Extra Virgin Olive Oil Polyphenols: Modulation of Cellular Pathways Related to Oxidant Species and Inflammation in Aging

The olive-oil-centered Mediterranean diet has been associated with extended life expectancy and a reduction in the risk of age-related degenerative diseases. Extra virgin olive oil (EVOO) itself has been proposed to promote a “successful aging”, being able to virtually modulate all the features of the aging process, because of its great monounsaturated fatty acids content and its minor bioactive compounds, the polyphenols above all. Polyphenols are mostly antioxidant and anti-inflammatory compounds, able to modulate abnormal cellular signaling induced by pro-inflammatory stimuli and oxidative stress, as that related to NF-E2-related factor 2 (Nrf-2) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), which have been identified as important modulators of age-related disorders and aging itself. This review summarizes existing literature about the interaction between EVOO polyphenols and NF-κB and Nrf-2 signaling pathways. Reported studies show the ability of EVOO phenolics, mainly hydroxytyrosol and tyrosol, to activate Nrf-2 signaling, inducing a cellular defense response and to prevent NF-κB activation, thus suppressing the induction of a pro-inflammatory phenotype. Literature data, although not exhaustive, indicate as a whole that EVOO polyphenols may significantly help to modulate the aging process, so tightly connected to oxidative stress and chronic inflammation

Modulation of LPS-induced nitric oxide production in intestinal cells by hydroxytyrosol and tyrosol metabolites:Insight into the mechanism of action

At intestinal level, after acute or chronic exposure to iNOS-derived NO, a toxic mechanism of action leads to inflammation and degenerative diseases. The aim of this study was to investigate the effect of glucuronide and sulfate metabolites of the extra virgin olive oil phenols tyrosol (Tyr) and hydroxytyrosol (HT), in comparison with their parent compounds, on the release of NO following exposure to a pro-inflammatory stimulus, the bacterial lipopolysaccharide (LPS). Human colon adenocarcinoma cells (Caco-2), differentiated as normal enterocytes, were treated with pathological concentrations of LPS, in order to stimulate iNOS pathway, which involves NF-ĸB activation through IĸBα phosphorylation and subsequent degradation induced by Akt or MAPKs. All the tested metabolites inhibited NO release induced by LPS, acting as inhibitors of iNOS expression, with an efficacy comparable to that of the parent compounds. HT and Tyr metabolites were effective in the inhibition of IĸBα degradation. No one of the compounds was able to inhibit Akt activation, whereas they modulated p38 and ERK1/2 MAPK. Obtained data show that HT and Tyr metabolites are able to prevent a pathological NO overproduction at intestinal level, where they concentrate, thus significantly contributing to the protective activity exerted by their parent compounds against inflammation

Probiotic lactobacilli attenuate oxysterols-induced alteration of intestinal epithelial cell monolayer permeability: Focus on tight junction modulation

Oxidative stress and inflammation lead by dietary oxidised lipids, as oxysterols, have been linked to the loss of intestinal barrier integrity, a crucial event in the initiation and progression of intestinal disorders. In the last decade, probiotic lactobacilli have emerged as an interesting tool to improve intestinal health, thanks to their antioxidant and anti-inflammatory properties. The aim of the present study was to evaluate the ability of two commercial probiotic strains of lactobacilli (Lactiplantibacillus plantarum 299v® (DMS 9843) and Lacticaseibacillus casei DG® (CNCMI-1572)), both as live bacteria and intracellular content, to attenuate the oxysterols-induced alteration of intestinal epithelial Caco-2 cell monolayer permeability. Our investigation was focused on the modulation of tight junctions (TJs) proteins, occludin, ZO-1 and JAM-A, in relation to redox-sensitive MAPK p38 activation. Obtained results provided evidence on the ability of the two probiotics to counteract the alteration of monolayer permeability and loss of TJs proteins, at least in part, through the modulation of p38 pathway. The protective action was exerted by live bacteria, whose adhesion to Caco-2 cells was not altered by oxysterols, and bacterial intracellular components equally able to interact with the signaling pathway

Glutamine Starvation Affects Cell Cycle, Oxidative Homeostasis and Metabolism in Colorectal Cancer Cells

Cancer cells adjust their metabolism to meet energy demands. In particular, glutamine addiction represents a distinctive feature of several types of tumors, including colorectal cancer. In this study, four colorectal cancer cell lines (Caco-2, HCT116, HT29 and SW480) were cultured with or without glutamine. The growth and proliferation rate, colony-forming capacity, apoptosis, cell cycle, redox homeostasis and metabolomic analysis were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test (MTT), flow cytometry, high-performance liquid chromatography and gas chromatography/mass spectrometry techniques. The results show that glutamine represents an important metabolite for cell growth and that its deprivation reduces the proliferation of colorectal cancer cells. Glutamine depletion induces cell death and cell cycle arrest in the GO/G1 phase by modulating energy metabolism, the amino acid content and antioxidant defenses. Moreover, the combined glutamine starvation with the glycolysis inhibitor 2-deoxy-D-glucose exerted a stronger cytotoxic effect. This study offers a strong rationale for targeting glutamine metabolism alone or in combination with glucose metabolism to achieve a therapeutic benefit in the treatment of colon cancer

Periodontal microbiota of Sardinian children: comparing 200-year-old samples to present-day ones

Introduction: The microrganisms of the human oral cavity include more than 700 species or phenotypes of bacteria. Some “diseases of civilization” are strictly correlated to changes in the microbiome following the food revolution that occurred after WWII. For that reason, a precise recognition of the microbiome profile before and after this period should be useful to determine the health-compatible model of icrobiome. The aim of this study was to compare the microbiome profiles (number of total cells, and pathogen types) of dental samples obtained from two distinct groups of children, a 200-year-old retrieved one and a present one. Methods: Two different groups of samples have been studied. The first group was a set of 50 recent subgingival plaque samples obtained from children of age 2-8 years, 14 males and 36 females. They were enrolled by the Department of Dental Disease Prevention (University of Cagliari, in Sardinia, Italy) during standard dental care procedures. None reported periodontal disease and none had been under antibiotic therapy during the previous 6 months. The second group was an old retrieved group that included 24 teeth from 6 different 6- to 8-year-old crania fragments; they were obtained from a 200-year-old charnel-house located in Villaputzu, a city close to Cagliari. Representative periodontal bacteria have been identified by a previously published real-time PCR procedure (Sokransky et al., 1998) in which P. gingivalis and T. forsythia (red complex), A. Original article 2/5 www.jpnim.com Open Access Journal of Pediatric and Neonatal Individualized Medicine • vol. 6 • n. 1 • 2017 Orrù • Contu • Casula • Demontis • Blus • Szmukler-Moncler • Serreli • Maserati • Steri • Fanos • Coghe • Denotti actinomycetemcomitans (green complex) and F. nucleatum (orange complex) were detected. In addition, the title of each pathogen was expressed as a percentage of the total bacteria (biofilm) in the sample. Results and discussion: The profile of periodontal microbiomes, between recent/ancient samples showed a significant difference relative to Sokransky’s red complex bacteria (p < 0.05). In all analyzed periodontal strains, the pathogenic bacteria P. gingivalis and T. forsythia showed the highest title in the recent group. Conclusions: Our hypothesis is that the transfer of “commensal-pathogen” as an absolute number on the oral biofilm might be linked to the distinct alimentary habits of the two populations. Some diet rich in reducing agents, such as processed meat-based foods, might be able to increase the average number of pathogen anaerobic bacteria in the oral microbiota. The outcome would be an increase of the oral systemic diseases reported with these pathogens. Our data suggest that the ancient Sardinian population was able to control the pathogen oral anaerobic biofilm by some diet rich in antioxidant compounds. Further investigations are required to focus on the genetic profile and the health status of this ancient population but it appears that molecular microbiology might be considered as the “time machine” in oral biology

Exploring the Antiviral Potential of Esters of Cinnamic Acids with Quercetin

Severe acute respiratory syndrome-related Coronavirus 2 (SARS-CoV-2) has infected more than 762 million people to date and has caused approximately 7 million deaths all around the world, involving more than 187 countries. Although currently available vaccines show high efficacy in preventing severe respiratory complications in infected patients, the high number of mutations in the S proteins of the current variants is responsible for the high level of immune evasion and transmissibility of the virus and the reduced effectiveness of acquired immunity. In this scenario, the development of safe and effective drugs of synthetic or natural origin to suppress viral replication and treat acute forms of COVID-19 remains a valid therapeutic challenge. Given the successful history of flavonoids-based drug discovery, we developed esters of substituted cinnamic acids with quercetin to evaluate their in vitro activity against a broad spectrum of Coronaviruses. Interestingly, two derivatives, the 3,4-methylenedioxy 6 and the ester of acid 7, have proved to be effective in reducing OC43-induced cytopathogenicity, showing interesting EC50s profiles. The ester of synaptic acid 7 in particular, which is not endowed with relevant cytotoxicity under any of the tested conditions, turned out to be active against OC43 and SARS-CoV-2, showing a promising EC50. Therefore, said compound was selected as the lead object of further analysis. When tested in a yield reduction, assay 7 produced a significant dose-dependent reduction in viral titer. However, the compound was not virucidal, as exposure to high concentrations of it did not affect viral infectivity, nor did it affect hCoV-OC43 penetration into pre-treated host cells. Additional studies on the action mechanism have suggested that our derivative may inhibit viral endocytosis by reducing viral attachment to host cells

Vitamin C Cytotoxicity and Its Effects in Redox Homeostasis and Energetic Metabolism in Papillary Thyroid Carcinoma Cell Lines.

High-dose of vitamin C (L-ascorbic acid, ascorbate) exhibits anti-tumoral effects, primarily mediated by pro-oxidant mechanisms. This cytotoxic effect is thought to affect the reciprocal crosstalk between redox balance and cell metabolism in different cancer types. Vitamin C also inhibits the growth of papillary thyroid carcinoma (PTC) cells, although the metabolic and redox effects remain to be fully understood. To shed light on these aspects, PTC-derived cell lines harboring the most common genetic alterations characterizing this tumor were used. Cell viability, apoptosis, and the metabolome were explored by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test (MTT), flow cytometry, and UHPLC/MS. Changes were observed in redox homeostasis, with increased reactive oxygen species (ROS) level and perturbation in antioxidants and electron carriers, leading to cell death by both apoptosis and necrosis. The oxidative stress contributed to the metabolic alterations in both glycolysis and TCA cycle. Our results confirm the pro-oxidant effect of vitamin C as relevant in triggering the cytotoxicity in PTC cells and suggest that inhibition of glycolysis and alteration of TCA cycle via NAD+ depletion can play an important role in this mechanism of PTC cancer cell death

DIAGNOSI DI LABORATORIO PER INFEZIONE DA TREPONEMA PALLIDUM, VALUTAZIONE DELLE RESISTENZE AI MACROLIDI TRAMITE SEQUENZIAMENTO DEL GENE 23S rRNA.

Obiettivi: L’infezione sostenuta da Treponema pallidum (Tp) presenta, secondo i dati riportati dall’European Centre for Disease Control, ECDC e dell’OMS risultano allarmanti, sia per l’aumento di incidenza dei casi di sifilide, sia per la comparsa di ceppi resistenti o multiresistenti agli antibiotici. Lo scopo del lavoro è quello di utilizzare metodiche diagnostiche molecolari altamente specifiche che in tempi rapidi permettano di rilevare (anche a basse concentrazioni) T. pallidum e contemporaneamente rilevare le mutazioni genomiche a carico del gene 23S rRNA responsabili della resistenza ai macrolidi. Materiali e Metodi: Da campioni provenienti da lesioni cutanee è stato estratto e poi amplificato il DNA tramite nested real time PCR, utilizzando primer specifici disegnati su una porzione del gene 23s rRNA. Il controllo positivo era rappresentato da un frammento di DNA a titolo noto contenente l’amplicone di PCR (606 bp). I campioni risultati positivi sono stati sequenziati con metodo capillare e comparati con la sequenza di riferimento GenBank NR_076531. Risultati: La sensibilita’ del metodo e’ risultata pari a 100 genomi Tp/PCR, sono state rilevate 2 mutazioni principali nel target genico: (i) A2059G responsabile per resistenza alla Eritromicina e Azitromicina e (ii) A2058G per la Spiramicina. Conclusioni: La sperimentazione eseguita dimostra l’affidabilità del metodo molecolare di cui il clinico può trarre vantaggio per un approccio terapeutico personalizzato e qualora il farmaco di elezione, la penicillina, non possa essere somministrato. 1. European Centre for Disease Prevention and Control. Annual epidemiological report 2016. Syphilis. Stockholm: ECDC; 2016. 2. Baseline report on global sexually transmitted infection surveillance 2012. WHO Publication date: 2013 - ISBN: 978 92 4 150589 5 3. Stamm LV, Bergen HL. A point mutation associated with bacterial macrolide resistance is present in both 23S rRNA genes of an erythromycin-resistant Treponema pallidum clinical isolate. Antimicrob Agents Chemother 2000;44:806-7. 4. Centers for Disease Control and Prevention. Primary and secondary syphilis—United States, 2003–2004. MMWR Morb Mortal Wkly Rep 2006;55:269-73

DIAGNOSI DI LABORATORIO PER INFEZIONE DA NOCARDIA SPP. TRAMITE PCR REAL TIME

Obiettivi: Diversi microrganismi appartenenti al genere Nocardia spp. possono causare infezioni eterogenee nell’uomo quali: malattie respiratorie, ascessi cerebrali, lesioni meningee, infezioni cutanee, articolari e oculari e le specie più frequentemente isolate sono N. asteroides e N. brasiliensis. La prognosi è favorevole, tranne nei casi di nocardiosi disseminata nei pazienti immunocompromessi. La diagnosi di laboratorio tradizionale a volte non è semplice e in genere si basa sull’osservazione microscopica del campione e sull’esame colturale. Lo scopo del lavoro è stato quello di utilizzare metodiche molecolari, quale la PCR real time, per arrivare a una diagnosi di laboratorio più veloce e specifica. Materiali e metodi: Il DNA proveniente da diversi campioni, biopsie o tamponi cutanei prelevati da pazienti con sospetta Nocardiosi, e’ stato sottoposto ad amplificazione tramite PCR real time (light-Cycler Roche). La PCR amplificava un segmento di 228 bp lungo il gene rrs specifico per Nocardia spp; nei casi dubbi o dove è richiesta l’identificazione di specie, e’ stato possibile sequenziare l’amplicone con metodo capillare. La positività è stata valutata tramite l’analisi della curva di melting (Tm=91 °C) o tramite gel di agarosio. I risultati sono stati confrontati con il metodo colturale. Come controllo positivo è stato utilizzato e un ceppo di collezione di N. seriolae (ATCC 43993). Risultati: La procedura utilizzata è risultata più veloce (4 ore) e sensibile rispetto al metodo colturale tradizionale, il limite di rilevamento della PCR è risultato essere pari a (500 copie DNA/PCR). Conclusioni: La procedura descritta potrebbe dare dei benefici nella diagnosi di laboratorio per Nocardia spp. In particolare nei casi di campioni negativi alla coltura e positivi al microscopio per forme alcool resistenti. 1. Brown-Elliott BA, Brown JM, Conville PS, et al. Clinical and laboratory features of the nocardia spp. based on current molecular taxonomy. Clinical Microbiology Reviews 2006;19:259-82. 2. Wehrhahn MC, Xiao M, Kong F, et al. A PCR-based intergenic spacer region-capillary gel electrophoresis typing method for identification and subtyping of Nocardia species. J Clin Microbiol 2012;50:3478-84

Role of Dietary Polyphenols in the Activity and Expression of Nitric Oxide Synthases: A Review

Nitric oxide (NO) plays several key roles in the functionality of an organism, and it is usually released in numerous organs and tissues. There are mainly three isoforms of the enzyme that produce NO starting from the metabolism of arginine, namely endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), and neuronal nitric oxide synthase (nNOS). The expression and activity of these isoforms depends on the activation/deactivation of different signaling pathways at an intracellular level following different physiological and pathological stimuli. Compounds of natural origin such as polyphenols, which are obtainable through diet, have been widely studied in recent years in in vivo and in vitro investigations for their ability to induce or inhibit NO release, depending on the tissue. In this review, we aim to disclose the scientific evidence relating to the activity of the main dietary polyphenols in the modulation of the intracellular pathways involved in the expression and/or functionality of the NOS isoforms