Current protocols最新文献

Common problems in biological sample processing for scanning electron microscopy (SEM) include cell collapse and destruction. To overcome the challenges surrounding SEM micrograph preparation, dried rice stems were used to develop a specific set of protocols for processing dried plant samples. Dried stems are rehydrated with a glycerol solution and fixed in formalin-acetic-alcohol to avoid cell wall collapse or organ distortion. The protocols detailed here comprise the first published method for preparing SEM images of dried plant tissue. The protocols offer a cost-effective approach to obtaining high-quality micrographs, facilitating the reconstruction of growth processes and the study of plant cell wall features. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Pretreatment and preparation of rice straw samples at the heading stage

Basic Protocol 2: Paraffin infiltration and embedding

Basic Protocol 3: Preparation of microscopic sections

Basic Protocol 4: Transferring, adhering, and expanding sections on slides

Support Protocol: Preparation of gelatin slides before sectioning to affix samples

Basic Protocol 5: Preparation of samples for SEM imaging

Basic Protocol 6: SEM analysis

Basic Protocol 7: Processing and analysis of SEM images using ImageJ software

The cover image is based on the article Assays of Angiogenic Potential Using Quail and Chicken Chorioallantoic Membrane (CAM) by Ana Claudia Oliveira Carreira et al., https://doi.org/10.1002/cpz1.70223.

Targeted protein degradation using bifunctional molecules like proteolysis targeting chimeras (PROTACs) has revolutionized drug discovery but remains limited by E3 ligase availability and lack of cell selectivity. N-degron degraders provide a smaller, more drug-like alternative that harnesses endogenous N-terminal degradation signals independent of recruited ligases. However, they are prone to proteolysis and lack mechanisms for tissue-specific activation. To overcome these challenges, we developed a “caged” N-degron degrader selectively activated by the immunoproteasome (iCP), a proteasome isoform induced in cancer and inflammatory settings. By appending an iCP-recognition tetrapeptide (Ala-Thr-Met-Trp) capped with a morpholine group to the N-terminus, we shielded the degron from nonspecific cleavage and enabled selective activation in iCP-expressing cells. This design improved stability, enhanced degradation potency, and minimized activity in healthy cells. Our findings establish a generalizable strategy to spatially control N-degron activity through disease-specific proteolytic environments, advancing safer and more selective protein degradation therapeutics. © 2025 Wiley Periodicals LLC.

Basic Protocol 1: Synthesis and characterization of an N-degron degrader for Abl

Basic Protocol 2: Synthesis and characterization of an immunoproteasome N-degron prodrug

Basic Protocol 3: Assessing protein degradation in cells

DNA replication is often challenged by endogenous and exogenous sources of DNA damage, which can stall replication forks and result in single-stranded DNA (ssDNA) gaps, double-strand breaks (DSBs), and genomic instability. Detecting DNA damage specifically in newly synthesized DNA strands is essential for understanding how eukaryotic cells respond to replication stress and continue the cell cycle progression through DNA repair or DNA damage tolerance (DDT) mechanisms. Here, we present an optimized and accessible protocol for the alkaline BrdU comet assay—a single-cell technique that combines bromodeoxyuridine (BrdU; a thymidine analog) pulse-labeling of newly synthesized DNA with the alkaline comet assay (single-cell gel electrophoresis) followed by fluorescence immunodetection. This method enables the specific detection and measurement of DNA strand breaks occurring in newly replicated DNA during and immediately after the S phase, even without cell synchronization, allowing researchers to differentiate replication-associated DNA damage from overall genomic damage. We provide detailed instructions for performing the assay using human cells (RPE-1 h-TERT TP53 KO) in vitro after exposure to DNA replication-stress-inducing agents, such as hydroxyurea (HU) and ultraviolet-C (UV-C) radiation. We also demonstrate its application in translesion-synthesis-deficient (Pol eta-deficient) human fibroblasts in vitro. Importantly, this protocol supports time-course chase experiments (e.g., 0, 1, 2, and 4 hr post-treatment) to monitor the kinetics of DNA damage in nascent DNA strands. This BrdU-based protocol offers high specificity, single-cell resolution, and cost-effectiveness, making it particularly valuable for laboratories studying replication stress, post-replication DNA repair proficiency, DDT, and/or genotoxic responses in unsynchronized human cells in vitro. This protocol also adheres to the Minimum Information for Reporting Comet Assay (MIRCA) guidelines and is aligned with the objectives of the International Comet Assay Working Group (ICAW), ensuring high reproducibility and standardization. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol: Alkaline BrdU comet assay to assess replication-associated DNA damage in unsynchronized human cells (RPE-1 h-TERT TP53 KO) in vitro

Alternate Protocol 1: Alkaline BrdU comet assay to monitor replication-associated DNA damage dynamics in unsynchronized human cells (RPE-1 h-TERT TP53 KO) after HU and UV-C exposure and 0- to 2- hr chase

Alternate Protocol 2: Alkaline BrdU comet assay to compare replication-associated DNA damage dynamics in translesion synthesis polymerase η-deficient and complemented (XP-V comp) unsynchronized fibroblasts after UV-C exposure and 0- to 4-hr chase

To establish a standardized technical framework for large-scale production of recombinant collagen, we have employed Pichia pastoris GS115 as a host expression system and optimized the fermentation and purification processes for a transdermal peptide–type III collagen fusion protein (transdermal peptide–hCOL3A) at the 15-L bioreactor scale. A recombinant vector encoding the fusion gene was constructed through codon optimization and transformed into the GS115 strain. The seed culture was prepared via shake-flask cultivation in BMGY medium. High-cell-density fed-batch fermentation was conducted in a 15-L fermenter with an inoculum size ranging from 3% to 10%, achieving a final recombinant protein yield of 1.9 g/L. In the downstream purification, optimization of membrane-based concentration parameters and the incorporation of strong cation-exchange chromatography using an SP column enhanced the recovery rate of the target protein to 68%. Gel-filtration analysis demonstrated that transdermal peptide–hCOL3A exists as a trimer, and a CCK-8 assay revealed that it promotes proliferation of HaCaT cells in vitro, indicating biological functionality. This work successfully establishes a robust and scalable collagen production process suitable for pilot-scale manufacturing and provides a solid technical foundation for future industrial-scale translation. © 2025 Wiley Periodicals LLC.

Cancer drug resistance, whether intrinsic or acquired, underlies most relapses and treatment failures. Reliable preclinical models are crucial to define resistance mechanisms and test counterstrategies. This overview article concisely compares three model classes: (1) clinical, patient‑derived xenografts that retain tumor heterogeneity and can mirror patient resistance; (2) induced‑resistance models produced by prolonged drug selection in vivo or in vitro that recapitulate tumor evolution under therapy; and (3) engineered isogenic cell lines that isolate specific resistance drivers. We summarize some key resistance mechanisms revealed and potential therapeutic approaches informed by these models, including rational combinations, mutation‑targeted inhibitors/degraders, efflux/epigenetic modulators, and immune-related combinations. Each model has trade‑offs, but integrating them accelerates mechanistic insight and translational drug development. This overview guides selection of preclinical models and design of strategies to overcome cancer drug resistance. © 2025 Wiley Periodicals LLC.

Rheumatoid arthritis (RA) is a chronic disease involving inflammation of the joints. While the etiology of RA remains unknown, evidence exists for a significant contribution of the major histocompatibility complex (MHC). Environmental factors are also indicated as playing an etiological role. Since it is impossible to define the mechanisms contributing to disease onset and progression in humans, mouse models have been used widely. While many animal models have been generated by immunization with self-proteins to induce arthritis, type II collagen (CII)-induced arthritis is one of the most commonly used models utilized to understand the immunopathology of RA. CII constitutes 80% to 90% of total collagen content of hyaline cartilage found in joints and is a genetically conserved sequestered protein. Immunization with heterologous CII with an adjuvant in mice leads to cellular and humoral responses to heterologous and autoreactive CII-specific responses and collagen-induced arthritis (CIA). Mice immunized with CII develop inflammatory arthritis that shares many similarities in clinical, serological, and radiological features with RA in humans. However, selecting an antigen for inducing arthritis and the mouse strain are important, as not all strains are susceptible to CIA. A critical difference between RA and CIA is that in mice that lack the expression of human MHC II, the development of CIA is linked to the H2A locus, which is the homologue of HLA-DQ, while most human studies have linked RA susceptibility with HLA-DR alleles. Mice expressing HLA-DQ and HLA-DR molecules have been used to understand the role of MHC genes in susceptibility to RA. The protocols for inducing CIA in the HLA expressing transgenic mice described in this article can be used to understand how the different HLA molecules confer susceptibility to RA. © 2025 Wiley Periodicals LLC.

Basic Protocol 1: Use of mouse strains and humanized mice for modeling rheumatoid arthritis

Basic Protocol 2: Use of humanized mice for therapeutic protocols

Tumor-treating fields (TTFields) represent an innovative approach to cancer treatment that leverages low-intensity (1-3 V/cm) alternating electric fields operating at intermediate frequencies (100-300 kHz). These electric fields are specifically designed to disrupt the mitotic process, thereby inhibiting the proliferation of malignant cells. Recent research has shown that TTFields not only induce direct cytotoxic effects but also modulate the immune system by triggering immunogenic cancer cell death, thereby enhancing immune cell infiltration into tumor sites. This dual mechanism opens up new possibilities for synergistic integration with immunotherapy, which has already revolutionized oncology through the advent of immune-checkpoint inhibitors, adoptive cell therapies, and cancer vaccines. Given the immune-modulatory properties of TTFields, there is growing interest in exploring their potential synergistic effect to enhance immunotherapeutic efficacy. Recognizing the complementary role of TTFields in cancer treatment paves the way for innovative combinatorial strategies that may further improve patient outcomes by enhancing the effectiveness of both TTFields and immunotherapy. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

The lung is a structurally and immunologically complex organ, constantly exposed to airborne microbes, allergens, and pollutants. Understanding how diverse pulmonary immune cells respond to these challenges is critical for advancing respiratory disease research and identifying appropriate therapeutic interventions. Flow cytometry remains a cornerstone of immune profiling, and advances in high-parameter spectral cytometry have significantly expanded its analytical capabilities. However, challenges, such as poor tissue dissociation, spectral overlap, and loss of spatial information, can hinder the comprehensive interpretation of the lung environment. To address these limitations, we developed an optimized spectral flow cytometry platform, utilizing 5-laser Cytek Aurora spectral cytometers, for deep immunophenotyping of murine lung tissue. Our approach integrates in vivo CD45 antibody labeling—administered intravenously and oropharyngeally—to distinguish circulating, airway, and interstitial immune populations, preserving spatial context in single-cell suspensions. We utilize complementary 25+ parameter panels targeting myeloid and lymphoid compartments, built on a shared backbone to enable consistent classification across datasets. Refined tissue processing protocols support optimal recovery of representative lung cell populations, and overnight intracellular staining enhances marker resolution. Using this platform, we reliably resolve stromal, endothelial, and epithelial cells alongside immune subsets—including macrophages, monocytes, dendritic cells, eosinophils, neutrophils, T and B cells, innate lymphoid cells (ILCs), and natural killer (NK) cells—subclassified by activation, function, and tissue residency. Validation with an influenza A virus model confirmed expected dynamic immune responses and revealed previously unrecognized populations. This spatially informed approach enables high-resolution interrogation of pulmonary immunity in health and disease. © 2025 Wiley Periodicals LLC.

Basic Protocol 1: Differential in vivo labeling for spatial profiling of pulmonary immune cells by spectral cytometry

Basic Protocol 2: Preparation of single-cell suspensions from murine lung tissue for spectral cytometric analysis of immune cell populations

Basic Protocol 3: Optimized workflow and spectral flow cytometry panels for profiling of pulmonary immune cell populations in a single-cell suspension

JBrowse 2 is an open-source genome browser that provides unique features for visualizing syntenic relationships between multiple genomes. This article describes a protocol for setting up synteny views in JBrowse 2, using an assembly-to-assembly whole-genome alignment example. We detail data preparation steps, including the generation and formatting of whole-genome alignment data into formats compatible with JBrowse 2's synteny visualization capabilities, and show the GUI-driven process for setting up interactive synteny views and generating publication-quality figures. This protocol establishes methods for using JBrowse 2 to explore conserved sequences across multiple genomes. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol: Using JBrowse 2 to show genomic synteny