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Past Issue in 2025
Volume: 15, Issue: 7
Biochemistry
Ex Vivo Measurement of Stable Isotope-Labeled Fatty Acid Incorporation Into Phospholipids in Isolated Mice Muscle
With the advancement of liquid chromatography–mass spectrometry (LC–MS/MS), the quantification of glycerophospholipid (PL) molecules has become more accessible, leading to the discovery of numerous enzymes responsible for determining the acyl groups attached to these molecules. Metabolic tracer experiments using radioisotopes and stable isotopes are powerful tools for defining the function of metabolic enzymes and metabolic flux. We have established an ex vivo muscle experimental system using stable isotope–labeled fatty acids to evaluate fatty acid incorporation into PL molecules. Here, we describe a method to incorporate fatty acids with stable isotope labels into excised skeletal muscle and detect the PL molecules containing labeled acyl chains by LC–MS/MS.
Bioinformatics and Computational Biology
Development of a Novel Automated Workflow in Fiji ImageJ for Batch Analysis of Confocal Imaging Data to Quantify Protein Colocalization Using Manders Coefficient
Confocal microscopy is integral to molecular and cellular biology, enabling high-resolution imaging and colocalization studies to elucidate biomolecular interactions in cells. Despite its utility, challenges in handling large datasets, particularly in preprocessing Z-stacks and calculating colocalization metrics like the Manders coefficient, limit efficiency and reproducibility. Manually processing large numbers of imaging data for colocalization analysis is prone to observer bias and inefficiencies. This study presents an automated workflow integrating Python-based preprocessing with Fiji ImageJ's BIOP-JACoP plugin to streamline Z-stack refinement and colocalization analysis. We generated an executable Windows application and made it publicly available on GitHub (https://github.com/weiyue99/Yue-Colocalization), allowing even those without Python experience to directly run the Python code required in the current protocol. The workflow systematically removes signal-free Z-slices that sometimes exist at the beginning and/or end of the Z-stacks using auto-thresholding, creates refined substacks, and performs batch analysis to calculate the Manders coefficient. It is designed for high-throughput applications, significantly reducing human error and hands-on time. By ensuring reproducibility and adaptability, this protocol addresses critical gaps in confocal image analysis workflows, facilitating efficient handling of large datasets and offering broad applicability in protein colocalization studies.
Screening for Streptococcus agalactiae: Development of an Automated qPCR-Based Laboratory-Developed Test Using Panther Fusion® Open AccessTM
Laboratory-developed tests (LDTs) are optimal molecular diagnostic modalities in circumstances such as public health emergencies, rare disease diagnosis, limited budget, or where existing commercial alternatives are unavailable, limited in supply, or withdrawn, either temporarily or permanently. These tests reduce access barriers and enhance equitable clinical practice and healthcare delivery. Despite recommendations for the development of nucleic acid amplification tests, procedural details are often insufficient, inconsistent, and arbitrary. This protocol elucidates the methodology used in the development of a fully automated real-time polymerase chain reaction (qPCR)-based test, using the Panther Fusion® Open AccessTM functionality, for the detection of Streptococcus agalactiae in pregnant women, using selectively enriched rectovaginal swabs. In addition, guidelines are provided for oligonucleotide design (primers and TaqMan probes), in silico and in vitro evaluation of design effectiveness, optimization of the physicochemical conditions of the amplification reaction, and result analysis based on experimental designs and acceptance criteria. Furthermore, recommendations are provided for the analytical and clinical validation of the intended use. Our approach is cost-effective, particularly during the design and optimization phases. We primarily used open-source bioinformatics software and tools for in silico evaluations for the test design. Subsequently, the process was manually optimized using a CFX96 Dx analyzer, whose technical specifications and performance are homologous to that of the final platform (Panther Fusion®). Unlike Panther Fusion®, the CFX96 Dx does not require excess volumes of reagents, samples, and evaluation materials (dead volume) to accommodate potential robotic handling-associated imprecisions. The utilization of the CFX96 Dx analyzer represents a strategic approach to enhancing the efficiency of resources and the optimization of time during LDT optimization.
Pupillometry: A Simple and Automatic Way to Explore Implicit Cognitive Processing
Pupil size is a non-invasive and highly sensitive technique used to measure changes in pupil diameter. It not only responds to light but also reflects inner cognitive processes (e.g., attention and emotion perception). Recently, it has been introduced to the traditional cognitive neuroscience field as a useful tool to objectively and sensitively capture the current cognitive state and its temporal dynamics. Importantly, this index is automatic and requires no explicit reports, thus it could be used to investigate the rarely explored realm of implicit cognitive processing. Here, we describe a comprehensive protocol that records pupil responses during the passive viewing of emotional biological motion (BM). Our results reliably reveal the multi-level implicit processing mechanism of BM emotion, as indicated by the fine-grained emotion processing in intact BM and the rapid but rather coarse emotion processing in local BM. Moreover, the emotion modulation effects observed in intact BM are indicative of individual autistic tendencies. We believe this protocol could be adapted to unveil the automatic processing of emotions and other attributes in social signals and further assist the early detection of social-cognitive disorders (e.g., autism).
Biological Engineering
A Human Cervix Chip for Preclinical Studies of Female Reproductive Biology
Pathological conditions of the cervix ranging from cervical cancer to structural dysfunction associated with preterm labor all have limited treatment options. Thus, there is a need for physiologically relevant preclinical models that recapitulate the structure and function of this human organ. Here, we describe a protocol for engineering and studying a highly functional in vitro model of the human cervix that is composed of a commercially available, dual-channel, microfluidic, organ-on-a-chip (Organ Chip) device lined by primary cervical epithelial (CE) cells interfaced across a porous membrane with cervical stromal cells. The provision of dynamic and customized media flow through both the epithelial and stromal compartments results in cell growth and differentiation, including the accumulation of a thick mucus layer overlying the epithelium. The resulting model closely mimics the structure, epithelial barrier, mucus composition and structure, and biochemical properties of the in vivo human cervix, as well as its responsiveness to female hormones, pH, and microbiome. This Cervix Chip protocol also includes noninvasive techniques for longitudinal monitoring of the live 3D tissue model. The Cervix Chip offers a powerful preclinical platform for replicating in vivo cervical physiology, studying disease mechanisms, and facilitating the development of new therapeutics and diagnostics.
Cancer Biology
A Protocol for Laser-Assisted Microdissection and tRF & tiRNA Sequencing in Lung Adenocarcinoma
Laser-assisted microdissection (LAM) coupled with next-generation sequencing technologies offers a powerful approach to dissecting the complex cellular heterogeneity within lung adenocarcinoma (LUAD) tumors. This protocol outlines the method for isolating specific high-risk LUAD tissues containing micropapillary/solid (MIP/SOL) patterns, which is linked to poor prognosis. We detail the process of LAM, which involves tissue fixation, microtome sectioning, and the precise dissection and collection of cells of interest under microscopic guidance. The isolated cells are then subjected to RNA extraction, library preparation, and sequencing to profile transfer RNA–derived fragments (tRFs) and tRNA-derived stress-induced RNAs (tiRNAs), which are emerging as key regulators in cancer. This protocol enables researchers to obtain high-quality transcriptomic data from specific LUAD cell populations, aiming to uncover tRF-Val-CAC-024 and tiRNA-Gly-CCC as potential biomarkers for early diagnosis and therapeutic targets for LUAD treatment.
Assay for Site-Specific Homologous Recombination Activity in Adherent Cells, Suspension Cells, and Tumor Tissues
Homologous recombination (HR) is a major pathway to repair DNA double-strand breaks. Hereditary breast and ovarian cancer syndrome (HBOC) is caused by germline pathogenic variants of HR-related genes, such as BRCA1 and BRCA2 (BRCA1/2). Cancer cells with HR deficiency are sensitive to poly(ADP-ribose) polymerase (PARP) inhibitors. Therefore, accurate evaluation of HR activity is helpful to diagnose HBOC and predict the effects of PARP inhibitors. The direct-repeat GFP (DR-GFP) assay has been utilized to evaluate cellular HR activity. However, evaluation by the DR-GFP assay tends to be qualitative and requires the establishment of stable cell lines. Therefore, we developed an assay to quantitatively measure HR activity called Assay for Site-Specific HR Activity (ASHRA), which can be performed by transiently transfecting two plasmids. In ASHRA, we use Cas9 endonuclease to create DNA double-strand breaks at specific sites in the genome, enabling the targeting of any endogenous loci. Quantification of HR products by real-time PCR using genomic DNA allows HR activity evaluated at the DNA level. Thus, ASHRA is an easy and quantitative method to evaluate HR activity at any genomic locus in various samples. Here, we present the protocols for adherent cells, suspension cells, and tumor tissues.
Cell Biology
Optimizing Confocal Imaging Protocols for Muscle Fiber Typing in the Mouse Masseter Muscle
The masseter muscle, a key orofacial muscle, demonstrates unique anatomical and functional properties, including sexual dimorphism in myosin heavy chain (MyHC) expression and complex fiber architecture. Despite its importance in mastication and relevance to various disorders, phenotypic characterization of the masseter remains limited. Conventional fluorescence microscopy has been a cornerstone in muscle fiber typing, reliably identifying MyHC isoforms and measuring fiber cross-sectional areas. Building on this foundation, confocal microscopy offers complementary advantages, such as enhanced resolution, increased flexibility for multiplexing, and the ability to visualize complex structures in three dimensions. This study presents a detailed protocol for using confocal microscopy to achieve high-resolution imaging and molecular characterization of masseter muscle cryosections. By leveraging advanced technologies such as white light lasers and extended z-length imaging, this method ensures precise spectral separation, simultaneous multichannel fluorescence detection, and the ability to capture muscle architecture in three dimensions. The protocol includes tissue preparation, immunostaining for MyHC isoforms, and postprocessing for fiber segmentation and quantification. The imaging setup was optimized for minimizing signal bleed through, improving the signal-to-noise ratio, and enabling detailed visualization of muscle fibers and molecular markers. Image postprocessing allows for quantification of the cross-sectional area of individual fibers, nuclei location measurements, and identification of MyHC isoforms within each fiber. This confocal microscopy–based protocol provides similar resolution and contrast compared to conventional techniques, enabling robust multiplexed imaging and 3D reconstruction of muscle structures. These advantages make it a valuable tool for studying complex muscle architecture, offering broad applications in muscle physiology and pathology research.
Isolation of In Vitro Osteoblastic-Derived Matrix Vesicles by Ultracentrifugation and Cell-Free Mineralization Assay
Matrix vesicles (MVs) represent a heterogeneous group of spherical membrane-bound extracellular vesicles in the range of 100–200 nm in diameter secreted by mineralizing osteoblasts. The initial synthesis of the amorphous calcium phosphate occurs within the confines of the intracellular MVs, which are capable of transporting Pi and Ca2+ into the MV lumen. Thus, understanding the initial process of MV-mediated mineralization is critical in developing better therapeutic strategies for various bone-related disorders such as osteoporosis and addressing ectopic calcification of soft tissues. Although various techniques and commercially available kits are now available for isolating MVs, isolating a pure population of MVs is challenging mainly because of their variable size and lack of consensus protein markers. This ultracentrifugation-based protocol ensures high purity of isolated MVs by removing other contaminated extracellular vesicles and cellular debris through sequential centrifugation steps but also allows downstream functional mineralization assays of the isolated MVs.
Developmental Biology
Single Molecule Fluorescence In Situ Hybridization Using RNAscope to Study Hematopoietic and Vascular Interactions in the Zebrafish Embryo and Larva
Our goal is to understand how hematopoietic stem cell precursors emerge from vessels and to visualize their settling in developmental and more definitive niches that will persist in the adult. For this, we use as a biological model the zebrafish, which offers invaluable advantages owing to its transparency and small size, allowing high-resolution imaging and investigations of the entire animal. In vertebrate species, precursors of hematopoietic stem cells emerge from arterial vessels, mainly from the ventral side of the dorsal aorta. From there, they can either reside in the underlying vascular niche and/or pass through the vein to enter the blood circulation and conquer the caudal hematopoietic tissue, a functional equivalent to the fetal liver in mammals. Here, we provide experimental details of a protocol we have recently optimized to identify, based on mRNA in situ hybridization, precursors of hematopoietic stem cells while still embedded in the aortic wall (at the embryonic stage) as well as when they reside in specific niches a few days after emergence (at the early larval stage). Our experimental approach uses RNAscope technology, which allows combining high-sensitivity mRNA detection with high-resolution fluorescence confocal imaging to achieve spatial transcriptomics. Importantly, the small size of the probes allows better penetration inside tissues, which is a significant improvement in comparison to long mRNA probes; this is an invaluable advantage for reaching deeply embedded niches such as the ones of the pronephros region in the larva and, in addition, provides an increased signal-to-noise ratio.
Environmental science
Baculovirus and Plasmid Vector-Mediated Gene Transfer in Daphnia magna Cells in Primary Embryonic In Vitro Cultures in an Optimized Microenvironment: Methods and Protocols
Daphnia magna is a well-established model organism in ecotoxicology, environmental monitoring, and genetics due to its sensitivity to pollutants, its pivotal role in freshwater ecosystems, and its well-characterized genome. Despite its extensive use in these fields, there is a notable lack of established protocols for developing primary cell culture systems and conducting transgenic studies in Daphnia spp. This study addresses these gaps by optimizing a medium and standardizing protocols for primary cell culture and transgenic experiments in D. magna. Primary cell cultures were established from both D. magna embryos and whole organisms, with medium optimization verified using XTT assay. Cell viability was sustained for over two months using a modified Schneider’s insect medium enriched with FBS, glucose, MEM vitamin mix, and selenium. DNA replication and cell proliferation were confirmed through BrdU labeling. Both mechanical and enzymatic passaging methods were compared, resulting in 20% and 10% cell attachment, respectively. For transgenic applications, this study successfully standardized plasmid-mediated lipofection and baculovirus-mediated transduction, achieving success rates of 52% and 45%. These findings represent a pioneering effort in D. magna embryonic cell culture, offering a reliable in vitro platform for future biological research, including ecotoxicological and epigenetic investigations. The established protocols and optimized cell culture medium have significant implications for advancing crustacean cell line research and transgenic model development, enhancing our understanding of biological processes in controlled laboratory environments.
Immunology
A Participant-Derived Xenograft Mouse Model to Decode Autologous Mechanisms of HIV Control and Evaluate Immunotherapies
Human immunodeficiency virus (HIV) remains a global health challenge with major research efforts being directed toward the unmet needs for a vaccine and a safe and scalable cure. Antiretroviral therapy (ART) suppresses viral replication but does not cure infection and so requires lifelong adherence. HIV-specific CD8+ T-cell responses play a crucial role in long-term HIV control as demonstrated in elite controllers, highlighting their potential in HIV cure strategies. Various HIV mouse models—including the human-hematopoietic stem cell (Hu-HSC) mouse, the bone marrow, liver, and thymus (BLT) mouse, and the human peripheral blood leukocyte (Hu-PBL) mouse—have deepened the understanding of HIV dynamics and facilitated the development of therapeutics. We developed the HIV participant-derived xenograft (HIV PDX) mouse model to enable long-term in vivo evaluation of bona fide autologous T-cell mechanisms of HIV control, including the antiviral activity of primary memory CD8+ (mCD8+) T cells taken directly from people with or without HIV, as well as testing potential immunotherapies. Additionally, this model faithfully recapitulates virus escape mutations in response to sustained CD8+ T-cell pressure, enabling the assessment of strategies to curb virus escape. In this model, NSG mice are engrafted with purified memory CD4+ (mCD4+) cells and infected with HIV; then, they receive autologous CD8+ T cells or T-cell products. Key advantages of this model include the minimization of graft-versus-host disease (GvHD), which severely limits peripheral blood mononuclear cell (PBMC) or total CD4-engrafted mice, the ability to evaluate long-term natural donor-specific T-cell responses in vivo, and the lack of use of human fetal tissues required for most humanized mouse models of HIV.
Analysis of Vascular Permeability by a Modified Miles Assay
The endothelial barrier is a semipermeable cell layer covering the inside of blood vessels that regulates the flux of ions, macromolecules, and plasma from blood to tissues. Inflammation promotes an increase in vascular permeability, which can contribute to disease if not controlled properly. Thus, it is important to understand in detail the molecular mechanisms underlying inflammatory vascular hyperpermeability. While endothelial permeability can be measured in vitro, these assays do not recapitulate precisely the in vivo vasculature. Thus, in vivo assays are required to understand the full picture of vascular permeability regulation. Here, we describe an established assay that involves injection of Evans blue dye followed by intradermal injection of agents inducing vascular permeability. This assay is relatively easy to perform and provides reliable data on permeability regulation in vivo.
Microbiology
Detection and Analysis of S-Acylated Proteins via Acyl Resin–Assisted Capture (Acyl-RAC)
Protein palmitoylation is a lipid modification where a palmitoyl group is covalently attached via a thioester linkage to one or more cysteines on a substrate protein. This modification, catalyzed by a group of enzymes named DHHC enzymes after their conserved Asp-His-His-Cys motif, plays a significant role in regulating the localization, stability, and function of a wide range of cellular and viral proteins. By influencing how and where proteins interact within the cell, palmitoylation is essential for various cellular processes, including signaling pathways, membrane dynamics, and protein–protein interactions. Here, we describe the acyl-RAC assay, a biochemical technique designed to specifically enrich and analyze palmitoylated proteins from complex biological samples, such as cell lysates or tissue extracts. The assay begins by reducing and blocking free cysteine thiol groups on proteins, ensuring that only those thiols involved in thioester bonds with palmitates are accessible for downstream analysis. These thioester bonds are then cleaved to release the fatty acids from the cysteines, which are subsequently captured using thiopropyl Sepharose beads that bind to the newly exposed thiol groups. The captured proteins are eluted from the beads by breaking the bond between the thiol and the resin with reducing agents, and the proteins are then analyzed by SDS-PAGE followed by western blotting to identify and quantify them. The acyl-RAC assay's specificity for S-palmitoylated proteins makes it an invaluable tool for exploring this modification. It not only allows for the identification of previously unknown palmitoylated proteins, thereby deepening our understanding of palmitoylation in cellular processes and viral infections, but it also enables quantitative comparisons of protein palmitoylation under different experimental conditions or treatments.
Flotation Assay With Fluorescence Readout to Study Membrane Association of the Enteroviral Peripheral Membrane Protein 2C
Enteroviruses are abundant pathogens of humans and animals. Their replication is strictly dependent on the conserved, viral AAA+ ATPase 2C. 2C is an oligomerizing, peripheral membrane protein, and its low solubility as recombinant protein has hampered functional studies of the full-length, recombinant protein bound to a membrane. Here, we describe a modification of the classical, ultracentrifugation-based liposome flotation assay optimized to study the interaction of recombinant 2C with membranes and the functions of membrane-bound, full-length recombinant 2C. The assay takes advantage of the high solubility of recombinant 2C while fused to a maltose-binding protein. Removing this solubility-enhancing tag by specific protease cleavage in the presence of liposomes allows 2C to associate with membranes prior to aggregating. Fluorophore labeling of protein and liposomes allows rapid and precise quantitation of 2C’s association with membranes. This assay is adaptable to any peripheral membrane protein that can be fluorophore-labeled and expressed as a solubility-enhancing fusion protein.
Plant Science
Enzymatic Starch Quantification in Developing Flower Primordia of Sweet Cherry
Starch is a carbohydrate widely used in the plant kingdom as a fuel for different physiological processes. While different techniques are available for the quantification of starch stored in seeds and bark tissues, they have hardly been used to quantify starch content in developing flower buds, where starch has been reported to accumulate in different reproductive organs. Here, we detail a quantitative enzymatic method to measure starch concentration in developing flower primordia in sweet cherry (Prunus avium L.). First, starch is enzymatically hydrolyzed to D-glucose, which was then quantified by an enzyme-coupled assay involving hexokinase (HK) and glucose-6-phosphate dehydrogenase (G6PD) and spectrophotometric quantification of NADH absorbance at 340 nm. This method is a sensitive, rapid, and affordable protocol specifically optimized for tiny flower buds with low starch content. The technique is revealed to successfully determine starch content in non-freshly harvested samples—frozen and stored at -20 °C or stored in fixatives—allowing a temporal separation of sampling and quantification and making the protocol suitable for high-throughput experimental designs in different fields of plant research.
Analysis of Modified Plant Metabolites Using Widely Targeted Metabolite Modificomics
Metabolite modifications play a critical role in enhancing plants’ adaptability to environmental changes and serve as a major source of functional diversity in metabolites. However, current metabolomics approaches are limited to targeted analyses of a small number of known modified metabolites and lack comprehensive, large-scale studies of plant metabolite modifications. Here, we describe a widely targeted metabolite modificomics (WTMM) strategy, developed using ultra-high-performance liquid chromatography–quadrupole linear ion trap (UHPLC-Q-Trap) and ultra-high-performance liquid chromatography–Q-Exactive Orbitrap (UHPLC-QE-Orbitrap) technologies. This strategy enables high-throughput identification and sensitive quantification of modified metabolites. Using tomato as a model, we conducted a metabolite modificomics study and constructed a WTMM database, identifying 165 novel modified metabolites. The WTMM strategy is broadly applicable and can be extended to the study of other plant species.
A Detailed Guide to Recording and Analyzing Arabidopsis thaliana Leaf Surface Potential Dynamics Elicited by Mechanical Wounding
Recordings of electric potential changes on plant surfaces have been utilized to identify the components and mechanisms involved in the formation and transmission of systemic signals elicited by stimuli such as herbivory, wounding, or burning. The recorded responses, commonly referred to as slow wave or variation potentials, exhibit striking variability in their waveform. The extent to which this variability is due to differences in experimental procedures or plant biological variability remains unclear. Here, we provide a detailed and robust protocol refined from years of experience in conducting leaf surface potential recordings of Arabidopsis thaliana in response to mechanical wounding. This protocol serves as a comprehensive tutorial covering plant growth, procedures for reproducible mechanical wounding, critical aspects of electrophysiological recordings, and statistical analysis of surface potential recordings. It particularly emphasizes the construction and maintenance of electrodes, placement of the reference or ground electrode, mechanisms for wounding, and data analysis. This protocol aims to promote and facilitate the adoption, standardization, and interoperability of plant surface potential recordings among research groups, thereby increasing the reproducibility and comparability of data within the field.
High-Performance Liquid Chromatography Quantification of Glyphosate, Aminomethylphosphonic Acid, and Ascorbate in Culture Medium and Microalgal Cells
Glyphosate (GLY) is a widely used herbicide that can induce oxidative stress in microalgae and other non-target organisms. The quantification of GLY in surface water is a difficult task, especially in trace-level concentrations, due to its high polarity and susceptibility to biotic and abiotic degradation. Several analytical methods have been developed for GLY quantification. Most of them use high-performance liquid chromatography (HPLC) coupled with detection by mass spectrometry (MS) and include a derivatization step to decrease the polarity of the herbicide to improve detection. This protocol describes an adaptation of an existing protocol for the quantification of GLY and its metabolite aminomethylphosphonic acid (AMPA) in a water-based microalgae culture medium using ultra-high-pressure liquid chromatography (UHPLC) coupled with fluorescence detection (FLR). The principal advantage of this protocol compared with other analytical methods that employ HPLC–MS is its low cost and accessibility since it does not require an MS detector nor radioactively labeled analytical standards. Ascorbic acid (AH-) is one of the most important hydrosoluble non-enzymatic antioxidants in eukaryotic cells and plays a key role in many metabolic pathways of critical importance in plants and algae. In this protocol, we also describe an adaptation of a previously published protocol to quantify AH- in blood samples to be used in microalgal cells exposed to GLY and GLY-based herbicides. The sample preparation procedure for this last protocol is fast, easy, and does not require expensive equipment. It uses an HPLC system coupled with an electrochemical detector (EC) for AH- quantification but may be adapted to be used with a UV-Vis detector.