Ivana Caputo

Celiac disease (CD) is an autoimmune inflammatory enteropathy triggered by consuming gluten-containing cereals. A key role in its pathogenesis is played by type 2 transglutaminase, an enzyme that causes an increase in gluten immunogenicity. Celiac cells seem to present constitutive characteristics, even recognizable under a gluten-free diet, such as defects in vesicular trafficking and autophagy, protein hyperphosphorylation, and cytoskeleton rearrangement. In this work, by using an omics approach, we attempted to identify those proteins differentially expressed or differentially phosphorylated in a cell model suitable to study cell behavior in the absence of inflammation, i.e., primary cultures of dermal fibroblasts from control or CD subjects. By performing mass spectrometry analyses, we found several up- and-down expressed or phosphorylated proteins in CD samples, mainly involved in signaling, homeostatic responses, cytoskeleton organization, vesicular trafficking, and extracellular vesiculation. These proteins may represent a molecular signature of the celiac cellular phenotype and may contribute to adding new insight into the comprehension of the complex mechanisms of CD pathogenesis.

Background: Quercetin (Q) can reduce cellular oxidative stress, though it is susceptible to degradation in physiological conditions. Through adsorption and protection of Q, mesoporous silica nanoparticles (MSNs) could enhance its bioactivity. This work aimed to determine the effect of Q loading in MSN and in its aminated (A-MSN), carboxylated (C-MSN) or thiolated (T-MSN) derivatives on its Caco-2-cytoprotective effect against H₂O₂-induced oxidative stress. Methods: The mesoporous silica materials were characterized (FT-IR, ζ-potential, TGA), and their cytotoxicity was assessed; then, they were loaded with Q and incubated with Caco-2 cells prior to oxidative stress induction, and the cytoprotective effect was evaluated through measurement of cell viability. Results: None of the nanoparticles showed toxicity to Caco-2 cells. A-MSN showed the highest Q loading capacity (5.26% ± 0.06%), due to hydrogen-bonding interactions. C-MSN clearly enhanced the Q cellular uptake compared to the other nanoparticles. Oxidative stress decreased Caco-2 cell viability, which was prevented by 100 µM free Q after 18 h incubation. In contrast, higher cell viability than in non-stressed cells was observed with the same Q concentration loaded across all nanoparticle types. Conclusions: Despite the high instability of free quercetin under cell culture conditions, it exerted a time-dependent cytoprotective effect against H₂O₂-induced oxidative stress that was enhanced upon loading into nanoparticles. Prior release of the Q molecule in the medium is ineffective, and the presence of the loaded material is required.

Alkylphenols are byproducts of anthropogenic activities that widely contaminate waters, soils and air; among them, the most represented are 4-nonylphenol (4-NP) and 4-octylphenol (4-OP). These compounds tend to bioaccumulate in animal and plant tissues and also represent a risk to human health. Indeed, humans are constantly exposed to alkylphenols through ingestion of contaminated water and food, inhalation and dermal absorption. In the present work, we characterized the cytotoxic ability of 4-OP towards several human cell lines, representing the potential main targets in the human body, also comparing its effect with that of 4-NP and of a mixture of both 4-OP and 4-NP in a range of concentrations between 1 and 100 μM. Viability assays demonstrated that each cell type had a peculiar sensitivity to 4-OP and that, in some cases, a combination of the two alkylphenols displayed a higher cytotoxic activity with respect to the single compound. Then, we focused our attention on a liver cell line (HepG2) in which we observed that 4-OP increased cell death and also caused interference with protective physiological cell processes, such as the unfolded protein response, autophagy and the antioxidant response. Finally, our experimental data were compared and correlated with ADMET properties originating from an in silico analysis. Altogether, our findings highlight a possible contribution of this pollutant to deregulation of the normal homeostasis in human liver cells.

Novel Resorcin[4]arene-based, octacationic Quaternary Ammonium Compounds (ResQAC^R) were obtained by linking tetraalkylammonium groups on the macrocycle wider rim to give multivalent multi-QACs. Their structures were elucidated through 1D and 2D NMR experiments, HR-MS analysis, and crystallographic studies. Additionally, the conformational dynamics of these octacationic-resorcinarenes were investigated using ¹H variable-temperature (VT) NMR experiments. The antimicrobial properties of ResQAC^R derivatives were studied by in vitro biological investigations. We identify the ResQAC^butyl derivative which shows an impressive bacteriostatic activity against S. aureus. A remarkable multivalent effect was observed for this bacteriostatic activity. Interestingly, cytotoxic studies indicate that ResQAC^butyl showed no adverse impact on cell viability of a human cell line, even at concentrations 30 times greater than the MIC for S. aureus and approximately 3 times higher than the MIC for E. coli. ResQAC^undecyl exhibits greater bacteriostatic activity against E. coli than S. aureus, but it proves cytotoxic at lower concentrations (IC₅₀ of 12.1 µM) compared to its MIC. ResQAC^phenyl shows scarce bacteriostatic activity against both S. aureus and E. coli, while no multivalent effect was observed. This is likely attributed to the conformational rigidity of the boat C_2v conformation of ResQAC^phenyl, which hinders an optimal matching between the the cationic chains and the negatively charged bacterial surface

The widespread use of drugs is a global problem which affects not only humans but also the environment around them, as research is showing the presence of these substances in different environmental matrices, like air, water, and soil. Above all, due to the remarkable pharmacological properties of drugs, it is discovered that organisms accidentally exposed to them, as aquatic organisms, undergo behavioral and physiological changes that can compromise their health, survival, and reproduction ability. In addition to this, we must consider the ability of some drugs to accumulate within these organisms, thus entering the food chain, and the possible interactions that drugs in water can establish with each other and with other possible pollutants, making the final effects on exposed organisms unpredictable. This article is an overview of the effects of one of these drugs, cocaine, one of the drugs commonly found in the aquatic environment, on European eel, an endangered species and known biomonitor of aquatic contamination.

Fibrous membranes of polycaprolactone (PCL)-polyvinylpyrrolidone (PVP) encapsulating 15% wt of quercetin are fabricated by a uniaxial electrospinning technique. Morphological analysis of the electrospun systems proved the fabrication of micrometric fibers (1.58 µm for PCL/PVP and 2.34 µm for quercetin-loaded membrane). The liquid retention degree of the electrospun membranes is evaluated by testing four different liquid media. The contact angle estimation is performed by testing three liquids: phosphate buffer solution, basic solution (pH = 13) and acidic solution (pH = 3), showing high hydrophobicity degree (contact angles > 90°) in all cases. The release of quercetin from the nanofibers in PBS (phosphate buffer solution) and pH = 3 medium, modeled through different models, shows the possibility of a fine tuning of drug release (up to 7 days) for the produced materials. The release profiles attained a plateau regime after roughly 50 h up to 82% and 71% for PBS and pH = 3 media, respectively. Then, since quercetin is known to undergo photooxidation upon UV radiation, release tests after different UV treatment times are carried out and compared with the untreated membrane, demonstrating that the release of the active drug changes from 82% for no-irradiated sample up to 57% after 10 h of UV exposure. The biology activity of released quercetin is evaluated on two human cell lines. The reported results demonstrate the ability of the quercetin-loaded membranes to reduce cell viability of human cell lines in two different conditions: direct contact between cells and quercetin-loaded membranes and cells treatment with culture medium previously conditioned with quercetin-loaded membranes. Therefore, the reported preliminary data confirm the possibility of applying the electrospun quercetin-loaded PCL-PVP membranes for health applications.

Celiac disease (CD) is an inflammatory intestinal disease caused by the ingestion of gluten-containing cereals by genetically predisposed individuals. Constitutive differences between cells from CD patients and control subjects, including levels of protein phosphorylation, alterations of vesicular trafficking, and regulation of type 2 transglutaminase (TG2), have been reported. In the present work, we investigated how skin-derived fibroblasts from CD and control subjects responded to thapsigargin, an endoplasmic reticulum ER stress inducer, in an attempt to contribute to the comprehension of molecular features of the CD cellular phenotype. We analyzed Ca²⁺ levels by single-cell video-imaging and TG2 activity by a microplate assay. Western blots and PCR analyses were employed to monitor TG2 levels and markers of ER stress and autophagy. We found that the cytosolic and ER Ca²⁺ level of CD cells was lower than in control cells. Treatments with thapsigargin differently activated TG2 in control and CD cells, as well as caused slightly different responses regarding the activation of ER stress and the expression of autophagic markers. On the whole, our findings identified further molecular features of the celiac cellular phenotype and highlighted that CD cells appeared less capable of adapting to a stress condition and responding in a physiological way.

Pergularia tomentosa L., a milkweed tropical plant belonging to the family Asclepiadaceae, is a rich source of unusual cardiac glycosides, characterised by transfused A/B rings and a sugar moiety linked by a double link, generating a dioxanoid structure. In the present report, five cardenolides isolated from the aerial parts of the plant (calactin, calotropin, 12β-hydroxycalactin, 12β,6′-dihydroxycalotropin, and 16α-hydroxycalotropin) were investigated for their biological effects on a human hepatocarcinoma cell line. Cell viability was monitored by an MTT assay. The occurrence of apoptosis was evaluated by detecting caspase-3 activation and chromatin fragmentation. The ability of these compounds to induce autophagy was analysed by monitoring two markers of the autophagic process, LC3 and p62. Our results indicated that all cardenolides had cytotoxic effects, with IC₅₀ ranging from 0.127 to 6.285 μM. All compounds were able to induce apoptosis and autophagy, calactin being the most active one. Some of them also caused a reduction in cell migration and a partial block of the cell cycle into the S-phase. The present study suggests that selected cardenolides from aerial parts of P. tomentosa, particularly calactin, possess potentially desirable properties for further investigation as anticancer agents.