Human Amniotic Mesenchymal Stem Cells and Fibroblasts Accelerate Wound Repair of Cystic Fibrosis Epithelium.

Cystic fibrosis (CF) airways are affected by a deranged repair of the damaged epithelium resulting in altered regeneration and differentiation. Previously, we showed that human amniotic mesenchymal stem cells (hAMSCs) corrected base defects of CF airway epithelial cells via connexin (CX)43-intercellular gap junction formation. In this scenario, it is unknown whether hAMSCs, or fibroblasts sharing some common characteristics with MSCs, can operate a faster repair of a damaged airway epithelium. A tip-based scratch assay was employed to study wound repair in monolayers of CFBE14o- cells (CFBE, homozygous for the F508del mutation). hAMSCs were either co-cultured with CFBE cells before the wound or added to the wounded monolayers. NIH-3T3 fibroblasts (CX43+) were added to wounded cells. HeLa cells (CX43-) were used as controls. γ-irradiation was optimized to block CFBE cell proliferation. A specific siRNA was employed to downregulate CX43 expression in CFBE cells. CFBE cells showed a delayed repair as compared with wt-CFTR cells (16HBE41o-). hAMSCs enhanced the wound repair rate of wounded CFBE cell monolayers, especially when added post wounding. hAMSCs and NIH-3T3 fibroblasts, but not HeLa cells, increased wound closure of irradiated CFBE monolayers. CX43 downregulation accelerated CFBE wound repair rate without affecting cell proliferation. We conclude that hAMSCs and fibroblasts enhance the repair of a wounded CF airway epithelium, likely through a CX43-mediated mechanism mainly involving cell migration.

Lipidomics profile of irradiated ground meat to support food safety.

Meat irradiation is considered as an effective treatment that expose the advantageous effects on meat preservation. This research, based on untargeted LC-HR orbitrap MS-based lipidomics strategy was meant to estimate the alterations in lipid profile of irradiated chicken, turkey and mixed (chicken, turkey and pork) ground meat in order to evaluate if exists any food safety issue concerning the lipidome alteration. Special attention was paid on oxidation triggered by irradiation. All three matrices exhibited a characteristic lipidome profile which was affected differently by five levels of irradiation intensity. Overall, 345 lipids categorized into 14 subclasses were identified. Remarkably, the oxidized glycerophosphoethanolamines and oxidized glycerophosphoserines were identified in irradiated turkey meat, while for all three categories a characteristic diacylglycerols profile was recognised. Our analytical approach highlighted that the estimation of qualitative variations in lipid portion might be valuable in food inspection purposes, especially when the samples from animal origin are suspected on irradiation treatment.

Innovative approaches for identifying a mechanically separated meat: evaluation of radiostrontium levels and development of a new tool of investigation.

Due to food safety concerns, the European Food Safety Authority (EFSA) asked to identify useful parameters for identifying the mechanically separated meat (MSM). In this work, the 90Sr levels of MSM products were assessed for evaluating the suitability of this parameter for MSM identification. Indeed, this contaminant may accumulate in animal bones and bone fragments may be present in MSM. One hundred samples of fresh meats and meat products composed of different MSM percentages and without MSM were analyzed by ultra-low-level liquid scintillation counting. The 90Sr activity concentrations detected in meat products with high percentage of MSM were significantly higher (p ≥ 0.05) (0.099 Bq kg-1) than those of fresh meats (0.024 Bq kg-1) and meat products containing low percentage of MSM (0.011 Bq kg-1). The percentage of correct MSM identification was 61%. In order to increase this percentage and to develop a new tool of investigation, the 90Sr was elaborated in combination with other three parameters: 88Sr, Ca and ash percentage, through a multivariate approach. The precision of this tool of investigation was significantly increased (87%), higher than the reference method (Ca level) (76%), representing a valid novel approach in the identification of MSM products.

Chromatographic determination of 12 dyes in meat products by HPLC-UV-DIODE array detection.

The use of food dyes in meat is regulated by the current European and non-European legislation, due to several food safety concerns. A reliable method for the quali-quantitative determination of 12 food dyes (Amaranth, Ponceau 4R, Carmine, Ponceau SX, Ponceau 3R, Allura Red AC, Carmoisine, Erythrosine, Sudan I, Sudan II, Sudan III and Sudan IV) in meat products, by high performance liquid chromatography coupled to UV diode array detection is presented. The extraction was accomplished by using acetonitrile, methanol, water, and ammonia, 50:40:9:1 (v/v/v/v) as the solvent and ultrasonic bath. The chromatographic separation was obtained with a C18 RP column eluted by a gradient of acetate buffer/acetonitrile. Good analytical performances characterized this method (Table 1), in terms of selectivity, sensitivity, accuracy and ruggedness. Both method precision (CV% range: 6%-15%) and recovery percentages (range: 86%-105%) resulted in compliance with Decision 2002/657/EC, and the expanded measurement uncertainties, estimated by a bottom-up approach, were in the range 6%-20%. All these results demonstrated that the procedure can be applied successfully for confirmation analyses of commercial meat products. •12 food dyes were determined in meat by new HPLC/UV-DAD method.•The analytical method was fully validated for accurate confirmation analyses.•Method accuracy, sensitivity, selectivity and ruggedness resulted satisfactory.

Altered morphokinetics in equine embryos from oocytes exposed to DEHP during IVM.

Di-(2-ethylhexyl) phthalate (DEHP) is a commonly used plasticizer with endocrine-disrupting properties. In this study, we used an equine model to investigate DEHP concentrations in ovarian follicular fluid (FF), and to determine the effects of exposure of oocytes to potentially toxic concentrations of DEHP during in vitro maturation (IVM) on embryo development and quality. Embryo development was evaluated using time-lapse monitoring (TLM), a photomicroscopic tool that reveals abnormalities in cleavage kinetics unobservable by conventional morphology assessment. Blastocyst bioenergetic/oxidative status was assessed by confocal analysis. The possibility that verbascoside (VB), a bioactive polyphenol with antioxidant activity, could counteract DEHP-induced oocyte oxidative damage, was investigated. DEHP was detected in FF and in IVM media at concentrations up to 60 nM. Culture of oocytes in the presence of 500 nM DEHP delayed second polar body extrusion, reduced duration of the second cell cycle, and increased the percentage of embryos showing abrupt multiple cleavage, compared with controls. Mitochondrial activity and intracellular levels of reactive oxygen species were reduced in blastocysts from DEHP-exposed oocytes. VB addition during IVM limited DEHP-induced blastocyst damage. In conclusion, DEHP is detectable in equine FF and culture medium, and oocyte exposure to increased concentrations of DEHP during IVM affects preimplantation embryo development. Moreover, TLM, reported for the first time in the horse in this study, is an efficient tool for identifying altered morphokinetic parameters and cleavage abnormalities associated with exposure to toxic compounds.

Simultaneous determination of twelve dyes in meat products: Development and validation of an analytical method based on HPLC-UV-diode array detection.

The use of food dyes in meat is subject to regulations, due to food safety concerns. A reliable method for the determination of 12 food dyes (Amaranth, Ponceau 4R, Carmine, Ponceau SX, Ponceau 3R, Allura Red AC, Carmoisine, Erythrosine, Sudan I, Sudan II, Sudan III and Sudan IV) in meat products using high performance liquid chromatography coupled to UV-diode array detection was developed, optimized and fully validated. The extraction was accomplished using acetonitrile, methanol, water, ammonia, 50:40:9:1 (v/v/v/v) as the solvent, and an ultrasonic bath. Chromatographic separation was achieved using a C18 RP column and samples eluted with a gradient acetate-acetonitrile mobile phase. Good analytical performance was obtained, in terms of selectivity, sensitivity, accuracy and ruggedness. Both method precision (CV% range: 6.2%-18.0%) and recovery (range: 86.4%-105.0%) complied with Decision 657/2002/EC, suggesting the procedure could be applied successfully for analyses of meat products in the European Union.

Radiostrontium accumulation in animal bones: development of a radiochemical method by ultra low-level liquid scintillation counting for its quantification.

Strontium-90 (90Sr) is a fission product, resulting from the use of uranium and plutonium in nuclear reactors and weapons. Consequently, it may be found in the environment as a consequence of nuclear fallouts, nuclear weapon testing, and not correct waste management. When present in the environment, strontium-90 may be taken into animal body by drinking water, eating food, or breathing air. The primary health effects are bone tumors and tumors of the blood-cell forming organs, due to beta particles emitted by both 90Sr and yttrium-90 (90Y). Moreover, another health concern is represented by inhibition of calcification and bone deformities in animals. Actually, radiometric methods for the determination of 90Sr in animal bones are lacking. This article describers a radiochemical method for the determination of 90Sr in animal bones, by ultra low-level liquid scintillation counting. The method precision and trueness have been demonstrated through validation tests (CV% = 12.4%; mean recovery = 98.4%). Detection limit and decision threshold corresponding to 8 and 3 mBecquerel (Bq) kg-1, respectively, represent another strong point of this analytical procedure. This new radiochemical method permits the selective extraction of 90Sr, without interferences, and it is suitable for radiocontamination surveillance programs, and it is also an improvement with respect to food safety controls.

Characterization, chemometric evaluation, and human health-related aspects of essential and toxic elements in Italian honey samples by inductively coupled plasma mass spectrometry.

Concentration values of 24 elements (Al, As, Ba, Be, Ca, Cd, Co, Cr, Cu, Fe, Ge, Hg, Mn, Mo, Pb, Sb, Se, Sn, Sr, Ti, Tl, U, V, and Zn) were determined in 72 honey samples produced in Italy by inductively coupled plasma mass spectrometry (ICP-MS). Considering the recommended established heavy metal daily intakes for humans, in this perspective, an equilibrated and ordinary honey consumption should not be considered matter of concerns for human health, even if particular attention should be addressed if honey is consumed by children, due to different maximum daily heavy metal intakes. Chemometric analysis of the results obtained highlights heavy metal content differences in honey samples obtained from notoriously polluted zones, confirming then that honey can be considered a bio-indicator of environmental pollution. Finally, Pearson coefficients highlighted correlations among element contents in honey samples.

Environmental monitoring of the area surrounding oil wells in Val d'Agri (Italy): element accumulation in bovine and ovine organs.

In this work, environmental heavy metal contamination in the Val d'Agri area of Southern Italy was monitored, measuring the accumulation of 18 heavy metals (U, Hg, Pb, Cd, As, Sr, Sn, V, Ni, Cr, Mo, Co, Cu, Zn, Ca, Mn, Fe, and Al) in the organs of animals raised in the surrounding area (kidney, lung, and liver of bovine and ovine species). Val d'Agri features various oil processing centers which are potentially a significant source of environmental pollution, making it essential to perform studies that will outline the state of the art on which any recovery plans and interventions may be developed. The analysis was carried out using official and accredited analytical methods based on inductively coupled plasma mass spectrometry, and the measurements were statistically processed in order to give a contribution to risk assessment. Even though five samples showed Pb and Cd concentrations above the limits defined in the European Commission Regulation (EC) No 1881/2006, the mean concentrations of most elements suggest that contamination in this area is low. Consequently, these results also suggest that there is no particular risk for human exposure to toxic trace elements. Nevertheless, the findings of this work confirm that element accumulation in ovine species is correlated with geographical livestock area. Therefore, ovine-specific organs might be used as bioindicators for monitoring contamination by specific toxic elements in exposed areas.

Transperineal ultrasonography: First level exam in IBD patients with perianal disease.

BACKGROUND: A pelvic magnetic resonance imaging (MRI) represents the front-line method for evaluating perianal disease in patients with inflammatory bowel disease (IBD). Recently, transperineal ultrasonography (TPUS) has been proposed as a simple, safe, time-sparing and useful diagnostic technique to assess different pathological conditions of the pelvic floor. AIM: The aim of this prospective single centre study was to evaluate the accuracy of TPUS versus MRI for the detection and classification of perineal disease in IBD patients. METHODS: From November 2013 to November 2014, 28 IBD patients underwent either TPUS or MRI. Fistulae and abscesses were classified according to Parks' and AGA's classification methods. A concordance was assessed by k statistics. RESULTS: Overall, 33 fistulae and 8 abscesses were recognized by TPUS (30 and 7 by MRI, respectively). The agreement between TPUS and MRI was 75% according to Parks' classification (k=0.67) and 86% according to AGA classification (k=0.83), while it was 36% (k=0.34) for classifying abscesses. CONCLUSIONS: TPUS proved to be as accurate as MRI for detecting superficial and small abscesses and for classifying perianal disease. Both examinations may be performed at the initial presentation of the patient, but TPUS is a cheaper, time-sparing procedure. The optimal use of TPUS might be in follow-up patients.

Radiostrontium levels in foodstuffs: 4-Years control activity by Italian reference centre, as a contribution to risk assessment.

(90)Sr is considered an important contaminant relating to food supply chains. In this study, 176 liquid and 260 solid foods, were analysed in order to quantify (90)Sr. Through ruggedness tests, the application field of radiochemical methods used was extended successfully to all most important types of foodstuffs. Regarding liquid matrices, milk samples resulted the most important indicator about (90)Sr contamination, with mean (90)Sr activity concentration equal to 0.058BqL(-1). Among other liquid foods, wine/spirits and livestock watering resulted the most contaminated, with mean contamination levels equal to 0.022 and 0.035BqL(-1), respectively. Concerning solid matrices, cheeses produced from sheep's milk and animal feeds resulted the most contaminated (mean levels: 1.237 and 1.557Bqkg(-1), respectively). Meat products and seafood showed contamination levels not significant within this survey; while, among vegetables, cacao/chocolate and spices resulted in contamination levels comparable with those of cheese obtained from milk of cows origin.

Determination of radiostrontium in milk samples by ultra-low-level liquid scintillation counting: a validated approach.

Strontium-90 is a hazardous radionuclide derived from nuclear fission processes, and it decays emitting high-energy beta-particles forming yttrium-90. It may be easily absorbed in humans through several food supply chains, since it is chemically similar to some important biological elements such as potassium and calcium. In this work an analytical method for the determination of radiostrontium in milk by liquid scintillation counting was developed in order to obtain a precise quantification at low activity concentration levels. Moreover, a full validative approach was applied to confirm method reliability. Good analytical performance was obtained by developing an in-house validation model according to Regulation 882/2004/EC and Decision 657/2002/EC with a good selectivity, a decision threshold and detection limit corresponding to 0.003 and 0.006 Bq l⁻¹ (α = ß = 0.05) respectively, a repeatability value, expressed as CV%, equal to 13%, a mean recovery value equal to 102.5% and a measurement uncertainty equal to 11.5%. Strontium and yttrium chemical yields were determined by a reliable analytical method by inductively coupled mass spectrometry, assuring a suitable correction of final result. The results of the method validation, combined with a quality control assurance, performed on a reference material, demonstrated that the procedure represents a substantial improvement in the determination of 9°Sr relating to accurate confirmation analyses.

Dietary exposure to trace elements and radionuclides: the methodology of the Italian Total Diet Study 2012-2014.

This article presents the methodology of the Italian Total Diet Study 2012-2014 aimed at assessing the dietary exposure of the general Italian population to selected nonessential trace elements (Al, inorganic As, Cd, Pb, methyl-Hg, inorganic Hg, U) and radionuclides (40K, 134Cs, 137Cs, 90Sr). The establishment of the TDS food list, the design of the sampling plan, and details about the collection of food samples, their standardized culinary treatment, pooling into analytical samples and subsequent sample treatment are described. Analytical techniques and quality assurance are discussed, with emphasis on the need for speciation data and for minimizing the percentage of left-censored data so as to reduce uncertainties in exposure assessment. Finally the methodology for estimating the exposure of the general population and of population subgroups according to age (children, teenagers, adults, and the elderly) and gender, both at the national level and for each of the four main geographical areas of Italy, is presented.

Identifying irradiated oysters by luminescence techniques (TL & PSL).

This study shows an exhaustive comparison of different methods, based on luminescence techniques, to identify X-ray irradiated oysters at five different dose levels in the range 0.1-2 kGy and suggests a simple, fast and sequential routine analysis protocol. A total number of 50 oysters from North Sea, including 10 control samples, were analysed by using two photo-stimulated luminescence (PSL) methods (named A and B) and three thermoluminescence (TL) methods (named C, D and E), either on constituents or contaminating minerals from shells and intestines. Setting a lower threshold value T1 (1000 counts/60s) all of control specimens gave negative screening results while photo counts from irradiated samples were found to be higher than upper threshold T2 (4000 counts/60s), except at the lowest dose level 0.1 kGy for procedure A. All PSL calibrated analysis were successful and a sensitivity index was also determined to better classify obtained data according to the revised European Standard (EN 13751:2009). TL ratios, Glow 1 over second Glow 2, the latter after irradiating at 1 kGy and remeasuring the same minerals for each sample, showed values less than 0.1 related to untreated samples or higher than 0.1 for irradiated ones. Reported procedures were also tested over 60 days, longer than oyster shelf life confirming the applicability and feasibility of the proposed methods.

Chemometric data analysis application to Sparus aurata samples from two offshore farming plants along the Apulian (Italy) coastline.

The levels of polychlorodibenzo-p-dioxins (PCDDs), polychlorodibenzofurans (PCDFs), dioxin-like polychlorobiphenyls (DL-PCBs), non-dioxin-like polychlorobiphenyls (NDL-PCBs), and polybromodiphenyl ethers (PBDEs) in fish collected from two marine offshore farming plants were determined. Each sample was constituted by specimens of the same size collected at the same time in four different seasons along the farming year. The feeds given were of industrial origin and the plants were positioned in two different sites respectively exposed to different environmental characteristics. A chemometric approach was applied to interpret the subtle differences observed in fish body burdens across the three chemical groups taken into consideration. The approach consisted in a stepwise multivariate process including a hierarchical cluster analysis (CA) and a linear discriminant analysis (DA). The two main clusters determined by CA were subjected to the canonical DA, backward and forward selection procedures to select the best discriminative functions. A clear temporal and spatial discrimination was found among the samples. Across the three chemical groups, the monthly separation seemed to depend on the growth process and the main exposure was due to the feed. In addition, the two plants differed significantly from the environmental point of view and the most important discriminating group of chemicals were the NDL-PCBs. The approach resulted really effective in discriminating the subtle differences and in individuating suggestions to improve the quality of culturing conditions.

Treating meats with ionising radiations. An EPR approach to the reconstruction of the administered dose and its reliability.

Treatment of food with ionising radiations in order to increase its shelf-life is largely employed in many countries. Because of restrictions issued by different governments on the use of this technique, in addition to the identification of irradiated foodstuffs, it may be important to determine the radiation dose administered to the foodstuffs. An EPR based protocol to reconstruct the administered dose from samples of irradiated meats (rabbit, pork and duck) within an uncertainty of ± 25% is reported.