Studio del meccanismo di sintesi e rilascio della dopamina in cellule PC 12 TET-ON contenenti la dardarina (LRRK2) WT e mutata

Mutations in the gene coding for dardarin (LRRK2) cause autosomal-dominant Parkinson’s disease. Using PC12 as a model for studying in vitro dopamine (DA) metabolism,we studied the effect of LRRK2 on DA secretion and their metabolites in PC12 cells with human LRRK2, in order to distinguish between the effects of transgene expression, activated in presence of doxycycline (DOX),and the potential effects of nicotine. At the start of experiment,50x103 cells/cm2 were plated and treated 24h later (time 0) with DOX (0.2-1.0 µg/ml); after 48h,through microdialysis procedure, was treated with nicotine 5 mM and DA and metabolites (DOPAC, HVA and 3-MT) were assayed by HPLC-EC. Four PC12-ON cells lines were used for experiments and in 3 PC12-ON cell lines,expressing human LRRK2, pathological mutation was induced as follow: PC12-ON, PC12-ON LRRK2 WT,PC12-ON LRRK2 G2019S and PC12-ON LRRK2 R1441C. Changes in baseline levels of DA secretion were observed, in PC12-ON expressing either mutant LRRK2 or WT,48h after treatment with 0.2 µg/ml DOX.DA was higher in mutant LRRK2G2019S,and lower in WT-LRRK2, when compared with the control. A reduction of DA secretion was detected,48h after treatment with 1 µg/ml DOX,only in mutant LRRK2G2019S. In all the experiments with PC12 cell with mutant or WT-LRRK2,the baseline of DA metabolites was lower than the control. The results on DA secretion and their metabolites were confirmed when the cells were treated with different concentrations of DOX and infused with nicotine

Exploring interaction modes between polysaccharide-based selectors and biologically active 4,4′-bipyridines by experimental and computational analysis

In the last few years, chiral 4,4′-bipyridine derivatives have been developed for different applications in catalysis, enantioseparation science, supramolecular and theoretical chemistry by modulating the activity of the molecular system through the introduction of specific substituents in the heteroaromatic scaffold. More recently, the biological activity of 2′-substituted-3,3′,5,5′-tetrachloro-2-iodo-4,4′-bipyridines has been explored in the field of transthyretin (TTR) fibrillogenesis inhibition, and the anticancer cytotoxicity of some derivatives is currently under systematic investigation. In this frame, the high-performance liquid chromatography (HPLC) enantioseparation of four atropisomeric 2,2′-disubstituted-4,4′-bipyridines (R, R’ = Ar, I), which contain multiple interaction sites, such as hydrogen bonding (HB) donors and acceptors, halogen bond (XB) donors, and -extended electronic clouds, was explored by using n-hexane (Hex)/2-propanol (2-PrOH) 90:10 v/v as a mobile phase (MP), and eight chiral columns with coated and immobilized amylose- and cellulose-based relectors. The impact of subtle structural variations of analytes and selectors on their mutual intermolecular interactivity was evaluated in terms of retention (k) and selectivity () factors. On this basis, chromatographic analysis based on systematic screening of analytes and selectors was integrated with electrostatic potential (V) analysis and molecular dynamics (MD) simulations as computational techniques. The effect of temperature on retention, selectivity, and enantiomer elution order (EEO) of the analytes with coated and immobilized amylose tris(3,5-dimethylphenylcarbamate) was also considered by comparing the variation of the thermodynamic profile associated with each enantioseparation. Chromatographic responses proved to be strictly dependent on specific regions within the analyte, and functions of different interactions sites of the analytes as the structure of the chiral selector changes were significantly disclosed

Functionalization of Screen-Printed Sensors with a High Reactivity Carbonaceous Material for Ascorbic Acid Detection in Fresh-Cut Fruit with Low Vitamin C Content

In this study, carbon screen-printed sensors (C-SPEs) were functionalized with a high reactivity carbonaceous material (HRCM) to measure the ascorbic acid (AA) concentration in fresh-cut fruit (i.e., watermelon and apple) with a low content of vitamin C. HRCM and the functionalized working electrodes (WEs) were characterized by SEM and TEM. The increases in the electroactive area and in the diffusion of AA molecules towards the WE surface were evaluated by cyclic voltammetry (CV) and chronoamperometry. The performance of HRCM-SPEs were evaluated by CV and constant potential amperometry compared with the non-functionalized C-SPEs and MW-SPEs nanostructured with multi-walled carbon nanotubes. The results indicated that SPEs functionalized with 5 mg/mL of HRCM and 10 mg/mL of MWCNTs had the best performances. HRCM and MWCNTs increased the electroactive area by 1.2 and 1.4 times, respectively, whereas, after functionalization, the AA diffusion rate towards the electrode surface increased by an order of 10. The calibration slopes of HRCM and MWCNTs improved from 1.9 to 3.7 times, thus reducing the LOD of C-SPE from 0.55 to 0.15 and 0.28 μM, respectively. Finally, the functionalization of the SPEs proved to be indispensable for determining the AA concentration in the watermelon and apple samples

An Ascorbate Bluetooth© Analyzer for Quality Control of Fresh-Cut Parsley Supply Chain

This work provides companies in the fresh-cut produce sector with an Ascorbate Bluetooth© Analyzer (ABA), a screen-printed sensor-based device for ascorbic acid (AA) detection, for quality control all along the supply chain. The amperometric detection of AA on fresh and fresh-cut parsley, under correct and incorrect storage temperature, allowed us to investigate the kinetics of AA decay in response to oxidative stress. The role of ascorbate oxidase (AOx) and ascorbate peroxidase (APx) was studied. ABA was used in situ by unskilled personnel. Treatments influenced AA decay kinetics, which were linear in fresh parsley, and non-linear in fresh-cut. Two hours at 28◦C immediately after chopping, the resilience of the fresh-cut parsley was reduced, even though the cold chain was restored. Two hours at −2◦C caused a rapid loss of AA until its complete decay after 72 h. Significant differences between treatments were observed in both the expression and activity of AOx and APx. ABA registered sudden changes of parsley AA following unpredicted variations of temperature during processing or transport. It was useful to remedy the effects of unexpected flaws in the cold chain, which can be proposed for quality preservation of different fresh-cut produce

SUMOylation Protects FASN Against Proteasomal Degradation in Breast Cancer Cells Treated with Grape Leaf Extract

Existing therapeutic strategies for breast cancer are limited by tumor recurrence and drug-resistance. Antioxidant plant-derived compounds such as flavonoids reduce adverse outcomes and have been identified as a potential source of antineoplastic agent with less undesirable side effects. Here, we describe the novel regulation of fatty-acid synthase (FASN), the key enzyme in de novo fatty-acid synthesis, whereby Vitis vinifera L. cv Vermentino leaf hydroalcoholic extract lowers its protein stability that is regulated by small ubiquitin-like modifier (SUMO)ylation. The phenolic compounds characterization was performed by liquid chromatography–mass spectrometry (LC–MS), whereas mass spectrometry (LC–MS/MS), Western blotting/co-immunoprecipitation (Co-IP) and RT-PCR, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), clonogenicity assays, and FACS analysis were used to measure the expression of targets and tumorigenicity. Vermentino extract exhibits antitumorigenic effects, and we went on to determine that FASN and ubiquitin-conjugating enzyme 9 (UBC9), the sole E2 enzyme required for SUMOylation, were significantly reduced. Moreover, FASN was found SUMOylated in human breast cancer tissues and cell lines, and lack of SUMOylation caused by SUMO2 silencing reduced FASN protein stability. These results suggest that SUMOylation protects FASN against proteasomal degradation and may exert oncogenic activity through alteration of lipid metabolism, whereas Vermentino extract inhibits these effects which supports the additional validation of the therapeutic value of this compound in breast cancer.This research was supported by a grant from Cedars-Sinai Medical Center, ACB&P Division

Biotelemetric Monitoring of Brain Neurochemistry in Conscious Rats Using Microsensors and Biosensors

In this study we present the real-time monitoring of three key brain neurochemical species in conscious rats using implantable amperometric electrodes interfaced to a biotelemetric device. The new system, derived from a previous design, was coupled with carbon-based microsensors and a platinum-based biosensor for the detection of ascorbic acid (AA), O2 and glucose in the striatum of untethered, freely-moving rats. The miniaturized device consisted of a single-supply sensor driver, a current-to-voltage converter, a microcontroller and a miniaturized data transmitter. The redox currents were digitized to digital values by means of an analog-to-digital converter integrated in a peripheral interface controller (PIC), and sent to a personal computer by means of a miniaturized AM transmitter. The electronics were calibrated and tested in vitro under different experimental conditions and exhibited high stability, low power consumption and good linear response in the nanoampere current range. The in-vivo results confirmed previously published observations on striatal AA, oxygen and glucose dynamics recorded in tethered rats. This approach, based on simple and inexpensive components, could be used as a rapid and reliable model for studying the effects of different drugs on brain neurochemical systems

Pseudomonas aeruginosa sulfur volatile organic compounds useful as contamination markers in packed ready-to-eat leafy greens

Ready to eat leafy greens are consumed raw and represent a potential transmission vehicle for human chronic infections. The outbreak of resistant strains and emerging foodborne pathogens evidence the need to set up checkpoints along the chain with handy contamination markers. In this respect, to ascertain a contamination marker for Pseudomonas aeruginosa on minimally processed leafy greens, we fingerprinted the sulfur volatile organic compounds (SVOCs) released into packed lettuce and parsley. - Leafy greens were processed and packed in industrial plants under a strict HACCP governance, and 24 specimens of lettuce and parsley were immediately moved to the laboratory and kept at 5 °C. Three mL of a P. aeruginosa (strain ATTC 2422) water suspension (106 cfu) was injected into half of the bags by means of an insulin syringe, and the same volume of sterile water was injected in the left ones (control). Following 12 h of cold storage, half (n=12) of lettuce and parsley bags were removed from storage and 0.5 mL headspace gas was analyzed by means of a portable SVOC dedicated GC. Then, bags were opened and 2 g (n=3) of lettuce and parsley tissue were employed to determine P. aeruginosa development by molecular probes. The same protocol was performed 12 h later on the remaining packed greens. - Three main sulfur compounds were detected (H2S; CH3S and (CH3)2S). In parsley, (CH3)2S was present only when contaminated (4.00±0,003 ppm) while, H2S and CH3S were in the control, and not in contaminated parsley. In lettuce, all SVOCs were detected in the control while, CH3S was the sole missing in contaminated lettuce. The pabulum used in this experiment did not influence P. aeruginosa growth. - Microbial SVOCs are influenced by the pabulum and only (CH3)2S may be considered as a marker for parsley contamination by P. aeruginosa. Keywords: Microbial Volatile Organic Compounds, Foodborne pathogens, contamination risk assessment, food safety management syste

Use of screen-printed sensors for quality loss determination in vegetables rich in vitamin C or with high ascorbate oxidative metabolism: a fresh-cut parsley and iceberg lettuce case study

Introduction: The use of screen-printed sensors for ascorbic acid (AA) electrochemical determination in fresh-cut fruit and vegetables is the core of the Real Time Check IV Gamma project since when AA has been recognized as rapid and reliable quality indicator in minimally processed produce. Methods: A carbon screen-printed electrode, coupled with an innovative telemetry device based on IoT (Internet of Things) technology, was used for a real time assessing of quality loss of fresh-cut parsley and iceberg lettuce, by recording the AA content, at harvest, during the processing and transport, and all along 5 days of shelf life, both when the cold chain was strictly respected and interrupted. The sensors work at an applied potential of + 120 mV, are specific for AA, and have been proven to be free of interferences. The sensitivity is 0.3001 ± 0.029 nA*µM-1 and the LOD 0.72 ± 0.117 µM. Results: AA content in parsley is high and was recorded by the sensors as a baselinesubtracted currents, expressed in nanoamperes, and transformed in mg*kg-1 of fresh weight. Differently, a direct amperometric real time detection of AA content in iceberg lettuce was not possible since a strong enzymatic activity, consistent with ascorbate peroxidase and ascorbate oxidase activity, prevailed. This enzymatic activity was recorded by the sensor system as an AA decreasing current and the half-life of exponential decay of the current curves was used as an indicator of the speed of quality loss. All the results were confirmed by traditional methods, AA with the titrimetric one and enzymatic activity by specific assays. Discussion: the proposed device is built with simple and inexpensive disposable components. It works as a portable workstation and was designed to be used by non-specialized personnel, at a farm level, in all the companies involved in the productive chain

Antioxidant and Anticancer Activity of Pericarp Water Extracts of Mediterranean Ancient Chestnut Accessions

The residue of chestnut processing generates a large amount of waste material, a resource not adequately exploited. The antioxidant and antitumoral properties of cold and hot water extracts from discarded pericarp of four chestnut Sardinian accessions and one marron variety were studied. The antioxidant capacity of the extracts was determined by spectrophotometric and electrochemical tests. The 1,1-diphenyl-2-pic-rylhydrazyl (DPPH) and 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) results were highly correlated with each other; likewise, a good correlation was found between Ferric Reducing Antioxidant Power (FRAP) and cyclic voltammetry (CV) values, both based on the direct transfer of electrons. The antiproliferative effect on normal cells (fibroblasts), and on colon (RKO and SW48) and breast (MCF7) cancer cells was evaluated. Additionally, this paper marks the first application of chestnut extracts to investigate their effects on melanoma (B16F10) cells. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test demonstrated that temperature and different extraction times significantly influenced the growth of cells, both normal and tumor. The fibroblast growth was significantly inhibited by moderate doses of cold extracts, while the GI50 values calculated for hot extracts were high, regardless of the accession or cultivar. An even more marked inhibitory action of the cold extracts was observed both on the growth of RKO and SW48 cells and on B16F10 melanoma cells. Otherwise, an extract concentration, both cold and hot, of no less than 243 µg mL−1 is required to achieve a 50% inhibition of MCF7 cell growth

Development and characterization of an ascorbate oxidase-based sensor–biosensor system for telemetric detection of AA and antioxidant capacity in fresh orange juice

A new carbon ascorbate oxidase-based sensor–biosensor system (SB) was coupled to a dual-channel telemetric device for online simultaneous electrochemical detection of ascorbic acid (AA) and antioxidant capacity in Hamlin, Sanguinello, and Moro orange varieties. The electrocatalytic performances of the SB were investigated by cyclic voltammetry and amperometric techniques. The phenol composition of orange juice of each variety, and the cyclic voltammetries of the most represented phenols, were provided. The in vitro calibrations were performed in PBS (pH 5.6), applying a constant potential of +500 mV. A standard mixture of phenols, based on orange juice composition, was used as reference material for studying SB behavior. SB works at an applied potential of +500 mV, in a concentration range comprised between the LOD 0.26 μM and 20 μM. In this concentration range, limiting the data acquisition time to 2 min, the problems of electrode passivation due to phenols polymerization were overcome. AA calibration showed that the biosensor registered statistically lower currents than the sensor since the enzyme oxidized AA before it reached the electrode surface. Standard mixture calibration showed that currents registered by sensor and biosensor did not statistically differ. The difference between sensor and biosensor AA registered currents was used to calculate an AA selectivity index and, consequently, to determine the AA content and the antioxidant capacity in the juices. The novelty of the SB is its ability to distinguish between AA and phenols contribution to antioxidant capacity. The obtained results were in accordance with reference methods