Setting out the frame conditions for feasible use of FFPE derived RNA

Introduction

The usage of formalin-fixed paraffin embedded (FFPE) tissue is characterized by its long shelf-life and simple handling. Therefore it is the most commonly available tissue specimen in routine diagnostics and histological studies. Formaldehyde fixation may result in RNA degradation and cross linking with proteins, while storage conditions also affect RNA integrity. The present study was designed to investigate the influence of these factors on RNA analysis.

Developmental regulation of type 1 and type 3 interferon production and risk for infant infections and asthma development

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Colonic In Vitro Model Assessment of the Prebiotic Potential of Bread Fortified with Polyphenols Rich Olive Fiber

The use of olive pomace could represent an innovative and low-cost strategy to formulate healthier and value-added foods, and bakery products are good candidates for enrichment. In this work, we explored the prebiotic potential of bread enriched with Polyphenol Rich Fiber (PRF), a defatted olive pomace byproduct previously studied in the European Project H2020 EcoProlive. To this aim, after in vitro digestion, the PRF-enriched bread, its standard control, and fructo-oligosaccharides (FOS) underwent distal colonic fermentation using the in vitro colon model MICODE (multi-unit colon gut model). Sampling was done prior, over and after 24 h of fermentation, then metabolomic analysis by Solid Phase Micro Extraction Gas Chromatography Mass Spectrometry (SPME GCMS), 16S-rDNA genomic sequencing of colonic microbiota by MiSeq, and absolute quantification of main bacterial species by qPCR were performed. The results indicated that PRF-enriched bread generated positive effects on the host gut model: (i) surge in eubiosis; (ii) increased abundance of beneficial bacterial groups, such as Bifidobacteriaceae and Lactobacillales; (iii) production of certain bioactive metabolites, such as low organic fatty acids; (iv) reduction in detrimental compounds, such as skatole. Our study not only evidenced the prebiotic role of PRF-enriched bread, thereby paving the road for further use of olive by-products, but also highlighted the potential of the in vitro gut model MICODE in the critical evaluation of functionality of food prototypes as modulators of the gut microbiota.     

Effect of acidic conditions on the growth and expression of two virulence genes of Listeria monocytogenes serotype 4b

In this work, the effect of the usual acidic conditions of dry-cured fermented foods (pH values between 4.5 and 6), on the growth and expression of the virulence genes, hly and inlA, of Listeria monocytogenes serotype 4b, was evaluated. To analyse the expression of the inlA gene, a novel real-time PCR (qPCR) method using SYBR® Green methodology was developed. L. monocytogenes levels increased as the pH did and they were kept constant throughout incubation time at pH 4.5. However, a significant increase in the relative expression of the virulence genes was detected in most of the acidic conditions in all the incubation times. The most pronounced upregulation of the relative expression of the virulence genes was found at pH 4.5. The efficient inlA-based qPCR method could be of interest to check changes in the expression of such virulence gene of this pathogenic bacterium in acidic environments.     

Detection of Highly Poisonous Nerium oleander Using Quantitative Real-Time PCR with Specific Primers

Nerium oleander is one of the most poisonous plants, and its accidental ingestion has frequently occurred in humans and livestock. It is vital to develop a rapid and accurate identification method for the timely rescue of oleander-poisoned patients and the investigation of poisoning cases. In this study, a specific and highly sensitive quantitative real-time PCR (qPCR)-based method was developed to identify oleander in mixture systems and simulated forensic specimens (SFS). First, a new pair of oleander-specific primers, JZT-BF/BR, was designed and validated. Then, a qPCR method was developed using the primers, and its detective sensitivity was examined. The results showed that JZT-BF/BR could specifically identify oleander in forage and food mixtures, and qPCR was capable of accurate authentication even at a low DNA concentration of 0.001 ng/μL. This method was further applied to the analysis of SFS containing different ratios of N. oleander. The method was confirmed to be applicable to digested samples, and the detection limit reached 0.1% (w/w) oleander in mixture systems. Thus, this study undoubtedly provides strong support for the detection of highly toxic oleander and the diagnosis of food poisoning in humans and animals.     

Multiplex quantitative polymerase chain reaction assay for the identification and quantitation of major vaginal lactobacilli

Lactobacilli play a key role in promoting vaginal health. Depletion of these bacteria is associated with bacterial vaginosis (BV), the most common vaginal disorder. Here we describe the development and laboratory validation of a novel single-tube multiplex TaqMan quantitative polymerase chain reaction (qPCR) assay for the identification and quantitative assessment of the four major vaginal Lactobacillus species: L. crispatus, L. jensenii, L. gasseri, and L. iners. The assay utility was evaluated by the analysis of lactobacilli in non-cultured clinical vaginal swab specimens collected from BV patients and healthy individuals. As confirmed by the assay, L. crispatus, L. jensenii, and to a lesser extent L. gasseri, are common in the vagina of healthy women, whereas L. iners dominance is associated with BV. The major assay limitation was preferential detection of dominant Lactobacillus species in samples with mixed lactobacilli resulting in lower sensitivity for minor species. The multiplex qPCR assay described here is an advance in the detection and quantitation of the major vaginal lactobacilli, potentially facilitating the molecular diagnosis of BV and post-therapy restoration of the vaginal microflora.