Methods in molecular biology
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β-D-glucan (Fungitell) and polymerase chain reaction-based (T2Candida) assays of blood samples are FDA-approved adjuncts to cultures for diagnosing candidemia and other types of invasive candidiasis, but their clinical roles are unclear. In this chapter, we describe laboratory protocols for performing Fungitell and T2Candida assays. We then discuss step-by-step methods for interpreting test results at the bedside using a Bayesian framework, and for incorporating assays into rational patient management strategies. ⋯ Using the thresholds, it is possible to assign windows of pretest likelihood for invasive candidiasis (and corresponding patient populations) in which tests are most likely to valuable. At the individual patient level, tests may be useful outside of the windows proposed for screening populations. The interpretive and clinical decision-making processes we discuss will be applicable to other diagnostic assays as they enter the clinic, and to existing assays as more data emerge from various populations.
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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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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.
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Titanium dioxide (TiO2) has very high affinity for phosphopeptides and in recent years it has become one of the most popular methods for phosphopeptide enrichment from complex biological samples. Peptide loading onto TiO2 resin in a highly acidic environment in the presence of 2,5-dihydroxybenzoic acid (DHB), phthalic acid, lactic acid, or glycolic acid has been shown to improve selectivity significantly by reducing unspecific binding of non-phosphorylated peptides. ⋯ TiO2 chromatography is extremely tolerant towards most buffers used in biological experiments, highly robust and as such it has become the method of choice in large-scale phosphoproteomics. Here we describe a batch mode protocol for phosphopeptide enrichment using TiO2 chromatographic material followed by desalting and concentration of the sample by reversed phase micro-columns prior to downstream MS and LC-MS/MS analysis.