Current protocols最新文献

Nonenzymatic genome replication is thought to be an important process for primitive lifeforms, but this has yet to be demonstrated experimentally. Recent studies on the nonenzymatic primer extension mechanism mediated by nucleoside 5′-monophosphates (NMPs) activated with 2-aminoimidazole have revealed that imidazolium-bridged dinucleotide intermediates (N*N) account for the majority of the chemical copying process. As a result, an efficacious synthetic pathway for producing substrates activated with an imidazoyl moiety is desirable. This article provides a detailed protocol for the standard dehydrative redox reaction between NMPs and 2-aminoimidazole to produce nucleotide phosphoroimidazolides. In addition, we describe a similar synthetic pathway to produce N*N in high yields for homodimers. Finally, a simple reversed-phase cation exchange step is described to increase NMP solubility, which significantly increases yields for certain substrates. This approach allows for an efficient and cost-effective methodology to prepare high-quality substrates utilized in origins-of-life studies. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Synthesis of 2-aminoimidazolephosphoroimidazolide-activated cytidine

Basic Protocol 2: Synthesis of 2-aminoimidazolium-bridged dicytidyl intermediate

Basic Protocol 3: Cation exchange of guanosine 5′-monophosphate disodium salt

Alternate Protocol: Synthesis of cytidine 5′-phosphoroimidazolide or 2-aminoimidazolium-bridged dicytidyl from cytidine 5′-monophosphate disodium salt

Antibody-mediated receptor activation is successfully used to develop medical treatments. If the activation induces a pathological response, such antibodies are also excellent tools for defining molecular mechanisms of target receptor malfunction and designing rescue therapies. Prominent examples are naturally occurring autoantibodies inducing the severe blistering disease pemphigus vulgaris (PV). In the great majority of patients, the antibodies bind to the adhesion receptor desmoglein 3 (Dsg3) and interfere with cell signaling to provoke severe blistering in the mucous membranes and/or skin. The identification of a comprehensive causative signaling network downstream of antibody-targeted Dsg3 receptors (e.g., shown by pharmacological activators or inhibitors) is currently being discussed as a basis to develop urgently needed first-line treatments for PV patients. Although polyclonal PV IgG antibodies have been used as proof of principle for pathological signal activation, monospecific anti-Dsg3 antibodies are necessary and have been developed to identify pathological Dsg3 receptor–mediated signal transduction. The experimental monospecific PV antibody AK23, produced from hybridoma cells, was extensively tested in our laboratory in both in vitro and in vivo models for PV and proved to recapitulate the clinicopathological features of PV when generated using the standardized production and purification protocols described herein. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Bovine IgG stripping from FBS and quality control

Basic Protocol 2: AK23 hybridoma expansion and IgG production

Basic Protocol 3: AK23 IgG purification

Basic Protocol 4: AK23 IgG quality control

Support Protocol 1: Detection of endotoxin levels

Support Protocol 2: Detection and removal of mycoplasma

Breast cancer is a prevalent malignancy affecting women worldwide. Currently, there are no precise molecular biomarkers with immense potential for accurately predicting breast cancer development, which limits clinical management options. Recent evidence has highlighted the importance of metastatic and tumor-infiltrating immune cells in modulating the antitumor therapy response. However, the prognostic value of using these features in combination, and their potential for guiding individualized treatment for breast cancer, remains vague. To address this challenge, we recently developed the metastatic and immunogenomic risk score (MIRS), a comprehensive and user-friendly scoring system that leverages advanced bioinformatics methods to facilitate transcriptomics data analysis. To help users become familiar with the MIRS tool and apply it effectively in analyzing new breast cancer datasets, we describe detailed protocols that require no advanced programming skills. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Calculating a MIRS score from transcriptomics data

Basic Protocol 2: Predicting clinical outcomes from MIRS scores

Basic Protocol 3: Evaluating treatment responses and guiding therapeutic strategies in breast cancer patients

Basic Protocol 4: Guidelines for utilizing the MIRS webtool

CAR-T cell therapy has emerged as a potent and effective tool in the immunotherapy of refractory cancers. However, challenges exist in their clinical application, necessitating extensive preclinical research to optimize their function. Various preclinical in vitro and in vivo models have been proposed for such purpose; among which immunocompetent mouse models serve as an invaluable tool in studying host immune interactions within a more realistic simulation of the tumor milieu. We hereby describe a standardized protocol for the generation of high-titer γ-retroviral vectors through transfection of the HEK293T packaging cell line. The virus-containing supernatant is further concentrated using an inhouse concentrator solution, titrated, and applied to mouse T cells purified via a convenient and rapid method by nylon-wool columns. Using the method presented here, we were able to achieve high titer γ-retrovirus and highly pure mouse T cells with desirable CAR transduction efficiency. The mouse CAR T cells produced through this protocol demonstrate favorable CAR expression and viability, thus making them suitable for further in vitro/in vivo assays. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Production of γ-retroviral vectors from retrovirus-backbone plasmids

Basic Protocol 2: Concentration of γ-retrovirus-containing supernatants

Basic Protocol 3: Titration of concentrated γ-retrovirus

Basic Protocol 4: Isolation and activation of mouse T cells

Basic Protocol 5: Transduction of activated mouse T cells, assessment of CAR expression, and expansion of CAR T cells for further in vitro/in vivo studies

Support Protocol: Surface staining of cells for flow cytometric assessment of CAR expression

In a scientific context, a suitable color choice is more than simple decoration. Color handling, as part of scientific visualization, is a scientific methodology that is one of the most widely used, given the importance of figures and images in conveying results. Yet, an expert-level understanding and application of proper scientific coloring is rare. Here, a concise overview of important color tools is provided and complemented by ready-to-apply resources for using color in science research, publishing, communication, tool development, editing, and teaching. This overview offers a guide to spot problems, master the methodology, and support accessible and accurate use of color for science figures in both short and long terms. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Acute liver injury is a life-threatening disease. Although immune responses are involved in the development and exacerbation of acute liver injury, the cellular and molecular mechanisms are not fully understood. Intravenous administration of the plant lectin concanavalin A (ConA) is widely used as a model of acute liver injury. ConA triggers T cell activation and cytokine production by crosslinking glycoproteins, including the T cell receptor, leading to the infiltration of myeloid cells into the liver and the subsequent amplification of inflammation in the liver. Thus, the pathogenesis of ConA-induced acute liver injury is considered a model of immune-mediated acute liver injury or autoimmune hepatitis in humans. However, the severity of the liver injury and the analyses of immune cells and non-hematopoietic cells in the liver following ConA injection are significantly influenced by the experimental conditions. This article outlines protocols for ConA-induced acute liver injury in mice and evaluation methods for liver injury, immune cells, and non-hematopoietic cells in the liver. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Induction of acute liver injury by ConA injection

Basic Protocol 2: Evaluation of inflammatory cytokines in mouse plasma

Basic Protocol 3: Preparation of liver sections and histological analysis of liver injury

Basic Protocol 4: Preparation of liver immune cells

Basic Protocol 5: Preparation of hepatocytes, endothelial cells, and hepatic stellate cells

Basic Protocol 6: Flow cytometry of immune and non-hematopoietic liver cells

Basic Protocol 7: Flow cytometric sorting of endothelial cells and hepatic stellate cells

Basic Protocol 8: Quantitative reverse transcription polymerase chain reaction

JBrowse 2 is a modular genome browser that can visualize many common genomic file formats. While JBrowse 2 supports a variety of different usages, it is particularly suited for deployment on websites, such as model organism databases or other web-based genomic data resources. This protocol provides detailed instructions for setting up JBrowse 2 on an Ubuntu Linux web server, loading a reference genome from a FASTA format file, and adding a gene annotation track from a GFF3 format file. By the end of the protocol, users will have a working JBrowse 2 instance that is accessible via the web. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol: Setting up JBrowse 2 on your web server

Identification of protein-protein interfaces is necessary for understanding and regulating biological events. Genetic code expansion enables site-specific photo-cross-linking by introducing photo-reactive non-canonical amino acids into proteins at defined positions during translation. This technology is widely used for analyzing protein-protein interactions and is applicable in mammalian cells. However, the identification of the cross-linked region still remains challenging. Our new protocol enables its identification by pre-installing a site-specific cleavage site, an α-hydroxy acid (N^ε-allyloxycarbonyl-α-hydroxyl-L-lysine acid, AllocLys-OH), into the target protein. Alkaline treatment cleaves the crosslinked complex at the position of the α-hydroxy acid residue and thus helps to identify which side of the cleavage site, either closer to the N-terminus or C-terminus, the crosslinked site is located on within the target protein. A series of AllocLys-OH introductions narrows down the crosslinked region. This combination of site-specific crosslinking and cleavage promises to be useful for revealing binding interfaces and protein complex geometries. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Search for crosslinkable sites

Basic Protocol 2: Site-specific photo-cross-linking/cleavage

Alopecia areata is the second most common form of hair loss in humans after androgenetic alopecia. Although a variety of animal models for alopecia areata have been described, currently the C3H/HeJ mouse model is the most commonly used and accepted. Spontaneous hair loss occurs in 15%-25% of older mice in which the lesions wax and wane, similar to the human disease, with alopecia being more common and severe in female mice. Full-thickness skin grafts from mice with spontaneous alopecia areata to young, normal-haired, histocompatible mice provide a highly reproducible model with progressive lesions that makes it useful for drug efficacy and mechanism-based studies. As alopecia areata is a cell-mediated autoimmune disease, transfer of cultured lymph node cells from affected mice to unaffected, histocompatible recipients also promotes disease development and provides an alternative, nonsurgical protocol. Protocols are presented to produce these models such that they can be used to study alopecia areata and to develop novel drug therapies. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Full-thickness skin grafts to reproducibly induce alopecia areata in C3H/HeJ mice

Basic Protocol 2: Adoptive transfer of cultured lymphoid cells provides a nonsurgical method to induce alopecia areata in C3H/HeJ mice

Postural control (PC) and sleep are critical in several aspects of health. Poor sleep negatively influences PC and balance, which is necessary for performing various tasks, from reaching to mobility. Moreover, sleep disturbances and consequent PC and balance deterioration are associated with job accidents, traffic accidents, falls, and injuries. Healthy adults who have inadequate sleep show a decline in optimal functioning, even in the absence of medical illnesses. This suggests that getting enough sleep, both in duration and quality, is essential to maintain optimal health. Moreover, inadequate sleep has also been observed to have a bidirectional relationship with stress levels. However, there is insufficient evidence regarding the impact of non-pharmacological treatments to improve PC, sleep, and stress in the sedentary young adult (YA) population. This article describes the protocol for a study to investigate the effects of sensorimotor training and relaxation therapy on various static and dynamic PC tests, balance measures, and subjective and objective indices of sleep and stress among sedentary YAs with impaired sleep quality. The protocol is also designed to evaluate the effect of these therapies on fatigue, salivary cortisol levels, anxiety, and depression. Methods for assessing the sleep architecture, static and dynamic PC, balance, and stress are described along with the methods of scoring with the primary goal of providing a standardized set of assessment and scoring procedures according to the latest guidelines and gold-standard techniques and measures that can be used reliably at different laboratories. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Postural control assessment

Basic Protocol 2: Balance assessment

Basic Protocol 3: Sleep architecture assessment

Basic Protocol 4: Salivary cortisol analysis