Michela Bruschi

Phenylketonuria (PKU) is a genetic metabolic disorder caused by the lack of functional phenylalanine hydroxylase (PAH). Elevated levels of phenylalanine (Phe) are known to be neurotoxic; however, the molecular mechanisms underlying Phe’s effects remain elusive. This study investigates the impact of PKU on proteostasis, redox balance, and metabolism in BTBR PAH ^enu2 mice, a severe disease animal model. Combined proteomics and metabolomics revealed impaired redox homeostasis in the brain and disrupted mitochondrial energy metabolism (ATP and TCA intermediates). The dysregulation was further supported by decreased levels of ATP, reduced glutathione (GSH), cysteine, and reduced catalase activity. Western blot analyses revealed substantial remodeling of protein degradation systems: the 19S regulatory (Rpt1) subunit and 26S proteasome content and activity were significantly increased, and ubiquitinated protein levels were elevated, indicating protein turnover and activation of the ubiquitin-proteasome system. Autophagy was also activated, as evidenced by a reduced LC3-II/LC3-I ratio, decreased p62 levels, unchanged ATG5 levels, and increased HSPA8 protein expression. By contrast, UPR markers remained stable despite an increase in the oxidized-to-reduced PDI ratio, suggesting a localized shift without activation of a full ER stress response. In parallel, systemic alterations were assessed in whole blood. Indeed, GSH, cysteine, ATP and ADP were decreased in PKU, whereas NADPH increased. These changes were accompanied by reduced activities of GSH reductase and GSH peroxidase, thereby confirming metabolic and redox disruption. Collectively, these findings indicate that PKU is associated with activation of protein degradation pathways as an adaptive response to cellular stress combined with redox imbalance and energy dysregulation. Highlights: - Proteasome complex levels and activity were upregulated in PAH ^enu2 mouse brains - Autophagy was also enhanced in PAH ^enu2 mouse brains - Despite increased PDI oxidation, the Unfolded Protein Response was not activated - Redox and energy metabolism were disrupted in both brain and blood

The persistence of P. aeruginosa infections is largely driven by the secretion of several factors during invasion, including the redox-active phenazine pyocyanin (PYO), which promotes biofilm formation and oxidative stress. Biofilms contribute to chronic infections and antibiotic resistance, limiting the efficacy of conventional therapies. We found that a synthetic compound, I-152, a conjugate of N-acetyl-L-cysteine (NAC) and S-acetylcysteamine (also known as S-acetyl-β-mercaptoethylamine; SMEA), effectively restored colistin susceptibility against P. aeruginosa by altering biofilm nanomechanical properties. These perturbations in matrix integrity were associated with I-152’s ability to hinder the phenazine redox cycle, shifting PYO to a reduced state as well as enabling S-conjugate formation. The compound decreased PYO accumulation in bacterial cultures and PYO-generated reactive oxygen species (ROS) in macrophage cells. Together with PYO, LPS is another driver of ROS-dependent inflammatory signaling in the host, which leads to an uncontrolled cytokine response and organ damage, especially in patients with cystic fibrosis. I-152 treatment downregulated the expression of LPS-induced inflammatory cytokines, i.e., IL-6 and TNF-α, in bone marrow-derived macrophages (BMDM) isolated from transgenic CFTR ^-/- and CFTR ^+/+ mice. Consistently, I-152 partially counteracted the inflammatory response in the P. aeruginosa LPS-induced acute lung injury murine model. Taken together, these results support I-152 as an adjunctive treatment for P. aeruginosa respiratory infections through a dual mechanism: combating antimicrobial resistance in biofilms and dampening host inflammation in the respiratory system. Highlights: I-152 potentiates colistin activity against P. aeruginosa by compromising the biofilm surface I-152 rewires the pyocyanin (PYO) redox state and forms covalent adducts with it PYO accumulation and PYO-induced ROS generation in macrophages is impaired by I-152 Ex vivo, I-152 dampens excessive pro-inflammatory response to P. aeruginosa LPS in CFTR ^- / ^- and CFTR ⁺ / ⁺ BM-derived macrophages I-152 (140 mg/Kg) attenuates LPS-driven inflammation and lung damage in CFTR ⁺ / ⁺ mice

This study aimed to estimate the ADME properties and safety of I-152, a conjugate of N-acetyl-l-cysteine (NAC) and S-acetylcysteamine (also known as S-acetyl-β-mercaptoethylamine; SMEA), linked by an amide bond. Its potent antioxidant and pro-glutathione effects make it of interest for a range of conditions linked to oxidative stress, such as infectious diseases and inflammation. I-152 was characterized in vitro for its stability in plasma, liver microsomes, and hepatocytes; its protein binding; and its AB BA (apical-to-basolateral and basolateral-to-apical) permeability using Caco-2 cells. Derisking and preliminary safety pharmacology assays were performed through a human ether-à-go-go-related gene assay (hERG) and in vitro cellular toxicity tests. The results demonstrated that I-152 is hydrolyzed in human plasma (half-life of about 9 min), human liver microsomes, and hepatocytes, as well as in rat liver microsomes and hepatocytes. In addition, I-152 was found to be permeable across the Caco-2 monolayer, indicating good intestinal absorption. Furthermore, I-152 did not produce detectable toxic effects at concentrations up to 1 mM in vitro assays using human keratinocytes, alveolar epithelial cells, and immortalized human embryonic kidney cells (HEK293T). These findings support further preclinical evaluation as a potential redox-modulating agent and thiol-based approach for viral infections and other conditions associated with oxidative stress.

Perturbation in redox status elicits multiple cellular pathways, including those involved in the inflammatory response. A thiol-based molecule (I-152), releasing N-acetyl-cysteine (NAC) and β-mercaptoethylamine (MEA), was exploited as a redox-modulating agent, and its effects on pro-inflammatory cytokine expression and secretion in lipopolysaccharide (LPS)-stimulated macrophages (MΦ) were investigated. I-152 inhibited cytokine gene expression as well as protein secretion of the most important inflammatory cytokines in three different MΦ models in vitro and ex vivo. It alleviated inflammation via the c-Jun/AP-1 and NF-κB signaling pathways, depending on the dose, and regulated NLRP3 inflammasome expression, leading to decreased IL-1β and IL-18 release and reduced pyroptotic cell death. Consequently, the influence of redox-modulated MΦ secretome on the crosstalk with endothelial cells was evaluated. Co-culture experiments between THP-1 MΦ, that had been pre-treated with I-152 before LPS stimulation, and Human Vascular Endothelial Cells (HUVECs) showed reduced VCAM/ICAM expression in these cells in concomitance with a less oxidized and inflamed MΦ proteomic portrait. Overall, our findings suggest that I-152 redox modulation could target the AP-1/NLRP3 axis, affecting LPS-induced inflammation in MΦ and influencing HUVEC responses, revealing a complex and bidirectional interchange.

Reduction in oxygen levels is a key feature in the physiology of the bone marrow (BM) niche where hematopoiesis occurs. The BM niche is a highly vascularized tissue and endothelial cells (ECs) support and regulate blood cell formation from hematopoietic stem cells (HSCs). While in vivo studies are limited, ECs when cultured in vitro at low O₂ (2 were treated with N‐(N‐acetyl‐l‐cysteinyl)‐S‐acetylcysteamine (I‐152). Metabolomic analyses revealed that I‐152 increased glutathione levels and influenced the metabolic profiles interconnected with the glutathione system and the redox couples NAD(P)+/NAD(P)H. mRNA analysis showed a lowered gene expression of HIF‐1α and VEGF following I‐152 treatment whereas TRX1 and 2 were stimulated. Accordingly, the proteomic study revealed the redox‐dependent upregulation of thioredoxin and peroxiredoxins that, together with the glutathione system, are the main regulators of intracellular ROS. Indeed, a time‐dependent ROS production under hypoxia and a quenching effect of the molecule were evidenced. At the secretome level, the molecule downregulated IL‐6, MCP‐1, and PDGF‐bb. These results suggest that redox modulation by I‐152 reduces oxidative stress and ROS level in hypoxic ECs and may be a strategy to fine‐tune the environment of an in vitro BM niche able to support functional HSC maintenance.

Sinusoidal endothelial cells are the predominant vascular surface of the bone marrow and constitute the functional hematopoietic niche where hematopoietic stem and progenitor cells receive cues for self-renewal, survival, and differentiation. In the bone marrow hematopoietic niche, the oxygen tension is usually very low, and this condition affects stem and progenitor cell proliferation and differentiation and other important functions of this region. Here, we have investigated in vitro the response of endothelial cells to a marked decrease in O₂ partial pressure to understand how the basal gene expression of some relevant biological factors (i.e., chemokines and interleukins) that are fundamental for the intercellular communication could change in anoxic conditions. Interestingly, mRNA levels of CXCL3, CXCL5, and IL-34 genes are upregulated after anoxia exposure but become downmodulated by sirtuin 6 (SIRT6) overexpression. Indeed, the expression levels of some other genes (such as Leukemia Inhibitory Factor (LIF)) that were not significantly affected by 8 h anoxia exposure become upregulated in the presence of SIRT6. Therefore, SIRT6 mediates also the endothelial cellular response through the modulation of selected genes in an extreme hypoxic condition.

Hematopoietic stem cells (HSCs) reside in a subzone of the bone marrow (BM) defined as the hematopoietic niche where, via the interplay of differentiation and self-renewal, they can give rise to immune and blood cells. Artificial hematopoietic niches were firstly developed in 2D in vitro cultures but the limited expansion potential and stemness maintenance induced the optimization of these systems to avoid the total loss of the natural tissue complexity. The next steps were adopted by engineering different materials such as hydrogels, fibrous structures with natural or synthetic polymers, ceramics, etc. to produce a 3D substrate better resembling that of BM. Cytokines, soluble factors, adhesion molecules, extracellular matrix (ECM) components, and the secretome of other niche-resident cells play a fundamental role in controlling and regulating HSC commitment. To provide biochemical cues, co-cultures, and feeder-layers, as well as natural or synthetic molecules were utilized. This review gathers key elements employed for the functionalization of a 3D scaffold that demonstrated to promote HSC growth and differentiation ranging from 1) biophysical cues, i.e., material, topography, stiffness, oxygen tension, and fluid shear stress to 2) biochemical hints favored by the presence of ECM elements, feeder cell layers, and redox scavengers. Particular focus is given to the 3D systems to recreate megakaryocyte products, to be applied for blood cell production, whereas HSC clinical application in such 3D constructs was limited so far to BM diseases testing.

Despite the great strides in healthcare during the last century, some challenges still remained unanswered. The development of multi-drug resistant bacteria, the alarming growth of fungal infections, the emerging/re-emerging of viral diseases are yet a worldwide threat. Since the discovery of natural antimicrobial peptides able to broadly hit several pathogens, peptide-based therapeutics have been under the lenses of the researchers. This review aims to focus on synthetic peptides and elucidate their multifaceted mechanisms of action as antiviral, antibacterial and antifungal agents. Antimicrobial peptides generally affect highly preserved structures, e.g., the phospholipid membrane via pore formation or other constitutive targets like peptidoglycans in Gram-negative and Gram-positive bacteria, and glucan in the fungal cell wall. Additionally, some peptides are particularly active on biofilm destabilizing the microbial communities. They can also act intracellularly, e.g., on protein biosynthesis or DNA replication. Their intracellular properties are extended upon viral infection since peptides can influence several steps along the virus life cycle starting from viral receptor-cell interaction to the budding. Besides their mode of action, improvements in manufacturing to increase their half-life and performances are also taken into consideration together with advantages and impairments in the clinical usage. Thus far, the progress of new synthetic peptide-based approaches is making them a promising tool to counteract emerging infections.