Articles in press

Plant proximity labeling proteomics enables the identification of transient and weak intracellular protein interactions that are undetectable via traditional biochemical assays. Conventional enrichment pipelines suffer severe quantitative bias caused by urea-derived chemical artifacts and high mass spectrometry background signals from sample- and carrier-derived impurities. This protocol describes a complete standardized workflow for biotinylated protein extraction, enrichment, and LC-MS/MS sample preparation optimized for Arabidopsis seedlings. The procedure integrates controlled urea dilution and pre-desalting to suppress non-enzymatic protein modifications, introduces acetylation blocking of streptavidin magnetic beads to mitigate streptavidin degradation during on-bead digestion, and applies two-step on-bead trypsin digestion to improve peptide coverage. Multi-point sample retention and dual protein quantification are implemented throughout all experimental stages to ensure full-process quality control. Subsequent data processing pipelines using Spectronaut for data-independent acquisition (DIA) data and MaxQuant for data-dependent acquisition (DDA) data are also detailed for consistent proteome quantification. This workflow delivers higher protein recovery and better signal-to-noise ratios than standard protocols while offering flexible adaptation for various crop proximity labeling and affinity enrichment proteomic research.

While advances in omics technologies have greatly improved our understanding of cellular heterogeneity, there is an increasing need for complementary approaches that capture the spatial organization and structural dynamics of cells. Image-based single-cell phenotypic profiling provides quantitative information on cell morphology and organelle organization, offering valuable insights into cellular function and regulation. Although numerous image analysis tools are available, establishing a complete analysis workflow, from image preprocessing and segmentation to feature extraction and multivariate analysis, often requires substantial computational expertise and software integration. Here, we describe a Python-based workflow for image-based single-cell phenotypic profiling from immunofluorescence microscopy images and provide a detailed protocol for its implementation. Using synchronized HeLa cells with drug-induced mitotic spindle defects as an example, the workflow covers image loading, cell segmentation, quantitative feature extraction, profile integration, dimensionality reduction, clustering, and data visualization. The protocol is accompanied by example datasets, annotated Jupyter Notebooks, and instructions for execution in either a local Python environment or Google Colab, facilitating straightforward implementation and customization. By integrating the entire analysis pipeline within a single coding environment, this workflow enables reproducible and accessible single-cell morphological profiling without requiring specialized imaging equipment or extensive programming expertise. The workflow therefore provides a practical platform for studying cell morphology, organelle organization, and cellular dynamics across a broad range of biological applications.

The release and updating of whole-genome sequences of several representatives of the human opportunistic pathogen Pseudomonas aeruginosa have laid the ground for investigating the mechanisms of antibiotic resistance, biofilm formation, and virulence, while also offering opportunities for researchers to find new therapeutic targets to control this bacterium using a functional genomics approach. However, there is still a lack of detailed protocols describing gene inactivation methods in P. aeruginosa, resulting in failures and extra time spent designing in-house protocols. Here, we introduce a rapid, efficient, and unmarked deletion mutagenesis method combining overlap extension PCR, efficient conjugation, and the traditionally used sacB-based counter-selection procedure. Efficient generation of deletion mutants using this detailed protocol can be easily completed in one week using standard lab reagents. Importantly, this method may be adaptable to other bacteria where the sacB-based counter-selection system works.

Zebrafish is an excellent in vivo model for high-throughput antiangiogenic drug testing, commonly known as the zebrafish angiogenesis assay. Conventional zebrafish angiogenesis assays are performed in wild-type zebrafish to evaluate the vascular changes in intersegmental and subintestinal vessel regions of the zebrafish larvae at 2 and 3 dpf stages, respectively. However, wild-type zebrafish larvae do not adequately mimic the hypoxia microenvironment and ectopic vessel branching characteristics of cancer. To overcome this limitation, we developed a genetically engineered zebrafish model with constitutive activation of the hypoxia signaling pathway by targeting the vhl, a tumor suppressor, by negative regulation of the hypoxia pathway, using CRISPR mutagenesis. This model exhibits robust ectopic blood vessel branching throughout the larval body, thereby recapitulating pathological angiogenesis. The utility of this zebrafish model system for drug screening was validated with the sorafenib treatment, a known antiangiogenic tyrosine kinase inhibitor. Overall, this proangiogenic hypoxia zebrafish model provides a physiologically relevant platform for testing antiangiogenic drugs.

Spatial transcriptomics enables genome-wide gene expression profiling while preserving tissue architecture, making it a powerful approach for studying plant developmental transitions. However, preparing small and structurally complex plant tissues for spatial transcriptomics remains technically challenging because samples must be rapidly preserved, precisely oriented, serially sectioned, and accurately positioned within the limited capture area of the Visium slides. Here, we describe an optimized workflow for cryo-embedding, serial cryosectioning, section placement, and data analysis of small plant samples for 10x Genomics Visium spatial transcriptomics. Using maize seedling shoot apices as the target, this protocol includes preparation of custom molds for optimal cutting temperature embedding, rapid fresh sample embedding, serial cryosectioning, section-position marking for Visium HD workflows, and morphological quality assessment of replicate tissue slides before transcript capture. The associated data analysis workflow includes Space Ranger processing, Seurat-based normalization and Harmony integration, anatomical domain annotation, pseudobulk and developmental trend analyses, RNA velocity, pseudotime analysis, transcription factor network analysis, single-cell reference mapping, and 3D transcriptome reconstruction. This computational workflow was developed and tested using maize Visium V1 data, but not Visium HD data. This protocol was used to generate serial spatial transcriptomes of maize shoot apices and developing leaf primordia, enabling reconstruction of gene expression transitions from the shoot apical meristem to sequential leaf developmental stages. The approach is also applicable to other small plant tissues, including Arabidopsis first true leaves and Marchantia thalli.

Region-specific RNA sequencing is a powerful approach for investigating tissue differentiation and dynamic changes in gene expression during embryonic development. The chicken embryo has long served as an important model system in developmental biology. However, the limited availability of tissue-specific reporter lines makes region-specific RNA-seq approaches particularly valuable in this organism. Here, we applied photo-isolation chemistry-based RNA sequencing (PIC-RNA-seq) to the somatic lateral plate mesoderm (sLPM) before the emergence of limb bud progenitor cells (LPCs) and to early LPCs in chicken embryos. These analyses revealed the upregulation of multiple genes, including Hox genes, in LPCs, suggesting the initiation of their regional patterning program. This workflow enables visualization of dynamic changes in the gene expression profile of LPCs and should also be applicable to other tissues in avian embryos.

Leptospirosis is a widespread zoonotic disease caused by pathogenic bacteria of the genus Leptospira. The isolation and comprehensive characterization of circulating strains within a region are essential for understanding the local epidemiology and improving public health surveillance. Historically, whole blood has been the specimen of choice for isolation; however, its efficiency can be limited by the presence of inhibitory substances in the sample, and Leptospira viability may depend on rapid processing and inoculation. Here, we present an in-house culture protocol for the isolation of Leptospira from serum samples previously maintained under refrigeration (i.e., 4–8 °C) for up to 10 days. The protocol employs a real-time PCR-guided strategy by first screening specimens for the lipL32 gene. Positive samples are then inoculated into specialized EMJH media supplemented with AFAS and EMJH+AFAS supplemented with STAFF antibiotic cocktail, followed by incubation at 30 °C for up to six months. Growth is monitored weekly through visual inspection and, once turbid, the presence of Leptospira is determined via dark-field microscopy prior to downstream serogroup and genomic characterization. A significant advantage of this method is the successful recovery of viable Leptospira from non-fresh serum specimens stored under refrigeration, even in samples with low bacterial loads. Additionally, the protocol facilitates broader surveillance by repurposing serum samples already collected for routine serology, increasing the probability of identifying diverse strains without further clinical collection

Human bone marrow organoids provide a tractable three-dimensional platform for modeling hematopoiesis and hematologic disease in a human niche–like context. Here, we describe a stepwise protocol for utilizing human induced pluripotent stem cell (iPSC)-derived bone marrow organoids that support autonomous hematopoiesis for hematopoietic disease modeling, mouse xenograft hematopoiesis, and drug sensitivity testing. The workflow combines embryoid body formation, early mesoderm/angiogenic induction under hypoxia, hemogenic endothelial commitment, maturation within a collagen-containing hydrogel, and subsequent suspension culture as individual organoids. The resulting organoids contain endothelial, stromal, and hematopoietic components and reproduce key structural and cellular features of human marrow. We further describe procedures for engraftment of normal donor- or patient-derived CD34+ cells and implantation of mature organoids under the renal capsule of immunodeficient mice to assess in vivo hematopoietic maintenance. In prior applications of this platform, donor-derived CD34+ cells were shown to engraft within the organoid niche and undergo multilineage differentiation, enabling detection of selective erythroid defects caused by DDX41 deficiency and assessment of therapeutic suppression of JAK2V617F-mutant patient-derived hematopoietic cells in a human marrow–like microenvironment. This protocol, therefore, enables disease modeling, in vivo xenograft assessment, and ex vivo functional analysis of patient-derived hematopoietic cells using relatively small input samples.

Reliable DNA extraction is essential for genetic research on marine species; however, obtaining sufficient DNA from single fish eggs remains challenging. Existing protocols often require optimization to achieve high PCR efficiency. The optimized phenol–chloroform–isoamyl extraction protocol presented in this paper improves DNA yield and quality from individual eggs of Atlantic bluefin tuna (Thunnus thynnus), bogue (Boops bops), saddled seabream (Oblada melanura), and painted comber (Serranus scriba) by modifying buffer volumes, incubation times, and washing steps, following prior micropuncturing of eggs on a glass slide. DNA quality is confirmed by spectrophotometry, PCR amplification of the mitochondrial COI gene, electrophoresis, and Sanger sequencing. This method provides a low-cost and effective approach for species identification from individual fish eggs.

Spatial organization of macromolecules is fundamental to cellular function, with colocalization providing key insights into molecular interactions and biological processes. However, quantification remains challenging due to diverse localization patterns and irregular sample geometries. Here, we present a protocol for analyzing colocalization between two fluorescent probes using coAnalyzer, a MATLAB-based software package. coAnalyzer features a user-friendly graphical interface and supports region of interest (ROI) selection, image merging, line scanning, signal isolation, scatterplot generation, and quantitative analysis. coAnalyzer enables colocalization analysis of any two fluorophores, regardless of the proteins or dyes involved. Its broad applicability across a wide range of organisms and sample types demonstrates the robustness, flexibility, and versatility of the platform.

Most existing preclinical models have been limited in their predictive value to mimic patients’ responses, which is a major drawback in drug development and the identification of predictive biomarkers. To overcome these limitations, patient-derived three-dimensional in vitro models have been proposed. One of them is the organoid model, which preserves the original cellular heterogeneity and recapitulates epithelial architecture and functionality. Recently, studies using patient-derived organoids for drug screening applications have increased in quantity, and organoids have already been applied to pancreatic, colon, and lung cancers and female gynecological malignancies. Here, we established a multiplex workflow to analyze longitudinal therapeutic effects of anti-cancer therapeutics on organoid growth, viability, and cytotoxicity by combining state-of-the-art viability measurement with automated live cell imaging. This workflow can be used for the prediction of patient-specific treatment response, high-throughput screening of potential anticancer drugs, and downstream analysis to identify novel therapeutic targets.