Polymerase chain reaction products (PCR) on “DNA barcode zone” resolved by temporal temperature gradient electophoresis: A tool for species identification of mixed meat specimens – A technical note on preliminary results

The capability of PCR-TTGE to detect meat species in mixed animal samples was investigated as a necessary step in developing a method where the identification will be performed matching on the “DNA barcode” zone the sequences of resolved PCR products obtained from a limited set of “universal” primers. Exemplary mixtures from five important meat species were analyzed. At this stage more PCR reactions have to be applied on a sample but this should be easily improved using primers simultaneously (as a “cocktail”) in a single reaction.     

Carcinogenesis of Pancreatic Adenocarcinoma: Precursor Lesions

Pancreatic adenocarcinoma displays a variety of molecular changes that evolve exponentially with time and lead cancer cells not only to survive, but also to invade the surrounding tissues and metastasise to distant sites. These changes include: genetic alterations in oncogenes and cancer suppressor genes; changes in the cell cycle and pathways leading to apoptosis; and also changes in epithelial to mesenchymal transition. The most common alterations involve the epidermal growth factor receptor (EGFR) gene, the HER2 gene, and the K-ras gene. In particular, the loss of function of tumor-suppressor genes has been documented in this tumor, especially in CDKN2a, p53, DPC4 and BRCA2 genes. However, other molecular events involved in pancreatic adenocarcinoma pathogenesis contribute to its development and maintenance, specifically epigenetic events. In fact, key tumor suppressors that are well established to play a role in pancreatic adenocarcinoma may be altered through hypermethylation, and oncogenes can be upregulated secondary to permissive histone modifications. Indeed, factors involved in tumor invasiveness can be aberrantly expressed through dysregulated microRNAs. This review summarizes current knowledge of pancreatic carcinogenesis from its initiation within a normal cell until the time that it has disseminated to distant organs. In this scenario, highlighting these molecular alterations could provide new clinical tools for early diagnosis and new effective therapies for this malignancy.     

X-Ray Induced DNA Damage and Repair in Germ Cells of PARP1?/? Male Mice

Poly(ADP-ribose)polymerase-1 (PARP1) is a nuclear protein implicated in DNA repair, recombination, replication, and chromatin remodeling. The aim of this study was to evaluate possible differences between PARP1^−/− and wild-type mice regarding induction and repair of DNA lesions in irradiated male germ cells. Comet assay was applied to detect DNA damage in testicular cells immediately, and two hours after 4 Gy X-ray irradiation. A similar level of spontaneous and radiation-induced DNA damage was observed in PARP1^−/− and wild-type mice. Conversely, two hours after irradiation, a significant level of residual damage was observed in PARP1^−/− cells only. This finding was particularly evident in round spermatids. To evaluate if PARP1 had also a role in the dynamics of H2AX phosphorylation in round spermatids, in which γ-H2AX foci had been shown to persist after completion of DNA repair, we carried out a parallel analysis of γ-H2AX foci at 0.5, 2, and 48 h after irradiation in wild-type and PARP1^−/− mice. No evidence was obtained of an effect of PARP1 depletion on H2AX phosphorylation induction and removal. Our results suggest that, in round spermatids, under the tested experimental conditions, PARP1 has a role in radiation-induced DNA damage repair rather than in long-term chromatin modifications signaled by phosphorylated H2AX.     

CHEMICAL AND TOXICOLOGICAL CHARACTERIZATION OF AIRBORNE TOTAL SUSPENDED PARTICULATE (TSP) AND PM 10 ORGANIC EXTRACTS

The relationship between chemical composition of airborne particulates and the genotoxicity has been investigated in the atmosphere of Rome, Italy. For this purpose, both total suspended particulate (TSP) and the PM ₁₀ fractions were collected daily inside a green park located in downtown, grouped on a weekly basis and speciated for their burdens of polycyclic aromatic hydrocarbons (PAH) and nitro-PAH. Concurrently, the genotoxicity of the organic extracts was evaluated by the Comet assay (SCGE: single cell gel electrophoresis) on human peripheral blood mononuclear cells (PBMC). The results indicate that organic extracts were able to induce DNA damage and a cytotoxic effect on PBMC. The TSP fraction was more cytotoxic than PM ₁₀ while the genotoxicity of both fractions was comparable. The genotoxic potential of the different samples was highly correlated to the amount of total PAH (correlation coefficient = 0.87), carcinogenic PAH (correlation coefficient = 0.88), B(a)P (correlation coefficient = 0.87) and to a less extend to the concentration of 1-nitropyrene (correlation coefficient = 0.66). The seasonal modulation suggests that in Rome the air is more toxic during winter, however in the warm season oxidized species including nitro-PAHs (evolving from secondary pollution) seem to balance the decrease of PAH concentration rates.     

Furosine as a Pasta Quality Marker: Evaluation by an Innovative and Fast Chromatographic Approach

A fast and simple high‐performance liquid chromatography method suitable for determining furosine level in heat‐treated food samples was developed. The analysis of furosine was performed by a novel mixed‐mode column that provides multiple and simultaneous retention mechanisms including cation‐exchange, anion‐exchange, reversed‐phase, or hydrophilic interaction. Each retention mechanism could be independently controlled by setting chromatographic conditions. Adequate retention and selectivity of polar charged furosine were achieved by adjusting mobile phase pH, buffer concentration, organic content, and ionic strength. The optimized method was successfully applied to determinate furosine in durum wheat semolina pasta samples. Furosine level in pasta may be used as a reliable marker of health and nutritional damage occurring during pasta manufacture. Indeed, a low content of furosine is generally related to high nutritional quality of food and application of mild heat treatments. A wide range of dry pasta samples, collected from both supermarkets (large‐scale retail trade) and shops selling local products, were analyzed. Variable amounts of furosine, ranging from 107 to 506 mg/100 g of protein, were found in pasta samples. The proposed method allows to discriminate products submitted to different time–temperature conditions during the drying process. At the same time, it may be used to highlight potential label fraud.     

Effect of inulin as prebiotic and synbiotic interactions between probiotics to improve fermented milk firmness

Inulin behaved as a prebiotic to improve firmness of skim milk fermented by (a) pure cultures of Lactobacillus acidophilus (La), Lactobacillus rhamnosus (Lr), Lactobacillus bulgaricus (Lb) and Bifidobacterium lactis (Bl), (b) binary co-cultures of them with Streptococcus thermophilus (St), or (c) a cocktail containing all them. Inulin addition to co-cultures and cocktail enhanced products firmness, either after 1 day (D1) or 7 days (D7) of cold storage, likely due to the increase in microbial growth induced by metabolic interactions among lactic acid bacteria and partial inulin metabolization. Co-culture firmness did in fact range from 0.33 N without inulin (St-Lb) after D1 and 0.55 N with inulin (St-Lr) after D7. Also cocktail cultures exhibited high values of firmness, ranging, as an average, from 0.43 N (D1) to 0.46 N (D7), which suggests that they could have been potentiated by the reciprocal synergistic effects of microorganisms in complex mixture.     

Extraction of phenolics from Vitis vinifera wastes using non-conventional techniques

Polyphenols, the well known naturally occurring antioxidants, are the most abundant secondary metabolites in grape wastes. Herein we investigate several non-conventional extraction methods vs classic solid–liquid extraction (SLE) to obtain phenolic compounds from grape seeds and skins. We compared SLE, ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE) and high pressure and temperature extraction (HPTE) in term of extraction yield and antioxidant power of the extract. Solvent of choice between methanol and ethanol was the former, both for skins and seeds. Quali-quantitative analyses were performed using colorimetric and HPLC methods. The highest content in total polyphenols, o-diphenols and flavonoids, both for seeds (108.3, 47.0 mg_GAE g_DW⁻¹, 47.2 mg_CE g_DW⁻¹) and skins (34.2, 10.1 mg_GAE g_DW⁻¹, 21.6 mg_CE g_DW⁻¹) was obtained with HPTE working in a Parr reactor. While the highest antiradical power was determined in seeds extracts from MAE (78.6 μl_extract μg_DPPH⁻¹). Prolonged extraction times (over 30 min) further increased the amount of total polyphenols, while progressively decreased the amount of flavonoids and the antiradical power.     

Effect of the lactic acid bacteria on the control of listerial activity and shelf life of smoked salmon scraps

The potential protective activity of lactic acid bacteria (LAB) from cold smoked salmon scraps was evaluated towards Listeria monocytogenes. Seventy‐three LAB strains were isolated and identified by biomolecular methods; Lactobacillus curvatus and Lactobacillus sakei prevailed. Three of the strains tested, identified as L. sakei, profile O, had a significant inhibitory activity against L. monocytogenes ATCC 19115 and clones DUP‐1042 and DUP‐18596. The evolution of microbial populations and chemical parameters were determined at time intervals to verify the shelf life. Listeria monocytogenes was isolated in half the packages also exceeding the legal limit. The shelf life of scraps was set at 30 days. Clonal characterisation of L. monocytogenes was performed by ribotyping. DUP‐1042, one of the human pathogen clones, was the most represented pattern. The results suggest further studies aimed at the selection of autochthonous nonspoilage LAB strains as bioprotective agents for cold smoked salmon.