Current protocols最新文献

FET proteins are large multifunctional proteins that have several key roles in biology. The FET family of proteins, including FUS, EWSR1, and TAF15, play critical roles in transcription regulation, RNA processing, and DNA damage repair. These multifunctional RNA- and DNA-binding proteins are ubiquitously expressed and conserved across vertebrate species. They contain low-complexity (LC) domains that allow them to assemble and phase separate but also makes the proteins prone to aggregation. Aberrations in FET proteins, such as point mutations, aggregation, or translocations leading to fusion proteins, have been implicated in several pathologies, including frontotemporal lobar degeneration (FTLD), amyotrophic lateral sclerosis (ALS), and Ewing sarcoma. In vitro study of FET proteins is hampered by their propensity to aggregate, their disordered structure, and their susceptibility to proteolysis, making high-yield production difficult. Here, we present optimized methods for the purification of full-length FUS, EWSR1, and their fusion proteins. These protocols enable researchers to overcome issues related to aggregation and solubility, facilitating biochemical and biophysical studies of these critical yet complex proteins. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol: Purification of EWSR1 and FUS proteins

Alternate Protocol: Purification for fusion proteins

Cryptococcus neoformans is an opportunistic fungal pathogen that heads the Fungal Priority Pathogen List published by the World Health Organization (WHO) in 2022. This pathogen is a primary cause of death for immunocompromised individuals (e.g., those with HIV/AIDS, the elderly, immunotherapy recipients), causing approximately 118,000 deaths yearly worldwide. C. neoformans relies on virulence factors that include a polysaccharide capsule, melanin, extracellular enzymes, and thermotolerance to initiate and sustain host infection. Additionally, similar to other fungal pathogens (e.g., Candida albicans), C. neoformans may develop a biofilm organization linked to more persistent cryptococcal infections. Cryptococcal biofilms are highlighted in cases of cryptococcal meningitis, in which biofilm-like structures form that are highly resistant to host immune response and to antifungal therapies. In this regard, fungal biofilm formation has become an important area of study as a means to improve our understanding of the mechanisms regulating biofilm formation and infection and to advance the discovery of antibiofilm therapeutics. To assess biofilm properties and compare across treatments, quantification and evaluation of cell viability are important. Herein, we describe a standardized method to establish a cryptococcal biofilm and quantify total biomass and cell viability. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Culturing and biofilm formation

Basic Protocol 2: Biofilm quantification

Alternate Protocol: Biofilm viability assay

Mouse models are essential for understanding gene function, environmental interaction, and brain structure and function. This is reinforced by the ability of mice to perform complex behavioral tasks. Still, their cognitive assessments often rely on aversive paradigms, such as fear conditioning and the Morris water maze. A promising alternative is the automated touchscreen platform, which enables cognitive tests comparable to those used in humans, such as the Cambridge Neuropsychological Test Automated Battery (CANTAB). This approach enhances standardization and reduces stress by employing appetitive reinforcement. Although widely used in non-human primates, touchscreen testing remains underutilized in rodents despite its potential for cross-species cognitive research. Motivation is key to successful touchscreen tasks, often achieved through water restriction, which mice tolerate well. However, water restriction is a stressful condition, combining negative and positive reinforcement. Here, we propose an alternative that uses citric acid (CA) water to avoid classical food privation in the touchscreen paradigm to mitigate mice's stress. By creating a strong contrast with the reward, we increase the reward's positive valence. We used the touchscreen visual discrimination task to assess the effectiveness of CA water in enhancing motivation. Our results show that administering CA water on training days while allowing access to plain water on weekends reduces the learning phase duration without causing significant weight loss in wild-type C57BL/6J mice. In addition, we observed a strong commitment to performing the pattern dissociation task. This approach offers a welfare-friendly alternative for maintaining motivation in touchscreen-based cognitive tasks while minimizing stress. © 2025 Wiley Periodicals LLC.

Basic Protocol: Pattern dissociation paradigm using sour water

This article presents a comprehensive guide for using the MethMotif platform, which includes the MethMotif database, the TFregulomeR R package, and a new R library, Forked-TF, designed specifically for analyzing leucine-zipper transcription factors (TFs) that bind DNA as dimers. The MethMotif platform integrates transcription factor binding site (TFBS) motifs with DNA methylation profiles, providing an in-depth analysis of how methylation modulates TF binding across different cell types and conditions. The protocols are organized into three main workflows: (1) Exploration of transcription factor dimerization partners, (2) visualization of methylation-specific TF motifs using TFregulomeR, and (3) characterization of leucine-zipper TF binding patterns with a focus on dimerization. Using the platform's MethMotif database, users can retrieve ChIP-seq and DNA methylation data, intersect TFBS peak regions, and generate TFBS-methylation-informed motif logos. A case study of CEBPB in K562 cells is included to demonstrate the use of the platform, showing how to identify TF dimers, analyze their co-binding behavior, and visualize the impact of DNA methylation on binding specificity. The protocols also provide step-by-step instructions for software installation, data input formats, and interpretation of results, making it accessible to researchers with varying levels of computational expertise. Through these protocols, users can uncover how DNA methylation and TF dimerization influence gene regulatory networks, with a focus on leucine-zipper TFs in a cell-type-specific context. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Exploration of transcription factor dimerization partners

Support Protocol 1: Software installation

Support Protocol 2: Docker installation

Support Protocol 3: Verifying installation

Basic Protocol 2: Visualization of alternative cofactors

Basic Protocol 3: Characterization of bZIP partners/cofactors

Basic Protocol 4: Context-independent and context-dependent analysis

Intracellular water content, W, and protein concentration, P, are essential characteristics of living cells. Healthy cells maintain them within a narrow range, but often become dehydrated under severe stress; moreover, persistent loss of water (an increase in P) can lead to apoptotic death. It is very likely that protein concentration affects cellular metabolism and signaling through macromolecular crowding (MC) effects, to which P is directly related, but much remains unknown in this area. Obviously, in order to study the biological roles and regulation of MC in living cells, one needs a method to measure it. Simple and accurate measurements of P in adherent cells can be based on its relationship to refractive index. The latter can be derived from two or more (depending on the algorithm) mutually defocused brightfield images processed by the transport-of-intensity equation (TIE) that must be complemented by a determination of volume. Here, we describe the experimental considerations for both TIE imaging and for a particular method of cell volume measurement, transmission-through-dye (TTD). We also introduce an ImageJ plugin for solving TIE. TIE and TTD are fully compatible with each other as well as with fluorescence. A similar approach can be applied to subcellular organelles; however, in this case, the volume must be determined differently.© 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Sample preparation for TIE with or without TTD

Basic Protocol 2: Acquisition of TIE and TTD images

Basic Protocol 3: Calibration of TIE

Basic Protocol 4: Measurement of the absorption coefficient of the medium used for TTD

Basic Protocol 5: Image processing using Fiji

Support Protocol 1: Installation and use of TIE plugin

Support Protocol 2: Automation of the double TTD/TIE processing using a Fiji macro

Machine learning (ML) is rapidly gaining traction in many areas of experimental molecular science for elucidating relationships and patterns in large or complex data sets. Historically, ML was largely the preserve of those with specialized training in fields such as statistics or cheminformatics. Increasingly, however, ML methodologies are becoming part of the standard toolkit for experimental scientists across a range of disciplines. For scientists without a significant background in computer science or statistics, lowering the barrier of entry to these ML techniques is important to broadening access to these powerful methods. Here we provide detailed, step-by-step protocols for performing four ML methods that are particularly useful for applications in biochemistry, cell biology, and drug discovery: hierarchical clustering, principal component analysis (PCA), partial least squares discriminant analysis (PLSDA), and partial least squares regression (PLSR). The protocols are written for the widely used software MATLAB, but no prior experience with MATLAB is required to use them. We include an explanation of each step, pitched at a level to be understood by investigators without any prior experience with ML, MATLAB, or any kind of coding. We also highlight the scientific issues pertaining to selecting and scaling the data to be analyzed. Throughout, we emphasize the relationship between the scientific question and how to choose data and methods that will allow it to be addressed in a meaningful way. Our aim is to provide a basic introduction that will equip experimental chemical biologists, chemists, and other biomedical scientists with the knowledge required to use ML to aid in the design of experiments, the formulation and data-driven testing of hypotheses, and the analysis of experimental data. © 2025 Wiley Periodicals LLC.

Basic Protocol 1: Clustering

Basic Protocol 2: Principal component analysis

Basic Protocol 3: Partial least squares-discriminant analysis

Basic Protocol 4: Partial least squares regression

An assay and protocol framework are provided for utilizing reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) at the point-of-care for diagnosing and monitoring a hypothetical zoonotic viral outbreak in a resource-limited area. This manuscript utilizes a previously published decision tree algorithm to determine an appropriate molecular diagnostic point-of-care test that can effectively address the outbreak presented in the hypothetical case study. © 2025 Wiley Periodicals LLC.

Myelodysplastic neoplasms (MDS) are acquired heterogeneous clonal hematopoietic stem cell neoplasms, clinically characterized by progressively ineffective hematopoiesis and an increased risk of acute myeloid leukemia. MDS are accompanied by an inflammatory microenvironment and genome instability. Signs of dysplasia can occur in the erythroid, myeloid, monocytic, and megakaryocytic cell lineages and result in anemia, neutropenia, and thrombocytopenia. Multi-parameter flow cytometry can be used to detect aberrant antigen expression patterns typical of MDS, which correlate with cytomorphologically identified dysplasias and provide important information for diagnosis and prognosis. Characteristic findings include an increase in myeloid progenitor cells; aberrant myeloid and erythroid maturation; aberrant marker expression on progenitor cells, granulocytes, and monocytes, which corresponds to lineage infidelity, under-/overexpression, or asynchronous expression; and an increase in monocytes and progenitor cells in chronic myelomonocytic leukemia. The latter represents an independent disease entity with a similar phenotype. In addition, flow cytometry can rule out other causes of cytopenia, such as lymphoma, acute leukemias, paroxysmal nocturnal hemoglobinuria, or systemic mastocytosis with associated hematologic neoplasm. To analyze those features, the European LeukemiaNet recommends a set of markers together with important technical aspects. At least three distinct aberrations in at least two lineages are associated with a high likelihood of MDS. © 2025 Wiley Periodicals LLC.

Basic Protocol: Flow cytometric bone marrow evaluation in suspected myelodysplastic neoplasms

Natural killer (NK) cells are critical components of the immune system, recognized for their ability to identify and eliminate target cells undergoing various forms of stress. Beyond their direct cytotoxic activity, NK cells secrete extracellular vesicles (EVs), which serve as mediators of intercellular communication. These vesicles play an essential role in modulating immune responses and influencing tumoral and post-infection microenvironments. In this study, we present a detailed methodology for the isolation and expansion of human primary NK cells, the accumulation of their EVs, and the subsequent procedures for their isolation and characterization. © 2025 Wiley Periodicals LLC.

Basic Protocol 1: NK cell isolation and expansion

Basic Protocol 2: NK-EV purification using ultracentrifugation

Alternate Protocol: NK-EV isolation using size exclusion chromatography

Basic Protocol 3: Characterization of NK-EVs

This protocol describes procedures for the preparation of a modified thymidine conjugated with galactose via a self-immolative linker at the O4-position, and the synthesis of β-galactosidase-responsive prodrug-type oligodeoxynucleotides (ONs) containing these modified thymidines. These prodrug-ONs are designed to be activated in response to β-galactosidase, enabling targeted activation in specific cells or tissues and potentially contributing to the reduction of adverse effects. © 2025 Wiley Periodicals LLC.

Basic Protocol 1: Preparation of O4-modified thymidine phosphoramidite 7

Basic Protocol 2: Preparation of O4-modified thymidine phosphoramidite 15

Support Protocol 1: Preparation of triazolyl thymidine derivative 1

Support Protocol 2: Preparation of benzyl alcohol derivative 2

Basic Protocol 3: Preparation of β-galactosidase-responsive ODNs 1 to 5