Current protocols最新文献

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.

Oligonucleotide-based therapeutics are now widely used in clinical settings. From the late 1980s to the mid-1990s, efforts to improve therapeutic efficacy focused on imparting drug-like properties to oligonucleotides, emphasizing nuclease stability and target sequence affinity. These efforts resulted in the standard gapmer design for RNase H–mediated antisense and the prevalent use of chemical modification such as phosphorothioate and 2′-substituted oligoribonucleotides in oligonucleotide therapeutics. Progress made in the antisense field also enabled the development of splice-modulating oligonucleotide therapeutics and later siRNA therapies. All three modes of action are now widely employed in >25 approved drugs. Since then, we have learned that oligonucleotides and their chemical modifications can interact with pattern recognition receptors as well as various other proteins. This can have both positive and negative effects, such as aiding in oligonucleotide delivery or activating the intracellular innate immune system. My current work aims to optimize the drug-like properties of oligonucleotides by combining the early chemical advances with the more recent insights into off-target protein binding. The present article describes how this resulted in several different cyclic structured oligonucleotide designs, in which 3′ and 5′ ends are transiently held together via Watson-Crick base pairing. The transient nature of these cyclic structures protects the functional parts of the structure against nucleases during delivery and cell entry while allowing effective release of the oligonucleotide drug into the intracellular environment. These cyclic designs demonstrate significant improvements in potency and specificity over gapmer antisense and are broadly applicable to potentially all types of RNA therapeutics, irrespective of their mechanism of action. © 2026 Wiley Periodicals LLC.

The IntelliCage is a home-cage-based radiofrequency identification (RFID) test system for studying learning behavior of group-housed mice. Specifically, the mice must learn where and how to get water within the IntelliCage. The protocol describes how to set up the IntelliCage, how to set up and start a learning experiment, what to consider before starting an experiment, and how to clean the system. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: General instructions for using the IntelliCage with mice

Basic Protocol 2: IntelliCage cleaning

A major, serendipitous psychiatric discovery is monoamine-transporter reuptake inhibition as an antidepressant mechanism of action. Chronic treatment with such antidepressants is efficacious, with onset requiring 1-2 weeks, in many but by no means all patients with major depressive or another stress-related neuropsychiatric disorder. The forced swim test (FST) in rats and mice involves the acute, moderate stressor of placement in a container of water: at test onset, the predominant reaction is swimming, interpreted cautiously as active “struggling”; over minutes, this is replaced by floating, described objectively as immobility. Acute administration of monoamine transporter inhibitors immediately prolongs “struggling.” Although this readout is of behavioral pharmacological interest, the FST has no (back-)translational relevance to the neurobiological and neuropsychological symptoms/states of stress-related psychiatric disorders. The persistent adoption of the FST to measure “depression-like state,” based on interpretation of immobility as “despair,” “helplessness,” or “passive coping,” is a major weakness in applied behavioral neuroscience. Rodents do have a concept of learned uncontrollability, such that tests showing this depict an adaptive, not a “depression-like,” state. Recent psychiatry-neuroscience initiatives, such as the Research Domain Criteria framework, increase the accessibility of specific, transdiagnostic symptoms/states to behavioral neuroscience methods, thereby facilitating the establishment of animal models. Such animal models must incorporate clinically valid (1) etiological factors, such as prolonged psychosocial stress, and (2) neurobehavioral readouts with face and construct validity for specific symptoms/states. Increased reactivity to an acute threat, measured as increased Pavlovian aversion learning-memory (PALM), is a neurobehavioral state common in major depression and other disorders. Mice that have undergone chronic social stress exhibit generalized excessive PALM. Therefore, although stating that the FST measures “depression-like state” is erroneous, mouse models of specific symptoms/states can be achieved by back-translating etiology and neurobehavioral readouts. Though complex and moderately severe, such models have the potential to provide much-needed benefits in terms of preclinical neuropharmacological target discovery and validation. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Multiple myeloma is a bone marrow–derived neoplastic proliferation of plasma cells with low levels of circulating tumor cells. Cytogenetic abnormalities are present in >90% of patients, when assessed by fluorescence in situ hybridization (FISH) on bone marrow, and the specific abnormalities provide vital prognostic information. Provided is a protocol for assessing the high-risk abnormalities del(17p) and amp(1q21) in the circulating plasma cells of myeloma patients by flow cytometry. This utilizes positive plasma cell identification by standard immunophenotyping and then chromosomal analysis by FISH using an imaging flow cytometer. Integrating cell phenotype and FISH in one test, a method called “immuno-flowFISH” allows for the detection of cytogenetic abnormalities in plasma cells identified by their antigenic profile. This method can be applied to both bone marrow and blood samples to detect primary abnormalities [i.e., hyperdiploidy; immunoglobulin heavy locus (IGH) translocation] and secondary abnormalities [i.e., del(17p) found in 10% of patients, and gain(1q) present in ∼40% of patients with myeloma]. Here we describe the protocol for the simultaneous detection of these secondary abnormalities in blood and bone marrow samples from myeloma patients that enables detection of single or “double hit” abnormalities, with the latter classified as ultra high–risk disease. The protocol includes the data analysis strategy, as well as the statistics used to confirm the presence of these abnormalities. Applying this method will facilitate blood-based monitoring for the presence and evolution of these critical cytogenetic abnormalities. © 2026 Wiley Periodicals LLC.

Basic Protocol: Immuno-flowFISH assessment of del(17p) and amp(1q21) in multiple myeloma

Support Protocol: Validation of single fluorophore for positive markers in multiple myeloma

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

Mice, as one of the most important model organisms in life science research, rely heavily on assisted reproductive technologies, particularly in vitro fertilization (IVF) and embryo transfer (ET), which serve as indispensable platforms for genetic modification, strain preservation and expansion, as well as studies in reproductive biology. This article provides a detailed description of an optimized and validated experimental system encompassing the entire workflow from superovulation, sperm collection and capacitation, in vitro fertilization, embryo culture, and vasectomy of male mice, to embryo transfer and related auxiliary procedures. © 2026 Wiley Periodicals LLC.

Basic Protocol 1: IVF in mice

Basic Protocol 2: Oviduct ET in mice

Support Protocol: Preparation of vasectomized males and pseudopregnant females

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