Methods in molecular biology
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Rapid diagnostic methods for fungal infections are long awaited and are expected to improve outcomes through early initiation of targeted antifungal therapy. T2Candida panel is a novel qualitative diagnostic platform that was recently approved by the US Food and Drug Administration (FDA) for diagnosis of candidemia with a mean time to species identification of less than 5 h. ⋯ By combining magnetic resonance with molecular diagnostics, T2Candida panel amplifies DNA and detects the amplified product by amplicon-induced agglomeration of supermagnetic particles and T2 Magnetic Resonance (T2MR) measurement. Here we describe the materials and methods needed to diagnose candidemia with the T2Candida panel.
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The smallpox vaccine based on the vaccinia virus was successfully used to eradicate smallpox, but although very effective, it was a very reactogenic vaccine and responsible for the deaths of one to two people per million vaccinated. Modified Vaccinia virus Ankara (MVA) is an attenuated derivative, also used in the smallpox eradication campaign and now being developed as a recombinant viral vector to produce vaccines against infectious diseases and cancer. MVA can encode one or more foreign antigens and thus can function as a multivalent vaccine. ⋯ Many clinical trials of these new vaccines have been conducted, and the safety of MVA is now well documented. Immunogenicity is influenced by the dose and vaccination regimen, and information on the efficacy of MVA-vectored vaccines is now beginning to accumulate. In this chapter, we provide protocols for generation, isolation, amplification, and purification of recombinant MVA for preclinical and clinical evaluation.
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Even though it is a pandemic health problem worldwide, the pathogenesis of obesity is poorly understood. Recently, emerging studies verified that microRNAs (miRNAs) are involved in complicated metabolic processes including adipocyte differentiation, fat cell formation (adipogenesis), obesity-related insulin resistance and inflammation. ⋯ MiRNAs may play an important part in regulating metabolic functions in adipose tissues and, by extension, obesity and its associated disorders. Consequently, they may be potential candidates for therapeutic targets and biomarkers.
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Direct oral anticoagulants (DOACs) can be quantified using methods that can be performed in any clinical or research laboratory using manual or automated instrument platforms. Dabigatran etexilate, the oral direct thrombin inhibitor, can be quantified by drug-calibrated clot or chromogenic-based assays using either thrombin or ecarin as substrates. Oral direct anti-Xa inhibitors, such as rivaroxaban, apixaban, and edoxaban, can be quantified with drug-calibrated anti-Xa kits or reagents as typically used for measuring heparins (unfractionated, low molecular weight, or pentasaccharides).
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A true and accurate bottom-up global proteomic measurement will only be achieved when all proteins in a sample can be digested efficiently and at least some peptides recovered on which to base an estimate of abundance. Integral membrane proteins make up around one-third of the proteome and require specialized protocols if they are to be successfully solubilized for efficient digestion by the enzymes used in bottom-up proteomics. ⋯ A subset of peptides is purified by reverse-phase solid-phase extraction and fractionated by strong-cation exchange prior to nano-liquid chromatography with data-dependent tandem mass spectrometry. For quantitative proteomics experiments a protocol is described for stable-isotope coding of peptides using dimethylation of primary amines allowing for three-way sample multiplexing.