Methods in molecular biology最新文献

Respiratory nasal or lung epithelial cells serve as a valuable in vitro model for studying respiratory viral infections due to their physiological relevance and ability to recapitulate key aspects of the nasal or lung mucosa. In this chapter, we discuss the use of primary nasal epithelial cell cultures in studying viral infections, including their advantages, production methods, quality control, and identifiable disadvantages. Different methods for quantifying infection are presented with a special emphasis on how to adapt automated imaging methods and image analysis tools to the pseudostratified nasal epithelial cell models where cells are grown at the air-liquid interphase.

Negative staining electron microscopy is one of the easiest ways to determine the shape and dimensions of multimeric protein complexes over 100 kDa molecular weight. This method requires small volumes (< 10 μL) of dilute protein (0.01-0.1 mg/mL). Here we describe a method for quickly crosslinking a protein sample and preparing negative stained grids, and we also describe how to label a biotinylated protein subunit with avidin to determine its position within a complex using negative staining EM. This method should be generally applicable for most soluble protein complexes.

Identifying metabolites in NMR-based plant metabolomics is challenging due to the complexity of plant metabolites. This complexity stems from the abundance and diverse chemical properties of compounds, which vary in concentration across plant specimens. Additionally, the lack of automated identification software complicates the analysis process. Primary metabolites such as amino acids and sugars are widespread in plants, yet their identification is not straightforward due to various stereoisomeric forms and dynamic equilibria. Our protocol offers a manual approach to identify these metabolites, particularly focusing on amino acids and sugars. Through step-by-step guidance, we aim to empower researchers to navigate plant metabolomics complexities effectively.

The Golden Gate cloning technique is used to assemble DNA parts into higher-order assemblies. Individual parts containing compatible overhangs generated by type IIS restriction enzymes are joined together using DNA ligase. The technique enables users to assemble custom transcription units (TUs) for a wide array of experimental assays. Several Golden Gate cloning systems have been developed; however, they are typically used with a narrow range of organisms. Here we describe the Multi-Kingdom (MK) cloning system that allows users to generate DNA plasmids for use in a broad range of organisms.

Efficient DNA assembly methods are an essential prerequisite in the field of synthetic biology. Modular cloning systems, which rely on Golden Gate cloning for DNA assembly, are designed to facilitate assembly of multigene constructs from libraries of standard parts through a series of streamlined one-pot assembly reactions. Standard parts consist of the DNA sequence of a genetic element of interest such as a promoter, coding sequence, or terminator, cloned in a plasmid vector. Standard parts for the modular cloning system MoClo, also called level 0 modules, must be flanked by two BsaI restriction sites in opposite orientations and should not contain internal sequences for two type IIS restriction sites, BsaI and BpiI, and optionally for a third type IIS enzyme, BsmBI. We provide here a detailed protocol for cloning of level 0 modules. This protocol requires the following steps: (1) defining the type of part that needs to be cloned, (2) designing primers for amplification, (3) performing polymerase chain reaction (PCR) amplification, (4) cloning of the fragments using Golden Gate cloning, and finally (5) sequencing of the part. For large standard parts, it is preferable to first clone sub-parts as intermediate level -1 constructs. These sub-parts are sequenced individually and are then further assembled to make the final level 0 module.

Vibrio natriegens is a gram-negative bacterium, which has received increasing attention due to its very fast growth with a doubling time of under 10 min under optimal conditions. To enable a wide range of projects spanning from basic research to biotechnological applications, we developed NT-CRISPR as a new method for genome engineering. This book chapter provides a step-by-step protocol for the use of this previously published tool. NT-CRISPR combines natural transformation with counterselection through CRISPR-Cas9. Thereby, genomic regions can be deleted, foreign sequences can be integrated, and point mutations can be introduced. Furthermore, up to three simultaneous modifications are possible.

Golden Gate cloning allows rapid and reliable assembly of multiple DNA fragments in a defined orientation. Golden Gate cloning requires careful design of the restriction fragment overhangs to minimize undesired products and to generate the desired junctions. The ApE (A plasmid Editor) software package can assist in silico design of input fragments or to generate expected assembly products.

Trapped ion mobility spectrometry (TIMS) using parallel accumulation serial fragmentation (PASEF^®) is an advanced analytical technique that offers several advantages in mass spectrometry (MS)-based lipidomics. TIMS provides an additional dimension of separation to mass spectrometry and accurate collision cross-section (CCS) measurements for ions, aiding in the structural characterization of molecules. This is especially valuable in lipidomics for identifying and distinguishing isomeric or structurally similar compounds. On the other hand, PASEF technology allows for fast and efficient data acquisition by accumulating ions in parallel and then serially fragmenting them. This accelerates the analysis process and improves throughput, making it suitable for high-throughput applications. Moreover, the combination of TIMS and PASEF reduces co-elution and ion coalescence issues, leading to cleaner and more interpretable mass spectra. This results in higher data quality and more confident identifications. In this chapter, a data-dependent TIMS-PASEF^® workflow for lipidomics analysis is presented.

The thyroid hormones, thyroxine (T4) and triiodothyronine (T3), are pivotal in regulating various physiological processes including growth, development, and metabolism. The biological actions of thyroid hormones are primarily initiated by binding to nuclear thyroid hormone receptors (TRs). These receptors, belonging to the superfamily of nuclear receptors, act as ligand-dependent transcription factors. Transcriptional regulation by TRs is mediated by the recruitment of coregulators, governing activation and repression of target genes, thereby modulating cellular responses to thyroid hormones. Beyond this canonical genomic pathway, TH can regulate the expression of genes not directly bound by TRs through cross-talk mechanisms with other transcription factors and signaling pathways. Thyroid hormones can also elicit rapid non-genomic effects, potentially mediated by extranuclear TR proteins or by interactions with membrane receptors such as integrin αvβ3. This non-genomic mode of action adds another layer of complexity to the diverse array of physiological responses orchestrated by thyroid hormones, expanding our understanding of their multifaceted actions.

The generation of hypothyroid and hyperthyroid mouse models is one of the approaches used to investigate the complex interplay between thyroid hormones and the immune system. We present a detailed protocol describing how to induce endotoxic shock by lipopolysaccharide (LPS) administration, and how to investigate the role of immune populations, specifically macrophages, responding to endotoxemia.This book chapter provides the use of different molecular techniques, such as Western Blotting, Immunohistochemistry, q-PCR, Luciferase assays, or ChIP assays, with which researchers can gain valuable insights into the immune system's interaction with hormonal signaling pathways, for instance, examining the effect of thyroid hormones on signaling of STAT3, NF-κB, and ERK in response to LPS, and inflammatory mediators, such as interleukin-6 (IL-6) or tumor necrosis factor-alpha (TNFα) within these cells. The signaling pathways involved and the exploration of the relationship between thyroid hormones and the immune system can be analyzed using several molecular biology technologies in order to clarify their interplay in various disease states.