Jessica Fiori

Protective Effects of Cyanidin-3-O-β-glucopyranoside Against UVA-induced Oxidative Stress in Human Keratinocytes¶

Ultraviolet‐A (UVA) radiation causes significant oxidative stress because it leads to the generation of reactive oxygen species (ROS), leading to extensive cellular damage and eventual cell death either by apoptosis or necrosis. We evaluated the protective effects of cyanidin‐3‐O‐β‐glucopyranoside (C‐3‐G) against UVA‐induced apoptosis and DNA fragmentation in a human keratinocyte cell line (HaCaT). Treatment of HaCaT cells with C‐3‐G before UVA irradiation inhibited the formation of apoptotic cells (61%) and DNA fragmentation (54%). We also investigated antioxidant properties of C‐3‐G in HaCaT cells against ROS formation at apoptotic doses of UVA; C‐3‐G inhibited hydrogen peroxide (H2O2) release (an indicator of cellular ROS formation) after UVA irradiation. Further confirmation of the potential of C‐3‐G to counteract UVA‐induced ROS formation comes from our demonstration of its ability to enhance the resistance of HaCaT cells to the apoptotic effects of both H2O2 and the superoxide anion (O2•−), two ROS involved in UVA‐oxidative stress. Furthermore, in terms of Trolox Equivalent Antioxidant Activity, C‐3‐G treatment led to a greater increase in antioxidant activity in the membrane‐enriched fraction than in the cytosol (55%vs 19%). The protective effects against UVA‐induced ROS formation can be attributed to the higher membrane levels of C‐3‐G incorporation. These encouraging in vitro results support further research into C‐3‐G (and other anthocyanins) as novel agents for skin photoprotection.

GC-MS analysis of the lipophilic principles of Echinacea purpurea and evaluation of cucumber mosaic cucumovirus infection.

An analytical GC-MS method based on nonpolar fused silica capillary column was developed to analyze the lipophilic constituents, mainly alkamides, from the root extracts of Echinacea purpurea (L.) Moench. In particular, the proposed method was applied to evaluate the phytochemical impacts of cucumber mosaic cucumovirus (CMV) infection on the plants lipophilic marker phytochemicals. Methanolic (70% v/v) extracts, obtained from root materials by ultrasonic treatments, were subjected to liquid-liquid extraction with n-hexane-ethyl acetate (1:1 v/v) to recover the lipophilic, volatile to semivolatile, principles. Seventeen components, including the 11 alkamides known to E. purpurea roots, were identified in the GC-MS traces of the analyzed fractions and efficiently separated in a turnaround time of 25 min. CMV infection was found to be responsible for significant variations in the relative compositions of the major constituents, in particular germacrene D, Dodeca-2E, 4E, 8Z, 10Z(E)-tetraenoic acid isobutylamide cis/trans isomers, Undeca-2Z, 4E-diene-8, 10-diynoic acid isobutylamide and Dodeca-2E, 4Z-diene-8, 10-diynoic acid isobutylamide.

Fecal metabolome of the Hadza hunter-gatherers: A host-microbiome integrative view

The recent characterization of the gut microbiome of traditional rural and foraging societies allowed us to appreciate the essential co-adaptive role of the microbiome in complementing our physiology, opening up significant questions on how the microbiota changes that have occurred in industrialized urban populations may have altered the microbiota-host co-metabolic network, contributing to the growing list of Western diseases. Here, we applied a targeted metabolomics approach to profile the fecal metabolome of the Hadza of Tanzania, one of the world’s few remaining foraging populations, and compared them to the profiles of urban living Italians, as representative of people in the post-industrialized West. Data analysis shows that during the rainy season, when the diet is primarily plant-based, Hadza are characterized by a distinctive enrichment in hexoses, glycerophospholipids, sphingolipids, and acylcarnitines, while deplete in the most common natural amino acids and derivatives. Complementary to the documented unique metagenomic features of their gut microbiome, our findings on the Hadza metabolome lend support to the notion of an alternate microbiome configuration befitting of a nomadic forager lifestyle, which helps maintain metabolic homeostasis through an overall scarcity of inflammatory factors, which are instead highly represented in the Italian metabolome.

Separation of alkamides from Echinacea purpurea extracts by cyclodextrin-modified micellar electrokinetic chromatography

Separation of nine important alkyl methylbutyl‐ and isobutylamides (known as alkamides) obtained from Echinacea purpurea extracts was investigated by using cyclodextrin‐modified micellar electrokinetic chromatography (CD‐MEKC). Hydrophobic alkamides interact strongly with the micelles from the most common surfactants used in MEKC and this lead to predominant partition of the analytes into the micellar phase, resulting in poor resolution. The addition of neutral CDs to the alkaline (10 mM phosphate buffer pH 8.0) micellar system of sodium dodecyl sulfate (SDS), sodium cholate (SC) and sodium deoxycholate (SDC) was found to improve the separation of the studied alkamides. Among the several combinations surfactant/CD, three different systems showed to be particularly effective: SDS/hydroxypropyl‐β‐CD (110 mM/100 mM) and SC/heptakis (2, 3, 6‐tri‐O‐methyl)‐β‐CD (200 mM/40 mM) which provided a complete separation of the studied compounds, and SDC/heptakis (2, 6‐di‐O‐methyl)‐β‐CD. The importance of appropriate surfactant vs. CD concentration ratio as well as that of total concentration of both surfactant and CD was considered. The optimization of the separation was performed by focussing the need for a rapid separation of nine alkamides diagnostically useful to define the fingerprint of Echinacea species.

Nature-Inspired Multifunctional Ligands: Focusing on Amyloid-Based Molecular Mechanisms of Alzheimer's Disease.

The amyloidogenic pathway is a prominent feature of Alzheimers disease (AD). However, growing evidence suggests that a linear disease model based on β‐amyloid peptide (Aβ) alone is not likely to be realistic, which therefore calls for further investigations on the other actors involved in the play. The pro‐oxidant environment induced by Aβ in AD pathology is well established, and a correlation among Aβ, oxidative stress, and conformational changes in p53 has been suggested. In this study, we applied a multifunctional approach to identify allyl thioesters of variously substituted trans‐cinnamic acids for which the pharmacological profile was strategically tuned by hydroxy substituents on the aromatic moiety. Indeed, only catechol derivative 3 [(S)‐allyl (E)‐3‐(3,4‐dihydroxyphenyl)prop‐2‐enethioate] inhibited Aβ fibrilization. Conversely, albeit to different extents, all compounds were able to decrease the formation of reactive oxygen species in SH‐SY5Y neuroblastoma cells and to prevent alterations in the conformation of p53 and its activity mediated by soluble sub‐lethal concentrations of Aβ. This may support an involvement of oxidative stress in Aβ function, with p53 emerging as a potential mediator of their functional interplay.

Photomutagenic Properties of Terfenadine as Revealed by a Stepwise Photostability, Phototoxicity and Photomutagenicity Testing Approach¶

Administration of the second‐generation antihistamine, terfenadine, is sometimes associated with photosensitivity and other skin reactions. To obtain information on its photoreactivity, we used a stepwise experimental approach involving tests for photostability, phototoxicity (PT) (mouse fibroblast cell line [3T3] neutral red uptake [NRU] test) and photomutagenicity (with standard Ames salmonella tester strains TA98, TA100 and TA102). Terfenadine was not phototoxic to cultured mammalian cells under the conditions used (i.e. 5000/161 mJ cm−2 UVA–UVB). Natural sunlight and UV radiations caused considerable drug decomposition and formation of several photoproducts. Addition of the irradiated terfenadine solution (i.e. a mixture of photoproducts) to the tester did not significantly increase background mutation frequency. Irradiation of terfenadine coplated with the TA102 strain induced a clear‐cut photomutagenic response, the magnitude of which was dependent upon the precursor compound concentration and the UV dose (212/7 to 339/11 mJ cm−2 UVA–UVB). These findings demonstrate that in vitro terfenadine is photomutagenic in absence of PT. Further in vitro and in vivo studies are therefore needed to provide an adequate safety assessment of the photochemical genotoxicity–carcinogenicity potential of terfenadine. In the meantime, patients should be advised to avoid excessive exposure to sunlight.

Application of an ESI-QTOF method for the detailed characterization of GSK-3β inhibitors

HIGHLIGHTSDetermination of GSK‐3&bgr; activity and inhibition by monitoring GSM phosphorylation by ESI‐MS.Identification of a structural modification on a single amino acidic residual by ESI‐QTOF.GSKreaction characterizationby simultaneously monitoringphosphorylated and native substrate.Inhibitors potency and mechanism of action resulted in agreement with literature.The lowest incubation and analysis time make this method very fast.The potential automation makes this method suitable for theHTSof GSK‐3&bgr; inhibitors. ABSTRACT The crucial role of Glycogen Synthase Kinase 3 (GSK‐3&bgr;) as a pivotal player in Alzheimers Disease (AD) has recently inspired significant attempts to design and synthesize potent kinase inhibitors. In fact GSK‐3&bgr; is considered the main kinase which catalyzes the microtubule‐associated protein tau hyper‐phosphorylation and the neurofibrillary tangles (NFT) in vitro and in vivo, The first classes of GSK‐3&bgr; inhibitors were classified as ATP‐competitive and, therefore, they lack of an efficient degree of selectivity over other kinases. In light of this consideration, many efforts are devoted to characterize new non ATP‐competitive GSK‐3&bgr; inhibitors, endowed with high selectivity. In parallel, there is an urgent need to develop new analytical methodologies for the hit selection (highthroughput screening) and ligand binding characterization in terms of potency, affinity and mechanism of action. The new methodology for GSK‐3&bgr; enzymatic activity determination can be adopted as a realistic alternative to the currently used radioactive, luminescence and fluorescence detection methods, each showing limitations in terms of safety and interferences. Herein, we propose an alternative and selective electrospray ionization quadrupole time‐of‐flight (ESI‐QTOF) method, based on the direct quantification of phosphorylated substrate muscle glycogen synthase GSM, a peptide resembling the high affinity sequence of natural substrate muscle glycogen synthase 1, for the detailed characterization of GSK‐3&bgr; inhibitors. The method was validated in terms of accuracy and reproducibility of GSM signal intensity with a relative standard deviation RSD% value of 3.55%; Limit of Detection (LOD): 0.006 &mgr;M; Lower Limit of Quantification (LLOQ): 0.02 &mgr;M; linearity r2 0.9951. The kinetic constants (KM and vmax) of the GSK‐3&bgr; catalyzed kinase reaction and the inhibitory potency of known ligands (IC50), were determined. All the obtained results were in agreement with those reported in literature or obtained in house by the standard reference luminometric approach. The proposed method was applied to the elucidation of well known inhibitors mechanism of action by the construction of a Lineweaver–Burk plot and the Ki determination. Furthermore, the potency, affinity and mechanism of action of a new non ATP‐competitive compound were established. We demonstrated the ESI‐QTOF method to be more feasible than the classic kinase assays since it avoids drawbacks inherently connected with radioisotope labeling or the indirect detection of kinase activity, so far. It is also scalable to the screening of large library collections and suitable for pharmaceutical industries purposes.

Gut microbiome response to short-term dietary interventions in reactive hypoglycemia subjects

Reactive hypoglycemia is a metabolic disorder that provokes severe hypoglycemic episodes after meals. Over recent years, the gut microbiota has been recognized as potential target for the control of metabolic diseases, and the possibility to correct gut microbiota dysbioses through diet, favouring the recovery of metabolic homeostasis, has been considered.

Direct determination of GSK-3β activity and inhibition by UHPLC-UV–vis diode arrays detector (DAD)

Altered GSK-3β activity can contribute to a number of pathological processes including Alzheimers disease (AD). Indeed, GSK-3β catalyzes the hyperphosphorylation of tau protein by transferring a phosphate moiety from ATP to the protein substrate serine residue causing the formation of the toxic insoluble neurofibrillary tangles; for this reason it represents a key target for the development of new therapeutic agents for AD treatment. Herein we describe a new selective UHPLC methodology developed for the direct characterization of GSK-3β kinase activity and for the determination of its inhibition, which could be crucial in AD drug discovery. The UHPLC-UV (DAD) based method was validated for the very fast determination of ATP as reactant and ADP as product, and applied for the analysis of the enzymatic reaction between a phosphate primed peptide substrate (GSM), resembling tau protein sequence, ATP and GSK-3β, with/without inhibitors. Analysis time was ten times improved, when compared with previously published chromatographic methods. The method was also validated by determining enzyme reaction kinetic constants (KM and vmax) for GSM and ATP and by analyzing well known GSK-3β inhibitors. Inhibition potency (IC50) values for SB-415286 (81 ± 6 nM) and for Tideglusib (251 ± 17 nM), found by the newly developed UHPLC method, were in good agreement with the luminescence method taken as independent reference method. Further on, the UHPLC method was applied to the elucidation of Tideglusib mechanism of action by determining its inhibition constants (Ki). In agreement with literature data, Tideglusib resulted a GSM competitive inhibitor, whereas SB-415286 was found inhibiting GSK-3β in an ATP competitive manner. This method was applied to the determination of the potency of a new lead compound and was found potentially scalable to inhibitor screening of large compounds collections.

Development of a high-performance affinity chromatography-based method to study the biological interaction between whole micro-organisms and target proteins

Aims:u2002 The bacteria–host molecular cross‐talk is the matter of primary importance both in pathogenesis and in commensalism. Principally based on immunological methods, the methodologies commonly utilized for these studies are laborious and require specific antibodies. Here, we developed a new high‐performance affinity chromatography (HPAC)‐based approach that allows a direct measure of the interaction between whole bacterial cells and host molecules.