Methods in molecular biology最新文献

Targeted mass spectrometric analysis is widely employed across various omics fields as a validation strategy due to its high sensitivity and accuracy. The approach has been successfully employed for the structural analysis of proteins, glycans, lipids, and metabolites. Multiple reaction monitoring (MRM) and parallel reaction monitoring (PRM) have been the methods of choice for targeted structural studies of biomolecules. These target analyses simplify the analytical workflow, reduce background interference, and increase selectivity/specificity, allowing for a reliable quantification of permethylated N-glycans in complex biological matrices.

MicroRNAs represent an interesting group of regulatory molecules with the unique ability of a single miRNA able to regulate the expression of potentially hundreds of target genes. In that regard, their utility has been demonstrated as a strategy to improve the cellular phenotypes important in the biomanufacturing of recombinant proteins. Common approaches to stably deplete miRNAs are the use of sponge decoy transcripts or shRNA inhibitors, both of which require the introduction and expression of extra genetic material in the cell. As an alternative, we implemented the CRISPR/Cas9 system in our laboratory to generate CHO cells which lack the expression of a specific miRNA for the purpose of functional studies. To implement the system, miR-27a/b was chosen as it has been shown to be upregulated during hypothermic conditions and therefore may be involved in influencing CHO cell growth and recombinant protein productivity. In this chapter, we present a protocol for targeting miRNAs in CHO cells using CRISPR/Cas9 and the analysis of the resulting phenotype, using miR-27 as an example. We show that it is possible to target miRNAs in CHO cells and achieved ≥80% targeting efficiency. Indel analysis and TOPO-TA cloning combined with Sanger sequencing showed a range of different indels. Furthermore, it was possible to identify clones with no detectable expression of mature miR-27b. Depletion of miR-27b led to improved viability in late stages of batch and fed-batch cultures, making it a potentially interesting target to improve bioprocess performance of CHO cells.

Polyclonal antibodies are relatively easy to produce and may supplement monoclonal antibodies for some applications or even have some advantages.The choice of species for production of (peptide) antisera is based on practical considerations, including availability of immunogen (vaccine) and animals. Two major factors govern the production of antisera: the nature of adaptive immune responses, which take place over days/weeks and ethical guidelines for animal welfare.Here, simple procedures for immunization of mice, rabbits, sheep, goats, pigs, horses, and chickens are presented.

Pathogen proliferation and virulence depend on available nutrients, and these vary when the pathogen moves from outside of the host cell (extracellular) to the inside of the host cell (intracellular). Nuclear Magnetic Resonance (NMR) is a versatile analytical method, which lends itself for metabolic studies. In this chapter, we describe how ¹H NMR can be combined with a cellular infection model to study the metabolic crosstalk between a bacterial pathogen and its host both in the extracellular and intracellular compartments. Central carbon metabolism is highlighted by using glucose labeled with the stable isotope ¹³C.

One hundred years have passed since the death of Élie Metchnikoff (1845-1916). He was the first to observe the uptake of particles by cells and realized the importance of this process, named phagocytosis, for the host response to injury and infection. He also was a strong advocate of the role of phagocytosis in cellular immunity, and with this, he gave us the basis for our modern understanding of inflammation and the innate immune response. Phagocytosis is an elegant but complex process for the ingestion and elimination of pathogens, but it is also important for the elimination of apoptotic cells and hence fundamental for tissue homeostasis. Phagocytosis can be divided into four main steps: (i) recognition of the target particle, (ii) signaling to activate the internalization machinery, (iii) phagosome formation, and (iv) phagolysosome maturation. In this chapter, we present a general view of our current knowledge on phagocytosis performed mainly by professional phagocytes through antibody and complement receptors and discuss aspects that remain incompletely understood.

Classic in vitro coculture assays of pathogens with host cells have contributed significantly to our understanding of the intracellular lifestyle of several pathogens. Coculture assays with pathogens and eukaryotic cells can be analyzed through various techniques including plating for colony-forming units (CFU), confocal microscopy, and flow cytometry. However, findings from in vitro assays require validation in an in vivo model. Several physiological conditions can influence host-pathogen interactions, which cannot easily be mimicked in vitro. Intravital microscopy (IVM) is emerging as a powerful tool for studying host-pathogen interactions by enabling in vivo imaging of living organisms. As a result, IVM has significantly enhanced the understanding of infection mediated by diverse pathogens. The versatility of IVM has also allowed for the imaging of various organs as sites of local infection. This chapter specifically focuses on IVM conducted on the lung for elucidating pulmonary immune response, primarily involving alveolar macrophages, to pathogens. Additionally, in this chapter we outline the protocol for lung IVM that utilizes a thoracic suction window to stabilize the lung for acquiring stable images.

D-amino acid-containing peptides (DAACPs) in animals are a class of bioactive molecules formed via the posttranslational modification of peptides consisting of all-L-amino acid residues. Amino acid residue isomerization greatly impacts the function of the resulting DAACP. However, because isomerization does not change the peptide's mass, this modification is difficult to detect by most mass spectrometry-based peptidomic approaches. Here we describe a method for the identification of DAACPs that can be used to systematically survey peptides extracted from a tissue sample in a nontargeted manner.

Artificial intelligence underwent remarkable advancement in the past decade, revolutionizing our way of thinking and unlocking unprecedented opportunities across various fields, including drug development. The emergence of large pretrained models, such as ChatGPT, has even begun to demonstrate human-level performance in certain tasks.However, the difficulties of deploying and utilizing AI and pretrained model for nonexpert limited its practical use. To overcome this challenge, here we presented three highly accessible online tools based on a large pretrained model for chemistry, the Uni-Mol, for drug development against CNS diseases, including those targeting NMDA receptor: the blood-brain barrier (BBB) permeability prediction, the quantitative structure-activity relationship (QSAR) analysis system, and a versatile interface of the AI-based molecule generation model named VD-gen. We believe that these resources will effectively bridge the gap between cutting-edge AI technology and NMDAR experts, facilitating rapid and rational drug development.

The NMDAR is a heterotetramer composed of two GluN1 subunits and two GluN2 and/or GluN3 subunits, with the GluN2 subunits exhibiting significant diversity in their structure and function. Recent studies have highlighted the importance of characterizing the specific roles of each GluN2 subunit across central nervous system regions and developmental stages, as well as their unique contributions to NMDAR-mediated signaling and plasticity. Understanding the distinct functions of GluN2 subunits is critical for the development of targeted therapeutic strategies for NMDAR-related disorders. However, measuring the functional contribution of individual GluN2 subtypes in ex vivo slices is challenging. Conventionally, pharmacological or genetic approaches are used, but, in many cases, this is not possible or is restricted to population-level NMDAR responses. Here, we describe a technique for using biophysical properties of miniature synaptic NMDAR responses as a proxy to measure the functional contribution of specific GluN2-NMDAR subunits to individual synapses within a neuron.

In the mammalian central nervous system (CNS), fast excitatory transmission relies primarily on the ionic fluxes generated by ionotropic glutamate receptors (iGluRs). Among iGluRs, NMDA receptors (NMDARs) are unique in their ability to pass large, Ca²⁺-rich currents. Importantly, their high Ca²⁺ permeability is essential for normal CNS function and is under physiological control. For this reason, the accurate measurement of NMDA receptor Ca²⁺ permeability represents a valuable experimental step in evaluating the mechanism by which these receptors contribute to a variety of physiological and pathological conditions. In this chapter, we provide a theoretical and practical overview of the common methods used to estimate the Ca²⁺ permeability of ion channels as they apply to NMDA receptors. Specifically, we describe the principles and methodology used to calculate relative permeability (P_Ca/P_Na) and fractional permeability (P_f), along with the relationship between these two metrics. With increasing knowledge about the structural dynamics of ion channels and of the ongoing environmental fluctuations in which channels operate in vivo, the ability to quantify the Ca²⁺ entering cells through specific ion channels remains a tool essential to delineating the molecular mechanisms that support health and cause disease.