Chemoenzymatic Synthesis of Glycopeptides to Explore the Role of Mucin†1 Glycosylation in Cell Adhesion.

Post-translational modifications affect protein biology under physiological and pathological conditions. Efficient methods for the preparation of peptides and proteins carrying defined, homogeneous modifications are fundamental tools for investigating these functions. In the case of mucin 1 (MUC1), an altered glycosylation pattern is observed in carcinogenesis. To better understand the role of MUC1 glycosylation in the interactions and adhesion of cancer cells, we prepared a panel of homogeneously O-glycosylated MUC1 peptides by using a quantitative chemoenzymatic approach. Cell-adhesion experiments with MCF-7 cancer cells on surfaces carrying up to six differently glycosylated MUC1 peptides demonstrated that different glycans have a significant impact on adhesion. This finding suggests a distinct role for MUC1 glycosylation patterns in cancer cell migration and/or invasion. To decipher the molecular mechanism for the observed adhesion, we investigated the conformation of the glycosylated MUC1 peptides by NMR spectroscopy. These experiments revealed only minor differences in peptide structure, therefore clearly relating the adhesion behaviour to the type and number of glycans linked to MUC1.

Visual comet scoring revisited: a guide to scoring comet assay slides and obtaining reliable results.

Measurement of DNA migration in the comet assay can be done by image analysis or visual scoring. The latter accounts for 20%-25% of the published comet assay results. Here we assess the intra- and inter-investigator variability in visual scoring of comets. We include three training sets of comet images, which can be used as reference for researchers who wish to use visual scoring of comets. Investigators in 11 different laboratories scored the comet images using a five-class scoring system. There is inter-investigator variation in the three training sets of comets (i.e. coefficient of variation (CV) = 9.7%, 19.8%, and 15.2% in training sets I-III, respectively). However, there is also a positive correlation of inter-investigator scoring in the three training sets (r = 0.60). Overall, 36% of the variation is attributed to inter-investigator variation and 64% stems from intra-investigator variation in scoring between comets (i.e. the comets in training sets I-III look slightly different and this gives rise to heterogeneity in scoring). Intra-investigator variation in scoring was also assessed by repeated analysis of the training sets by the same investigator. There was larger variation when the training sets were scored over a period of six months (CV = 5.9%-9.6%) as compared to 1 week (CV = 1.3%-6.1%). A subsequent study revealed a high inter-investigator variation when premade slides, prepared in a central laboratory, were stained and scored by investigators in different laboratories (CV = 105% and 18%-20% in premade slides with comets from unexposed and hydrogen peroxide-exposed cells, respectively). The results indicate that further standardization of visual scoring is desirable. Nevertheless, the analysis demonstrates that visual scoring is a reliable way of analysing DNA migration in comets.

Inter-laboratory variation in measurement of DNA damage by the alkaline comet assay in the hCOMET ring trial.

The comet assay is a simple and versatile method for measurement of DNA damage in eukaryotic cells. More specifically, the assay detects DNA migration from agarose gel-embedded nucleoids, which depends on assay conditions and the level of DNA damage. Certain steps in the comet assay procedure have substantial impact on the magnitude of DNA migration (e.g. electric potential and time of electrophoresis). Inter-laboratory variation in DNA migration levels occurs because there is no agreement on optimal assay conditions or suitable assay controls. The purpose of the hCOMET ring trial was to test potassium bromate (KBrO3) as a positive control for the formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay. To this end, participating laboratories used semi-standardized protocols for cell culture (i.e. cell culture, KBrO3 exposure, and cryopreservation of cells) and comet assay procedures, whereas the data acquisition was not standardized (i.e. staining of comets and image analysis). Segregation of the total variation into partial standard deviation (SD) in % Tail DNA units indicates the importance of cell culture procedures (SD = 10.9), comet assay procedures (SD = 12.3), staining (SD = 7.9) and image analysis (SD = 0.5) on the overall inter-laboratory variation of DNA migration (SD = 18.2). Future studies should assess sources of variation in each of these steps. On the positive side, the hCOMET ring trial demonstrates that KBrO3 is a robust positive control for the Fpg-modified comet assay. In conclusion, the hCOMET ring trial has demonstrated a high reproducibility of detecting genotoxic effects by the comet assay, but inter-laboratory variation of DNA migration levels is a concern.

DNA strand break levels in cryopreserved mononuclear blood cell lines measured by the alkaline comet assay: results from the hCOMET ring trial.

The comet assay is widely used in biomonitoring studies for the analysis of DNA damage in leukocytes and peripheral blood mononuclear cells. Rather than processing blood samples directly, it can be desirable to cryopreserve whole blood or isolated cells for later analysis by the comet assay. However, this creates concern about artificial accumulation of DNA damage during cryopreservation. In this study, 10 laboratories used standardized cryopreservation and thawing procedures of monocytic (THP-1) or lymphocytic (TK6) cells. Samples were cryopreserved in small aliquots in 50% foetal bovine serum, 40% cell culture medium, and 10% dimethyl sulphoxide. Subsequently, cryopreserved samples were analysed by the standard comet assay on three occasions over a 3-year period. Levels of DNA strand breaks in THP-1 cells were increased (four laboratories), unaltered (four laboratories), or decreased (two laboratories) by long-term storage. Pooled analysis indicates only a modest positive association between storage time and levels of DNA strand breaks in THP-1 cells (0.37% Tail DNA per year, 95% confidence interval: -0.05, 0.78). In contrast, DNA strand break levels were not increased by cryopreservation in TK6 cells. There was inter-laboratory variation in levels of DNA strand breaks in THP-1 cells (SD = 3.7% Tail DNA) and TK6 reference sample cells (SD = 9.4% Tail DNA), whereas the intra-laboratory residual variation was substantially smaller (i.e. SD = 0.4%-2.2% Tail DNA in laboratories with the smallest and largest variation). In conclusion, the study shows that accumulation of DNA strand breaks in cryopreserved mononuclear blood cell lines is not a matter of concern.

Long-term cryopreservation of potassium bromate positive assay controls for measurement of oxidatively damaged DNA by the Fpg-modified comet assay: results from the hCOMET ring trial.

The formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay is widely used for the measurement of oxidatively generated damage to DNA. However, there has not been a recommended long-term positive control for this version of the comet assay. We have investigated potassium bromate as a positive control for the Fpg-modified comet assay because it generates many Fpg-sensitive sites with a little concurrent generation of DNA strand breaks. Eight laboratories used the same procedure for the treatment of monocytic THP-1 cells with potassium bromate (0, 0.5, 1.5, and 4.5 mM) and subsequent cryopreservation in a freezing medium consisting of 50% foetal bovine serum, 40% RPMI-1640 medium, and 10% dimethyl sulphoxide. The samples were analysed by the Fpg-modified comet assay three times over a 3-year period. All laboratories obtained a positive concentration-response relationship in cryopreserved samples (linear regression coefficients ranging from 0.79 to 0.99). However, there was a wide difference in the levels of Fpg-sensitive sites between the laboratory with the lowest (4.2% Tail DNA) and highest (74% Tail DNA) values in THP-1 cells after exposure to 4.5 mM KBrO3. In an attempt to assess sources of inter-laboratory variation in Fpg-sensitive sites, comet images from one experiment in each laboratory were forwarded to a central laboratory for visual scoring. There was high consistency between measurements of %Tail DNA values in each laboratory and the visual score of the same comets done in the central laboratory (r = 0.98, P < 0.001, linear regression). In conclusion, the results show that potassium bromate is a suitable positive comet assay control.

Olive phenols preserve lamin B1 expression reducing cGAS/STING/NFκB-mediated SASP in ionizing radiation-induced senescence.

Senescence occurs upon critical telomere shortening, or following DNA damage, oncogenic activation, hypoxia and oxidative stress, overall referred to stress-induced premature senescence (SIPS). In response to DNA damage, senescent cells release cytoplasmic chromatin fragments (CCFs), and express an altered secretome, the senescence-associated secretory phenotype (SASP), which contributes to generate a pro-inflammatory and pro-tumoral extracellular milieu. Polyphenols have gained significant attention owing to their anti-inflammatory and anti-tumour activities. Here, we studied the effect of oleuropein aglycone (OLE) and hydroxytyrosol (HT) on DNA damage, CCF appearance and SASP in a model of irradiation-induced senescence. Neonatal human dermal fibroblasts (NHDFs) were γ-irradiated and incubated with OLE, 5 µM and HT, 1 µM. Cell growth and senescence-associated (SA)-ß-Gal-staining were used as senescence markers. DNA damage was evaluated by Comet assay, lamin B1 expression, release of CCFs, cyclic GMP-AMP Synthase (cGAS) activation. IL-6, IL-8, MCP-1 and RANTES were measured by ELISA assay. Our results showed that OLE and HT exerted a protective effect on 8 Gy irradiation-induced senescence, preserving lamin B1 expression and reducing cGAS/STING/NFκB-mediated SASP. The ability of OLE and HT to mitigate DNA damage, senescence status and the related SASP in normal cells can be exploited to improve the efficacy and safety of cancer radiotherapy.

Colon fibroblasts from Pirc rats (F344/NTac-Apcam1137 ) exhibit a proliferative and inflammatory phenotype that could support early stages of colon carcinogenesis.

The role of fibroblast APC mutation in carcinogenesis is not clear. Apc+/- colon fibroblasts have been previously characterized: however, little is known about their behavior at very early-stage of colon carcinogenesis. We cultured colon mucosa fibroblasts (PCF, Apc+/- ) of Pirc rats (F344/NTac-Apcam1137 ) at an early stage of tumorigenesis, in absence of preneoplastic lesions, and of age-matched wt (WCF): DNA damage levels, inflammatory phenotype and the expression of known markers of CAFs were analyzed. The latter were also assessed by microarray analysis on colon normal mucosa of Pirc and wt animals. PCF exhibited higher proliferative rates (P < .001) and delayed replicative senescence onset (P < .05) compared to WCF, along with a lower level of oxidative DNA damage (P < .05). Furthermore, a constitutively higher expression of COX-2 and sensitivity to inflammatory stimuli was found in PCF compared to WCF (P < .05), accompanied by higher invasive capability (P < .05) and presence of cytoplasmic chromatin foci (cytoplasmic chromatin foci, P < .05). However, they neither expressed CAFs markers (α-SMA, IL-6) nor responded to CAFs activating stimuli (TGF-ß). Accordingly, CAFs markers and activating stimuli resulted down-regulated in Pirc normal mucosa compared to wt, whereas DNA damage response and tolerance pathways were overexpressed. These data show for the first time that a proliferative and inflammatory phenotype characterizes Apc+/- colon fibroblasts since very early stages of colon tumorigenesis, and indicate a role of Apc mutation in driving fibroblast phenotypic alterations that could support the establishment of a protumorigenic environment. Early pharmacological targeting of these dysfunctions might impact on tumor prevention in FAP patients.

Characterization of substituted piperazines able to reverse MDR in Escherichia coli strains overexpressing resistance-nodulation-cell division (RND) efflux pumps.

BACKGROUND: MDR in bacteria is threatening to public health. Overexpression of efflux pumps is an important cause of MDR. The co-administration of antimicrobial drugs and efflux pump inhibitors (EPIs) is a promising approach to address the problem of MDR. OBJECTIVES: To identify new putative EPIs and to characterize their mechanisms of action. METHODS: The effects of four selected piperazine derivatives on resistance-nodulation-cell division (RND) pumps was evaluated in Escherichia coli strains overexpressing or not expressing RND pumps by assays aimed at evaluating antibiotic potentiation, membrane functionality, ethidium bromide accumulation and AcrB expression. The cytotoxicity of selected piperazines towards primary cultures of human dermal fibroblasts was also investigated. RESULTS: Four molecules enhanced levofloxacin activity against strains overexpressing RND efflux pumps (AcrAB-TolC and AcrEF-TolC), but not against RND pump-deficient strains. They had little effects on membrane potential. Molecule 4 decreased, whereas the other three increased, membrane permeability compared with untreated control cells. The four molecules showed differences in the specificity of interaction with RND efflux pumps, by inactivating the transport of one or more antibiotics, and in the levels of ethidium bromide accumulation and of acrB expression inhibition. CONCLUSIONS: Piperazine derivatives are good candidates as inhibitors of RND efflux pumps. They decreased the activity of RND pumps by mixed mechanisms of action. Small structural differences among the molecules can be critical in defining their behaviour.

Parvovirus B19 induces cellular senescence in human dermal fibroblasts: putative role in systemic sclerosis-associated fibrosis.

OBJECTIVE: Emerging evidence demonstrates that excessive accumulation of senescent cells is associated with some chronic diseases and suggests a pathogenic role of cellular senescence in fibrotic processes, such as that occurring in ageing or in SSc. Recently we demonstrated that parvovirus B19 (B19V) activates normal human dermal fibroblasts and induces expression of different profibrotic/pro-inflammatory genes. This observation prompted us to investigate whether it is also able to induce fibroblast senescence as a potential pathogenetic mechanism in B19V-induced fibrosis. METHODS: Primary cultures of fibroblasts were infected with B19V and analysed for the acquisition of senescence markers, such as morphological modifications, senescence-associated ß-galactosidase (SA-ß-gal) activity, DNA damage response and expression of senescence-associated secretory phenotype (SASP)-related factors. RESULTS: We demonstrated that B19V-infected fibroblasts develop typical senescence features such as enlarged and flat-shaped morphology and SA-ß-gal activity similar to that observed in SSc skin fibroblasts. They also developed an SASP-like phenotype characterized by mRNA expression and release of some pro-inflammatory cytokines, along with activation of the transcription factor nuclear factor κB. Moreover, we observed B19V-induced DNA damage with the comet assay: a subpopulation of fibroblasts from B19V-infected cultures showed a significantly higher level of DNA strand breaks and oxidative damage compared with mock-infected cells. An increased level and nuclear localization of γH2AX, a hallmark of DNA damage response, were also found. CONCLUSIONS: B19V-induced senescence and production of SASP-like factors in normal dermal fibroblasts could represent a new pathogenic mechanism of non-productive B19V infection, which may have a role in the fibrotic process.

Collection and storage of human white blood cells for analysis of DNA damage and repair activity using the comet assay in molecular epidemiology studies.

DNA damage and repair activity are often assessed in blood samples from humans in different types of molecular epidemiology studies. However, it is not always feasible to analyse the samples on the day of collection without any type of storage. For instance, certain studies use repeated sampling of cells from the same subject or samples from different subjects collected at different time-points, and it is desirable to analyse all these samples in the same comet assay experiment. In addition, flawless comet assay analyses on frozen samples open up the possibility of using this technique on biobank material. In this article we discuss the use of cryopreserved peripheral blood mononuclear cells (PBMCs), buffy coat (BC) and whole blood (WB) for analysis of DNA damage and repair using the comet assay. The published literature and the authors' experiences indicate that various types of blood samples can be cryopreserved with only a minor effect on the basal level of DNA damage. There is evidence to suggest that WB and PBMCs can be cryopreserved for several years without much effect on the level of DNA damage. However, care should be taken when cryopreserving WB and BCs. It is possible to use either fresh or frozen samples of blood cells, but results from fresh and frozen cells should not be used in the same dataset. The article outlines detailed protocols for the cryopreservation of PBMCs, BCs and WB samples.

Effects of an Olive By-Product Called Pâté on Cardiovascular Risk Factors.

BACKGROUND: The nutraceutical effects of Olea europaea L. products are mainly due to phenolic compounds. During olive milling, most of the total phenols remain in the process by-products. AIM: We aimed to evaluate the effects of a specific by-product of olive oil called "pâté" (OlP) administered as tablets, on cardiovascular and metabolic risk factors. METHODS: The study was a crossover trial with 2 intervention periods. Nineteen participants (mean age: 38 years) took 4 tablets/day of either olive pâté (corresponding to 30 mg/day of hydroxytyrosol) or placebo for 2 months followed by a 2-month washout and another 2 months of crossed over treatment. RESULTS: After the intervention with pâté, participants showed a statistically significant reduction in plasma levels of total cholesterol (-10.8 mg/dL), LDL cholesterol (-10.8 mg/dL) and urea (-4.1 mg/dL), and a significant increase in calcium levels (+0.3 mg/dL). Leukocyte response to exogenous oxidative stress was significantly reduced (-12.8%) and levels of the antioxidant transcription factor Nrf-2 increased by 88.9%. Plasma levels of the pro-inflammatory protein MCP-1 were significantly reduced (-9.0 pg/mL). CONCLUSION: In conclusion, the intake of OlP showed positive effects on several cardiovascular risk factors, demonstrating the nutraceutical potential of a widely available but, to date, underestimated olive oil by-product.

Synthesis of functionalised organochalcogenides and in vitro evaluation of their antioxidant activity.

Differently substituted ß-hydroxy- and ß-amino dialkyl and alkyl-aryl tellurides and selenides have been prepared through ring-opening reactions of epoxides and aziridines with selenium- or tellurium-centered nucleophiles. The antioxidant properties and the cytotoxicity of such compounds have been investigated on normal human dermal fibroblasts. Most of the studied compounds exhibited a low cytotoxicity and a number of them proved to be non-toxic, not showing any effect on cell viability even at the highest concentration used (100 µM). The obtained results showed a significant antioxidant potential of the selected organotellurium compounds, particularly evident under conditions of exogenously induced oxidative stress. The antioxidant activity of selenium-containing analogues of active tellurides has also been evaluated on cells, highlighting that the replacement of Se with Te brought about a significant increase in the peroxidase activity.

Enhanced Vasculogenic Capacity Induced by 5-Fluorouracil Chemoresistance in a Gastric Cancer Cell Line.

Chemotherapy is still widely used as a coadjutant in gastric cancer when surgery is not possible or in presence of metastasis. During tumor evolution, gatekeeper mutations provide a selective growth advantage to a subpopulation of cancer cells that become resistant to chemotherapy. When this phenomenon happens, patients experience tumor recurrence and treatment failure. Even if many chemoresistance mechanisms are known, such as expression of ATP-binding cassette (ABC) transporters, aldehyde dehydrogenase (ALDH1) activity and activation of peculiar intracellular signaling pathways, a common and universal marker for chemoresistant cancer cells has not been identified yet. In this study we subjected the gastric cancer cell line AGS to chronic exposure of 5-fluorouracil, cisplatin or paclitaxel, thus selecting cell subpopulations showing resistance to the different drugs. Such cells showed biological changes; among them, we observed that the acquired chemoresistance to 5-fluorouracil induced an endothelial-like phenotype and increased the capacity to form vessel-like structures. We identified the upregulation of thymidine phosphorylase (TYMP), which is one of the most commonly reported mutated genes leading to 5-fluorouracil resistance, as the cause of such enhanced vasculogenic ability.

Potassium bromate as positive assay control for the Fpg-modified comet assay.

The comet assay is a popular assay in biomonitoring studies. DNA strand breaks (or unspecific DNA lesions) are measured using the standard comet assay. Oxidative stress-generated DNA lesions can be measured by employing DNA repair enzymes to recognise oxidatively damaged DNA. Unfortunately, there has been a tendency to fail to report results from assay controls (or maybe even not to employ assay controls). We believe this might have been due to uncertainty as to what really constitutes a positive control. It should go without saying that a biomonitoring study cannot have a positive control group as it is unethical to expose healthy humans to DNA damaging (and thus potentially carcinogenic) agents. However, it is possible to include assay controls in the analysis (here meant as a cryopreserved sample of cells i.e. included in each experiment as a reference sample). In the present report we tested potassium bromate (KBrO3) as a positive comet assay control for the formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay. Ten laboratories used the same procedure for treatment of monocytic THP-1 cells with KBrO3 (0.5, 1.5 and 4.5 mM for 1 h at 37°C) and subsequent cryopreservation. Results from one laboratory were excluded in the statistical analysis because of technical issues in the Fpg-modified comet assay. All other laboratories found a concentration-response relationship in cryopreserved samples (regression coefficients from 0.80 to 0.98), although with different slopes ranging from 1.25 to 11.9 Fpg-sensitive sites (%DNA in tail) per 1 mM KBrO3. Our results demonstrate that KBrO3 is a suitable positive comet assay control.

miR-210-3p mediates metabolic adaptation and sustains DNA damage repair of resistant colon cancer cells to treatment with 5-fluorouracil.

Chemoresistance is the primary cause of chemotherapy failure. Compelling evidence shows that micro RNAs (miRNAs) contribute to reprogram cancer cells toward a resistant phenotype. We investigate the role of miRNAs in the response to acute treatment with 5-FU in colon cancer-resistant cells. We performed a global gene expression profile for the entire miRNA genome and found a change in the expression of four miRNAs following acute treatment with 5-FU. Among them, we focused on miR-210-3p, previously described as a key regulator of DNA damage repair mechanisms and mitochondrial metabolism. We show that miR-210-3p downregulation enables resistant cells to counteract the toxic effect of the drug increasing the expression of RAD-52 protein, responsible for DNA damage repair. Moreover, miR-210-3p downregulation enhances oxidative phosphorylation (OXPHOS), increasing the expression levels of succinate dehydrogenase subunits D, decreasing intracellular succinate levels and inhibiting HIF-1α expression. Altogether, these adaptations lead to increased cells survival following drug exposure. These evidence suggest that miR-210-3p downregulation following 5-FU sustains DNA damage repair and metabolic adaptation to counteract drug treatment.

Opioid response in paediatric cancer patients and the Val158Met polymorphism of the human catechol-O-methyltransferase (COMT) gene: an Italian study on 87 cancer children and a systematic review.

BACKGROUND: Genetic polymorphisms in genes involved in pain modulation have been reported to be associated to opioid efficacy and safety in different clinical settings. METHODS: The association between COMT Val158Met polymorphism (rs4680) and the inter-individual differences in the response to opioid analgesic therapy was investigated in a cohort of 87 Italian paediatric patients receiving opioids for cancer pain (STOP Pain study). Furthermore, a systematic review of the association between opioid response in cancer patients and the COMT polymorphism was performed in accordance with the Cochrane Handbook and the Prisma Statement. RESULTS: In the 87 paediatric patients, pain intensity (total time needed to reach the lowest possible level) was significantly higher for G/G than A/G and A/A carriers (p-value = 0.042). In the 60 patients treated only with morphine, the mean of total dose to reach the same pain intensity was significantly higher for G/G than A/G and A/A carriers (p-value = 0.010). Systematic review identified five studies on adults, reporting that opioid dose (mg after 24 h of treatment from the first pain measurement) was higher for G/G compared to A/G and A/A carriers. CONCLUSIONS: Present research suggests that the A allele in COMT polymorphism could be a marker of opioid sensitivity in paediatric cancer patients (STOP Pain), as well as in adults (Systematic Review), indicating that the polymorphism impact could be not age-dependent in the cancer pain context. TRIAL REGISTRATION: Registration number: CRD42017057831 .

Serpin A1 and the modulation of type I collagen turnover: Effect of the C-terminal peptide 409-418 (SA1-III) in human dermal fibroblasts.

The pharmacological modulation of collagen turnover is a strategy potentially useful in different skin conditions. The serine protease inhibitor Serpin A1 and portions of its C-terminal region have been investigated as collagen modulators. To clarify the mechanisms by which the C-terminal 409-418 peptide SA1-III increases extracellular type I collagen levels, to compare its activities range with that of the originator molecule Serpin A1, and to evaluate its efficacy in primary cultures from adult and aged human subjects. The different forms of type I collagen were analyzed by means of western blot in cell lysates and cell-conditioned media of primary human dermal fibroblasts obtained from subjects of different ages. Gelatin zymography was used to investigate the degrading enzymes. Cell viability and in vitro wound healing tests were used to evaluate cell proliferation. The SA1-III peptide increased extracellular collagen levels by reducing degradation, with no effect on cellular biosynthesis or cell proliferation mechanisms. A reduced level of MMP-2 and MMP-9 was also found in cell media upon peptide treatment. No peptide effect was detected on inflammatory mediators gene expression in resting and LPS-stimulated fibroblasts, or in the wound healing test. The SA1-III peptide is a good collagen modulator candidate, protecting collagen against degradation without detectable actions on biosynthesis, acting at reasonably low concentrations, and non-interfering with cell proliferation. It is effective in primary fibroblasts from young and aged subjects. These effects can prove useful in pathological and physiological skin conditions in which collagen degradation is excessive compared to the synthetic capacity.

A nutrigenomics approach for the study of anti-aging interventions: olive oil phenols and the modulation of gene and microRNA expression profiles in mouse brain.

PURPOSE: Middle-aged C57Bl/6J mice fed for 6 months with extra-virgin olive oil rich in phenols (H-EVOO, phenol dose/day: 6 mg/kg) showed cognitive and motor improvement compared to controls fed the same olive oil deprived of phenolics (L-EVOO). The aim of the present study was to evaluate whether these behavioral modifications were associated with changes in gene and miRNA expression in the brain. METHODS: Two brain areas involved in cognitive and motor processes were chosen: cortex and cerebellum. Gene and miRNA profiling were analyzed by microarray and correlated with performance in behavioral tests. RESULTS: After 6 months, most of the gene expression changes were restricted to the cerebral cortex. The genes modulated by aging were mainly down-regulated, and the treatment with H-EVOO was associated with a significant up-regulation of genes compared to L-EVOO. Among those, we found genes previously associated with synaptic plasticity and with motor and cognitive behavior, such as Notch1, BMPs, NGFR, GLP1R and CRTC3. The agrin pathway was also significantly modulated. miRNAs were mostly up-regulated in old L-EVOO animals compared to young. However, H-EVOO-fed mice cortex displayed miRNA expression profiles similar to those observed in young mice. Sixty-three miRNAs, out of 1203 analyzed, were significantly down-regulated compared to the L-EVOO group; among them, we found miRNAs whose predicted target genes were up-regulated by the treatment, such as mir-484, mir-27, mir-137, mir-30, mir-34 and mir-124. CONCLUSIONS: We are among the first to report that a dietary intervention starting from middle age with food rich in phenols can modulate at the central level the expression of genes and miRNAs involved in neuronal function and synaptic plasticity, along with cognitive, motor and emotional behavior.

Modulation of the Senescence-Associated Inflammatory Phenotype in Human Fibroblasts by Olive Phenols.

Senescent cells display an increase in the secretion of growth factors, inflammatory cytokines and proteolytic enzymes, termed the "senescence-associated-secretory-phenotype" (SASP), playing a major role in many age-related diseases. The phenolic compounds present in extra-virgin olive oil are inhibitors of oxidative damage and have been reported to play a protective role in inflammation-related diseases. Particularly, hydroxytyrosol and oleuropein are the most abundant and more extensively studied. Pre-senescent human lung (MRC5) and neonatal human dermal (NHDF) fibroblasts were used as cellular model to evaluate the effect of chronic (4-6 weeks) treatment with 1 µM hydroxytyrosol (HT) or 10 µM oleuropein aglycone (OLE) on senescence/inflammation markers. Both phenols were effective in reducing ß-galactosidase-positive cell number and p16 protein expression. In addition, senescence/inflammation markers such as IL-6 and metalloprotease secretion, and Ciclooxigenase type 2 (COX-2) and α-smooth-actin levels were reduced by phenol treatments. In NHDF, COX-2 expression, Nuclear Factor κ-light-chain-enhancer of activated B cells (NFκB) protein level and nuclear localization were augmented with culture senescence and decreased by OLE and HT treatment. Furthermore, the inflammatory effect of Tumor Necrosis Factor α (TNFα) exposure was almost completely abolished in OLE- and HT-pre-treated NHDF. Thus, the modulation of the senescence-associated inflammatory phenotype might be an important mechanism underlying the beneficial effects of olive oil phenols.