Current protocols最新文献

It is crucial that experiments dealing with plant–pathogen interactions are robust and reproducible. For evaluating the disease-resistance ability of a host plant against any pathogen, controlled conditions are a prerequisite that cannot be obtained with soil-based systems. Variation in environmental conditions during challenge inoculation of a pathogen, when performed in fields or a polyhouse, may lead to spurious results. This is of particular importance when dealing with disease resistance in a fruit tree like pomegranate, as determined after years of research efforts. Due to lack of uniformity and environment variability, the possibility of pathogen escapes and false positives is large under field or polyhouse conditions. To address these limitations, we have used tissue-cultured plants and developed an easy, reliable, rapid, and efficient in vitro bioassay for bacterial blight on pomegranate. Owing to its performance under a closed system, this system minimizes the risk of pathogen escapes, reduces environmental variability, and provides a robust high-throughput screening platform for disease resistance and tissue sampling for downstream functional genomics studies. The protocols enable reproducible experiments to study plant response to bacterial blight infection. Stepwise protocols and detailed instructions are provided to carry out the screening experiment in pomegranate or other similar plant species. © 2025 Wiley Periodicals LLC.

Basic Protocol 1: Preparation of nodal explants and in vitro rooting

Basic Protocol 2: In vitro blight bioassay

Basic Protocol 3: Pathogen re-isolation and confirmation

This paper, the second in a two-part series, outlines a low-cost, open-source, pseudo-randomization approach for measuring cutaneous reflexes during human walking. The protocol guides users through integrating an Arduino Uno microcontroller, a VICON data acquisition system, and an electrical stimulation device. A custom code was developed in C++, and steps are provided to guide users in downloading and integrating it with an Arduino. This setup provides researchers the ability to define key experimental parameters such as the number of gait cycles to include in quantifying the average time of the subject's walking gait cycle, the number of bins to partition the human walking gait cycle, and the number of stimuli per bin—all while maintaining real-time stimulation capability. During a walking trial, the system is designed to (1) deliver stimuli pseudo-randomly using instances when the foot makes initial contact with the treadmill surface and (2) deliver an even distribution of stimuli across all bins. Real-time feedback mechanisms and data monitoring are enabled via an integrated Arduino and VICON framework, allowing researchers to conduct controlled, repeatable experiments that aim to measure cutaneous reflexes during human walking. This protocol provides a flexible and accessible solution for researchers aiming to investigate phase-dependent modulation of reflexes during normal and perturbed walking conditions. Its modularity and integration with existing motion capture systems make it a powerful tool for both basic and clinical research within the fields of neuroscience, biomechanics, and gait rehabilitation. © 2025 Wiley Periodicals LLC.

Basic Protocol 1: Integrating Arduino to VICON data acquisition and stimulator

Basic Protocol 2: Downloading and running pseudo-randomized Arduino code

Angiogenesis is crucial in tissue repair, wound healing, and embryo development. Maintaining a balance between pro-angiogenic and anti-angiogenic factors is crucial for tissue homeostasis, thereby preventing the development of pathological conditions. In addition, the tissue microenvironment constantly influences molecular signaling, potentially altering this delicate balance. The chorioallantoic membrane (CAM) assay has emerged as a dynamic, cost-effective, and ethically favorable alternative for studying angiogenesis. This work presents a detailed and accessible approach to the CAM assay, focusing on the evaluation of the angiogenic potential of applied stimuli (such as conditioned media, extracellular vesicles, or bioscaffolds) through various techniques, including hematoxylin and eosin, PicroSirius Red, Alcian Blue, and resorcin-fuchsin staining; immunofluorescence; immunohistochemistry; and scanning electron microscopy (SEM). Our protocol uses the in ovo CAM assay approach, utilizing a simple incubator setup that eliminates the need for expensive equipment. The primary objective is to provide a comprehensive methodology for researchers to efficiently analyze angiogenesis, offering insights into vessel morphology and protein expression. The experiment comprises three major steps: (1) egg opening, stimulation, and sample collection; (2) histological processing and paraffin blocking; and (3) microscopic analyses. The first and second procedures take approximately 15 days, after which each analysis can be conducted whenever the researcher judges appropriate. The expected results include several types of data concerning the proliferation rate, vessel presence and integrity, measurement of angiogenic-specific proteins, and investigation of structural proteins. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol: Angiogenesis quail and chicken chorioallantoic membrane assay analyses

This paper, the first in a two-part series focused on measuring cutaneous reflexes during human walking, provides a detailed step-by-step methodology for reliably eliciting cutaneous reflexes during human treadmill walking. The procedure addresses the technical challenges of eliciting reflexes from cutaneous nerves in a consistent and reproducible manner throughout the gait cycle. Building on approaches used in previous studies, we integrate practical guidance on equipment setup, electrode placement, configuration of a foot-sensitive resistor for quantifying gait cycle parameters, and reflex measurements to enable successful implementation across laboratories with varying levels of expertise. The custom development and use of a pseudorandomized stimulation approach is a novel feature of our broader methodology and is described in detail in the second paper. The present protocol focuses on the experimental setup required to obtain high-quality reflex measurements during walking, thereby providing the basis for advanced stimulation paradigms in human sensorimotor research. © 2025 Wiley Periodicals LLC.

Basic Protocol: Evoking cutaneous reflexes during human walking using a pseudorandomized approach

The oncogenic Epstein Barr virus (EBV) is an exclusively human pathogen with related lymphocryptoviruses (γ1-herpesviruses) only present in monkeys. Therefore, experimentation with EBV infection in a small animal model requires reconstitution or adoptive transfer of human lymphocyte populations, primarily EBV's main host cell, the human B cell. In this protocol we describe human immune system reconstitution after neonatal transfer of CD34⁺ hematopoietic progenitor cells in lymphodeplete immune compromised mouse strains, using NOD-scid γ_c^–/– (NSG) mice as a commonly used example. Such reconstituted humanized mice allow intraperitoneal and intranasal infection with EBV and we describe injection of 10⁵ infectious particles of the prototypic EBV strain B95-8 that can be produced from a recombinant bacmid (p2089) in HEK293 cells. Infection with this dose mimics symptomatic primary EBV infection, infectious mononucleosis (IM), with high viral loads plateauing 4 weeks after infection, with CD8⁺ T-cell lymphocytosis at week 5 and 6 after infection. This IM-like primary EBV infection in humanized mice leads to clonal EBV-induced B-cell lymphoproliferations that resemble large B-cell lymphomas with the latency III program of EBV infection. We describe Basic Protocols to monitor viral loads, immunohistochemistry of infected tissues and spectral flow cytometry to characterize protective T-cell expansion. The described mouse model has been used by us and others to characterize mutant EBV infections, cell-mediated immune control of EBV, modulation of EBV pathogenesis by co-infections with human immunodeficiency virus (HIV) and Kaposi sarcoma associated herpesvirus (KSHV), as well as passive transfer of vaccine elicited antibodies to test their protection against EBV infection. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: CD34⁺ human hematopoietic progenitor cell isolation and characterization

Basic Protocol 2: Human immune system reconstitution and characterization

Basic Protocol 3: Recombinant EBV production and humanized mouse infection

Basic Protocol 4: Viral load quantification, lymphoma assessment, and immunohistochemistry after EBV infection of humanized mice

Basic Protocol 5: Human T-cell response analysis after EBV infection of humanized mice

This article provides a comprehensive set of protocols for the in vitro selection of threose nucleic acid (TNA) enzymes capable of catalyzing RNA cleavage or ligation reactions. The article first outlines the generation of random-sequence TNA libraries (typically comprising ∼10¹⁴ unique molecules) covalently attached to their RNA substrates via engineered DNA polymerases. It then describes the procedure for isolating catalytically active TNA sequences through affinity chromatography or gel electrophoresis. These sequences are subsequently reverse-transcribed to cDNA for amplification or sequencing. This set of protocols is primarily designed for selecting RNA-cleaving and RNA ligase TNA enzymes but can be adapted for the discovery of TNA enzymes that act on DNA substrates or enzymes derived from other xeno-nucleic acids, provided that requisite nucleoside triphosphates and compatible tool enzymes are available.© 2025 Wiley Periodicals LLC.

Basic Protocol 1: TNA library synthesis

Basic Protocol 2: Selection of RNA-cleaving TNA enzyme

Alternate Protocol: Selection of RNA ligase TNA enzyme

Basic Protocol 3: Reverse transcription of enriched TNA library

Basic Protocol 4: Preparation of template DNA for the next round

An estimated 40 million people are infected with human immunodeficiency virus type 1 (HIV-1) and there is no effective cure. Humanized mice are a useful model to study transmission, pathogenesis, prevention, and treatment of HIV-1, which only infects human cells. Here we provide detailed protocols to humanize immunodeficient mice, e.g., NSG or MISTRG6 strains, using human hematopoietic stem cells isolated from cord blood and a flow cytometry panel of antibodies to assess human immune cell reconstitution. Furthermore, we describe how to produce, concentrate, and titer HIV-1 in vitro, which can be used to challenge humanized mice either systemically or mucosally. Finally, we adapted a sensitive quantitative reverse transcription–polymerase chain reaction (RT-qPCR) assay with internal control to detect HIV-1 RNA in longitudinal plasma samples to measure viremia over time in the mice. © 2025 Wiley Periodicals LLC.

Basic Protocol 1: Isolation of CD34+ cells from human cord blood

Basic Protocol 2: Injection of human CD34+ cells in immunodeficient mice

Basic Protocol 3: Flow cytometry of peripheral blood mononuclear cells from mouse blood

Basic Protocol 4: Production and titration of HIV-1

Basic Protocol 5: Systemic HIV-1 challenge of humanized mice

Alternate Protocol: Mucosal HIV-1 challenge of humanized mice

Basic Protocol 6: Single-copy HIV-1 RNA detection in mouse plasma

Support Protocol 1: Retrovirus internal control for plasma virus isolation and RT-qPCR

Support Protocol 2: Production of HIV-1 standard for RT-qPCR

Current Protocols is issuing a correction for the following protocol article.

Mueller, E. J., Rahimi, S., Sauta, P., Shojaeian, T., Durrer, L., Quinche, S., Francois, M., Locher, E., Edler, M., Illi, M., Gentinetta, T., Lau, K., Pojer, F., Borradori, L., & Hariton, W. V. J. (2024). Standardized production of anti-desmoglein 3 antibody AK23 for translational pemphigus vulgaris research. Current Protocols, 4, e1118. doi: 10.1002/cpz1.1118

In the above-referenced article:

The author name “Mueller” has been corrected to “Müller” throughout the article including the Biblography.

The current version online now includes this correction and may be considered the authoritative version of record.

This protocol presents methods for isolating and identifying novel nucleic acid–binding proteins. We focus on bacterial DNA-binding proteins, although the methods can be readily adapted to identify nucleic acid–binding proteins of eukaryotes or archaea, and proteins that bind to RNAs. Briefly, the DNA sequence of interest is affixed to beads and then incubated with bacterial cytoplasmic extract. Washes with buffers containing nonspecific DNA and low salt concentrations will remove non-adhering and low-specificity DNA-binding proteins, while subsequent washes with higher salt concentrations will elute DNA-binding proteins that have greater specificity. Eluted proteins are then identified by standard proteomic techniques, such as mass spectrometry. © 2025 Wiley Periodicals LLC.

Basic Protocol 1: Lysis procedure

Basic Protocol 2: DNA affinity chromatography: Borrelia burgdorferi and Leptospira interrogans

Basic Protocol 3: Visualization and identification of DNA-binding proteins

We present a reproducible protocol for isolating and analyzing natural killer (NK) cells from freshly resected human glioblastoma multiforme (GBM) tissue using flow cytometry. The workflow yields viable single-cell suspensions suitable for downstream applications, including cell sorting, immunophenotyping, functional assays, and single-cell RNA-seq.© 2025 Wiley Periodicals LLC.

Basic Protocol 1: Dissociation of glioblastoma multiforme tissue to single-cell suspension

Support Protocol 1: Preparation of digestion dissociation mix for dissociation of glioblastoma multiforme tissue

Alternate Protocol 1: Mechanical-only dissociation of glioblastoma multiforme tissue (no-enzyme control)

Basic Protocol 2: Mononuclear cell enrichment and red blood cell lysis of glioblastoma multiforme single-cell suspension

Support Protocol 2: Removal of cellular debris from glioblastoma multiforme single-cell suspension

Basic Protocol 3: Flow cytometry staining and acquisition for identification of tumor-infiltrating NK cells from glioblastoma multiforme tissue

Alternate Protocol 2: Panel splitting for flow cytometry using instruments with limited numbers of channels