Reply to Rayman et al. Comment on "Ambra et al. Could Selenium Supplementation Prevent COVID-19? A Comprehensive Review of Available Studies. Molecules 2023, 28, 4130".

We thank the Authors of the Comment (AC) [...].

The Fate of Chlorophylls in Alkali-Treated Green Table Olives: A Review.

This paper reviews the current knowledge regarding modifications to chlorophylls during the processing of green table olives treated with alkali. Particular attention is given to the pheophytinization reactions (substitution of Mg2+ by 2H+ in the chlorophyll chromophore group) that can take place because of pH and/or temperature changes and the possible sequential substitution of the 2H+ with Cu2+ within the chlorophyll porphyrin ring. These reactions may have a direct impact on the commercial value of olive productions as some naturally forming Cu-chlorophylls complexes (i) are identical to strictly forbidden colorants for table olives (E141) and (ii) have been identified as responsible for the unwelcome appearance of the so-called green staining alteration (characterized by bluish-green zones distributed over the olive skin of the drupes).

Could Selenium Supplementation Prevent COVID-19? A Comprehensive Review of Available Studies.

The purpose of this review is to systematically examine the scientific evidence investigating selenium's relationship with COVID-19, aiming to support, or refute, the growing hypothesis that supplementation could prevent COVID-19 etiopathogenesis. In fact, immediately after the beginning of the COVID-19 pandemic, several speculative reviews suggested that selenium supplementation in the general population could act as a silver bullet to limit or even prevent the disease. Instead, a deep reading of the scientific reports on selenium and COVID-19 that are available to date supports neither the specific role of selenium in COVID-19 severity, nor the role of its supplementation in the prevention disease onset, nor its etiology.

The Fate of the Chlorophyll Derivatives in Olives Preserved and/or Packaged in Presence of Exogenous Copper.

Chlorophyll pigments are thought to be responsible for the highly appreciated green color of unfermented Castelvetrano-style table olives, but no studies have considered the effects of a controlled addition of copper during storage or packaging at the industrial level. For this purpose, chlorophyll derivatives were analyzed in Nocellara cultivar table olives debittered industrially using the Castelvetrano method, via means of HPLC and MS analyses, following the addition of copper in alkaline brines stored at 4 °C for 3 months in 220 L barrels, and during the subsequent storage in acid brines in commercial 400 g packages at 4 °C for up to 18 months. The presence of copper in storage or in packaging brines both contributed significantly to maintaining the green color of the olives, which was associated with a specific pattern of chlorophyll derivatives, as evidenced by principal component analysis. Notably, re-greening was rapidly achievable also for olives that had yellowed for 18 months at a copper concentration below the limit of EU legislation. Finally, by means of PCA, we also demonstrated that a short-term thermic treatment can work as an accelerated predictive tool in determining the fate of chlorophyll derivatives.

A Review of the Effects of Olive Oil-Cooking on Phenolic Compounds.

The fate of phenolic compounds in oil and food during cooking vary according to the type of cooking. From a nutritional point of view, reviews largely suggest a preference for using extra-virgin olive oil at a low temperature for a short time, except for frying and microwaving, for which there appears to be no significant advantages compared to olive oil. However, due to the poorly pertinent use of terminology, the different protocols adopted in studies aimed at the same objective, the different type and quality of oils used in experiments, and the different quality and quantity of PC present in the used oils and in the studied vegetables, the evidence available is mainly contradictory. This review tries to reanalyse the main experimental reports on the fate, accessibility and bioavailability of phenolic compounds in cooking oils and cooked vegetables, by considering different cooking techniques and types of oil and foods, and distinguishing experimental findings obtained using oil alone from those in combination with vegetables. The re-analysis indicates that incomplete and contradictory observations have been published in the last few years and suggests that further research is necessary to clarify the impact of cooking techniques on the phenolic compounds in oil and vegetables during cooking, especially when considering their nutritional properties.

The Role of Bioactive Phenolic Compounds on the Impact of Beer on Health.

This review reports recent knowledge on the role of ingredients (barley, hop and yeasts), including genetic factors, on the final yield of phenolic compounds in beer, and how these molecules generally affect resulting beer attributes, focusing mainly on new attempts at the enrichment of beer phenols, with fruits or cereals other than barley. An entire section is dedicated to health-related effects, analyzing the degree up to which studies, investigating phenols-related health effects of beer, have appropriately considered the contribution of alcohol (pure or spirits) intake. For such purpose, we searched Scopus.com for any kind of experimental model (in vitro, animal, human observational or intervention) using beer and considering phenols. Overall, data reported so far support the existence of the somehow additive or synergistic effects of phenols and ethanol present in beer. However, findings are inconclusive and thus deserve further animal and human studies.

MAPK signalling pathway in cancers: Olive products as cancer preventive and therapeutic agents.

The mitogen-activated protein kinases (MAPKs) are fundamental in inflammation and cancer control, through the crosstalk between the redox regulated nuclear factor E2-related factor 2 (Nrf2) and nuclear factor-kB (NFκB) gene expression. MAPKs regulate various cellular activities involved in cancer progression, including proliferation, apoptosis and immune escape and blockade of upstream kinases is a current therapeutic strategy. However, these therapies are associated with some adverse effects and with the paradoxical activation of the MAPKs pathway. In the context of cancer prevention and treatment, it has been suggested that dietary factors are able to modulate cancer initiation and progression by interacting with the MAPKs. Within these dietary factors, virgin olive oil (VOO) is of particular interest due to its content in squalene, already used as drug delivery system in cancer therapy. The aim of this review is to discuss the studies pointing to the effects of olive-derived foodstuff and nutraceuticals on MAPKs signalling cascades. The reviewed experimental studies suggest that the stress-activated JNK and p38 MAPKs could be targets of olive-derived nutraceuticals. The latter, including phytochemicals from olive cultivation and processing wastes, could be adjuvants in chemotherapies, whereas VOO could be considered a "natural delivery system" of bioactive phytochemicals due to its high content in squalene.

Involvement of miR-190b in Xbp1 mRNA Splicing upon Tocotrienol Treatment.

We previously demonstrated that apoptosis induced by tocotrienols (γ and δT3) in HeLa cells is preceded by Ca2+ release from the endoplasmic reticulum. This event is eventually followed by the induction of specific calcium-dependent signals, leading to the expression and activation of the gene encoding for the IRE1α protein and, in turn, to the alternative splicing of the pro-apoptotic protein sXbp1 and other molecules involved in the unfolded protein response, the core pathway coping with EndoR stress. Here, we showed that treatment with T3s induces the expression of a specific set of miRNAs in HeLa cells. Data interrogation based on the intersection of this set of miRNAs with a set of genes previously differentially expressed after γT3 treatment provided a few miRNA candidates to be the effectors of EndoR-stress-induced apoptosis. To identify the best candidate to act as the effector of the Xbp1-mediated apoptotic response to γT3, we performed in silico analysis based on the evaluation of the highest ∆ in Gibbs energy of different mRNA-miRNA-Argonaute (AGO) protein complexes. The involvement of the best candidate identified in silico, miR-190b, in Xbp1 splicing was confirmed in vitro using T3-treated cells pre-incubated with the specific miRNA inhibitor, providing a preliminary indication of its role as an effector of EndoR-stress-induced apoptosis.

α-Tocopherol, ß-carotene, lutein, squalene and secoiridoids in seven monocultivar Italian extra-virgin olive oils.

Fatty acids and bioactive lipophilic and hydrophilic compounds (tocopherols, ß-carotene, lutein, squalene, total polyphenols and secoiridoids) in monocultivar Italian extra-virgin olive oil (EVOO) samples produced from the Leccino cultivar and six other yet uncharacterised cultivars (Rustica, Carpinetana, Dritta, Gentile di Chieti and Intosso) were analysed, also taking into account the effect of the type of decanter used for the oil extraction. Significant differences among cultivars were found for α-tocopherol and squalene, but not for carotenoids. Among phenols, cultivars significantly influenced oleuropein and ligstroside aglycones contents, but not those of the dialdehydic form of decarboxymethyl elenolic acid linked neither to tyrosol nor hydroxytyrosol. As previously reported, phenol is quantitatively affected by the type of decanter used for oil extraction. Accordingly, we found that the two-phases decanter preserved in the oil 1.5 times more phenolic compounds as compared with three-phases, whereas it did not influence the amount of lipophilic compounds. Moreover, our data statistically support the finding that type of decanter affects phenols also qualitatively. In fact, the two-phases decanter preferably preserved more the hydroxytyrosol secoiridoid derivatives than the tyrosol ones. Our results from one hand characterise for the first time oils previously unreported, from the other give some new indications on the relative role of factors relevant for the achievement of biologically active extra-virgin oil, i.e. the cultivar and technological ones.

Development of a Sensitive, Easy and High-Throughput Compliant Protocol for Maize and Soybean DNA Extraction and Quantitation Using a Plant-Specific Universal Taqman Minor Groove Binder Probe.

We report the optimization of a high-throughput, compliant DNA extraction method that uses standard format 96-well plates and a commercial automated DNA purification system (ABI PRISM® 6100 Nucleic Acid PrepStation). The procedure was set up for maize and soybean, the most common GMO crops and the main ingredients of several foodstuffs, and compared with an EU-validated CTAB-based method. Optimization of the DNA extraction was achieved by applying self-prepared buffers (for DNA extraction, binding, and washing) on the PrepStation loaded with proprietary glass-fiber-coated purification plates. Quantification of extracted DNA was performed by real-time PCR using previously reported endogenous soybean lectin and maize starch synthase genes and a novel plant-specific universal TaqMan MGB probe that targets the 18S rRNA multiple copy gene. Using serial dilutions of both maize and soybean genomic DNAs, we show low PCR sensitivity and efficiency for the official TransPrep DNA extraction protocol compared to the CTAB-based one. On the other hand, using serial dilutions of a standard reference plasmid containing a 137 bp sequence cloned from the 18S rRNA plant-specific ribosomal gene, we demonstrate the high PCR sensitivity and efficiency of the optimized DNA extraction protocol setup with self-prepared buffers. The limits of detection and quantification of the 18S rDNA reiteration were consistent with the calculated values, supporting the suitability of the DNA extraction procedure for high-throughput analyses of large populations and small amounts of tissue.

Unraveling the Effects of Carotenoids Accumulation in Human Papillary Thyroid Carcinoma.

Among the thyroid cancers, papillary thyroid cancer (PTC) accounts for 90% of the cases. In addition to the necessity to identify new targets for PTC treatment, early diagnosis and management are highly demanded. Previous data indicated that the multivariate statistical analysis of the Raman spectra allows the discrimination of healthy tissues from PTC ones; this is characterized by bands typical of carotenoids. Here, we dissected the molecular effects of carotenoid accumulation in PTC patients by analyzing whether they were required to provide increased retinoic acid (RA) synthesis and signaling and/or to sustain antioxidant functions. HPLC analysis revealed the lack of a significant difference in the overall content of carotenoids. For this reason, we wondered whether the carotenoid accumulation in PTC patients could be related to vitamin A derivative retinoic acid (RA) biosynthesis and, consequently, the RA-related pathway activation. The transcriptomic analysis performed using a dedicated PCR array revealed a significant downregulation of RA-related pathways in PTCs, suggesting that the carotenoid accumulation in PTC could be related to a lower metabolic conversion into RA compared to that of healthy tissues. In addition, the gene expression profile of 474 PTC cases previously published in the framework of the Cancer Genome Atlas (TGCA) project was examined by hierarchical clustering and heatmap analyses. This metanalysis study indicated that the RA-related pathways resulted in being significantly downregulated in PTCs and being associated with the follicular variant of PTC (FV-PTC). To assess whether the possible fate of the carotenoids accumulated in PTCs is associated with the oxidative stress response, the expression of enzymes involved in ROS scavenging was checked. An increased oxidative stress status and a reduced antioxidant defense response were observed in PTCs compared to matched healthy thyroids; this was possibly associated with the prooxidant effects of high levels of carotenoids. Finally, the DepMap datasets were used to profile the levels of 225 metabolites in 12 thyroid cancer cell lines. The results obtained suggested that the high carotenoid content in PTCs correlates with tryptophan metabolism. This pilot provided novel possible markers and possible therapeutic targets for PTC diagnosis and therapy. For the future, a larger study including a higher number of PTC patients will be necessary to further validate the molecular data reported here.

Oxidative stress activation of miR-125b is part of the molecular switch for Hailey-Hailey disease manifestation.

Hailey-Hailey disease (HHD) is an autosomal dominant disorder characterized by suprabasal cutaneous cell separation (acantholysis) leading to the development of erosive and oozing skin lesion. Micro RNAs (miRNAs) are endogenous post-transcriptional modulators of gene expression with critical functions in health and disease. Here, we evaluated whether the expression of specific miRNAs may play a role in the pathogenesis of HHD. Here, we report that miRNAs are expressed in a non-random manner in Hailey-Hailey patients. miR-125b appeared a promising candidate for playing a role in HHD manifestation. Both Notch1 and p63 are part of a regulatory signalling whose function is essential for the control of keratinocyte proliferation and differentiation and of note, the expression of both Notch1 and p63 is downregulated in HHD-derived keratinocytes. We found that both Notch1 and p63 expression is strongly suppressed by miR-125b expression. Additionally, we found that miR-125b expression is increased by an oxidative stress-dependent mechanism. Our data suggest that oxidative stress-mediated induction of miR-125b plays a specific role in the pathogenesis of HHD by regulating the expression of factors playing an important role in keratinocyte proliferation and differentiation.

Covid-19 and diet: an evaluation of information available on internet in Italy.

BACKGROUND AND AIM: During Coronavirus disease 2019 (Covid-19) Italy experienced deep lockdown with closure of almost all activities, with the exception for food shops and few others. During this quarantine, alteration of dietary habits occurred, due to the changes in food availability and mandatory meal house-consumption. The change in dietary habits could somehow be directed by freely accessible information available on internet.  Thus, s we evaluated the type and the scientific quality of the information provided to the Italian population by the most visible web sites found on google, relating diet in Covid-19 time. METHODS: we systematically perform a reliability and content analysis of Italian language websites using google as search engine and a combinations of diet/nutrition/Covid-19 as search terms Results: 88 webpages were included in the study, the great part representing newspaper webpages. Institutional webpages, despite having high scientific quality, did not have high visibility. Generally, all the other webpages reported information of medium-quality scientific level. CONCLUSION: finding appropriate solutions to redirect the population's attention to more reliable and accessible information is mandatory.

Human Metabolites of Hamaforton™ (Hamamelis virginiana L. Extract) Modulates Fibroblast Extracellular Matrix Components in Response to UV-A Irradiation.

Hamamelis virginiana L. a rich source of both condensed and hydrolyzable tannins, utilized to treat dermatological disorders. Since no experimental and clinical data is available for its use as oral formulation in skin related disorders, the purpose of this study was to investigate the effects of Hamaforton™ (Hamamelis virginiana extract) metabolites on gene dysregulation induced by ultraviolet A radiation in cultured human dermal fibroblasts. A combination of in vivo and ex vivo experimental designs has been exploited in order to take into account the polyphenol metabolic transformation that occurs in humans. 12 healthy volunteers received either a capsule of Hamaforton™ or a placebo in a randomized, blinded crossover trial. After Hamaforton™ ingestion, the kinetic of appearance of galloyl derivatives was measured in plasma. Then, in the ex vivo experiment, the serum isolated after supplementation was used as a source of Hamaforton™ metabolites to enrich the culture medium of dermal fibroblasts exposed to ultraviolet A radiation. Three different gallic acid metabolites (4-O-methyl gallic acid, 4-O-methyl gallic acid sulphate and trimethyl gallic acid glucuronide) were identified in volunteer plasma. While, ultraviolet A irradiation of dermal fibroblasts affected the expression of extracellular matrix genes, the presence of Hamaforton™ metabolites in the culture media did not affect the expression of most of those genes. However, the activation of the expression of 10 different genes involved in repair processes for the maintenance of skin integrity, suggest that the metabolites can play a role in damage recovery. To our knowledge, this is the first study that demonstrates the bioavailability of Hamaforton™ phenolic compounds, and the effects of its metabolites on cultured dermal fibroblast response to ultraviolet A irradiation.

Red wine metabolites modulate NF-kappaB, activator protein-1 and cAMP response element-binding proteins in human endothelial cells.

We have studied the effect of human serum, collected after red wine consumption (RWS), on TNF-alpha-dependent activation of transcription factors (NF-kappaB, activator protein-1 (AP-1) and cAMP response element-binding proteins) and on the expression of selected genes involved in cell adhesion or fibrinolysis processes in human primary endothelial cells (human umbilical vein endothelial cells (HUVEC)). Our data indicate that RWS containing RW metabolites, isolated after 40 min from an acute consume of wine (5 ml/kg body weight), induces nuclear translocation of NF-kappaB and AP-1 in the absence of any further stimulus. On the other hand, TNF-alpha treatment in the presence of RWS is associated with a delay in transcription factor activation and to a negative modulation on the expression of specific genes. Moreover, RWS stimulates c-jun binding to the tissue-type plasminogen activator cAMP responsive element consensus site modulating the expression of the specific gene downstream. These results confirm that RW metabolites affect the activity of different transcription factors playing an important preconditioning role in the modulation of the inflammatory pathway in endothelial cells. This is the first report on the effects of a complex food matrix, on the molecular mechanisms associated with inflammatory response in HUVEC cultured in condition that reproduces the physiological environment occurring in vivo.

A novel mechanism of natural vitamin E tocotrienol activity: involvement of ERbeta signal transduction.

Vitamin E is a generic term used to indicate all tocopherol (TOC) and tocotrienol (TT) derivates. In the last few years, several papers have shown that a TT-rich fraction (TTRF) extracted from palm oil inhibits proliferation and induces apoptosis in a large number of cancer cells. However, the molecular mechanism(s) involved in TT action is still unclear. In the present study, we proposed for the first time a novel mechanism for TT activity that involves estrogen receptor (ER) signaling. In silico simulations and in vitro binding analyses indicated a high affinity of TTs for ERbeta but not for ERalpha. In addition, in ERbeta-containing MDA-MB-231 breast cancer cells, we demonstrated that TTs increase the ERbeta translocation into the nucleus, which in turn activates estrogen-responsive genes (MIC-1, EGR-1 and cathepsin D), as demonstrated by cell preincubation with the ER inhibitor ICI-182,780. Finally, we observed that TT treatment is associated with alteration of cell morphology, DNA fragmentation, and caspase-3 activation. Altogether, these experiments elucidated the molecular mechanism underling gamma- and delta-TT effects.

Antiangiogenic Effect of Alkaloids.

Alkaloids are among the natural phytochemicals contained in functional foods and nutraceuticals and have been suggested for the prevention and/or management of oxidative stress and inflammation-mediated diseases. In this review, we aimed to describe the effects of alkaloids in angiogenesis, the process playing a crucial role in tumor growth and invasion, whereby new vessels form. Antiangiogenic compounds including herbal ingredients, nonherbal alkaloids, and microRNAs can be used for the control and treatment of cancers. Several lines of evidence indicate that alkaloid-rich plants have several interesting features that effectively inhibit angiogenesis. In this review, we present valuable data on commonly used alkaloid substances as potential angiogenic inhibitors. Different herbal and nonherbal ingredients, introduced as antiangiogenesis agents, and their role in angiogenesis-dependent diseases are reviewed. Studies indicate that angiogenesis suppression is exerted through several mechanisms; however, further investigations are required to elucidate their precise molecular and cellular mechanisms, as well as potential side effects.

S. Giovanni Varieties (Pyrus communis L.): Antioxidant Properties and Phytochemical Characteristics.

In order to evaluate and identify the antioxidant properties and the phytochemical characteristics, as well as the role of the genetic background and the different fruit parts in the definition of fruit quality, we characterized the existing germplasm through nuclear simple sequence repeats (SSRs) and evaluated the genetic divergence between ancient S. Giovanni varieties (Pyrus communis L.) and different types of grafting in relation to some variables of fruit quality. On the peel and pulp of pear varieties, the contents of flavanols, flavonols, and hydroxycinnamic acids as well as total antioxidant capacity were assessed. Their role in plant defences was confirmed by a significantly higher amount in the peel (206.66 ± 44.27, 48.45 ± 13.65, and 31.11 ± 11.94 mg/100 g, respectively) of S. Giovanni pears than in the pulp (71.45 ± 34.94, 1.62 ± 0.83, and 17.05 ± 5.04 mg/100 g, respectively). Data obtained using capillary analysis of SSR indicate unequivocally that the four samples of San Giovanni varieties can be divided into 3 different genetic groups. Cultivar and the different parts of the fruit can exert an action in the definition of the quality product. The use of local varieties and ecotypes can be considered a valid tool to improve food quality and at the same time to support local agrobiodiversity.

Redox Role of Lactobacillus casei Shirota Against the Cellular Damage Induced by 2,2'-Azobis (2-Amidinopropane) Dihydrochloride-Induced Oxidative and Inflammatory Stress in Enterocytes-Like Epithelial Cells.

In western societies where most of the day is spent in the postprandial state, the existence of oxidative and inflammatory stress conditions makes postprandial stress an important factor involved in the development of cardiovascular risk factors. A large body of evidence have been accumulated on the anti-inflammatory effects of probiotics, but no information is available on the mechanisms through which intestinal microbiota modulates redox unbalance associated with inflammatory stress. Here, we aimed to investigate the ability of Lactobacillus casei Shirota (LS) to induce an antioxidant response to counteract oxidative and inflammatory stress in an in vitro model of enterocytes. Our results show that pretreatment of enterocytes with LS prevents membrane barrier disruption and cellular reactive oxygen species (ROS) accumulation inside the cells, modulates the expression of the gastro-intestinal glutathione peroxidase (GPX2) antioxidant enzyme, and reduces p65 phosphorylation, supporting the involvement of the Nfr2 and nuclear factor kappa B pathways in the activation of antioxidant cellular defenses by probiotics. These results suggest, for the first time, a redox mechanism by LS in protecting intestinal cells from AAPH-induced oxidative and inflammatory stress.

Genetic Polymorphisms and Zinc Status: Implications for Supplementation in Metabolic Diseases.

BACKGROUND: Zinc is an essential component for all living organisms, representing the second most abundant trace element, after iron. This element is widely distributed in the tissues of a human body where it is involved in normal growth, reproduction and several biological functions including immunity, energy metabolism and antioxidant processes. Because of its essential role, zinc levels in the human body must remain constant, independently of dietary intake fluctuations. The homeostasis of zinc is a well-regulated cellular process and has been reported to be chiefly mediated by the expression and activity of zinc-binding proteins such as metallothioneins and zinc transporters. Genes encoding for these proteins are subjected to genetic variants. METHODS: We performed a multi-database electronic search to provide an overview on the relationship between specific polymorphisms (SNP) of genes encoding for metallothioneins and zinc transporters and their relationship with zinc status, immune function and some non-communicable diseases. RESULTS: A number of SNP are implicated in a range of metabolic disease. Some SNP may affect the impact of zinc supplementation on immune function, diabetes, and obesity. CONCLUSION: New studies are needed to clarify the interaction between individual genetic profile and zinc status. Moreover, there is a need for a better interaction between the scientific bodies and health professionals to allow better dietary and behavioural recommendations to promote human health, with particular concern to elderly people.