Current protocols最新文献

Identifying viral sequences from metagenomic datasets is critical for investigating their origins, evolutionary patterns, and ecological functions. Previously, we developed a novel deep learning software, DeepVirFinder, to predict viral sequences from shotgun metagenomic assemblies. This method employs a twin convolutional neural network model to extract features from known viral and prokaryotic host genomic sequences for binary classification of input query sequences. With the rapid accumulation of environmental metagenomic data, this approach has accelerated the discovery of novel viruses from diverse environments through an alignment-free and reference-free deep learning strategy. To facilitate the rapid adoption of this software for beginning users, here we have further improved DeepVirFinder by optimizing its runtime performance, while maintaining the essential user interface of the original version. This comprehensive guide provides basic workflows for the most common use cases of DeepVirFinder. Additionally, to assist users in downstream analyses, supplementary scripts were provided in the software for extracting viral sequences and inspecting the results, thereby helping researchers more effectively mine viral information from metagenomic datasets. © 2026 Wiley Periodicals LLC.

Basic Protocol 1: Predicting viral sequences in metagenomic assemblies

Basic Protocol 2: An integrated pipeline for viral sequence analysis: Prediction, extraction, and visualization

Basic Protocol 3: Retraining the DeepVirFinder model using a customized dataset

Virus-like particles (VLPs) are widely recognized as safe and versatile nanostructures with broad applications in vaccine development, gene delivery, and nanotechnology, but their production typically requires time-consuming procedures, incurs high costs, and yields products of limited purity. This article outlines a rapid, scalable, and cost-effective method for producing high-quality VLPs using the VP1s capsid protein from murine polyomavirus. The procedure comprises four principal stages: expression of the capsid protein in Escherichia coli, extraction and stabilization of the protein, purification to yield high-quality capsomeres, and controlled in vitro assembly of VLPs. Compared with conventional protocols that rely on in vivo assembly and labor-intensive purification procedures, such as ultracentrifugation- or affinity tag-based methods, the described approach provides a streamlined and rapid workflow that avoids affinity tags, achieves consistently high yields, and enables precise regulation of assembly conditions, resulting in a scalable and highly cost-efficient strategy for VLP production within 5 days. © 2026 Wiley Periodicals LLC.

Basic Protocol 1: Expression and preparation of polyomavirus VP1 protein in E. coli

Basic Protocol 2: Polymomavirus VP1 purification

Basic Protocol 3: VP1 assembly

Basic Protocol 4: Quality control

This article provides detailed methods for preparing membrane and cytosolic proteins isolated from human CD4⁺ T cells to identify biomarkers of oxidative stress (OS). Proteins important for T-cell metabolism, differentiation, and redox homeostasis can be identified using this untargeted approach. Proteins of interest contain cysteine thiols hypothesized to be susceptible to irreversible modification from reactive electrophiles generated under conditions of OS. Membrane-bound targets include receptors, ion channels, and enzymes that serve fundamental roles in sensing the redox microenvironment of T cells during activation and signaling pathways controlling responses to biochemical cues from their surroundings. Cytosolic targets include enzymes, chaperones, and transcription factors that can be separated from membrane proteins providing increased sequence coverage. This procedure includes detailed steps for isolating CD4⁺ T cells from peripheral blood samples and separating their membrane and cytosolic protein fractions for subsequent identification by LC-MS/MS. Proteomics data obtained from this procedure allows the relative abundance of proteins responsible for redox regulation of CD4⁺ T-cell activation and differentiation programs to be established. These data can be used to determine whether additional enrichment steps are necessary for discovering, characterizing, and quantifying novel irreversible modifications to specific thiols from proteins of interest that are expressed in low abundance. This protocol can be used for ex vivo studies where oxidation is experimentally induced to identify proteins diagnostic of OS associated with specific chemical exposures. Results from these studies can help determine the biochemical mechanisms of disease influenced by the human exposome and increased OS. © 2026 Wiley Periodicals LLC.

Basic Protocol 1: Separation of human peripheral blood mononuclear cells from whole blood

Basic Protocol 2: Isolation of CD4⁺ T cells by negative selection

Basic Protocol 3: Fractionation of CD4⁺ T-cell membrane and cytosolic proteins

Basic Protocol 4: Bradford assay and concentration of protein fractions

Traditional retroviral gene transfer protocols for the genetic modification of hematopoietic stem and progenitor cells (HSPC) include a multiday ex vivo culture period, which can negatively affect the biology of the cells and is costly. As an alternative approach, we outline here a method for gene transfer into quiescent human HSPCs using lentiviral (LV) vectors and gamma-retroviral virus-like particles (eVLP). To achieve this, we use LV vectors and eVLPs pseudotyped with the modified baboon endogenous retroviral glycoprotein BaEVRLess, which targets the neutral amino acid transporters ASCT1 and ASCT2 on HSPCs. This envelope enables immediate transduction of freshly isolated or thawed, unstimulated HSPCs with higher gene transfer efficiencies than those obtained with VSVg-pseudotyped LV vectors and enables the transplantation of transduced HSPCs within less than 24 hr of cell isolation. These protocols provide detailed guidance on the production and titration of BaEVRLess-pseudotyped LV vectors and eVLPs and the cultivation and transduction of quiescent HSPCs, and highlights critical steps and potential pitfalls of these processes. © 2026 Wiley Periodicals LLC.

Basic Protocol 1: Generation of BaEVRLess-pseudotyped lentiviral vectors

Alternate Protocol: Generation of BaEVRLess-pseudotyped virus-like particles

Support Protocol: Preparation and testing of polyethyleneimine solution for DNA transfection

Basic Protocol 2: Transduction of quiescent human CD34⁺ hematopoietic stem and progenitor cells

Bone regeneration depends on coordinated interactions among cells, extracellular matrices, and the dynamic microenvironment within engineered constructs. Mineralized collagen-glycosaminoglycan (MC-GAG) scaffolds are designed to replicate key features of native bone matrix, yet their mineral accumulation and mechanical performance remain lower than those of mature bone, making precise evaluation of mineral deposition essential. High-resolution microCT imaging provides nondestructive, three-dimensional (3D) visualization of mineral distribution, density, and microarchitecture with substantially improved fidelity. Through dynamic, angle-dependent calibration and scan-specific recalibration, our microCT scanner HiCT minimizes motion- and temperature-related artifacts and enables consistent, high-resolution imaging suitable for small scaffold systems. This protocol outlines an integrated workflow combining high-resolution microCT scanning with advanced image segmentation to assess mineralization within MC-GAG scaffolds. The method includes scaffold fabrication, chemical cross-linking, and human mesenchymal stem cell culture, followed by stable positioning in custom 3D-printed racks to ensure precise imaging geometry. Reconstruction and threshold-based segmentation in a DICOM viewer such as ORS Dragonfly allow separation of scaffold structure from higher-density mineral phases, providing detailed visualization and quantitative analysis of mineral deposition. This reproducible framework enhances the characterization of bone-regenerative biomaterials and supports efforts to engineer scaffolds that more closely mimic the structural and functional properties of native bone. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Fabrication of bone regenerative mineralized collagen scaffolds

Basic Protocol 2: High-resolution microCT scanning and microstructural analysis of the MC-GAG scaffolds

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

Mousavi, N., Zhou, E., Razavi, A., Ebrahimi, E., Varela-Castillo, P., & Yang, X.-J. (2025). Efficient site-directed mutagenesis mediated by primer pairs with 3’-overhangs. Current Protocols, 5, e70104. doi: 10.1002/cpz1.70104

In the above-referenced article:

In Steps 5 and 6 of Basic Protocol 1, the concentrations of the oligonucleotide stock and working concentrations, the forward and reverse primers have been corrected. The oligonucleotide stock and working concentrations as well as the forward and reverse primers concentrations have been changed from mM to µM.

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

Fecal DNA metabarcoding is a powerful tool for examining animal diets with unprecedented resolution, offering insights into ecological patterns shaped by trophic interactions. As a result, dietary metabarcoding has become widely applied across ecology, evolution, behavior, and conservation. This article provides a practical guide to the key steps involved in metabarcoding animal fecal samples, from field collection and storage through to laboratory processes, such as DNA extraction, PCR amplification, and sequencing library preparation. It also outlines a bioinformatics workflow using the open-source QIIME2 platform to filter, error-correct, and assign taxonomy to dietary DNA sequences. We present key considerations for study design, highlighting potential caveats and limitations to enable researchers to make informed methodological choices. In addition, we offer guidance on the statistical analysis of diet data, including generalized linear models, multivariate analyses, and network analyses. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Metabarcoding library preparation of animal fecal dietary DNA for Illumina MiSeq sequencing

Support Protocol 1: Making a SpeedBeads solution

Support Protocol 2: Calibrating the SpeedBeads solution

Basic Protocol 2: QIIME2 bioinformatics workflow for metabarcoded dietary DNA

In vitro modeling of human liver function is essential for assessing drug-induced toxicity, particularly in the context of metabolic dysfunction–associated steatotic liver disease (MASLD). However, standard liver organoid systems often lack xenobiotic-metabolizing enzyme activity and fail to replicate the pathological features of MASLD. Here, we present a method for generating functional, multi cell–type human liver organoids (HML) organoids from HepaRG cells, primary human macrophages, and LX-2 hepatic stellate cells. These three-dimensional (3D) organoids are cultured under defined conditions that support key hepatic functions. To mimic MASLD, we expose HML organoids to a mixture of stearic and oleic acids for 9 days, inducing steatosis and fibrogenic responses characteristic of the disease. These MASLD-HML organoids retain liver-specific functions and express key fibrosis markers. We further demonstrate the usefulness of this method to produce standardized organoids useful for the evaluation of drug-induced liver injury (DILI) through IC50 and benchmark dose calculations, particularly in the context of MASLD. Together, HML and MASLD-HML organoids provide a robust model for studying drug metabolism, toxicity, and adverse drug reactions in healthy and diseased liver states. © 2026 Wiley Periodicals LLC.

Basic Protocol 1: Culture of HepaRG cells

Basic Protocol 2: Culture of LX-2 cells

Basic Protocol 3: Culture of primary human unstimulated M1 macrophages

Basic Protocol 4: Organoid seeding procedure 1 in agarose molds

Basic Protocol 5: Organoid seeding procedure 2 in ultra-low attachment 96-well plate

Alternate Protocol: Organoid seeding procedure from cryopreserved cells

Basic Protocol 6: Preparation of the fatty acid mixture

Basic Protocol 7: MASLD induction

Site-directed mutagenesis is indispensable for protein, RNA, and plasmid engineering. It is ideal to carry out such mutagenesis at an efficiency close to 100%, but many current methods fail to reach this goal and thus require extensive screening efforts. We have recently optimized an innovative site-specific mutagenesis approach based on PCR with primer pairs possessing 3’-overhangs, thereby reaching the ideal efficiency of ∼100%. Such high efficiency and the incorporation of the “handshaking” feature of primer pairs with 3’-overhangs have led us to adapt this method for seamless cassette mutagenesis, thereby conferring highly efficient deletion (up to 5 kb), insertion (up to 0.4 kb) or replacement of DNA fragments. Conceptually, deletion and insertion are special cases of replacement mutations, where the respective sequences to be inserted and deleted are 0 bp. This is because replacement mutagenesis converts fragment A to fragment B; for deletion, fragment B is 0 bp in size, whereas for insertion, fragment A is 0 bp. Thus, this new method makes site-directed and cassette mutagenesis a highly efficient and reliable tool for protein, RNA and plasmid engineering in different types of biomedical research. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: P3a site-directed mutagenesis to introduce point mutations, deletions, insertions or replacements

Basic Protocol 2: Transformation of chemically competent DH5α cells to obtain bacterial colonies

Basic Protocol 3: Isolation of plasmids from bacterial colonies for sequence analysis to identify clones with designed mutations

Alternate Protocol: P3 site-directed mutagenesis to introduce deletions, insertions, or replacements

Dermatophytes are keratinophilic fungi that cause superficial infections of the skin, hair, and nails. The emergence of recalcitrant infections caused by Trichophyton mentagrophytes complex and T. indotineae, particularly in diabetic patients, has heightened the need for reproducible small-animal models to study disease pathogenesis and test antifungal therapies. Here, we describe a diabetic mouse model of dermatophytosis that faithfully mimics human infection. The protocol details (1) preparation of dermatophyte conidial inocula from laboratory or clinical isolates, (2) pharmacological induction of diabetes or immunosuppression in mice, (3) standardized infection of abraded dorsal skin, and (4) downstream assessments of fungal burden and pathology, including clinical evaluation and scoring, enumeration of colony-forming units, and histopathology. Critical parameters, troubleshooting guidance, and anticipated results are provided to ensure reproducibility. This system has been successfully applied to evaluate conventional and investigational antifungal drugs and can be readily adapted for emerging resistant species such as T. indotineae. By integrating diabetic predisposition with current epidemiological trends, this model provides a clinically relevant and cost-effective platform for dermatophyte research and antifungal development. © 2026 Wiley Periodicals LLC.

Basic Protocol 1: Preparation of dermatophyte inoculum

Basic Protocol 2: Establishment of immunosuppressed or diabetic mouse models of dermatophytosis