Rosa Carotenuto

Toxicity Effects of Functionalized Quantum Dots, Gold and Polystyrene Nanoparticles on Target Aquatic Biological Models: A Review

Nano-based products are widespread in several sectors, including textiles, medical-products, cosmetics, paints and plastics. Nanosafety and safe-by-design are driving nanoparticle (NP) production and applications through NP functionalization (@NPs). Indeed, @NPs frequently present biological effects that differ from the parent material. This paper reviews the impact of quantum dots (QDs), gold nanoparticles (AuNPs), and polystyrene-cored NPs (PSNPs), evidencing the role of NP functionalization in toxicity definition. Key biological models were taken into consideration for NP evaluation: Saccharomyces cerevisiae, fresh- (F) and saltwater (S) microalgae (Raphidocelis subcapitata (F), Scenedesmus obliquus (F) and Chlorella spp. (F), and Phaeodactylum tricornutum (S)), Daphnia magna, and Xenopus laevis. QDs are quite widespread in technological devices, and they are known to induce genotoxicity and oxidative stress that can drastically change according to the coating employed. For example, AuNPs are frequently functionalized with antimicrobial peptides, which is shown to both increase their activity and decrease the relative environmental toxicity. P-NPs are frequently coated with NH2− for cationic and COOH− for anionic surfaces, but when positively charged toxicity effects can be observed. Careful assessment of functionalized and non-functionalized NPs is compulsory to also understand their potential direct and indirect effects when the coating is removed or degraded.

Fructose supplementation worsens the deleterious effects of short-term high-fat feeding on hepatic steatosis and lipid metabolism in adult rats.

What is the central question of this study? In humans, ‘Western‐style’ diet is characterized by high levels of both saturated fats and fructose. Lipid oversupply to the liver typical of high‐fat diets could be exacerbated by the coexistence of high levels of fat and fructose in the diet, thus accelerating the development of metabolic deregulation. What is the main finding and its importance? Short‐term consumption of a Western diet, rich in saturated fats and fructose, is more conducive to the development of liver steatosis and deleterious to glucose homeostasis than a high‐fat diet. This result points to the harmful effect of adding fructose to the usual Western, high‐fat diet.

Sperm-egg interaction in the painted frog (Discoglossus pictus): An ultrastructural study

The ultrastructure of sperm changes and penetration in the egg was studied in the anuran Discoglossus pictus, whose sperm have an acrosome cap with a typical tip, the apical rod. The first stage of the sperm apical rod and acrosome reaction (AR) consists in vesiculation between the plasma membrane and the outer acrosome membrane. The two components of the acrosome cap are released in sequence. The innermost component (component B) is dispersed first. The next acrosome change is the dispersal of the outermost acrosome content (component A). At 30 sec postinsemination, when the loss of component B is first observed, holes are seen in the innermost jelly coat (J1), surrounding the penetrating sperm. Therefore, this acrosome constituent might be related to penetration through the innermost egg investments. At 1 min postinsemination, during sperm penetration into the egg, a halo of finely granular material is observed around the inner acrosome membrane of the spermatozoon, suggesting a role for component A at this stage of penetration.

p27(BBP)/eIF6 acts as an anti-apoptotic factor upstream of Bcl-2 during Xenopus laevis development

p27BBP/eIF6 (β4-binding protein/eukaryotic initiation factor 6) regulates the joining of 40S and 60S ribosomal subunits, on receptor for activated C kinase 1 binding and protein kinase C phosphorylation in serine 235. In Xenopus, p27BBP/eIF6 is abundantly expressed in the majority of the embryonic anlagen. Although p27BBP/eIF6 abundance may be required for a general regulation of protein synthesis, our data suggest that p27BBP/eIF6 may target the translation of specific mRNAs. We injected Xp27BBP/eIF6 mRNA in one blastomere of two-cell-stage embryos and obtained a bent phenotype, the curvature being lateral with respect to the embryo antero-posterior axis. The injected side had fewer apoptotic cells than the uninjected side, whereas cell proliferation appeared unaffected. Accordingly, in Xp27BBP/eIF6 morphants, endogenous apoptosis increased. Injection of Xp27BBP/eIF6 point mutants indicated that the anti-apoptotic action of Xp27BBP/eIF6 requires the conserved S235. The bent phenotype was also obtained with B-cell lymphoma gene-2 (Bcl-2) overexpression and was rescued by Bcl-2-associated X protein (Bax)/Xp27BBP/eIF6 co-injection. In addition, embryos overexpressing Xp27BBP/eIF6 had a higher amount of Bcl-2 and an unchanged amount of Bax with respect to controls. In Xp27BBP/eIF6 morphants, Bcl-2 levels were unaffected and Bax levels were higher than in the controls. Thus, we propose that Xp27BBP/eIF6 is part of a mechanism acting on the specific translation of messengers regulating cell survival. In particular, we suggest that Xp27BBP/eIF6 may regulate the translation of factors upstream of Bcl-2/Bax.

Raman spectroscopy of Xenopus laevis oocytes

This work reports on the application of Raman spectroscopy for the analysis of Xenopus laevis oocytes (stage-I). A two-color home-made microscope has been used for this investigation. In particular, a 785nm Raman probe has been used to acquire the spontaneous Raman scattering from the oocyte cytoplasm, while a 532nm probe has been employed to detect carotenoids through Resonant Raman Scattering. Finally, the distribution of beta-carotene along a diameter of a single oocyte has been investigated.

Multiple-Particle-Tracking to investigate viscoelastic properties in living cells

Cell mechanical properties play an important role in determining many cellular activities. Passive microrheology techniques, such as Multiple-Particle-Tracking (MPT) give an insight into the structural rearrangements and viscoelastic response of a wide range of materials, in particular soft materials and complex fluids like cell cytoplasm in living cells. The technique finds an important field of application in large cells such as oocytes where, during their growth, several organelles and molecules are displaced in specific territories of the cell instrumental for later embryonic development. To measure cell mechanics, cells are usually deformed by many techniques that are slow and often invasive. To overcome these limits, the MPT technique is applied. Probe particles are embedded in the viscoelastic sample and their properties are extracted from the thermal fluctuation spectra measured using digital video-microscopy. The Brownian motion of a probe particle immersed in a network is directly related to the networks mechanical properties. Particles exhibit larger motions when their local environments are less rigid or less viscous. The mean-square-displacement (MSD) of the particles trajectory is used to quantify its amplitude of motions over different time scales.

α-spectrin has a stage-specific asymmetrical localization during Xenopus oogenesis

Xenopus oocyte organization largely depends upon the cytoskeleton distribution, which is dynamically regulated during oogenesis. An actin‐based cytoskeleton is present in the cortex starting from stage 1. At stages 4–6, a complex and polarized cytoskeleton network forms in the cytoplasm. In this paper, we studied the distribution of spectrin, a molecule that has binding sites for several cytoskeletal proteins and is responsible for the determination of regionalized membrane territories. The localization of α‐spectrin mRNA was analyzed during Xenopus oogenesis by in situ hybridization on both whole mount and sections, utilizing a cDNA probe encoding a portion of Xenopus α‐spectrin. Furthermore, an antibody against mammalian α‐spectrin was used to localize the protein. Our results showed a stage‐dependent mRNA localization and suggested that spectrin may participate in the formation of specific domains in oocytes at stages 1 and 2 and 4–6. Mol. Reprod. Dev. 55:229–239, 2000.

Spectrin and Ankyrin‐like Proteins in the Egg of Discoglossus pictus (Anura): Their Identification and Localization in the Site of Sperm Entrance versus the Rest of the Egg

The Discoglossus pictus egg has a specific site of sperm‐egg interaction, the dimple, which has a well‐defined cytoskeleton. We studied whether there are cytoskeletal and cytoskeleton‐related proteins typically involved in the polarization of plasma membrane proteins. The identity and the localization of the molecules cross‐reacting with antispectrin, antifodrin and antiankyrin antiobodies were investigated by immunofluoresecence and immunoblotting of the proteins of the dimple (D) and of the rest of the egg (dimple‐less‐egg; DLE). Two polypeptides of about 254‐and 246‐kD were detected in the D and DLE, and localized in the egg cortex. A third molecule, weakly cross‐reacting with antispectrin and antifodrin, was found in the subcortical cytoplasm. The 246‐kD polypeptide was labile in samples prepared for SDS‐PAGE; a mild prefixation of eggs prevented its dispersion. Mild fixation was also needed to retain antispectrin reactivity in cryostat sections of the DLE cortex, while this is not necessary in D. A molecule of about 204‐kD, cross‐reacting with antiankyrin, was detected in the cortex of the whole egg. These data and the finding that the concentrations of both the 254‐kD polypeptide and ankyrin are about 12‐fold higher in D than in the DLE, suggest that, in D, spectrin has a specific organization.

Following passage through the oviduct, the coelomic envelope of Discoglossus pictus (amphibia) acquires fertilizability upon reorganization, conversion of gp 42 to gp 40, extensive glycosylation, and formation of a specific layer.

This paper describes the morphological and biochemical changes in Discoglossus pictus coelomic oocyte envelope (CE) following passage through the oviduct. As in other anurans, in this species, the transformation of the envelope into vitelline envelope (VE) leads to the acquisition of fertilizability and involves the cleavage of a glycoprotein. In addition, several features, typical of Discoglossus pictus, were observed. A new layer, VE‐D, forms underneath the VE region facing the site of sperm entrance, the dimple. In the VE, arrowhead‐like bundles of fibrils are perpendicularly oriented toward the dimple. Ultrastructural observations and staining with UEA‐I suggested that VE‐D might have a role in supporting sperm penetration into the dimple by orienting VE bundles and exposing sugar residues such as fucose. In ‘in vitro’ tests, VE binding of sperm occurs only if sperm are exposed to A23187, in agreement with previous data (Campanella et al., 1997 : Mol Reprod Dev 47:323–333). Sperm binding occurs all over the VE. Accordingly, extracts of the VE covering the animal or the vegetal hemisphere have the same affinity to lectins (DBA, DSA, GNA, MAA, SBA, SNA, UEA‐I, WGA). The CE contains six main glycoproteins. Peptide mapping indicated that during CE transformation into VE, gp 42 shifts to an apparent Mr of 40 and gp 61 is converted to an apparent Mr of 63 kDa. Lectin blot analyses showed extensive changes in cross‐reactivity of most glycoproteins during the CE→VE transition. The fact that DBA and UEA‐I stain gp 63 rather than gp 61 and that this change is related only to gp 63, suggested that O‐glycosylation and terminal fucose might be acquired by gp 63 in preparation of fertilization. Gp 63 has recently been cloned (Vaccaro et al., submitted) and shown to exhibit high homology to Xenopus gp 69/64, a VE sperm ligand (Tian et al., 1997a : J. Cell Biol. 136: 1099–1108; Tian et al., 1997b : Dev Biol 187:143–153), and to ZP2 of mammals. Mol. Reprod. Dev. 58:318–329, 2001.

Polystyrene nanoparticles internalization in human gastric adenocarcinoma cells.

The increase in the use of nanoparticles, as a promising tool for drug delivery or as a food additive, raises questions about their interaction with biological systems, especially in terms of evoked responses. In this work, we evaluated the kinetics of uptake of 44 nm (NP44) and 100 nm (NP100) unmodified polystyrene nanoparticles (PS-NPs) in gastric adenocarcinoma (AGS) cells, as well as the endocytic mechanism involved, and the effect on cell viability and gene expression of genes involved in cell cycle regulation and inflammation processes. We showed that NP44 accumulate rapidly and more efficiently in the cytoplasm of AGS compared to NP100; both PS-NPs showed an energy dependent mechanism of internalization and a clathrin-mediated endocytosis pathway. Dose response treatments revealed a non-linear curve. PS-NPs also affected cell viability, inflammatory gene expression and cell morphology. NP44 strongly induced an up-regulation of IL-6 and IL-8 genes, two of the most important cytokines involved in gastric pathologies. Our study suggests that parameters such as time, size and concentration of NPs must be taken carefully into consideration during the development of drug delivery systems based on NPs and for the management of nanoparticles associated risk factors.