Autochthonous white grape pomaces as bioactive source for functional jams

Seeds and skins from grape pomaces of Pecorello and Mantonico cv underwent extraction (ultrasound–assisted or maceration), in order to obtain added-value ingredients for the production of a functional pear jam. The antioxidant features of the extracts were tested by in vitro colorimetric assays. Among seeds, Mantonico by maceration (MSC) showed the best results, as well as Mantonico by ultrasound-assisted extraction (MBs) among skin extracts. The selected extracts were fully characterised by NMR and MS techniques, confirming the presence of many polyphenols, flavonoids and tannins among others. Pectin was then derivatised by the grafting procedure with the active molecules of MBs and MSC. The latter produced the best antioxidant polymer also without toxicity evaluated using Caco-2 cells and was used for the jam preparation. The functional pear jam showed improved antioxidant performances in comparison with its non-functional counterparts as well as its maintenance over time (15 days).     

Fluorimetrische Bestimmung von Erythromycin-Rückständen in Lebensmitteln tierischer Herkunft nach Derivatisierung mit FMOC und HPLC-Trennung

A high-performance liquid Chromatographic (HPLC) method for the determination of the macrolide antibiotic erythromycin in eggs, milk, swine muscle, kidney and liver was developed. The drug was extracted from the matrix with acetonitrile. The raw extract was purified by liquid-liquid partitioning and fractionation by reversed-phase HPLC for additional cleanup. Erythromycin was reacted in a pre-column procedure with 9-fluorenylmethylchloroformate (FMOC) to enable fluorimetric detection (excitation 255 nm, emission 315 nm) after isocratic separation on an analytical RP-18 HPLC column. Mean recoveries ranged from 99% at fortification levels of 0.03 mg/kg in egg to 38% at 0.06 mg/kg in liver. With the exception of liver all detection limits were below 0.01 mg/kg and precision for all other matrices and tested concentrations (0.015–0.09 mg/kg) better than 20% (coefficient of variation).     

Release and absorption of carotenes from processed carrots (Daucus carota) using in vitro digestion coupled with a Caco-2 cell trans-well culture model

The release and absorption (bioavailability) of carotenoids is a prerequisite for their nutritional impact. This can be strongly affected by the processing conditions used to prepare the food matrix that contains them. To determine the effect of processing on carotenoid bioavailability, homogenized, raw, blanched and cooked carrots were exposed to an in vitro gastric and intestinal digestion model. Final digest samples were placed onto a Caco-2 cell trans-well monolayer culture to mimic intestinal absorption. The results show that the cooked carrot puree consisting of primarily single plant cell particles had the highest release of carotenes, followed by blanched consisting primarily of plant cell clusters and raw carrot puree consisting of larger plant cell clusters. Absorption through the Caco-2 cell layer was the highest from the digesta of cooked carrot puree followed by the digesta of blanched carrot puree. This study demonstrates that thermal processing and/or mechanical homogenization to disrupt plant cell wall matrix enhances the in vitro bioavailability of carotenes from carrots.     

Glycolaldehyde‐modified β‐lactoglobulin AGEs are unable to stimulate inflammatory signaling pathways in RAGE‐expressing human cell lines

Scope: Advanced glycation endproducts (AGEs) are suspected to stimulate inflammatory signaling pathways in target tissues via activation of the receptor for AGEs. Endotoxins are generally recognized as potential contamination of AGE preparations and stimulate biological actions that are very similar as or identical to those induced by AGEs. Methods and results: In our study, we used glycolaldehyde‐modified β‐lactoglobulin preparations as model AGEs and employed two methods to remove endotoxin using either affinity columns or extraction with Triton X‐114 (TX‐114). Affinity column‐purified AGEs retained their ability to stimulate inflammatory signaling as measured by mRNA expression of inflammatory cytokines in the human lung epithelial cell line Beas2b. However, glycolaldehyde‐modified AGEs purified by extraction with TX‐114 did not show any stimulation of mRNA expression of inflammatory cytokines. The presence of a cell stimulating endotoxin‐like activity was demonstrated in the detergent phase after extraction with TX‐114, thus indicating that not AGEs but a lipophilic contamination was responsible for the stimulation of inflammatory signaling. Conclusion: Our results demonstrate that glycolaldehyde‐modified AGEs are unable to induce inflammatory signaling in receptor for AGE‐expressing cells. The observed cell‐activating activity can be ascribed to an endotoxin‐like lipophilic contamination present in AGE preparations and affinity column purification was insufficient to remove this contamination.     

Hazard assessment of edible oil refining: Formation of flammable mixtures in storage tanks

This work presents a methodology for the assessment of hazards due to the formation of flammable mixtures in edible oil refineries, with particular focus on storage tanks. Safety issues in these plants are due to the presence of residual volatile flammable solvents deriving from the extraction processes, which may accumulate in the storage tanks and generate flammable vapour mixtures. The methodology, based on the application of a thermodynamic model for the estimation of vapour phase composition in oil storage tanks as a function of operating conditions (i.e. temperature and residual solvent content), allows the identification of potential hazards due to formation of flammable mixtures inside the tanks. The model was first validated using available experimental data and then extended to case-studies representative of state-of-the-art industrial storage applications. The results of the case-studies were then used to calculate specific hazard indexes, providing a quick tool for preliminary assessment of hazards due to the formation of flammable mixtures in edible oil storage plants.     

Micro-RNA and Proteomic Profiles of Plasma-Derived Exosomes from Irradiated Mice Reveal Molecular Changes Preventing Apoptosis in Neonatal Cerebellum

Cell communication via exosomes is capable of influencing cell fate in stress situations such as exposure to ionizing radiation. In vitro and in vivo studies have shown that exosomes might play a role in out-of-target radiation effects by carrying molecular signaling mediators of radiation damage, as well as opposite protective functions resulting in resistance to radiotherapy. However, a global understanding of exosomes and their radiation-induced regulation, especially within the context of an intact mammalian organism, has been lacking. In this in vivo study, we demonstrate that, compared to sham-irradiated (SI) mice, a distinct pattern of proteins and miRNAs is found packaged into circulating plasma exosomes after whole-body and partial-body irradiation (WBI and PBI) with 2 Gy X-rays. A high number of deregulated proteins (59% of WBI and 67% of PBI) was found in the exosomes of irradiated mice. In total, 57 and 13 miRNAs were deregulated in WBI and PBI groups, respectively, suggesting that the miRNA cargo is influenced by the tissue volume exposed to radiation. In addition, five miRNAs (miR-99b-3p, miR-200a-3p, miR-200a, miR-182-5p, miR-182) were commonly overexpressed in the exosomes from the WBI and PBI groups. In this study, particular emphasis was also given to the determination of the in vivo effect of exosome transfer by intracranial injection in the highly radiosensitive neonatal cerebellum at postnatal day 3. In accordance with a major overall anti-apoptotic function of the commonly deregulated miRNAs, here, we report that exosomes from the plasma of irradiated mice, especially in the case of WBI, prevent radiation-induced apoptosis, thus holding promise for exosome-based future therapeutic applications against radiation injury.     

Nitroreductase-Mediated Release of Inhibitors of Lysine-Specific Demethylase 1 (LSD1) from Prodrugs in Transfected Acute Myeloid Leukaemia Cells

Lysine-specific demethylase 1 (LSD1) has evolved as a promising therapeutic target for cancer treatment, especially in acute myeloid leukaemia (AML). To approach the challenge of site-specific LSD1 inhibition, we developed an enzyme-prodrug system with the bacterial nitroreductase NfsB (NTR) that was expressed in the virally transfected AML cell line THP1-NTR⁺. The cellular activity of the NTR was proven with a new luminescent NTR probe. We synthesised a diverse set of nitroaromatic prodrugs that by design do not affect LSD1 and are reduced by the NTR to release an active LSD1 inhibitor. The emerging side products were differentially analysed using negative controls, thereby revealing cytotoxic effects. The 2-nitroimidazolyl prodrug of a potent LSD1 inhibitor emerged as one of the best prodrug candidates with a pronounced selectivity window between wild-type and transfected THP1 cells. Our prodrugs are selectively activated and release the LSD1 inhibitor locally, proving their suitability for future targeting approaches.     

Lipidomic Approaches to Study HDL Metabolism in Patients with Central Obesity Diagnosed with Metabolic Syndrome

The metabolic syndrome (MetS) is a cluster of cardiovascular risk factors characterised by central obesity, atherogenic dyslipidaemia, and changes in the circulating lipidome; the underlying mechanisms that lead to this lipid remodelling have only been partially elucidated. This study used an integrated “omics” approach (untargeted whole serum lipidomics, targeted proteomics, and lipoprotein lipidomics) to study lipoprotein remodelling and HDL composition in subjects with central obesity diagnosed with MetS (vs. controls). Compared with healthy subjects, MetS patients showed higher free fatty acids, diglycerides, phosphatidylcholines, and triglycerides, particularly those enriched in products of de novo lipogenesis. On the other hand, the “lysophosphatidylcholines to phosphatidylcholines” and “cholesteryl ester to free cholesterol” ratios were reduced, pointing to a lower activity of lecithin cholesterol acyltransferase (LCAT) in MetS; LCAT activity (directly measured and predicted by lipidomic ratios) was positively correlated with high-density lipoprotein cholesterol (HDL-C) and negatively correlated with body mass index (BMI) and insulin resistance. Moreover, many phosphatidylcholines and sphingomyelins were significantly lower in the HDL of MetS patients and strongly correlated with BMI and clinical metabolic parameters. These results suggest that MetS is associated with an impairment of phospholipid metabolism in HDL, partially led by LCAT, and associated with obesity and underlying insulin resistance. This study proposes a candidate strategy to use integrated “omics” approaches to gain mechanistic insights into lipoprotein remodelling, thus deepening the knowledge regarding the molecular basis of the association between MetS and atherosclerosis.     

A statistical model for the failure of glass plates due to nickel sulfide inclusions

Nickel Sulfide (NiS) inclusions can provoke the rupture of thermally treated glass due to a phase transformation with volume increase that stresses the surrounding glass. Starting from a Pareto statistics for the population of inclusion sizes, from an assumed kinetics of the phase transformation, a micro-mechanically motivated model provides the statistical characterization of the probability of spontaneous failure of glass during lifetime. A distinction based upon the composition of NiS is used to discuss the effects of the heat soak test (HST), where glass remains at high temperature for a certain time to speed-up the phase transformation and destroy those elements with critical inclusions. Three functions à la Weibull for the probability of spontaneous rupture during lifetime are theoretically derived for the case of no HST, short HST, and long HST. In particular, the probability of collapse for long HSTs depends upon the holding time in the oven. An explanatory example shows the potentiality of the model for optimizing the HST parameters toward a target probability of failure, but experimental campaigns are needed for a proper calibration.     

Quantitative Assessment of Affinity Selection Performance by Using DNA-Encoded Chemical Libraries

DNA-encoded chemical libraries are often used for the discovery of ligands against protein targets of interest. These large collections of DNA-barcoded chemical compounds are typically screened by using affinity capture methodologies followed by PCR amplification and DNA sequencing procedures. However, the performance of individual steps in the selection procedures has been scarcely investigated, so far. Herein, the quantitative analysis of selection experiments, by using three ligands with different affinity to carbonic anhydrase IX as model compounds, is described. In the first set of experiments, quantitative PCR (qPCR) procedures are used to evaluate the recovery and selectivity for affinity capture procedures performed on different solid-phase supports, which are commonly used for library screening. In the second step, both qPCR and analysis of DNA sequencing results are used to assess the recovery and selectivity of individual carbonic anhydrase IX ligands in a library, containing 360 000 compounds. Collectively, this study reveals that selection procedures can be efficient for ligands with sub-micromolar dissociation constants to the target protein of interest, but also that selection performance dramatically drops if 10⁴ copies per library member are used as the input.