tumor, and different efforts have been employed in the search for new drugs and therapeutic protocols for GBM. A label-free, mass spectrometry–based quantitative proteomics has been developed to identify and characterize proteins that are differentially expressed in GBM to gain a better understanding of the interactions and functions that lead to the pathological state focusing on the extracellular matrix (ECM). The main challenge in GBM research has been to identify novel molecular therapeutic targets and accurate diagnostic/prognostic biomarkers. To better investigate the GBM secretome upon in vitro treatment with histone deacetylase inhibitor (iHDAC), we employed a high-throughput label-free methodology of protein identification and quantification based on mass spectrometry followed by in silico studies. Our analysis revealed significant changes in the ECM protein profile, particularly those associated with the angiogenic matrisome. Proteins such as decorin, ADAM10, ADAM12, and ADAM15 were differentially regulated upon in silico analysis. In contrast, key angiogenesis markers such as VEGF and ECM proteins like fibronectin and integrins did not display significant changes. These results suggest that iHDAC inhibitors may modulate or suppress tumor behavior growth by targeting ECM proteins’ secretion rather than directly inhibiting angiogenesis.
Immunology
Using the Sleeping Beauty Transposon System for Doxycycline-inducible Gene Expression in RAW264.7 Macrophage Cells to Study Phagocytosis
Macrophages are known for engulfing and digesting pathogens and dead cells through a specialized form of endocytosis called phagocytosis. Unfortunately, many macrophage cell lines are refractory to most reagents used for transient transfections. Alternative transient approaches, such as electroporation or transduction with lentiviral vectors, typically cause cell death (electroporation) or can be time-consuming to generate numerous lentivirus when using different genes of interest. Therefore, we use the Sleeping Beauty system to generate stably transfected cells. The system uses a “resurrected” transposase gene named Sleeping Beauty found in salmonid fish. Experimentally, the system introduces two plasmids: one carrying the Sleeping Beauty transposase and the other with an integration cassette carrying the gene of interest, a reverse-doxycycline controlled repressor gene, and an antibiotic resistance gene. The construct used in this protocol provides puromycin resistance. Stable integrations are selected by culturing the cells in the presence of puromycin, and further enrichment can be obtained using fluorescence-activated cell sorting (FACS). In this protocol, we use the Sleeping Beauty transposon system to generate RAW264.7 cells with doxycycline-inducible inositol polyphosphate 4-phosphatase B containing a C-terminal CaaX motif (INPP4B-CaaX). INPP4B-CaaX dephosphorylates the D-4 position of phosphatidylinositol 3,4-bisphosphate and inhibits phagocytosis. One benefit is that generating stable cell lines is substantially faster than selecting for random integrations. Without FACS, the method typically gives ~50% of the cells that are transfected; with sorting, this approaches 100%. This makes phagocytosis experiments easier since more cells can be analyzed per experiment, allowing for population-based measurements where a ~10% transient transfection rate is insufficient. Finally, using the doxycycline-promoter allows for low near endogenous expression of proteins or robust overexpression.
Microbiology
Identification of Mycobacterium tuberculosis and its Drug Resistance by Targeted Nanopore Sequencing Technology
Tuberculosis (TB) remains the leading cause of human mortality in infectious diseases. Drug-resistant TB, particularly multidrug-resistant TB and extensively drug-resistant TB, poses a pressing clinical and public health challenge. The main causative agents of TB are known as Mycobacterium tuberculosis (MTB), which exhibits a highly complex drug resistance profile. Traditional culture-based phenotypic drug susceptibility testing is time-consuming, and PCR-based assays are restricted to detecting known mutational hotspots. In this study, we present a protocol leveraging high-throughput nanopore sequencing technology in conjunction with multiplex PCR, termed targeted nanopore sequencing, for the identification of MTB and analysis of its drug resistance. Our method for MTB drug resistance assessment offers the benefits of being culture-free, efficient, high-throughput, and highly accurate, which could significantly aid in clinical patient management and the control of TB infections.
Development and Validation of Chlamydia muridarum Mouse Models for Studying Genital Tract Infection Pathogenesis
Animal infection models play significant roles in the study of bacterial pathogenic mechanisms and host–pathogen interactions, as well as in evaluating drug and vaccine efficacies. Chlamydia trachomatis is responsible for infections in various mucosal tissues, including the eyes and urogenital, respiratory, and gastrointestinal tracts. Chronic infections can result in severe consequences such as trachoma-induced blindness, ectopic pregnancy, and infertility. While intravaginal inoculation of C. muridarum mimics the natural route of sexual transmission between individuals, transcervical inoculation allows the organisms to directly infect endometrial epithelial cells without interference from host responses triggered by chlamydial contact or infection of vaginal and cervical cells. Therefore, in this study, we used mouse models to visualize pathologies in both the endometrium and oviduct following C. muridarum inoculation.
Molecular Biology
Versatile Click Chemistry-based Approaches to Illuminate DNA and RNA G-Quadruplexes in Human Cells
The existence and functional relevance of DNA and RNA G-quadruplexes (G4s) in human cells is now beyond debate, but how did we reach such a level of confidence? Thanks to a panoply of molecular tools and techniques that are now routinely implemented in wet labs. Among them, G4 imaging ranks high because of its reliability and practical convenience, which now makes cellular G4 detection quick and easy; also, because this technique is sensitive and responsive to any G4 modulations in cells, which thus allows gaining precious insights into G4 biology. Herein, we briefly explain what a G4 is and how they can be visualized in human cells; then, we present the strategy we have been developing for several years now for in situ click G4 imaging, which relies on the use of biomimetic G4 ligands referred to as TASQs (for template-assembled synthetic G-quartets) and is far more straightforward and modular than classically used immunodetection methods. We thus show why and how to illuminate G4s with TASQs and provide a detailed, step-by-step methodology (including the preparation of the materials, the methodology per se, and a series of notes to address any possible pitfalls that may arise during the experiments) to make G4 imaging ever easier to operate.
Protocol to Identify Unknown Flanking DNA Using Partially Overlapping Primer-based PCR for Genome Walking
Genome walking is a popular molecular technique for accessing unknown flanking DNAs, which has been widely used in biology-related fields. Herein, a simple but accurate genome-walking protocol named partially overlapping primer (POP)-based PCR (POP-PCR) is described. This protocol exploits a POP set of three POPs to mediate genome walking. The three POPs have a 10 nt 3' overlap and 15 nt heterologous 5' regions. Therefore, a POP can partially anneal to the previous POP site only at a relatively low temperature (approximately 50 °C). In primary POP-PCR, the low-temperature (25 °C) cycle allows the primary POP to partially anneal to site(s) of an unknown flank and many sites of the genome, synthesizing many single-stranded DNAs. In the subsequent high-temperature (65 °C) cycle, the target single-stranded DNA is converted into double-stranded DNA by the sequence-specific primer, attributed to the presence of this primer complement, while non-target single-stranded DNA cannot become double-stranded because it lacks a binding site for both primers. As a result, only the target DNA is amplified in the remaining 65 °C cycles. In secondary or tertiary POP-PCR, the 50 °C cycle directs the POP to the previous POP site and synthesizes many single-stranded DNAs. However, as in the primary PCR, only the target DNA can be amplified in the subsequent 65 °C cycles. This POP-PCR protocol has many potential applications, such as screening microbes, identifying transgenic sites, or mining new genetic resources.
Neuroscience
Isolation of Intact Mitochondria From Drosophila melanogaster and Assessment of Mitochondrial Respiratory Capacity Using Seahorse Analyzer
Analysis of mitochondrial function has broad applicability in many research specialties. Neurodegenerative disorders such as chemotherapy-induced peripheral neuropathy (CIPN) often exhibit damaged mitochondria or reduced mitochondrial respiratory capacity. Isolation of intact mitochondria for protein analysis or respiration measurements has been previously reported in numerous model organisms. Here, we describe an adaptation of previous protocols to isolate intact functional mitochondria from Drosophila melanogaster for use in a model of CIPN. Whole Drosophila are ground in isolation buffer, and mitochondria are purified using differential centrifugation through a sucrose and mannitol solution. The intact mitochondria are plated as a monolayer for measurements of mitochondrial oxygen consumption rates and response to inhibitor compounds on an Agilent Seahorse analyzer. This experimental protocol is quick and yields a purified population of intact mitochondria that may be used for functional assays for several hours after isolation. The isolated mitochondria may be used for respiration measurements, which reflect their health, and stored for protein or genetic analysis. Mitochondrial populations from multiple strains or treatment groups can be easily compared simultaneously. The rapid biochemical assessment of mitochondria, in combination with the utility of Drosophila as an in vivo genetic model system, offers great potential for researchers to probe the impact of genetics and pharmacologic interventions on mitochondrial respiratory capacity.
Quantitative, Dynamic Detection of Neuronal Na+ Transients Using Multi-photon Excitation and Fluorescence Lifetime Imaging (FLIM) in Acute Mouse Brain Slices
Fluorescence lifetime imaging microscopy (FLIM) is a highly valuable technique in the fluorescence microscopy toolbox because it is essentially independent of indicator concentrations. Conventional fluorescence microscopy analyzes changes in emission intensity. In contrast, FLIM assesses the fluorescence lifetime, which is defined as the time a fluorophore remains in an excited state before emitting a photon. This principle is advantageous in experiments where fluorophore concentrations are expected to change, e.g., due to changes in cell volume. FLIM, however, requires collecting a substantial number of photons to accurately fit distribution plots, which constrains its ability for dynamic imaging. This limitation has recently been overcome by rapidFLIM, which utilizes ultra-low dead-time photodetectors in conjunction with sophisticated rapid electronics. The resulting reduction in dead-time to the picosecond range greatly enhances the potential for achieving high spatio-temporal resolution. Here, we demonstrate the use of multi-photon-based rapidFLIM with the sodium indicator ION NaTRIUM Green-2 (ING-2) for the quantitative, dynamic determination of Na+ concentrations in neurons in acute rodent brain tissue slices. We describe the loading of the dye into neurons and present a procedure for its calibration in situ. We show that rapidFLIM not only allows the unbiased determination of baseline Na+ concentrations but also allows dynamic imaging of changes in intracellular Na+, e.g., induced by inhibition of cellular ATP production. Overall, rapidFLIM, with its greatly improved signal-to-noise ratio and higher spatio-temporal resolution, will also facilitate dynamic measurements using other FLIM probes, particularly those with a low quantum yield.
Identification of Neurons Containing Calcium-Permeable AMPA and Kainate Receptors Using Ca2+ Imaging
Calcium-permeable AMPA receptors (CP-AMPARs) and kainate receptors (CP-KARs) play crucial roles in synaptic plasticity and are implicated in various neurological processes. Current methods for identifying neurons expressing these receptors, such as electrophysiological recordings and immunostaining, have limitations in throughput or inability to distinguish functional receptors. This protocol describes a novel approach for the vital identification of neurons containing CP-AMPARs and CP-KARs using calcium imaging. The method involves loading neurons with Fura-2 AM, a calcium-sensitive fluorescent probe, KCl application to identify all neurons, and further addition of specific AMPAR agonists (e.g., 5-fluorowillardiine) in the presence of voltage-gated calcium channel blockers and NMDAR/KAR antagonists to identify CP-AMPAR-containing neurons. CP-KAR-containing neurons are identified using domoic acid applications in the presence and absence of NASPM (a CP-AMPAR antagonist). This technique offers several advantages over existing methods, including the ability to assess large neuronal populations simultaneously, distinguish between different receptor types, and provide functional information about CP-AMPAR and CP-KAR expression in living neurons, making it a valuable tool for studying synaptic plasticity and neurological disorders.
Detection of Amylin-β-amyloid Hetero-Oligomers by Enzyme-Linked Immunosorbent Assay
Amylin is an amyloidogenic neuroendocrine hormone co-synthesized and co-secreted with insulin from the pancreas. It readily crosses the blood–brain barrier and synergistically forms mixed amyloid plaques with β-amyloid (Aβ) in brain parenchyma. Parenchymal amylin-Aβ plaques are found in both sporadic and early-onset familial Alzheimer’s disease (AD), yet their (patho)physiological role remains elusive, particularly due to a lack of detection modalities for these mixed plaques. Previously, we developed an enzyme-linked immunosorbent assay (ELISA) capable of detecting amylin-Aβ hetero-oligomers in brain lysate and blood using a polyclonal anti-amylin antibody to capture hetero-oligomers and a monoclonal anti-Aβ mid-domain detection antibody combination. This combination allows for the recognition of distinct amylin epitopes, which remain accessible after amylin-Aβ oligomerization has begun, and precise detection of Aβ epitopes available after oligomer formation. The utility of this assay is evidenced in our previous report, wherein differences in hetero-oligomer content in brain tissue from patients with and without AD and patients with and without diabetes were distinguished. Additionally, using AD model rats, we provided evidence that our assay can be employed for the detection of amylin-Aβ in blood. This assay and protocol are important innovations in the field of AD research because they meet an unmet need to detect mixed amyloid plaques that, if targeted therapeutically, could reduce AD progression and severity.
Plant Science
Confocal Live Imaging of Reproductive Organs Development in Arabidopsis
Understanding how multicellular organisms are shaped requires high-resolution, quantitative data to unravel how biological structures grow and develop over time. In recent years, confocal live imaging has become an essential tool providing insights into developmental dynamics at cellular resolution in plant organs such as leaves or meristems. In the context of flowers, growth tracking has primarily been limited to sepals, the outermost floral organs, or the post-fertilization gynoecium, which are easily accessible for microscopy. Here, we describe a detailed pipeline for the preparation, dissection, and confocal imaging of the development of internal reproductive floral organs of Arabidopsis thaliana including both the stamen and gynoecium. We also discuss how to acquire high-quality images suitable for efficient 2D and 3D segmentation that allow the quantification of cellular dynamics underlying their development.
An Effective and Safe Maize Seed Chipping Protocol Using Clipping Pliers With Applications in Small-Scale Genotyping and Marker-Assisted Breeding
In applications such as marker-assisted breeding and positional cloning, tissue sampling and plant tracking are vital steps in the genotyping pipeline. They enable the identification of desirable seedlings, saving time and reducing the cost, space, and handling required for growing adult plants, especially for greenhouses and winter nurseries. Small-scale marker-assisted selection laboratories rely heavily on leaf-based genotyping, which involves over-planting large, segregating populations followed by leaf sampling, genotyping, and backtracking to identify desired individuals, which is costly and laborious. Thus, there is a need to adopt seed-based genotyping to reduce costs and save time. Therefore, we developed a safe and cheap seed-chipping protocol using clipping pliers to chip seeds to genotype before planting. To identify a cost-effective and high-throughput DNA extraction method, we tested four extraction methods and assessed the quality of the seed DNA using PCR. For three of the methods, seed-based DNA was of comparable quality to DNA extracted from leaf punches. We also compared seed- and leaf-derived DNA from the same individuals in a segregating population to test for genotyping miscalls that could arise due to the presence of maternally derived pericarp in the seed samples. Out of 43 potential instances, we found zero miscalled samples and, therefore, no evidence supporting consequential pericarp inclusion. Germination rates of chipped and unchipped seeds were the same for the inbreds tested, B73 and Mo17. However, chipped seeds grew slower until ~14 days after sowing. Overall, seed sampling using clipping pliers provides a simple, reliable, and high-throughput method to identify specific genotypes before planting.
Flood Inoculation of Fusarium eumartii in Tomato Seedlings: Method for Evaluating the Infectivity of Pathogen Spores
The Fusarium genus includes various fungi of great significance in agriculture. Fusarium solani f. sp. eumartii (F. eumartii), traditionally known as a potato pathogen, has also been identified as a cause of disease in tomatoes. This protocol provides a detailed, efficient, and robust flood-inoculation method for assessing F. eumartii infection of young tomato seedlings grown on MS medium plates. It includes the evaluation of the lesion area and the quantification of the remaining fungal inoculum in tomato seedlings. In summary, the straightforwardness and efficiency of this bioassay make it a powerful quantitative tool for selecting fungicidal compounds or defense response inducers in tomato plants, offering a promising approach with significant potential for preventing fungal diseases in crops.