Nucleobase-Derived Nitrones: Synthesis and Antioxidant and Neuroprotective Activities in an In Vitro Model of Ischemia–Reperfusion

Herein, we report the synthesis, antioxidant, and neuroprotective properties of some nucleobase-derived nitrones named 9a–i. The neuroprotective properties of nitrones, 9a–i, were measured against an oxygen-glucose-deprivation in vitro ischemia model using human neuroblastoma SH-SY5Y cells. Our results indicate that nitrones, 9a–i, have better neuroprotective and antioxidant properties than α-phenyl-N-tert-butylnitrone (PBN) and are similar to N-acetyl-L-cysteine (NAC), a well-known antioxidant and neuroprotective agent. The nitrones with the highest neuroprotective capacity were those containing purine nucleobases (nitrones 9f, g, B = adenine, theophylline), followed by nitrones with pyrimidine nucleobases with H or F substituents at the C5 position (nitrones 9a, c). All of these possess EC₅₀ values in the range of 1–6 μM and maximal activities higher than 100%. However, the introduction of a methyl substituent (nitrone 9b, B = thymine) or hard halogen substituents such as Br and Cl (nitrones 9d, e, B = 5-Br and 5-Cl uracil, respectively) worsens the neuroprotective activity of the nitrone with uracil as the nucleobase (9a). The effects on overall metabolic cell capacity were confirmed by results on the high anti-necrotic (EC_50′s ≈ 2–4 μM) and antioxidant (EC_50′s ≈ 0.4–3.5 μM) activities of these compounds on superoxide radical production. In general, all tested nitrones were excellent inhibitors of superoxide radical production in cultured neuroblastoma cells, as well as potent hydroxyl radical scavengers that inhibit in vitro lipid peroxidation, particularly, 9c, f, g, presenting the highest lipoxygenase inhibitory activity among the tested nitrones. Finally, the introduction of two nitrone groups at 9a and 9d (bis-nitronas 9g, i) did not show better neuroprotective effects than their precursor mono-nitrones. These results led us to propose nitrones containing purine (9f, g) and pyrimidine (9a, c) nucleobases as potential therapeutic agents for the treatment of cerebral ischemia and/or neurodegenerative diseases, leading us to further investigate their effects using in vivo models of these pathologies.     

Isolation of antimicrobial compounds from aniseed and techno-economic feasibility report for industrial-scale application

This work evaluates the antimicrobial activity of the fractionated aqueous extract obtained from the aerial parts of aniseed. Fifteen fractions were collected by column chromatography using ethanol and water as mobile phases. All fractions were tested as potential antimicrobial agents against three common food-spoilage microorganisms: a yeast (Saccharomyces cerevisiae), a mold (Aspergillus awamori) and a bacterium (Lactobacillus delbrueckii subsp. bulgaricus). Fraction 2 exhibited the highest antimicrobial activity in both types of susceptibility test, and therefore, it was further studied for the identification of bioactive compounds. GC-MS analysis of Fraction 2 revealed the presence of threo-Anethole glycol. Based on laboratory-scale experiments, a process flowsheet for potential industrial-scale application is proposed combined with a techno-economic feasibility report. Fraction 2 with the detected compound threo-Anethole glycol showed promising results encouraging the attempts for industrial-scale application of antimicrobial compounds produced from aniseed.     

Engineering volatile thiol release in Saccharomyces cerevisiae for improved wine aroma

Volatile thiols, such as 4-mercapto-4-methylpentan-2-one (4MMP), 3-mercaptohexan-1-ol (3MH) and 3-mercaptohexyl acetate (3MHA), are among the most potent aroma compounds found in wine and can have a significant effect on wine quality and consumer preferences. At optimal concentrations in wine, these compounds impart flavours of passionfruit, grapefruit, gooseberry, blackcurrant, lychee, guava and box hedge. The enzymatic release of aromatic thiols from grape-derived, non-volatile cysteinylated precursors (Cys-4MMP and Cys-3MH) and the further modification thereof (conversion of 3MH into 3MHA) during fermentation, enhance the varietal characters of wines such as Sauvignon Blanc. Wine yeast strains have limited and varying capacities to produce aroma-enhancing thiols from their non-volatile counterparts in grape juice. Even under optimal fermentation conditions, the most efficient thiol-releasing Saccharomyces cerevisiae wine strain known realizes less than 5% of the thiol-related flavour potential of grape juice. The objective of this study was to develop a wine yeast able to unleash the untapped thiol aromas in grape juice during winemaking. To achieve this goal, the Escherichia coli tnaA gene, encoding a tryptophanase with strong cysteine-beta-lyase activity, was cloned and overexpressed in a commercial wine yeast strain under the control of the regulatory sequences of the yeast phosphoglycerate kinase I gene (PGK1). This modified strain expressing carbon-sulphur lyase activity released up to 25 times more 4MMP and 3MH in model ferments than the control host strain. Wines produced with the engineered strain displayed an intense passionfruit aroma. This yeast offers the potential to enhance the varietal aromas of wines to predetermined market specifications.     

Sensory characteristics of human milk: Association between mothers' diet and milk for bitter taste

It is unknown how consumption of bitter foods and beverages in the maternal diet influences sensory properties of fresh human milk. The aims of this study were (1) to determine the sensory characteristics of fresh human fore and hind milk, (2) to establish relationships between sensory properties and composition of fresh human milk, and (3) to assess the relationship between bitterness of the maternal diet and human milk. Twenty-two lactating mothers generated sensory terms to describe perception of their milk and received training on sensory attribute intensity rating. Mothers kept a 24-h food diary followed by a sensory self-assessment of their fore and hind milks. The odor of human fresh milk was described as neutral, creamy, and sweet, taste as sweet and bitter, and mouthfeel as thin, watery, smooth, and fatty. Sweetness was equivalent to 1.53 g of sucrose/100 mL and was not significantly different between fore and hind milk. Fore milk was significantly less creamy, less fatty, thinner, more watery, and lower in vanilla flavor intensity than hind milk. Carbohydrate content of human milk was positively correlated with sweetness and glutamic acid content with umami. The bitterness of the diet consumed 24 h before lactation was moderately positively correlated with bitterness of fore milk, but not hind milk. We conclude that the consumption of bitter foods may influence the bitterness of human fore milk, which may be another way for?breastfed children to learn to accept bitter vegetables and, hence, develop healthier food preferences.     

Antioxidant activity of the essential oil and various extracts of Nepeta flavida Hub.-Mor. from Turkey

This study was designed to examine the chemical composition and in vitro antioxidant activity of the essential oil and various extracts (hexane, dichloromethane and methanol sub-fractions) of Nepeta flavida. GC and GC–MS analyses of the essential oil resulted in the identification of 68 compounds, representing 96.4% of the oil; 1,8-cineole (38.9%) and linalool (25.1%) were the main components, comprising 64.0% of the total oil. The samples were subjected to a screening for their possible antioxidant activities by using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and β-carotene-linoleic acid assays. In the first case, the IC₅₀ value of the N. flavida essential oil was determined to be 42.8 ± 2.19 μg/ml. Among the extracts, the strongest activity was exhibited by the polar sub-fraction of the methanol extract with an IC₅₀ value of 63.2 ± 1.75 μg/ml. In the β-carotene-linoleic acid system, N. flavida essential oil exhibited 86.3% ± 1.69 inhibition against linoleic acid oxidation. Among the extracts prepared with various solvents, a correlation was observed between the polarity and antioxidant activity. The extracts exhibited the same activity pattern in this system the most active one is the polar sub-fraction, 79.7% ± 0.89. On the other hand, 1,8-cineole, a major compound of the essential oil, exhibited marked antioxidant activity in both systems, whereas the other compound, linalool, did not show any activity. The amount of total phenolics was highest in the polar and non-polar sub-fractions. Particularly, a positive correlation was observed between the total phenolic content and the antioxidant activity of the extracts. As estimated from the results, amounts of phenolic compounds were less in hexane and dichloromethane extracts than in the others. In conclusion, antioxidant potentials of polar and non-polar methanol sub-fractions could be attributed to their high phenolic contents. In both systems, antioxidant capacities of BHT, ascorbic acid, curcumin and α-tocopherol were also determined in parallel experiments.     

Light microscopy image de-noising using optimized LPA-ICI filter

Microscopic images are often corrupted by noise, where Poisson noise is one of the major types that can damage them. The local polynomial approximation (LPA) filter supported by the intersection confidence interval (ICI) rule was considered as an efficient filter for image de-noising. However, this filter depends on several parameters that affect its performance. In order to determine the optimal parameters, the present study employed the classic LPA-ICI (C-LPA-ICI) filter supported by optimization algorithms, namely the genetic algorithm (GA) and the particle swarm optimization (PSO) in the context of light microscopy imaging systems. Nevertheless, inclusion of the optimization algorithms increased the computational time. A novel automatic technique entitled “Standard Optimized LPA-ICI” (SO-LPA-ICI) is proposed. In this context, the average of the optimized ICI parameters was calculated, which obtained from both LPA-ICI-based GA (G-LPA-ICI) and LPA-ICI-based PSO (P-LPA-ICI). Thus, the proposed SO-LPA-ICI is included the optimal ICI parameters without optimization iterations. This procedure is proposed to speed up the optimized filter. A pool of 50 rats’ renal microscopic images is involved to test the proposed approach. A comparative study was conducted to compare the effectiveness of the four methods, namely C-LPA-ICI, G-LPA-ICI, P-LPA-ICI, and the SO-LPA-ICI for de-noising in the presence of Poisson noise. The experimental results established the outstanding performance of the SO-LPA-ICI in terms of the PSNR, MAE, and MSSIM with 28.27, 7.65, and 0.93 values, respectively. In addition, the proposed approach achieved fast de-noising compared to the G-LPA-ICI and the P-LPA-ICI.

     

Pepsin Promotes Activation of Epidermal Growth Factor Receptor and Downstream Oncogenic Pathways,at Slightly Acidic and Neutral pH,in Exposed Hypopharyngeal Cells

Pepsin refluxate is considered a risk factor for laryngopharyngeal carcinogenesis. Non-acidic pepsin was previously linked to an inflammatory and tumorigenic effect on laryngopharyngeal cells in vitro. Yet there is no clear evidence of the pepsin-effect on a specific oncogenic pathway and the importance of pH in this process. We hypothesized that less acidic pepsin triggers the activation of a specific oncogenic factor and related-signalling pathway. To explore the pepsin-effect in vitro, we performed intermittent exposure of 15 min, once per day, for a 5-day period, of human hypopharyngeal primary cells (HCs) to pepsin (1 mg/mL), at a weakly acidic pH of 5.0, a slightly acidic pH of 6.0, and a neutral pH of 7.0. We have documented that the extracellular environment at pH 6.0, and particularly pH 7.0, vs. pH 5.0, promotes the pepsin-effect on HCs, causing increased internalized pepsin and cell viability, a pronounced activation of EGFR accompanied by NF-κB and STAT3 activation, and a significant upregulation of EGFR, AKT1, mTOR, IL1β, TNF-α, RELA(p65), BCL-2, IL6 and STAT3. We herein provide new evidence of the pepsin-effect on oncogenic EGFR activation and its related-signaling pathway at neutral and slightly acidic pH in HCs, opening a window to further explore the prevention and therapeutic approach of laryngopharyngeal reflux disease.