Current protocols最新文献

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

Martin-Solana, E., Frendi, S., Ning, J., Banks-Tibbs, T., Vockley, J., & Freyberg, Z. (2025). Preparation of vitrified mammalian cells for in situ cryo-electron tomography. Current Protocols, 5, e70253. doi: 10.1002/cpz1.70253

In the above-referenced article:

The Acknowledgments section has been revised to include a funding statement for a key source of equipment. The revised version now reads as below:

African trypanosomes exhibit significant genetic diversity but share similar morphological characteristics, making effective surveillance and diagnosis challenging with conventional microscopy and other detection methods. While polymerase chain reaction (PCR) offers enhanced specificity and sensitivity, it requires DNA extraction, making it time-consuming and costly, especially for resource-limited regions such as ours (Africa). Additionally, amplification using DNA from the host's whole blood often fails due to the presence of polymerase inhibitors or low parasite DNA, particularly during chronic infections. This study introduces a novel PCR method that overcomes these challenges by using purified trypanosome cells as templates, bypassing the need for DNA extraction. Trypanosoma brucei brucei cells were isolated from mice blood using DEAE–cellulose anion-exchange chromatography and directly used in PCR amplification of the ITS-1 ribosomal RNA gene. Successful amplification was achieved with as few as 1.25 × 10⁵ isolated trypanosome cells, while gDNA extracted from parasitized blood failed to amplify. This cost-effective and highly sensitive method improves species differentiation and enhances diagnostic capabilities, especially in cases of low parasitemia. The approach represents a significant advancement in trypanosome diagnosis, offering a faster, affordable, and reliable alternative to traditional methods. These findings support improved epidemiological surveillance and may contribute to the World Health Organization's goal of eliminating African trypanosomiasis, a disease that continues to present major economic and public health challenges in Africa. A graphical abstract of this study is presented in Figure 1. © 2026 Wiley Periodicals LLC.

Basic Protocol 1: Isolation of Trypanosoma brucei brucei from parasitized blood

Basic Protocol 2: PCR characterization of isolated T. brucei brucei

With antimicrobial resistance emerging as a top global health concern, new approaches to prevent and treat infections are urgently needed. Antimicrobial peptides are recognized as a promising alternative to conventional antibiotics. Considering the costly, labor-intensive, and time-consuming nature of wet lab screening, in recent years, proteomics research has increasingly relied on in silico methods to predict physicochemical, structural, and biological properties of candidate peptides. Here, we present the AMPSeek protocol to enable scientists to screen peptides and predict their potential antimicrobial activity, toxicity, and associated three-dimensional structure with a single command. The AMPSeek framework integrates AMPlify, tAMPer, and LocalColabFold, scales efficiently with increasing sample size, and produces an interactive HyperText Markup Language report to facilitate result interpretation. We provide step-by-step instructions for installing and running AMPSeek on a ten-peptide test set, along with two additional datasets (n = 20 and n = 100; mean peptide length ≈121.3) to demonstrate AMPSeek's near-linear scalability. AMPSeek is available on GitHub under the GPLv3 software license agreement: https://github.com/bcgsc/AMPSeek. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol: Prediction of antimicrobial peptide activity, three-dimensional structure, and toxicity

Nucleic acid therapeutics (NATs), including antisense oligonucleotides and small interfering RNAs, represent an expanding class of therapeutic modalities with distinctive physicochemical, pharmacokinetic, pharmacodynamic, and biodistribution properties. Naturally, their bioanalysis requires platforms that can accurately quantify intact analytes of interest and metabolites across diverse biological matrices. Modifications ranging from 2′-modifications, alterations of the phosphate backbones, and varied ligands conjugated for targeted delivery, influence extraction recovery, matrix effects, and assay selectivity and sensitivity. Historically, ligand-binding assays and PCR-based methods were adopted due to exceptional sensitivity. However, these approaches often lacked structural resolution and overestimated intact analyte when “sequence-similar” metabolites prevailed. Conversely, two complementary methods emerged, providing higher structural resolution, i.e., peptide-nucleic acid (PNA)-based hybridization in conjunction with anion exchange high-performance liquid chromatography (PNA-HPLC assay), and liquid chromatography–tandem mass spectrometry (LC–MS/MS), enabling separation of “sequence-similar” metabolites from the parent, and additionally, metabolite identification by LC–MS and LC–MS/MS. Recent methodological advances in LC–MS/MS workflows combining sequence-specific enrichment have substantially bridged the previously observed sensitivity gap. The introduction of high-affinity capture probes has improved assay robustness and recovery for challenging analytes and enhanced signal response while minimizing matrix-effect and ion suppression. Comparative evaluation demonstrates that both the PNA-HPLC and the hybrid LC–MS/MS assays are comparably superior for metabolite profiling and tissue distribution studies. This article integrates the analytical principles, strengths, and limitations of those two assays with exemplary case studies for NATs. Practical guidance is provided for method selection, probe selection, sample preparation, assay validation, and cross-platform harmonization. Emerging trends include PNA probe engineering and high-resolution MS for structural elucidation. The integration of capture probe-based hybridization enrichment with modern LC–MS/MS detection now enables combined sensitivity and specificity. Together, these developments support increasingly robust, convergent, regulatory-compliant bioanalytical strategies for next-generation oligonucleotide therapeutics. © 2026 Wiley Periodicals LLC.

Basic Protocol 1: PNA hybridization-based HPLC assay for the detection and quantification of therapeutic oligonucleotide in biological tissue samples

Basic Protocol 2: Hybrid LC–MS/MS quantitative assay for identification and evaluation of NAT in biological tissue samples

A marked global decline in seasonal flu due to circulation of influenza virus was observed during the COVID-19 pandemic. Although public health interventions played a role, potential immunological cross-reactivity between influenza and SARS-CoV-2 viruses remains a possibility. In this article, we describe methods to determine if SARS-CoV-2 infection has resulted in the induction of antibodies that cross-react with seasonal influenza viruses. To determine if such cross-reactivity does in fact occur, we examined plasma samples from 311 individuals with confirmed SARS-CoV-2 infection for evidence of ability to neutralize influenza virus. Antibody responses against SARS-CoV-2 were estimated by enzyme-linked immunosorbent assay, and responses to seasonal influenza viral strains—A/H3N2, A/H1N1, B/Victoria, and B/Yamagata—were assessed by performing the hemagglutination inhibition assay. Sixteen samples that were positive for SARS-CoV-2 antibodies but negative for influenza antibodies were selected for further analysis by microneutralization (MN) assay to evaluate their ability to neutralize influenza viruses. Among these, five samples exhibited high MN titers (≥20), and six exhibited moderate titers (≥10) against influenza A viruses. To elucidate the molecular basis of this cross-reactivity, genomes of SARS-CoV-2 strains were sequenced by Illumina next-generation sequencing and subjected to in silico structural modeling. Antibodies generated against the SARS-CoV-2 Delta variant demonstrated strong binding affinities with influenza A glycoproteins, including hemagglutinin from H1N1 (−12.4 kcal/mol) and H3N2 (−9.3 kcal/mol) and neuraminidase from H1N1 (−10.1 kcal/mol) and H3N2 (−11.7 kcal/mol). These results indicate that SARS-CoV-2 infection, particularly with the Delta variant, can induce antibodies with cross-neutralizing activity against influenza A viruses. This immunological cross-reactivity may have contributed to the reduced seasonal influenza activity during the COVID-19 pandemic. © 2026 Wiley Periodicals LLC.

Basic Protocol 1: Hemagglutination inhibition assay

Basic Protocol 2: Evaluation of SARS-CoV-2-specific antibodies via enzyme-linked immunosorbent assay

Basic Protocol 3: Pseudovirus titration and neutralization assays of SARS-CoV-2

Basic Protocol 4: Microneutralization assay

Elevated arterial pressure is a key contributor to cardiovascular disease, yet experimental models that isolate the effects of pressure from confounding systemic factors remain limited. We describe a reproducible surgical protocol to induce mid-thoracic aortic coarctation in mice, generating a stable and quantifiable arterial pressure gradient within the same animal. Using a biocompatible rubber O-ring, a partial stenosis is applied to the descending thoracic aorta of anesthetized C57BL/6J mice via thoracotomy. The resulting model establishes upstream hypertension while preserving distal perfusion, enabling the investigation of pressure-specific effects on vascular structure and perivascular adipose tissue function. Hemodynamic assessment by radio telemetry and high-frequency Doppler ultrasound confirms significant and sustained gradients in mean and systolic blood pressure across the coarctation site. This model provides a valuable tool for studying pressure-induced remodeling and vascular biology in a controlled and physiologically relevant context. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Induction of mid-thoracic aortic coarctation using a rubber O-ring

Basic Protocol 2: Measurement of arterial pressure gradient using two PA-C10 radio telemeters

Basic Protocol 3: Measurement of blood flow velocity in the descending thoracic aorta using doppler ultrasound

Unconventional protein secretion (UcPS) enables the export of cytosolic proteins through pathways that bypass the canonical endoplasmic reticulum–Golgi secretory route. Although increasingly recognized as essential for intercellular communication, stress responses, and tissue homeostasis, UcPS remains difficult to quantify due to low secretion efficiency, high intracellular background, and the challenge of distinguishing active secretion from passive leakage. Recent methodological advances, including NanoLuc split luciferase–based reporters and the Retention Using Selective Hooks (RUSH) system for synchronized protein transport, have improved sensitivity and temporal control of trafficking. Here, we present complementary protocols integrating these tools to provide a highly sensitive, quantitative workflow centered on a split NanoLuc (HiBiT/LgBiT) complementation assay for monitoring UcPS in mammalian cells. The Basic Protocol describes a robust luminescence-based secretion assay, while the Support Protocols detail the generation of stable HiBiT reporter cell lines, approaches for probing UcPS mechanisms using siRNA-mediated gene knockdown and pharmacological perturbation, and the incorporation of the RUSH system to synchronize cargo release and identify potential trafficking intermediates. Together, these protocols provide a sensitive, scalable, high-throughput toolkit that enables analysis of UcPS mechanisms across diverse cargo proteins, cell types, and perturbations. This methodological framework allows for rigorous dissection of UcPS pathways in both physiological and disease-relevant contexts. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol: Split luciferase complementation assay for quantifying UcPS in mammalian cells

Support Protocol 1: Generation of stable cell lines expressing HiBiT-tagged cargo proteins for the split luciferase assay

Support Protocol 2: siRNA-mediated knockdown to assess the role of candidate genes in UcPS

Support Protocol 3: Pharmacological perturbation of UcPS

Support Protocol 4: Integration of the RUSH system to synchronize UcPS

This article describes a chemically defined stepwise protocol to differentiate human induced pluripotent stem cells (hiPSCs) into mature oligodendrocytes via harvestable developmental intermediates. The workflow recapitulates in vitro neural lineage specification through embryoid body formation, neuroectoderm induction, the generation of glial-restricted progenitors, and the transition through oligodendrocyte progenitor cells (OPCs) to terminally differentiated, myelinating oligodendrocytes. Lineage progression is driven exclusively by small molecules and growth factors selected based on developmental and pharmacological evidence, without the use of exogenous transcription factor overexpression. The protocol enables isolation and expansion of physiologically relevant intermediate populations, making it suitable for mechanistic studies, disease modeling, and drug screening. Differentiation efficiency and purity can be validated by immunocytochemistry, flow cytometry, and co-culture-based myelination assays. The method reliably yields high-purity oligodendroglial cultures within approximately 90 days and is readily adaptable across hiPSC lines. © 2026 Wiley Periodicals LLC.

Basic Protocol 1: Thawing and plating of human iPSCs on Geltrex-coated plates

Support Protocol 1: Passaging of human iPSCs using ReLeSR

Basic Protocol 2: Dissociation of iPSC colonies into clusters for suspension embryoid body formation and early basal differentiation

Basic Protocol 3: Embryoid body differentiation for enhancing ectoderm by suppressing mesoderm and endoderm using specific growth factor cocktail

Basic Protocol 4: Selection of embryoid bodies and plating for neuroectoderm differentiation

Basic Protocol 5: Differentiation of neuroectodermal cells to glial-restricted progenitors

Basic Protocol 6: Magnetic-activated cell sorting of A2B5⁺ cells for differentiation to oligodendrocyte progenitor cells

Basic Protocol 7: Optimization of culture protocol for differentiation of oligodendrocyte progenitor cells to pre-oligodendrocytes

Basic Protocol 8: Optimization of culture protocol for differentiation of pre-oligodendrocytes to immature oligodendrocytes

Basic Protocol 9: Optimization of culture protocol for differentiation of immature oligodendrocytes to mature oligodendrocytes

Basic Protocol 10: Co-culture of iPSC-derived neurons with iPSC-derived oligodendrocytes for myelination assay

Support Protocol 2: Differentiation of iPSCs to neurons

Support Protocol 3: Immunocytochemistry analysis

Support Protocol 4: Flow cytometry analysis

Drosophila melanogaster has been an indispensable model for dissecting out innate immune pathways in a controlled and genetically tractable system. Two modes of infection, oral and systemic, in flies reveal distinct facets of host defense. Oral infection captures how the gut senses and responds to ingested microbes, whereas systemic challenges expose the mechanisms that act once pathogens breach external barriers and multiply systemically. Here, we provide a set of protocols that together offer a framework for studying infection biology in flies. They comprise a step-by-step guide to studying infection biology in adult flies using the two routes of infection—systemic infection via needle pricking/injection into the hemolymph and oral infection via pathogen-contaminated food or filter disks. We also outline assays to track infection outcomes, both oral and systemic, including survival rates, bacterial load assessments, gut function evaluations, and wound-repair responses. All experiments in this article are described for wild-type flies, but the workflow can be readily applied to different genetic backgrounds or mutant strains as a means to compare post-infection phenotypes and probe shifts in underlying immune signaling. © 2026 Wiley Periodicals LLC.

Basic Protocol 1: Preparation of fly food vials

Basic Protocol 2: Starvation of adult Drosophila melanogaster flies

Basic Protocol 3: Preparation of pathogenic pellets

Basic Protocol 4: Systemic infection of adult Drosophila melanogaster flies

Basic Protocol 5: Oral infection of adult Drosophila melanogaster flies

Basic Protocol 6: Assessment of survival in infected adult Drosophila melanogaster flies

Basic Protocol 7: Scoring of melanization in adult Drosophila melanogaster flies

Basic Protocol 8: Tracking bacterial load and pathogen clearance in adult Drosophila melanogaster flies

Basic Protocol 9: Monitoring defecation patterns in adult Drosophila melanogaster flies

The mouse is currently the most widely used animal model for scientific research worldwide. In Europe, statistical data show that half of all mice used in research are genetically modified, and that 60% of the transgenic mice produced are ultimately not used. Optimizing production strategies is therefore essential to minimize the production of animals that will not be included in research. In this article, we examine the key components required for the rigorous management of genetically modified lines, organized under the acronym DOPPUME: Define, Obtain, Protect, Produce, Use, Manage, and Exchange. The first step is to clearly define the model in accordance with the scientific objective and strictly control its genetic characteristics, including both the genetic background and the engineered modification. Ensuring the stability of these parameters is crucial for maintaining continuity between studies and preventing genetic drift. The article also provides tools to support the clear definition of production goals based on planned experiments and to estimate production needs as accurately as possible, accounting for the types of crosses and breeding schemes involved. Emphasis is placed on producing only what is necessary and on implementing proactive, rather than reactive, colony management. This framework is fully aligned with the 3Rs principles (Replace, Reduce, Refine) by promoting a responsible, rational, and optimized use of mouse models in research. © 2026 Wiley Periodicals LLC.