Current protocols最新文献

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

In addition to current challenges in food production arising from climate change, soil salinization, drought, flooding, and human-caused disruption, abrupt sunlight reduction scenarios (ASRS), e.g., a nuclear winter, supervolcano eruption, or large asteroid or comet strike, are catastrophes that would severely disrupt the global food supply and decimate normal agricultural practices. In such global catastrophes, teragrams of particulate matter, such as aerosols of soot, dust, and sulfates, would be injected into the stratosphere and block sunlight for multiple years. The reduction of incident sunlight would cause a decrease in temperature and precipitation and major shifts to climate patterns leading to devastating reductions in agricultural production of traditional food crops. To survive a catastrophic ASRS or endure current and future disasters and famines, humans might need to rely on post-catastrophic foods, or those that could be foraged, grown, or produced under the new climate conditions to supplement reduced availability of traditional foods. These foods have sometimes been referred to as emergency, alternate, or resilient foods in the literature. While there is a growing body of work that summarizes potential post-catastrophic foods and their nutritional profiles based on existing data in the literature, this article documents a list of protocols to experimentally determine fundamental nutritional properties of post-catastrophic foods that can be used to assess the relative contributions of those foods to a balanced human diet that meets established nutritional requirements while avoiding toxic levels of nutrients. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Total digestible glucans

Basic Protocol 2: Apparent protein digestibility

Basic Protocol 3: Vitamins B₁, B₃, B₉, C, and D₂ by HPLC

Basic Protocol 4: Total antioxidant activity (DPPH-scavenging activity)

Basic Protocol 5: Total phenolic compounds (Folin–Ciocalteu reagent method)

Basic Protocol 6: Mineral content by ICP-OES

The innate immune system is the first line of host defense. Innate immune activation utilizes pattern recognition receptors to detect pathogens, pathogen-associated and damage-associated molecular patterns (PAMPs and DAMPs), and homeostatic alterations and drives inflammatory signaling pathways and regulated cell death. Cell death activation is critical to eliminate pathogens and aberrant or damaged cells, while excess activation can be linked to inflammation, tissue damage, and disease. Therefore, there is increasing interest in studying cell death mechanisms to understand the underlying biology and identify therapeutic strategies. However, there are significant technical challenges, as many cell death pathways share key molecules with each other, and genetic models where these cell death molecules are deleted remain the gold standard for evaluation. Furthermore, extensive crosstalk has been identified between the cell death pathways pyroptosis, apoptosis, necroptosis, and the more recently characterized PANoptosis, which is defined as a prominent, unique innate immune, lytic, and inflammatory cell death pathway initiated by innate immune sensors and driven by caspases and RIPKs through PANoptosomes. PANoptosomes are multi-protein complexes assembled by innate immune sensor(s) in response to pathogens, PAMPs, DAMPs, cytokines, and homeostatic changes that drive PANoptosis. In this article, we provide methods for molecularly defining distinct cell death pathways, including PANoptosis, using both genetic and chemical approaches through western blot, LDH assay, and microscopy readouts. This procedure allows for the assessment of cell death on the cell population and single-cell levels even without access to genetic models. Having this comprehensive workflow that is more accessible to all labs will improve our ability as a scientific community to accelerate discovery. Using these protocols will help identify new innate immune sensors that drive PANoptosis and define the molecular mechanisms and regulators involved to establish new targets for clinical translation. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Induction and quantification of cell death using live cell imaging

Alternate Protocol 1: Quantification of cell death using LDH

Alternate Protocol 2: Assessment of cell death complexes in single cells using immunofluorescence staining

Basic Protocol 2: Analysis of cell death mechanisms by immunoblots (western blots)

Image data from a single animal in neuroscientific experiments can be comprised of terabytes of information. Full studies can thus be challenging to analyze, store, view, and manage. What follows is an updated guide for preparing and sharing big neuroanatomical image data. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Naming and organizing images and metadata

Basic Protocol 2: Preparing and annotating images for presentations and figures

Basic Protocol 3: Assessing the internet environment and optimizing images

Interactions between proteins and small molecules or nucleic acids play a pivotal role in numerous biological processes critical for human health and are fundamental for advancing our understanding of biological systems. Proteins are the workhorses of the cell, executing various functions ranging from catalyzing biochemical reactions to transmitting signals within the body. Small molecules, including drugs and metabolites, can modulate protein activity, thereby impacting cellular processes and disease pathways. Similarly, nucleic acids, such as DNA and RNA, regulate protein synthesis and function through intricate interactions. Understanding these interactions is crucial for drug discovery and development and can shed light on gene regulation, transcriptional control, and RNA processing, providing insights into genetic diseases and developmental disorders. Moreover, studying protein–small molecule and protein–nucleic acid interactions enhances our comprehension of fundamental biological mechanisms. A wide array of methods to study these interactions range in cost, sensitivity, materials usage, throughput, and complexity. Notably in the last decade, new techniques have been developed that enhance our understanding of these interactions. In this review, we aim to summarize the new state-of-the-art methods for detecting interactions between proteins and small molecules or nucleic acids, as well as discuss older methods that still hold value today. © 2024 Wiley Periodicals LLC.

Sulfate-reducing bacteria (SRB) are crucial players in global biogeochemical cycling and some have been implicated in the anaerobic biodegradation of organic pollutants, including recalcitrant and hazardous polycyclic aromatic hydrocarbons (PAHs). Obtaining PAH-degrading SRB cultures for laboratories is of paramount importance in the development of the young field of anaerobic biodegradation of PAHs. SRB grow exceptionally slowly on PAH substrates and are highly sensitive to oxygen. Consequently, enrichment and maintenance of PAH-degrading SRB cultures and characterization of the biodegradation process remain a tedious and formidable task, especially for new researchers. To address these technical constraints, we have developed robust and effective protocols for obtaining and characterizing PAH-degrading SRB cultures. In this set of protocols, we describe step-by-step procedures for preparing inocula from contaminated soil or sediment, preparing anoxic medium, establishing enrichment cultures with PAHs as substrates under completely anaerobic sulfate-reducing conditions, successive culture transfers to obtain highly enriched cultures, rapid verification of the viability of SRB in slow-growing cultures, assessment of PAH degradation by extracting residuals using organic solvent and subsequent analysis by gas chromatography–mass spectrometry, and spectrophotometric determination of sulfate and sulfide in miniaturized, medium-throughput format. These protocols are expected to serve as a comprehensive manual for obtaining and characterizing PAH-degrading sulfate-reducing cultures. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Obtaining PAH-degrading strictly anaerobic sulfate-reducing enrichment cultures from contaminated soil and sediment

Support Protocol 1: Operation and maintenance of an anaerobic workstation

Support Protocol 2: Setup of gas purging systems for preparing anoxic solutions

Support Protocol 3: Verification of viability in slow-growing SRB enrichment cultures

Support Protocol 4: Extraction of genomic DNA from low-biomass cultures

Basic Protocol 2: Extraction of residual PAH from liquid culture and analysis by GC-MS

Basic Protocol 3: Spectrophotometric determination of sulfate concentration in SRB cultures

Basic Protocol 4: Spectrophotometric determination of sulfide concentrations in SRB cultures by the methylene blue method

Alternate Protocol: Spectrophotometric determination of sulfide concentrations in SRB cultures by the colloidal copper sulfide method