Methods in molecular biology
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Bioluminescence resonance energy transfer (BRET) is a prominent biophysical technology for monitoring molecular interactions, and has been widely used to study protein-protein interactions (PPI) in live cells. This technology requires proteins of interest to be associated with an energy donor (i.e., luciferase) and an acceptor (e.g., fluorescent protein) molecule. Upon interaction of the proteins of interest, the donor and acceptor will be brought into close proximity and energy transfer of chemical reaction-induced luminescence to its corresponding acceptor will result in an increased emission at an acceptor-defined wavelength, generating the BRET signal. ⋯ We term this NLuc-based BRET platform "BRET(n)". BRET(n) has been demonstrated to have significantly improved assay performance, compared to previous BRET technologies, in terms of sensitivity and scalability. This chapter describes a step-by-step practical protocol for developing a BRET(n) assay in a multi-well plate format to detect PPIs in live mammalian cells.
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The central nervous system (CNS) is protected by blood-brain barrier (BBB) and blood-cerebrospinal-fluid (CSF) barrier that limit toxic agents and most molecules from penetrating the brain and spinal cord. However, these barriers also prevent most pharmaceuticals from entering into the CNS. Drug delivery to the CNS following neurotrauma is complicated. ⋯ And this likely leads to better dose selection of the drug, as well as recognition of any possible side effects, prior to transition into a clinical trial. Additionally, novel approach that is noninvasive and yet circumvents BBB, such as drug delivery through nerve pathways innervating the nasal passages, needs to be investigated in animal models, as it may provide a viable drug delivery method for patients who sustain mild CNS injury or require chronic treatments. Therefore, the focus of this chapter is to present rationales and methods for delivering drugs by IV infusion via the jugular vein, and intranasally in preclinical studies.
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Clostridium difficile is a challenging infection that can be difficult to treat with antibiotic therapy. This chapter outlines the processing material for fecal microbiota transplantation (FMT), also known as stool transplant. ⋯ FMT uses a stool sample collected from a healthy, screened donor to restore healthy microbiota in the colon of a patient with CDI for symptom resolution. Here, we describe a rapid method for FMT preparation that uses inexpensive and disposable materials.
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In recent years, mass spectrometry-based phosphoproteomics has propelled our knowledge about the regulation of cellular pathways. Nevertheless, typically applied bottom-up strategies have several limitations. Trypsin, the preferentially used proteolytic enzyme shows impaired cleavage efficiency in the vicinity of phosphorylation sites. ⋯ To overcome these limitations, we introduce an alternative and simple approach based on the usage of the nonspecific serine protease subtilisin, which enables a fast and reproducible digestion and provides access to "hidden" areas of the proteome. Thus, in a single LC-MS experiment >1800 phosphopeptides were confidently identified and localized from 125 μg of HeLa digest, compared to >2100 sites after tryptic digestion. While the overlap was less than 20 %, subtilisin allowed the identification of many phosphorylation sites that are theoretically not accessible via tryptic digestion, thus considerably increasing the coverage of the phosphoproteome.
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Autoantibodies against myelin oligodendrocyte glycoprotein (MOG) occur in a proportion of patients with different inflammatory demyelinating diseases of the central nervous system, such as childhood multiple sclerosis (MS), acute disseminated encephalomyelitis (ADEM), and neuromyelitis optica spectrum disorders (NMOSD). We describe here in detail a sensitive cell-based assay that allows the identification of autoantibodies against MOG in serum.