Salt elicitation to enhance phytochemicals in durum wheat seedlings.

BACKGROUND: Salt has been identified as an elicitor that can increase the accumulation of phytochemicals in seedlings during the germination process. However, the salinity level required to maximize the yield of phytochemicals, particularly phenolic compounds, needs further investigation for several plant species. To address this issue, we imposed increasing levels of salinity (NaCl solutions) on the sprouting substrate of Triticum durum (var. Platone) grains, at concentrations of 0, 50, 100, 150, 200, 250, and 300 mM (0_S, 50_S, 100_S, 150_S, 200_S, 250_S, and 300_S, respectively). RESULTS: The highest NaCl doses (250_S and 300_S) significantly impacted germination performance and were excluded from further analysis. The seedlings harvested at 8 days after sowing exhibited different growth stages depending on the salinity level: wheatgrass for 0_S, early wheatgrass for 50_S, intermediate between sprout and wheatgrass for 100_S, sprout for 150_S, and very early sprout for 200_S. Furthermore, salinity induced the concentration of phenolic compounds (PhCs) in the seedlings' tissues (i.e., both roots and shoots) in a salinity-dependent manner. The highest values were observed at 200_S, with an increase of 187% of the total investigated PhCs in comparison with 0_S, averaged over shoots and roots. In particular, in 200_S, the accumulation of phenolic acids was up to fourfold higher in roots, and that of flavonoids was up to twofold higher in shoots. CONCLUSION: Our findings suggest that the use of 200 mM NaCl applied to the sprouting substrate is excessive for producing edible sprouts but may be suitable for phytochemical extraction purposes. © 2023 Society of Chemical Industry.

Rapid pretreatment-free evaluation of antioxidant capacity in extra virgin olive oil using a laser-nanodecorated electrochemical lab-on-strip.

An electroanalytical lab-on-a-strip device for the direct extra-virgin olive oil (EVOO) antioxidant capacity evaluation is proposed. The lab-made device is composed of a CO2 laser nanodecorated sensor combined with a cutter-plotter molded paper-strip designed for EVOOs sampling and extraction. Satisfactory performance towards the most representative o-diphenols of EVOOs i.e., hydroxytyrosol (HY) and oleuropein (OL) were achieved; good sensitivity (LODHY = 2 µM; LODOL = 0.6 µM), extended linear ranges (HY: 10-250 µM; OL: 2.5-50 µM) and outstanding reproducibility (RSD < 5%, n = 3) were obtained in rectified oil. The device was challenged for the extraction-free analysis of 15 different EVOO samples, with satisfactory recoveries (90-94%; RSD < 5%, n = 3) and correlation with classical photometric assays (r ≥ 0.91). The proposed device includes all analysis steps, needs 4 µL of sample, and returns reliable results in 2 min, resulting portable and usable with a smartphone.

Nanofibrillar biochar from industrial waste as hosting network for transition metal dichalcogenides. Novel sustainable 1D/2D nanocomposites for electrochemical sensing.

Industrial wastes have become elective sustainable sources to obtain materials for electronic/electroanalytical purposes; on the other hand, easy and green strategies to include semiconductor 2D graphene-like materials in conductive networks are highly required. In this work, 1D/2D nanocomposites (NCs) based on nanofibrillar biochar (BH) from paper industry waste and transition metal dichalcogenides (TMDs: MoS2, WS2, MoSe2, and WSe2), were prepared in water via liquid phase exfoliation (LPE) using sodium cholate as bioderived surfactant. The TMD amount in the NCs has been carefully optimized, searching for the best compromise between electron transfer ability and electroanalytical performances. Four different water-dispersed BH-TMD NCs have been selected and comprehensively studied from the electrochemical point of view and morphologically characterized. The BH-TMDs potentiality have been demonstrated in model solutions and real samples towards different analytes of biological and agri-food interest. The most performing NCs have been selected and used for the simultaneous determination of the neurotransmitters dopamine (DP) and serotonin (SR), and the flavonoids quercetin (QR) and rutin (RT), obtaining good linearity (R2 ≥ 0.9956) with limits of detection ranging from 10 to 200 nM. Reproducible quantitative recovery values (90-112%, RSD ≤6%, n = 3) were obtained analyzing simultaneously DP and SR in synthetic biological fluid and drugs, and QR and RT in food supplements, proving the usability of the proposed materials for real analyses. This work proves that BH-nanofibers act as a sustainable conductive hosting network for 2D-TMDs, allowing full exploit their electroanalytical potential. The proposed BH-TMD NCs represent a sustainable, affordable, and captivating opportunity for the electrochemical and (bio)sensoristic field.

Optical plasmonic sensing based on nanomaterials integrated in solid supports. A critical review.

Noble metal nanoparticles (MNPs), have represented the keystone of a plethora of (bio)sensing analytical strategies because of their unique physicochemical features, becoming unique tools in the analytical scenario; in particular, MNPs localized surface plasmon resonance (LSPR) offers infinite analytical possibilities. In this work, the scaling-up from colloidal MNPs to their integration in solid substrates is overviewed, and the relative sensing and biosensing optical strategies based on LSPR changes are systematically treated in accordance with the supporting substrate employed. Recent literature and key papers reporting MNPs integration into solid substrates are considered, paying particular attention to the MNPs-based event into/onto the solid support and the related plasmonic change used as analytical signal. The review is organized in sections according to the solid support nature (glass, polymers, cellulose) and the papers are discussed according to the sensing strategy. The strategies have been classified in MNPs synthesis, growth, etching, displacement/aggregation directly or indirectly mediated by the analyte(s); only works that rely on plasmonic-transduction principles are taken into account, MNPs used as catalysts or in lateral flow systems are not considered. The review demonstrates that MNPs decorated/integrated substrates are now mature analytical tools, able to overcome the limitations of MNPs colloidal suspensions; this results in new analytical opportunities, particularly the realization of integrated systems, lab-on-chip/lab-on-strip and flexible devices, paving the way for a new generation of plasmonic (bio)sensors for point-of-need applications.

An electronic nose based on 2D group VI transition metal dichalcogenides/organic compounds sensor array.

Rapid volatile organic compounds (VOCs) detection is a hot topic today; in this framework nanomaterials and their tailorable chemistry offer a plethora of compelling opportunities. In this work, Group VI transition metal dichalcogenides (TMDs, i.e., MoS2, WSe2, MoSe2, and WSe2) were functionalized with organic compounds (ellagic acid, tannic acid, catechin, and sodium cholate) able to assist their sonochemical exfoliation in water. The 16 resulting water-dispersed 2D hybrid inorganic/organic TMDs resulted in a few-layer nanoflakes conformation and were used to modify quartz crystal microbalances (QCMs) to equip an e-nose for VOCs determination. The ability of the sensors for the detection of VOCs was assessed on alcohols, terpenes, esters, and aldehydes; the responses were significatively different, confirming the synergic effect of TMD and the organic compound in the interaction with VOCs. The 16 sensors exhibited quantitative responses for VOCs (R2≥0.978) with fast signals recovery (<100 s) and repeatable (RSD ≤9.3%, n = 5), reproducible (RSD ≤12.8%, n = 3) and stable (RSD ≤14.6%, 3 months) signals. As proof of applicability, in an e-nose format, banana aroma evolution during post-harvest ripening was successfully monitored using the 2D TMDs-based sensors array. These data demonstrate that TMDs exfoliated in water with different organic compounds are sustainable functional nanomaterials, able to offer new opportunities in nano-bioelectronic applications.

Effect of Wheat Crop Nitrogen Fertilization Schedule on the Phenolic Content and Antioxidant Activity of Sprouts and Wheatgrass Obtained from Offspring Grains.

This work was aimed at investigating the effects of rate and timing of nitrogen fertilization applied to a maternal wheat crop on phytochemical content and antioxidant activity of edible sprouts and wheatgrass obtained from offspring grains. We hypothesized that imbalance in N nutrition experienced by the mother plants translates into transgenerational responses on seedlings obtained from the offspring seeds. To this purpose, we sprouted grains of two bread wheat cultivars (Bologna and Bora) grown in the field under four N fertilization schedules: constantly well N fed with a total of 300 kg N ha-1; N fed only very early, i.e., one month after sowing, with 60 kg N ha-1; N fed only late, i.e., at initial shoot elongation, with 120 kg N ha-1; and unfertilized control. We measured percent germination, seedling growth, vegetation indices (by reflectance spectroscopy), the phytochemical content (total phenols, phenolic acids, carotenoids, chlorophylls), and the antioxidant activity (by gold nanoparticles photometric assay) of extracts in sprout and wheatgrass obtained from the harvested seeds. Our main finding is that grains obtained from crops subjected to late N deficiency produced wheatgrass with much higher phenolic content (as compared to the other N treatments), and this was observed in both cultivars. Thus, we conclude that late N deficiency is a stressing condition which elicits the production of phenols. This may help counterbalance the loss of income related to lower grain yield in crops subjected to such an imbalance in N nutrition.

Modular graphene mediator film-based electrochemical pocket device for chlorpyrifos determination.

In this work, a redox-graphene (Rx-Gr) film with electron-mediating ability has been integrated into a modular flexible pocket device, giving rise to a reusable biosensing platform. The Rx-Gr has been obtained in water from graphite taking advantage of catechin, a redox-antioxidant, able to assist the sonochemical layered-material exfoliation, conferring electron mediating feature. A film composed exclusively of Rx-Gr has been transferred via thermal rolling onto a flexible PET-support that was used as the biosensor base. The biosensing platform, composed of office-grade materials, was then fabricated using a cutter-plotter and assembled by thermal lamination; an interchangeable paper-based strip was used to host the enzymatic reaction and drive the capillary flow. An acetylcholinesterase-based inhibition assay has been optimized onboard the pocket device to determine chlorpyriphos, a widespread environmental pesticide. The proposed set-up allows the determination of chlorpyriphos at low overpotential (0.2 V) with satisfactory sensitivity (LOD = 0.2 ppb), thanks to the straightforward electroactivity of the Rx-Gr film towards thiocholine (enzymatic product). The modular design allows 5 consecutive complete inhibition assays (control + inhibition measure) retaining the performance (RSD = 5.4%; n = 5). The coupling of bench-top technologies and a new functional graphene film resulted in the development of a cost-effective, reusable, transportable, and within everyone's reach biosensing platform.

Oxidative stress on-chip: Prussian blue-based electrode array for in situ detection of H2O2 from cell populations.

A Prussian blue-based electrode array (PBEA) constituted by eight stencil-printed electrodes on a flexible PET (polyethylene terephthalate) substrate is proposed for in-situ HeLa cell culturing and real-time detection of the released H2O2. The array was suitably interfaced with a poly- (methyl methacrylate) (PMMA) well-containing holders resulting in a low cost multichambered chip. PBEA fabrication was carried out employing a xurography-based cost-effective benchtop microfabrication technology using just a desktop cutting plotter and office grade thermal-laminator. The hydrophobicity of the PET isolating layer allows to constrain cell-containing drops directly on top of the electrochemical cells. HeLa cells growth in the very close vicinity of the working electrode ensures in-situ cell seeding, incubation, and further electrochemical detection of the H2O2 released, enabling high-throughput analysis. Selective and sensitive electrochemical sensing of hydrogen peroxide was carried out at -100 mV vs Ag|AgCl; the resulting LOD was 1.9 µM. Remarkably, the analytical exploitability of the approach was demonstrated by detection of the hydrogen peroxide released from HeLa cells stimulated with N-Formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) and after pretreatment of the cells with cocoa polyphenols, that induced a decreased oxidative stress levels. These data make our approach a promising tool for oxidative stress evaluation in cell cultures and biological systems.

Cocoa powder and catechins as natural mediators to modify carbon-black based screen-printed electrodes. Application to free and total glutathione detection in blood.

In this work, for the first time, the direct usability of natural products, catechins (CT) and cocoa powder (CO), as electrochemical mediators able to modify a carbon black modified screen-printed electrode (SPE-CB) is proved, and, as proof of applicability, free (GSH) and total glutathione (GSH + GSSG) in blood samples is successfully determined. Noteworthy, the cocoa powder (naturally rich in catechins), dissolved in DMSO, was able to give rise to a useful highly redox-active catechol-quinone surface-confined system onto a carbon black nanoparticles modified screen-printed electrode (SPE-CB-CO - Cocoatrode), giving rise to a similar behaviour obtained with pure catechins (SPE-CB-CT). The electrodeposition process has been carefully studied, the resulting immobilized natural mediator (obtained using both CT and CO) features investigated, and the performance of the resulting sensors (SPE-CB-CT and Cocoatrode) tested and compared. Both modified electrodes (SPE-CB-CT and Cocoatrode) have a good inter-electrodes precision (RSD ip,a ≤ 4.2%, n = 3) and intra-electrodes repeatability (RSD ip,a ≤ 3.9%, n = 20), indicating a robust and stable 'fabrication' strategy. Finally, SPE-CB-CT and Cocoatrode have been employed for the detection of free (GSH) and total (GSH+GSSG) glutathione in blood samples, using differential pulse voltammetry decrease in the mediator's reduction peak (Δi %), as analytical signal. Analytical curves (R2 ≥ 0.998), for the GSH detection, have been determined both with SPE-CB-CT and Cocoatrode in the 2.5 × 10-8-6.0 × 10-5 M and 5.0 × 10-8-1.0 × 10-4 M concentration ranges, respectively. Limits of detections (LODs) were ≤2.6 × 10-8 M. GSH (free and total) determined in blood samples, by the proposed CT and CO sensors, showed a good intra-electrode repeatability (RSD ip,a ≤ 9.0%, n = 3), with good recoveries (from 88.3% to 117.7%). The values obtained were in agreement with a classical spectrophotometric method (GSH and GSSG concentration relative error between -4.7 and +9.8%). The SPE-CB-CT and the Cocoatrode platforms demonstrated high potentiality in sensing and biosensing scenario, opening new gates to the natural/food products employment as unconventional, eco-friendly and economically affordable analytical active tools.

Nanohybrid carbon black-molybdenum disulfide transducers for preconcentration-free voltammetric detection of the olive oil o-diphenols hydroxytyrosol and oleuropein.

A new hybrid nanomaterial is used in a screen-printed electrode (SPE) for sensing of the ortho-diphenols oleuropein (OLEU) and hydroxytyrosol (HYT) in extra virgin olive oil (EVOO) and related samples. The hybrid material consists of carbon black (CB) and molybdenum disulfide (MoS2). In comparison with individual nanomaterials, CB-MoS2 exhibits improved charge-transfer ability, low charge-transfer resistance, high electrical conductivity and enhanced electrocatalysis. The sensor is also characterized by (a) high sensitivity that avoids the need for adsorptive voltammetry, (b) reduced analysis time, and (c) high anti-fouling ability (electrode RSDOLEU < 8%, for n = 10). OLEU can be detected in the 0.3 to 30 µM concentration range with a 0.1 µM LOD, and HYT in the 2-100 µM range with a 1 µM LOD. A comparison of the data obtained by this sensor and by HPLC-UV exhibited high correlation (r = 0.995, p < 0.05). These data revealed the reliability of CB-MoS2 for analysis of complex EVOO and related samples. Graphical abstract CB-MoS2-based electrochemical sensor for fast and reliable assessment of total ortho-diphenols antioxidants in olive oils.

Affinity Sensing Strategies for the Detection of Pesticides in Food.

This is a review of recent affinity-based approaches that detect pesticides in food. The importance of the quantification and monitoring of pesticides is firstly discussed, followed by a description of the different approaches reported in the literature. The different sensing approaches are reported according to the different recognition element used: antibodies, aptamers, or molecularly imprinted polymers. Schemes of detection and the main features of the assays are reported and commented upon. The large number of affinity sensors recently developed and tested on real samples demonstrate that this approach is ready to be validated to monitor the amount of pesticides used in food commodities.

Nano carbon black-based screen printed sensor for carbofuran, isoprocarb, carbaryl and fenobucarb detection: application to grain samples.

An electrochemical screening assay for the detection of phenyl carbamates (i.e. carbaryl, carbofuran, isoprocarb and fenobucarb) was developed and applied to grains samples (i.e. durum wheat, soft wheat and maize). Nano carbon black (CB) was strategically employed to realize an effective, reproducible, fouling resistant, low cost, delocalisable screen printed sensor (CB-SPE). CB-SPEs morphology (SEM and FEM) and electrochemical property (CV and EIS) were studied. The final pesticides analysis protocol consist of: (i) extraction of the analyte (just by mixing), (ii) alkaline hydrolysis (10 min R.T.), (iii) DPV detection directly of 100 µL of extract on the CB-SPE surface. Linear range between 1.0 × 10-7 and 1.0 × 10-4 mol L-1, good determination coefficients (R2 ≥ 0.9971) and satisfactory sensitivity (≥ 3.90 × 10-1 A M-1 cm-2) and LODs (≤ 8.0 × 10-8 mol L-1) were obtained for all the analytes. Excellent recoveries (78-102%) and accuracy (relative error vs. HPLC-MS/MS between 9.0% and -7.8%) resulted from the analysis of grains samples. The proposed CB-SPE based approach has demonstrated to be able to detect carbaryl at Maximum residue limits levels (MRLs), allowing class selective detection of commonly employed phenyl carbamates in food samples.

Development of gold nanoparticles biosensor for ultrasensitive diagnosis of foot and mouth disease virus.

BACKGROUND: Nano-PCR is a recent tool that is used in viral diseases diagnosis. The technique depends on the fundamental effects of gold nanoparticles (AuNPs) and is considered a very effective and sensitive tool in the diagnosis of different diseases including viral diseases. Although several techniques are currently available to diagnose foot and mouth disease virus (FMDV), a highly sensitive, highly specific technique is needed for specific diagnosis of the disease. In the present work, a novel AuNPs biosensor has been designed using thiol-linked oligonucleotides that recognize the conserved 3D gene of FMDV. RESULTS: The AuNPs-FMDV biosensor specifically recognizes RNA standards of FMDV, but not that of swine vesicular disease virus (SVDV) isolates. The analytical sensitivity of the AuNPs-FMDV biosensor was 10 copy number RNA standards in RT-PCR and 1 copy number RNA standard in real-time rRT-PCR with a 94.5% efficiency, 0.989 R2, a - 3.544 slope and 100% specificity (no cross-reactivity with SVDV). These findings were confirmed by the specific and sensitive recognition of 31 Egyptian FMDV clinical isolates that represents the three FMDV serotypes (O, A, and SAT2). CONCLUSIONS: The AuNPs-FMDV biosensor presents in this study demonstrates a superior analytical and clinical performance for FMDV diagnosis. In addition, this biosensor has a simple workflow and accelerates epidemiological surveillance, hence, it is qualified as an efficient FMDV diagnosis tool for quarantine stations and farms particularly in FMDV endemic areas.

Electrochromic Molecular Imprinting Sensor for Visual and Smartphone-Based Detections.

Electrochromic effect and molecularly imprinted technology have been used to develop a sensitive and selective electrochromic sensor. The polymeric matrices obtained using the imprinting technology are robust molecular recognition elements and have the potential to mimic natural recognition entities with very high selectivity. The electrochromic behavior of iridium oxide nanoparticles (IrOx NPs) as physicochemical transducer together with a molecularly imprinted polymer (MIP) as recognition layer resulted in a fast and efficient translation of the detection event. The sensor was fabricated using screen-printing technology with indium tin oxide as a transparent working electrode; IrOx NPs where electrodeposited onto the electrode followed by thermal polymerization of polypyrrole in the presence of the analyte (chlorpyrifos). Two different approaches were used to detect and quantify the pesticide: direct visual detection and smartphone imaging. Application of different oxidation potentials for 10 s resulted in color changes directly related to the concentration of the analyte. For smartphone imaging, at fixed potential, the concentration of the analyte was dependent on the color intensity of the electrode. The electrochromic sensor detects a highly toxic compound (chlorpyrifos) with a 100 fM and 1 mM dynamic range. So far, to the best of our knowledge, this is the first work where an electrochromic MIP sensor uses the electrochromic properties of IrOx to detect a certain analyte with high selectivity and sensitivity.

Simple and rapid silver nanoparticles based antioxidant capacity assays: Reactivity study for phenolic compounds.

A single-step, rapid (10 min), sensitive silver nanoparticles (AgNPs) based spectrophotometric method for antioxidant capacity (AOC) assay has been developed. The assay is based on the ability of natural polyphenols to reduce Ag(I) and stabilize the produced AgNPs(0) at room temperature. Localized surface plasmon resonance (LSPR) of AgNPs at ≈420 nm is then measured. Using different conditions of pH (8.4) and temperature (45 °C) a further assay based on the production of AgNPs with selectivity for flavonols was also developed. The reactivity of the two AgNPs based assays vs. 15 polyphenols belonging to different chemical classes and 9 different samples has been studied and compared with ABTS, Folin and AuNPs based methods for AOC. The proposed assays had good reproducibility (RSD ≤ 13) and are simple, sensitive and cost effective. Moreover, used in conjunction with the classical AOC assays, can improve the information on the polyphenolic pool of food samples.

Press-Printed Conductive Carbon Black Nanoparticle Films for Molecular Detection at the Microscale.

Carbon black nanoparticle (CBNP) press-transferred film-based transducers for the molecular detection at the microscale level were proposed for the first time. Current-sensing atomic force microscopy (CS-AFM) revealed that the CBNP films were effectively press-transferred, retaining their good conductivity. A significant correlation between the morphology and the resistance was observed. The highest resistance was localized at the top of the press-transferred film protrusions, whereas low values are usually obtained at the deep crevices or grooves. The amount of press-transferred CBNPs is the key parameter to obtain films with improved conductivity, which is in good agreement with the electrochemical response. In addition, the conductivity of such optimum films was not only Ohmic; in fact, tunneling/hopping contributions were observed, as assessed by CS-AFM. The CBNP films acted as exclusive electrochemical transducers as evidenced by using two classes of molecules, that is, neurotransmitters and environmental organic contaminants. These results revealed the potential of these CBNP press-transferred films for providing new options in microfluidics and other related micro- and nanochemistry applications.

Gold Nanoparticles-based Extraction-Free Colorimetric Assay in Organic Media: An Optical Index for Determination of Total Polyphenols in Fat-Rich Samples.

In this work, a rapid and simple gold nanoparticle (AuNPs)-based colorimetric assay meets a new type of synthesis of AuNPs in organic medium requiring no sample extraction. The AuNPs synthesis extraction-free approach strategically involves the use of dimethyl sulfoxide (DMSO) acting as an organic solvent for simultaneous sample analyte solubilization and AuNPs stabilization. Moreover, DMSO works as a cryogenic protector avoiding solidification at the temperatures used to block the synthesis. In addition, the chemical function as AuNPs stabilizers of the sample endogenous fatty acids is also exploited, avoiding the use of common surfactant AuNPs stabilizers, which, in an organic/aqueous medium, rise to the formation of undesirable emulsions. This is controlled by adding a fat analyte free sample (sample blank). The method was exhaustively applied for the determination of total polyphenols in two selected kinds of fat-rich liquid and solid samples with high antioxidant activity and economic impact: olive oil (n = 28) and chocolate (n = 16) samples. Fatty sample absorbance is easily followed by the absorption band of localized surface plasmon resonance (LSPR) at 540 nm and quantitation is refereed to gallic acid equivalents. A rigorous evaluation of the method was performed by comparison with the well and traditionally established Folin-Ciocalteu (FC) method, obtaining an excellent correlation for olive oil samples (R = 0.990, n = 28) and for chocolate samples (R = 0.905, n = 16). Additionally, it was also found that the proposed approach was selective (vs other endogenous sample tocopherols and pigments), fast (15-20 min), cheap and simple (does not require expensive/complex equipment), with a very limited amount of sample (30 µL) needed and a significant lower solvent consumption (250 µL in 500 µL total reaction volume) compared to classical methods.

Antioxidant capacity index based on gold nanoparticles formation. Application to extra virgin olive oil samples.

A simple gold nanoparticles (AuNPs) based colorimetric assay for the antioxidant activity determination has been developed. The AuNP formation is mediated by extra virgin olive oil (EVOO's) endogenous polyphenols; the reaction is described by a sigmoidal curve. The ratio KAuNPs/Xc(50) (slope of the linear part of the sigmoid/concentration at half value of the absorbance) was found to be the optimal parameter to report the antioxidant capacity with respect to the single KAuNPs or Xc(50) values. The obtained data demonstrated that the compounds with ortho-diphenols functionality are most active in reducing gold (III) to gold (0). Thus, intermediate activity was found for gallic acid, while tyrosol (mono-phenols) had a significant lower activity than the others antioxidant compounds (at least one order of magnitude). In the analysis of olive oil samples, a significant correlation among classical methods used to determine antioxidant activity and the proposed parameter was found with R values in the 0.96-0.97 range.

Extravirgin olive oil up-regulates CB1 tumor suppressor gene in human colon cancer cells and in rat colon via epigenetic mechanisms.

Extravirgin olive oil (EVOO) represents the typical lipid source of the Mediterranean diet, an eating habit pattern that has been associated with a significant reduction of cancer risk. Diet is the more studied environmental factor in epigenetics, and many evidences suggest dysregulation of epigenetic pathways in cancer. The aim of our study was to investigate the effects of EVOO and its phenolic compounds on endocannabinoid system (ECS) gene expression via epigenetic regulation in both human colon cancer cells (Caco-2) and rats exposed to short- and long-term dietary EVOO. We observed a selective and transient up-regulation of CNR1 gene - encoding for type 1 cannabinoid receptor (CB1) - that was evoked by exposure of Caco-2 cells to EVOO (100 ppm), its phenolic extracts (OPE, 50 µM) or authentic hydroxytyrosol (HT, 50 µM) for 24 h. None of the other major elements of the ECS (i.e., CB2; GPR55 and TRPV1 receptors; and NAPE-PLD, DAGL, FAAH and MAGL enzymes) was affected at any time point. The stimulatory effect of OPE and HT on CB1 expression was inversely correlated to DNA methylation at CNR1 promoter and was associated with reduced proliferation of Caco-2 cells. Interestingly, CNR1 gene was less expressed in Caco-2 cells when compared to normal colon mucosa cells, and again this effect was associated with higher level of DNA methylation at CNR1. Moreover, in agreement with the in vitro studies, we also observed a remarkable (~4-fold) and selective increase in CB1 expression in the colon of rats receiving dietary EVOO supplementation for 10 days. Consistently, CpG methylation of rat Cnr1 promoter, miR23a and miR-301a, previously shown to be involved in the pathogenesis of colorectal cancer and predicted to target CB1 mRNA, was reduced after EVOO administration down to ~50% of controls. Taken together, our findings demonstrating CB1 gene expression modulation by EVOO or its phenolic compounds via epigenetic mechanism, both in vitro and in vivo, may provide a new therapeutic avenue for treatment and/or prevention of colon cancer.

Membrane lipids are key modulators of the endocannabinoid-hydrolase FAAH.

Lipid composition is expected to play an important role in modulating membrane enzyme activity, in particular if the substrates are themselves lipid molecules. A paradigmatic case is FAAH (fatty acid amide hydrolase), an enzyme critical in terminating endocannabinoid signalling and an important therapeutic target. In the present study, using a combined experimental and computational approach, we show that membrane lipids modulate the structure, subcellular localization and activity of FAAH. We report that the FAAH dimer is stabilized by the lipid bilayer and shows a higher membrane-binding affinity and enzymatic activity within membranes containing both cholesterol and the natural FAAH substrate AEA (anandamide). Additionally, co-localization of cholesterol, AEA and FAAH in mouse neuroblastoma cells suggests a mechanism through which cholesterol increases the substrate accessibility of FAAH.