Methods in molecular biology
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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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Protein phosphorylation, a process in which kinases modify serines, threonines, and tyrosines with phosphoryl groups is of major importance in eukaryotic biology. Protein phosphorylation events are key initiators of signaling responses which determine cellular outcomes after environmental and metabolic stimuli, and are thus highly regulated. ⋯ Peptides are separated on a C18 reversed-phase column under basic conditions and fractions collected in timed intervals followed by concatenation of the fractions. Each Fraction is subsequently enriched for phosphopeptides using TiO2 followed by LC/MS analysis.
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Posttraumatic epilepsy (PTE) is one of the most common and devastating complications of traumatic brain injury (TBI). Currently, the etiopathology and mechanisms of PTE are poorly understood and as a result, there is no effective treatment or means to prevent it. Antiepileptic drugs remain common preventive strategies in the management of TBI to control acute posttraumatic seizures and to prevent the development of PTE, although their efficacy in the latter case is disputed. ⋯ Although acute and chronic recurrent posttraumatic seizures are well-recognized phenomena in these models, there is only a limited number of studies focused on PTE. The most used mechanical TBI models with documented electroencephalographic and behavioral seizures with remote epileptogenesis include fluid percussion, controlled cortical impact, and weight-drop. This chapter describes the most popular models of PTE-induced TBI models, focusing on the controlled cortical impact and the fluid percussion injury models, the methods of behavioral and electroencephalogram seizure assessments, and other approaches to detect epileptogenic properties, and discusses their potential application for translational research.
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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.
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Immune checkpoint inhibitors (ICI) are a new class of drugs characterized by their ability to enhance antitumor immune responses through the blockade of critical cell surface receptors involved in the maintenance of peripheral tolerance. The recent approval of ICI targeting CTLA-4 or PD-1 for the treatment of cancer constitutes a major breakthrough in the field of oncology and demonstrates the potential of immune-mediated therapies in achieving durable cancer remissions. The identification of new immune regulatory pathways that could be targeted to reactivate or boost antitumor immunity is now a very active field of research. ⋯ In this chapter, we describe the general methodology to evaluate antitumor activity of ICI in immunocompetent mice. We outline protocols to reliably establish tumors in mice and generate lung metastasis through tail vein injections with the aim of testing the efficacy of ICI. We also present methods to analyze the composition of the tumor immune-infiltrate by multicolor flow cytometry.