The Challenge to Stabilize, Extract and Analyze Urinary Cell-Free DNA (ucfDNA) during Clinical Routine.

BACKGROUND: The "Liquid Biopsy" has become a powerful tool for cancer research during the last decade. Circulating cell-free DNA (cfDNA) that originates from tumors has emerged as one of the most promising analytes. In contrast to plasma-derived cfDNA, only a few studies have investigated urinary cfDNA. One reason might be rapid degradation and hence inadequate concentrations for downstream analysis. In this study, we examined the stability of cfDNA in urine using different methods of preservation under various storage conditions. METHODOLOGY: To mimic patient samples, a pool of healthy male and female urine donors was spiked with a synthetic cfDNA reference standard (fragment size 170 bp) containing the T790M mutation in the EGFR gene. Spiked samples were preserved with three different buffers and with no buffer over four different storage periods (0 h; 4 h; 12 h; 24 h) at room temperature vs. 4 °C. The preservatives used were Urinary Analyte Stabilizer (UAS, Novosanis, Wijnegem, Belgium), Urine Conditioning Buffer (UCB, Zymo, Freiburg, Germany) and a self-prepared buffer called "AlloU". CfDNA was extracted using the QIAamp MinElute ccfDNA Mini Kit (Qiagen, Hilden, Germany). CfDNA concentration was measured using the Qubit™ 4 fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). Droplet digital PCR (ddPCR) was used for detection and quantification of the T790M mutation. RESULTS: Almost no spiked cfDNA was recoverable from samples with no preservation buffer and the T790M variant was not detectable in these samples. These findings indicate that cfDNA was degraded below the detection limit by urinary nucleases. Stabilizing buffers showed varying efficiency in preventing this degradation. The most effective stabilizing buffer under all storage conditions was the UAS, enabling adequate recovery of the T790M variant using ddPCR. CONCLUSION: From a technical point of view, stabilizing buffers and adequate storage conditions are a prerequisite for translation of urinary cfDNA diagnostics into clinical routine.

Prophylactic Human Papillomavirus Vaccination: From the Origin to the Current State.

Immunization is the most successful method in preventing and controlling infectious diseases, which has helped saving millions of lives worldwide. The discovery of the human papillomavirus (HPV) infection being associated with a variety of benign conditions and cancers has driven the development of prophylactic HPV vaccines. Currently, four HPV vaccines are available on the pharmaceutical market: Cervarix, Gardasil, Gardasil-9, and the recently developed Cecolin. Multiple studies have proven the HPV vaccines' safety and efficacy in preventing HPV-related diseases. Since 2006, when the first HPV vaccine was approved, more than 100 World Health Organization member countries reported the implementation of HPV immunization. However, HPV vaccination dread, concerns about its safety, and associated adverse outcomes have a significant impact on the HPV vaccine implementation campaigns all over the world. Many developed countries have successfully implemented HPV immunization and achieved tremendous progress in preventing HPV-related conditions. However, there are still many countries worldwide which have not created, or have not yet implemented, HPV vaccination campaigns, or have failed due to deficient realization plans associated with establishing successful HPV vaccination programs. Lack of proper HPV information campaigns, negative media reflection, and numerous myths and fake information have led to HPV vaccine rejection in many states. Thus, context-specific health educational interventions on HPV vaccination safety, effectiveness, and benefits are important to increase the vaccines' acceptance for efficacious prevention of HPV-associated conditions.

Targeted Lipidomics for Characterization of PUFAs and Eicosanoids in Extracellular Vesicles.

Lipids are increasingly recognized as bioactive mediators of extracellular vesicle (EV) functions. However, while EV proteins and nucleic acids are well described, EV lipids are insufficiently understood due to lack of adequate quantitative methods. We adapted an established targeted and quantitative mass spectrometry (LC-MS/MS) method originally developed for analysis of 94 eicosanoids and seven polyunsaturated fatty acids (PUFA) in human plasma. Additionally, the influence of freeze-thaw (FT) cycles, injection volume, and extraction solvent were investigated. The modified protocol was applied to lipidomic analysis of differently polarized macrophage-derived EVs. We successfully quantified three PUFAs and eight eicosanoids within EVs. Lipid extraction showed reproducible PUFA and eicosanoid patterns. We found a particularly high impact of FT cycles on EV lipid profiles, with significant reductions of up to 70%. Thus, repeated FT will markedly influence analytical results and may alter EV functions, emphasizing the importance of a standardized sample pretreatment protocol for the analysis of bioactive lipids in EVs. EV lipid profiles differed largely depending on the polarization of the originating macrophages. Particularly, we observed major changes in the arachidonic acid pathway. We emphasize the importance of a standardized sample pretreatment protocol for the analysis of bioactive lipids in EVs.

Characterizationharacterization of Extremophilic Actinomycetes Strains as Sources of Antimicrobial Agents.

Extremophilic actinomycetes strains can survive extreme saline and alkaline environments and produce antimicrobial agents. In this chapter, we discuss laboratory methods that can be used to isolate and characterize actinomycetes strains capable of potentially producing novel antimicrobial agent(s) when cultured in conditions that mimic the environments from which they were isolated. Methods used to screen for antibacterial and antiviral activities from these producer strains, and microbiological and molecular approaches used to identify these strains are described. Here we describe three methods. Method 1 focuses on the strategy to select optimal conditions to synthesize and accumulate the antibiotics from the studied actinomycetes strains by preparing crude extracts. In Method 2, we describe the screening strategies used to test the actinomycetes strains against gram-negative and gram-positive bacteria, antifungal agents, multidrug-resistant pathogens (MDR), and viral pathogens. Thus, the specific techniques to test for MDR pathogens such as the disk diffusion assay and wells assay are outlined. We also describe the antiviral activity screening of the selected actinomycetes extracts in Method 2 of this chapter. Specifically, we concentrate on methods used to test for antiviral activities such as primary hemolytic, hemagglutination, neuraminidase, and specific virus-inhibitory activities. Finally, the Method 3 section reveals the microbiological techniques used to morphologically characterize the actinomycetes strains that depend on the culture medium utilized for growth. Additionally, the method used to perform a detailed characterization of the morphology that actinomycetes strains possess is specified by the protocol for sample preparation and visualization using the scanning electron microscopy (SEM). Finally, we summarize the molecular approaches used to characterize actinomycetes strains, focusing specifically on the PCR and sequencing techniques.