Giuseppe Paglia

Trehalose, a natural disaccharide with well-established neuroprotective effects in the central nervous system (CNS), has been extensively investigated for its anti-inflammatory, protein-stabilizing, and autophagy-inducing activities. Despite these beneficial CNS effects, its potential impact on the blood−brain barrier (BBB), a key regulator of brain homeostasis and neurological health, remains poorly understood. Here, we demonstrate that trehalose reinforces the in vitro BBB by increasing transendothelial electrical resistance and reducing dextran permeability in hCMEC/D3 monolayers. These functional improvements were associated with AMPK activation and upregulation of the tight junction protein ZO-1, where findings were corroborated in vivo by increased ZO-1 expression in trehalose-treated C57BL/6J mice. Molecular docking further suggested the preferential binding of trehalose to the ZO-1 PDZ3-SH3 domain, supporting a potential mechanism for tight junction stabilization. These findings establish trehalose as a promising BBB-protective agent through complementary molecular mechanisms. Nevertheless, the therapeutic exploitation of free trehalose is hindered by its unfavorable pharmacokinetic properties. To address these limitations and improve its delivery, we engineered four self-assembling trehalose nanoparticles (Treh-NAs) based on mono- or bis-betulinic acid and mono- or bis-squalene conjugates. Nanoparticle encapsulation preserved the barrier-enhancing activity of trehalose while markedly reducing the effective dose in vitro. Among the formulations, the mono-betulinic acid nanoparticles displayed the highest endothelial permeability and the strongest barrier-reinforcing effect. Collectively, these results provide a rationale for the development of trehalose-based nanomedicines aimed at reinforcing the BBB and improving therapeutic interventions for CNS diseases associated with BBB dysfunction, in which the neuroprotective properties of trehalose may provide additional therapeutic benefit.

The overlap between the geographic distribution of COVID-19 outbreaks and pollution levels suggested a strong correlation between exposure to atmospheric particulate matter and an increased risk of developing severe forms of disease. This correlation has been highlighted by several epidemiological studies, indicating the existence of shared molecular mechanisms. Emerging evidence has highlighted the important role of lipid rafts in facilitating viral entry into cells. Specifically, the receptor binding domain of the SARS-CoV-2 spike protein interacts with sialylated glycans of the monosialic ganglioside GM1 and GM2 that are particularly enriched in lipid rafts. This interaction has been proposed to facilitate ACE2 recognition by the spike protein and may contribute to early events involved in viral attachment and entry. Here, we reveal that A549 alveolar lung cells, after DEP exposure, exhibit a significant shift in ACE2 into lipid rafts, accompanied by an increase in the immature form of ADAM17, the sheddase responsible for ACE2 cleavage. Additionally, DEP exposure results in a significant increase in IL-6 release, while no changes were observed in IL-8 and sACE2 release. This treatment does not cause significant alterations in protein levels or membrane redistribution of COX-2 and HO-1, proteins involved in the inflammatory response and oxidative stress following exposure to air pollution, and linked to COVID-19 pathogenesis. Finally, lipidomic analysis by UHPLC-MS revealed that DEP exposure induces a significant increase in GM2 levels, and a concomitant decrease in GM1 and GM3 levels. Together, these results indicate that DEP exposure remodels lipid raft-associated molecular features in A549 cells, including ACE2 membrane redistribution, altered ganglioside composition, and increased IL-6 release. Although these changes may be relevant to cellular mechanisms associated with SARS-CoV-2 susceptibility, the present study does not directly assess viral binding, viral entry, or infection, and further functional studies are required.

Nicotinamide adenine dinucleotide (NAD⁺) and its reduced form, NADH, are essential coenzymes that play central roles in cellular redox homeostasis, energy metabolism, DNA repair, and signaling. Cellular NAD⁺ levels are maintained by a dynamic balance between the de novo Preiss–Handler, and salvage synthesis pathways, and consumption by enzymes like sirtuins, PARPs, and CD38. Among these, the nicotinamide Phosphoribosyltransferase (NAMPT)-driven salvage pathway represents the predominant route of NAD+ synthesis. The specific regulation of NAD (NAD⁺ and NADH) levels across distinct subcellular compartments has emerged as a critical determinant of cellular function but it remains poorly understood. Dysregulation of NAD metabolism is a hallmark of aging and various pathologies, including cancer, neurodegenerative disorders, and metabolic diseases, making strategies to modulate NAD levels a promising therapeutic frontier. This review provides the first integrated overview of NAD concentrations across cellular compartments (cytosol, mitochondria, nucleus, endoplasmic reticulum, Golgi, peroxisomes, and the extracellular space) together with measurement and modulation strategies. We summarize current knowledge on NAD distribution within organelles, address key challenges in accurate quantification, and highlight established and emerging approaches for both global and compartment-specific analysis. Finally, we discuss therapeutic strategies, from NAD⁺ precursor supplementation to enzyme modulators and gene therapy, highlighting both their translational potential and current limitations in treating diverse diseases and prolonging life and health span.

GATA2 deficiency predisposes patients to recurrent infections, myelodysplastic neoplasms (MDSs), and malignancies through disrupted hematopoiesis and immune dysfunction. The role of the gut microbiome (GM) in this condition remains poorly defined. In this multicenter study, we analyzed GM composition, metabolomic, and lipidomic profiles in 12 Italian GATA2-deficient patients, comparing non-HSCT and post-HSCT GATA2-deficient individuals with healthy controls. Non-HSCT patients showed a relative enrichment of Proteobacteria-associated Gram-negative taxa, accompanied by increased levels of metabolites and lipids previously associated with inflammatory processes. Post-HSCT patients displayed profiles with a trend toward partial normalization of GM composition and metabolic features. Overall, our findings suggest the presence of microbiome and metabolic patterns in GATA2-deficient patients, which may reflect underlying immune and hematopoietic alterations, although these observations should be interpreted as descriptive and require validation in larger cohorts.

Cardiac rehabilitation (CR) is highly beneficial in postmyocardial infarction (MI) patients; however, its metabolic impact remains underexplored. This study investigated metabolic and lipidomic adaptations to an intensive CR program in 25 nondiabetic male patients (N-acetyl-l-tyrosine (NAT), suggesting a mitohormesis response to exercise-induced mitochondrial stress. The third training session exhibited the highest metabolic adaptation, primarily in energy metabolism pathways like the TCA cycle, indicating enhanced oxidative energy generation and improved exercise performance. The lipidome displayed an acute response to the first training, with upregulation of phosphatidylserines (PS). Predicted increased activity of phosphatidylserine synthase-1 (PSS1), enzymes vital for PS synthesis, underscores PS's protective role in myocardial damage and its contribution to muscle activity. These findings highlight CR's beneficial metabolic adaptations, potentially via mitohormesis, and suggest possible mechanistic targets and candidate biomarkers requiring investigation in future controlled intervention studies.

Mass spectrometry-based metabolomics and lipidomics are central analytical tools for characterizing cellular chemical composition, yet achieving subcellular resolution remains a major challenge.

Mesenchymal Stromal Cells (MSC)-derived Extracellular Vesicles (EV) represent innovative tools for drug delivery systems. However, their clinical use is limited by the lack of standardized good manufacturing practice (GMP)-compliant isolation and conservation protocols. In this study, we developed a GMP-compliant protocol for the preparation of MSC-EVs and investigated the feasibility of producing EVs loaded with paclitaxel (PTX) for clinical application as drug products. Adipose tissues from 13 donors were used to obtain MSC-EVs via culture supernatant ultracentrifugation. EVs loaded with PTX were manufactured by adding the drug to the culture medium of MSCs before supernatant collection. EV identity was verified in terms of concentration/size, protein content, morphology, and expression of EV surface markers. The anti-proliferative activity, accumulation ability in tumor cells and PTX content, as well as their stability over time, were also evaluated. High numbers of EV/EV-PTX compliant in terms of integrity/identity were obtained and can be successfully stored for up to one year at -80 °C. Cellular studies have shown that EVs are capable of accumulating in tumor cells and, when loaded with PTX, inhibiting the proliferation of a pleural mesothelioma cell line. These results support the potential future clinical use of EVs as carriers for drug delivery to improve cancer treatment strategies.

Microsampling, especially dried blood spots (DBS), emerged in recent years as a viable alternative to conventional blood collection since it is rapid, simple, minimally invasive, and has user-friendly characteristics. Moreover, DBS are able to avoid analyte degradation thanks to their great stability. Due to their versatility, clinical applications with DBS have increased, including mass spectrometry–based metabolomics and lipidomics studies. In this work, we evaluated and optimized extraction protocols testing five different extraction solutions to perform metabolomics and lipidomics studies on the same spot considering three commercially available microsampling devices, Capitainer, Whatman, and Telimmune. Parallelly, we also evaluated the short-term stability of the three devices at room temperature for up to 5 days. Our results showed that pure methanol was the best compromise to simultaneously extract from the same spot both the lipidome and polar metabolome. However, we also propose a two-step protocol combining methanol and water extraction that improves polar metabolite extraction and shows improved reproducibility in Capitainer and Whatman. Short-term stability results highlighted that both polar metabolites and lipids were stable for up to 6 days using the Capitainer device, while with Whatman and Telimmune, some significant variations were observed after 3 days for some classes of metabolites/lipids, suggesting the need for cold-chain storage when working with these devices.