Current protocols最新文献

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

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

Wu, J., Liu, T., Tang, M., Liu, Y., Wang, W., Wang, C., Ju, Y., Zhao, Y., & Zhang, Y. (2024). Ex Vivo evaluation of the function of hematopoietic stem cells in toxicology of metals. Current Protocols, 4, e1038. doi: 10.1002/cpz1.1038

In the above-referenced article:

The status of Dr. Yifan Zhao has been corrected to co-corresponding author, and the contact email address “[email protected]” has been added.

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

In the heart in vivo, vasculature forms a semi-permeable endothelial barrier for selective nutrient and (immune) cell delivery to the myocardium and removal of waste products. Crosstalk between the vasculature and the heart cells regulates homeostasis in health and disease. To model heart development and disease in vitro it is important that essential features of this crosstalk are captured. Cardiac organoid and microtissue models often integrate endothelial cells (ECs) to form microvascular networks inside the 3D structure. However, in static culture without perfusion, these networks may fail to show essential functionality. Here, we describe a protocol to generate an in vitro model of human induced pluripotent stem cell (hiPSC)-derived vascularized cardiac microtissues on a microfluidic organ-on-chip platform (VMToC) in which the blood vessels are perfusable. First, prevascularized cardiac microtissues (MT) are formed by combining hiPSC-derived cardiomyocytes, ECs, and cardiac fibroblasts in a pre-defined ratio. Next, these prevascularized MTs are integrated in the chips in a fibrin hydrogel containing additional vascular cells, which self-organize into tubular structures. The MTs become vascularized through anastomosis between the pre-existing microvasculature in the MT and the external vascular network. The VMToCs are then ready for downstream structural and functional assays and basic characterization. Using this protocol, cardiac MTs can be efficiently and robustly vascularized and perfused within 7 days. In vitro vascularized organoid and MT models have the potential to transition current 3D cardiac models to more physiologically relevant organ models that allow the role of the endothelial barrier in drug and inflammatory response to be investigated. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol: Generation of VMToC

Support Protocol 1: Functional Characterization of VMToC

Support Protocol 2: Structural Characterization of VMToC

Optical imaging technologies and cell targeting have played a major role in detecting and treating diseases such as cancer. Bioharmonophores are optical imaging nanoprobes composed of biodegradable polymer–encapsulated, self-assembling triphenylalanine peptides. They produce a strong second harmonic generation (SHG) signal, a non-linear optical process in which two photons directed at a non-centrosymmetric medium combine to form a new photon with twice the energy. Bioharmonophores demonstrate superior optical properties compared to fluorescent probes and, unlike previously developed inorganic SHG nanoprobes, are both biocompatible and biodegradable. Here, we present a protocol providing five detailed procedures that describe (1) synthesis of bioharmonophores; (2) embedding and imaging of the synthesized SHG nanoprobes in polyacrylamide gel; (3) functionalization of bioharmonophores with thiol-containing polyethyleneglycol; (4) subsequent click chemistry to target cancer cells; and (5) imaging of functionalized bioharmonophores endocytosed by cancer cells using two-photon microscopy. Bioharmonophores hold great potential as clinical contrast agents due to their optical features and could be used in the future as an innovative approach to cancer treatment using targeted high-resolution optical imaging. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Synthesis of bioharmonophores

Basic Protocol 2: Imaging of bioharmonophores in polyacrylamide gel

Basic Protocol 3: Functionalization of bioharmonophores with thiol-PEG

Basic Protocol 4: Functionalization of thiol-PEGylated bioharmonophores with peptides

Basic Protocol 5: Targeting of cancer cells with functionalized bioharmonophores

Durable cellular immunity against pathogens is dependent upon a coordinated recall response to antigen by memory CD8⁺ T cells, involving their proliferation and the generation of secondary cytotoxic effector cells. Conventional assays measuring ex vivo cytotoxicity fail to capture this secondary cytolytic potential, especially in settings where cells have not been recently exposed to their cognate antigen in vivo. Here we describe the expanded antigen-specific elimination assay (EASEA), a flow cytometric endpoint assay to measure the capacity of human CD8⁺ T cells to expand in vitro upon antigen re-exposure and generate secondary effector cells capable of selectively eliminating autologous antigen-pulsed target cells across a range of effector-to-target ratios. Unlike alternative assays, EASEA avoids the hazards of radioactive labeling and viral infection and can be used to study responses to individual or pooled antigens of interest. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol: Expanded antigen-specific elimination assay

With the ever-expanding toolkit of molecular viewers, the ability to visualize macromolecular structures has never been more accessible. Yet, the idiosyncratic technical intricacies across tools and the integration complexities associated with handling structure annotation data present significant barriers to seamless interoperability and steep learning curves for many users. The necessity for reproducible data visualizations is at the forefront of the current challenges. Recently, we introduced MolViewSpec (homepage: https://molstar.org/mol-view-spec/, GitHub project: https://github.com/molstar/mol-view-spec), a specification approach that defines molecular visualizations, decoupling them from the varying implementation details of different molecular viewers. Through the protocols presented herein, we demonstrate how to use MolViewSpec and its 3D view–building Python library for creating sophisticated, customized 3D views covering all standard molecular visualizations. MolViewSpec supports representations like cartoon and ball-and-stick with coloring, labeling, and applying complex transformations such as superposition to any macromolecular structure file in mmCIF, BinaryCIF, and PDB formats. These examples showcase progress towards reusability and interoperability of molecular 3D visualization in an era when handling molecular structures at scale is a timely and pressing matter in structural bioinformatics as well as research and education across the life sciences. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Creating a MolViewSpec view using the MolViewSpec Python package

Basic Protocol 2: Creating a MolViewSpec view with reference to MolViewSpec annotation files

Basic Protocol 3: Creating a MolViewSpec view with labels and other advanced features

Support Protocol 1: Computing rotation and translation vectors

Support Protocol 2: Creating a MolViewSpec annotation file