Alessandro Attanzio

Neuroinflammation is a hallmark of numerous neurodegenerative diseases and is characterized by profound inflammatory, metabolic, and redox alterations in activated microglia. Although lipopolysaccharide (LPS)-stimulated BV-2 cells represent one of the most widely used in vitro models of neuroinflammation, a comprehensive understanding of the coordinated molecular adaptations underlying this model remains incomplete. The aim of this study was to characterize the proteomic remodeling of BV-2 cells exposed to LPS 100 ng/mL for 24 h and investigate the relationship between antioxidant and inflammatory responses across increasing levels of LPS exposure. Proteomic analysis identified 89 differentially expressed proteins involved in cytoskeletal organization, clathrin-mediated endocytosis, lysosomal function, immunometabolism, and redox regulation. Validation experiments confirmed changes in cytoskeletal and endocytic proteins, including Vimentin, Gelsolin, Disabled homolog-2, and clathrin light chain A, together with modulation of proteins involved in the lysosomal pathways and marked induction of the immunometabolic enzyme aconitate decarboxylase 1. Functional enrichment analysis identified Nuclear factor erythroid 2-related factor 2 (NRF2) as the most significantly activated upstream regulator, a prediction consistent with engagement of an adaptive antioxidant program. Interestingly, analysis of increasing LPS concentrations revealed that antioxidant responses were not proportional to the inflammatory stimulus. Expression of NRF2-controlled antioxidant enzymes progressively increased for LPS concentrations up to 500 ng/mL, but declined at 1000 ng/mL LPS, whereas pro-inflammatory mediators remained continuously upregulated. This pattern is consistent with the attenuation of antioxidant adaptation under the highest inflammatory burden tested. Together, these findings demonstrate that the BV-2/LPS model is characterized by a dynamic interplay between inflammatory, redox, metabolic, lysosomal, endocytic, and cytoskeletal pathways, providing a more comprehensive characterization to support the interpretation and experimental use of this neuroinflammatory model.

Inflammatory bowel disease (IBD) involves intestinal barrier dysfunction and chronic inflammation. Manna, derived from the solidified phloem sap of Fraxinus species, is rich in mannitol and polyphenols and valued for its laxative, antioxidant, and anti-inflammatory properties. In this study, manna was digested in vitro to obtain its bioaccessible fraction (BFM), whose anti-inflammatory activity was tested in a Caco-2/RAW264.7 co-culture model. Caco-2 cells were pretreated with BFM (1/20 v/v, 6 mg/mL) 90 min before LPS stimulation (1 µg/mL, 24 h) of macrophages, using budesonide (1 μM) as reference. BFM pretreatment significantly reduced IL-8 secretion (70.8%) in Caco-2 cells, and IL-6 (43.1%) and TNF-α (83.1%) in RAW264.7 macrophages. It also improved redox balance in Caco-2 cells by decreasing iNOS (48.2%), NOx (33.2%), and ROS (26.4%), while stabilizing tight junctions through occludin upregulation (18.3%). Mechanistically, BFM downregulated NF-κB-COX-2-PGE₂ signaling in macrophages, reducing NF-κB p65 nuclear translocation (65.6%), COX-2 levels (79.3%), and PGE₂ production (50.8%). Co-treatment with budesonide showed antagonism for most markers (Combination Index (CI), 0.41–0.76), but additive/synergistic effects on ROS (CI, 1.06 ± 0.06) and NOx (CI, 1.10 ± 0.04). These findings highlight manna’s strong anti-inflammatory activity at a low, non-laxative dose (3.8 g/day), supporting its nutraceutical potential in IBD management.

Intensive broiler chicken farming is one of the most important livestock sectors globally. However, intensive production systems raise concerns about farm sustainability, as well as ensuring animal welfare and product quality. For this reason, identifying novel, high-value-added feed ingredients is crucial. Winery by-products (WBPs) are a valuable source of bioactive compounds and can be utilized as functional feed ingredients. This study evaluated the effects of dietary supplementation with grape seed meal and grape pomace meal in diets for broilers up to 42 days of age. Three dietary treatments were formulated—grape seed meal (3% and 6%), grape pomace meal (3% and 6%), and a combination (3% seed meal + 3% pomace meal)—along with a standard diet (control). The proximal composition (moisture, protein, fatty acid profile, fats, ash), antioxidant parameters (ROS, GSH, NO, POV), free radical scavenging activity (DPPH and ABTS^•+), and total phenolic content of the meat and physical characteristics (color) were assessed. While proximal composition of meat was not significantly influenced by the dietary treatment, some parameters, such as total phenolic content, PUFA levels, and antioxidant and free radical scavenging activity, were improved. These results demonstrate enhanced favorable traits improving chicken meat quality and confirm the potential of WBPs as functional feed ingredients, promoting a more sustainable production model aligned with the principles of the circular economy.

To date, there is extensive scientific evidence affirming that physical exercise plays a fundamental role in both the prevention and treatment of various pathological conditions in humans as well as in animals. It is understood that the advantages of movement and exercise have a multifactorial origin and they depend on a category of bioactive molecules vehicolated by extracellular microvesicles known as exosomes. The exosomes act as potential delivery systems for messages within the organism. These findings have drawn significant attention, leading researchers to further investigate the role of exosomes, delving into the study of microRNAs (miRNAs). In particular, these molecules are found inside exosomes and play a key role in cellular communication, with an impact on numerous physiological functions of the organism. It has been suggested that during physical exercise, the expression levels of miRNAs increase in parallel with those of exosomes, and their release enables intercellular communication in multicellular organisms, thereby regulating both cell growth and division. Studies have not only been carried out in humans, but also in laboratory animals and in mammals following exercise. Specifically, a change in exosome expression has been found in athletic horses following physical exercise. The aim of the current review was to highlight what is known about the role played by exosomes and miRNAs during physical exercise in equine species by considering, on a broad scale, the published data on this topic, including comparative data from humans and rodent models.

This research proposed a feasibility study for a qualitative and semiquantitative analysis of sheep serum using Raman Spectroscopy analysis as an alternative to standard methodologies. Raman Spectroscopy was used to obtain information about molecular vibrations that can provide information about the behavior of specific variations in the protein network. This study was conducted during the traditional vernal shearing procedure in Sicilian sheep breeding. Twenty female sheep were randomly chosen from a one-hundred Comisana-bred flock habituated to the handling required for shearing and venipuncture. Animals had a mean body weight of 52.35 ± 4.55 kg, were aged between 2 and 3 years old, and were clinically healthy with no evidence of disease and free from internal and external parasites. All animals were shorn on the same day by hand using traditional shearing scissors in a 15 m × 10 m pen. The animals were released into an adjacent pen at the end of the shearing procedure. For each animal, blood samples were collected through jugular venipuncture into a vacutainer tube with a clot activator (Terumo Corporation, Japan) immediately before and 5 and 60 min after the end of the shearing procedure. On the obtained sera, ELISA and Raman spectroscopy analyses were performed to evaluate cortisol concentrations. The band area corresponding to the cortisol vibration mode was identified in the 1300–1366 cm⁻¹ band. The Raman spectra obtained during the various protocol data points showed the same trend, with differences in the intensity of the band area 1300–1366 cm⁻¹. A positive correlation was found between ELISA and Raman assessment in all experimental conditions. The obtained results demonstrate that Raman spectroscopy analysis could be a suitable tool for biomolecule identification. This study demonstrated that this technique provides useful information for understanding sheep responses to stress induced by management conditions.

: Early studies have shown that erythrocytes have caspase-3 and caspase-8 and are capable of dying through an apoptotic-like cell death triggered by Ca²⁺ ionophores. This cell death is associated with apoptosis-like morphological signs, including cell shrinkage, membrane blebbing, and phosphatidylserine externalization. To emphasize that mature erythrocytes don’t have the apoptotic mitochondrial machinery and distinguish this unique cell death modality from apoptosis, it was named “eryptosis”. Over recent decades, our knowledge of eryptosis has been significantly expanded, providing more insights into the uniqueness of cell death pathways in erythrocytes. In this review, we aim to summarize our current understanding of eryptosis, formulate the nomenclature and guidelines to interpret results of eryptosis studies, provide a synopsis of morphological and biochemical features of eryptosis, and highlight the role of eryptosis in health and disease, including its druggability.

In recent years, the invasive Atlantic blue crab (Callinectes sapidus) has increased its spread throughout the Mediterranean Sea, threatening native biodiversity and local economies. This study aimed to valorize C. sapidus sampled in Sicily by utilizing its exoskeleton as a source of chitosan, astaxanthin, and bio-phenolic compounds. These biomolecules were evaluated for their reducing, radical scavenging, and antitumor activity. The ferric ion reducing antioxidant power (FRAP) and the free radical scavenging activity against radical 2,2-Diphenyl-1-picrylhydrazyl (DPPH) were significantly higher for chitosan (3.16 ± 0.10 mg AAE/g and 8.1 ± 0.10 µmol TE/g). No significant differences were observed among the tested biomolecules in their activity in scavenging the radical 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). Both bio-phenolic compounds and astaxanthin exhibited dose-dependent cytotoxicity on CaCo-2 (IC50 = 12.47 and 18 µg/mL) and HepG2 (IC50 = 10.25 and 1.26 µg/mL) cell lines, while only bio-phenols showed no cytotoxic effect on differentiated CaCo-2 cells up to 20 µg/mL. These findings highlight the value of blue crab by-products in supporting a circular economy, offering a sustainable approach to managing this invasive species while providing bioactive compounds with promising medical and nutraceutical applications.

Ferulago nodosa (L.) Boiss. is a species belonging to the Apiaceae family which grows spontaneously in the Balkan and southern Tyrrhenian areas of the Mediterranean Sea. Plants of the Ferulago genus are widely used in the ethnopharmacological field, mainly in the form of infusions, as remedies against panic attacks, to solve problems related to the gastrointestinal tract, and for ulcer complications. In this study, we assessed antioxidant and antiproliferative activities of the pressafonin‐A (PA), extracted from F. nodosa roots, following its spectroscopical characterization. Among the assays conducted, reducing capacity tests and free radical scavenging assays showed that PA can act as an antioxidant. PA also exhibited time‐ and concentration‐dependent antiproliferative activity against human cancer cell lines, including A‐375 (melanoma), HCT‐116 (colorectal carcinoma), and MCF‐7 (breast adenocarcinoma). Selectivity index (SI) was calculated using normal 16‐HBE cells. Among the cell lines tested, PA demonstrated an SI > 10 in A‐375 cells. Treatment with PA 6.2 μM (IC ₂₅ ) and 12.4 μM (IC ₅₀ ) for 24‐h induced apoptosis, as cytofluorimetrically evidenced by phosphatidylserine exposure, loss of mitochondrial membrane potential, and intracellular ROS production. Moreover, PA caused an arrest of cell cycle at the G1/S phase transition. Western blot analysis showed that PA caused an increase in protein levels of p53, p21 ^Waf1/Cip1 , cytochrome c, and BAX, along with a decrease in Bcl‐2 protein. In conclusion, natural PA demonstrated antioxidant and antiproliferative properties on A‐375 by inducing apoptosis via ROS‐dependent intrinsic mitochondrial pathway and cell cycle arrest. These results suggest that PA may have potential as a chemopreventive agent for skin diseases.

This study evaluates, for the first time, the reducing capacity, radical scavenger activity, and in vitro antitumor and anti-inflammatory effects of chitosan, astaxanthin, and bio-phenols extracted from the exoskeleton of Sicilian Procambarus clarkii, the most widespread species of invasive crayfish in the Mediterranean region. Among the extracted compounds, astaxanthin exhibited the highest antioxidant activity in all assays. Chitosan and polyphenols demonstrated reducing and radical scavenging activity; chitosan showed significant ferric ion reducing capacity in the FRAP test, while bio-phenolic compounds displayed notable radical scavenging activity in the DPPH and ABTS assays. Both astaxanthin and polyphenols showed dose-dependent cytotoxicity on two different cancer cell lines, with IC50 values of 1.45 µg/mL (phenolic extract) and 4.28 µg/mL (astaxanthin extract) for HepG2 cells and 2.45 µg/mL (phenolic extract) and 4.57 µg/mL (astaxanthin extract) for CaCo-2 cells. The bio-phenolic extract also showed potential anti-inflammatory effects in vitro by inhibiting nitric oxide production in inflamed RAW 264.7 macrophages, reducing the treated/control NO ratio to 77% and 74% at concentrations of 1.25 and 1.5 μg/mL, respectively. These results suggest that P. clarkii exoskeletons could be a valuable source of bioactive molecules for biomedical, pharmaceutical, and nutraceutical application while contributing to the sustainable management of this invasive species.