Publications

Abstract Quorum sensing (QS) is a bacterial cell-to-cell communication induced at high cell density, commonly involved in regulating gene expression of spoilage and pathogenic virulence factors. In dairy products, Pseudomonas species grow under cold conditions and produce thermostable proteases. The QS pathway in Pseudomonas represents a potential target to limit protease synthesis during raw milk storage prior to heat processing. This study aims to characterize AHL-mediated QS system in highly proteolytic Pseudomonas isolated from raw milk and to explore the use of polyphenols as a strategy to control proteolytic activity. Six isolates with high proteolytic activity were identified as Pseudomonas gessardii by Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) and 16S rRNA sequencing. Biosensor strains, thin layer chromatographyoverlay assay, and ultra performance liquid chromatography-mass spectrometry (UPLC-MS) analysis were used to explore QS, revealing C4-HSL as the main type of AHL produced by P. gessardii. Salicylic acid (SA), cinnamaldehyde (CIN), and tannic acid (TA) were tested as QS inhibitors (QSIs) and the mechanism verified by in silico analysis. Various degrees of proteolytic activity inhibition were observed at 4 degrees C and 25 degrees C by using QSIs (15-58 % by SA, 10-60 % by CIN), with no antibacterial effect. The mechanism behind that is the competition with the C4-HSL to bind with the receptor protein (LuxR) as corroborated by the in silico analysis. The results highlight the potential to employ polyphenols to restrict proteolytic activity by psychrotrophic Pseudomonas in dairy products.

Abstract New psychoactive substances (NPS) are designed to evade legal regulation while mimicking the effects of classic illicit drugs such as 3,4-methylenedioxymethamphetamine (MDMA). This category includes phenethylamine derivatives, such as the psychedelic 2C and NBOMe drugs. Given the lack of data regarding the toxicological profile of these substances, the goal of this study was to evaluate the neurotoxicity of 2C-I and 25I-NBOMe and explore their neurotoxic pathways. Lower EC50 values, in both NR uptake and MTT reduction assays in differentiated SH-SY5Y cells and primary rat cortical cultures, revealed that 25I-NBOMe is significantly more cytotoxic than 2C-I, likely due to its higher lipophilicity. Both drugs triggered severe mitochondrial dysfunction, characterized by decreased intracellular ATP levels and mitochondrial membrane depolarization, although no significant changes in intracellular ROS/RNS levels were observed. Additionally, 25I-NBOMe increased the intracellular Ca2* levels. Apoptosis was an observed mechanism of cell death for both drugs, as demonstrated by a significant increase in the number of cells undergoing early apoptosis (AnV+/PI-) and late apoptosis/necrosis (AnV+/PI+). However, only 2C-I induced autophagy and strongly triggered caspase-3 activation. This suggests that 2C-I induces caspase-3-dependent apoptosis, whereas 25I-NBOMe may also induce apoptosis through a caspase-3-independent pathway, possibly involving increased intracellular Ca2* levels and direct mitochondrial damage. These findings underscore the complex interplay between mitochondrial dysfunction, calcium dysregulation, and cell death pathways, highlighting the central role of mitochondria in the cytotoxicity of 2C-I and 25INBOMe.

Abstract Polypharmacological approaches have significant potential for the treatment of various complex diseases, including infectious bacteria-related diseases. Actually, multitargeting agents can achieve better therapeutic effects and overcome the drawbacks of monotherapy. Although multidrug multitarget strategies have demonstrated the ability to inactivate infectious bacteria, several challenges have been pointed out. In this way, multitarget direct ligands approaches appear to be a rational and sustainable strategy to combat antibiotic resistance. By combining different pharmacophores, antibiotic hybrids stand out as a promising application in the field of bacterial infections. These new chemical entities can achieve synergistic interactions that allow to extend the spectrum of action and target multiple pathways. In addition, antibiotic hybrids can reduce the likelihood of resistance development and provide improved chemical stability. It is worth highlighting that despite the efforts of the scientific community to discover new solutions for the most complex diseases, there is a significant lack of studies on biofilm-associated infections. This review describes the different polypharmacological approaches that can be used to treat bacterial infections with a particular focus, whenever possible, on those promoted by biofilms. By exploring these innovative approaches, we aim to inspire further research and progress in the search for effective treatments for infectious bacteria-related diseases, including biofilm-related ones. Significance Statement: The importance of the proposed topic lies in the escalating challenge of antibiotic resistance, particularly in the context of infectious bacteria-related infections. Polypharmacological approaches, such as antibiotic hybrids, represent innovative strategies to combat bacterial infections. By targeting multiple signaling pathways, these approaches not only enhance therapeutic effect but also reduce the development of resistance while improving the drug's chemical stability. Despite the urgent need to combat bacterial infectious diseases, there is a notable research gap, in particular in biofilmrelated ones. This review highlights the critical importance of exploring polypharmacological approaches with the aim of motivating further research and advances in effective treatments for infectious bacteria, including biofilm related infections.

Abstract Antimicrobial photodynamic inactivation (aPDI), using photosensitisers in combination with antibiotics, is a promising multi-target strategy to address antibiotic resistance, particularly in wound infections. This study aimed to elucidate the antibacterial mode of action of combinations of berberine (Ber) or curcumin (Cur) with selected antibiotics (Ber-Ab or Cur-Ab) under blue light irradiation (420 nm) against Staphylococcus aureus, including methicillin-resistant (MRSA) and methicillin-susceptible (MSSA) strains. Multiple physiological parameters were assessed using complementary assays (fluorometry, epifluorescence microscopy, flame emission and atomic absorption spectroscopy, zeta potential, flow cytometry, and the plate agar method) to examine the effect on ROS production, membrane integrity, DNA damage, motility and virulence factors of S. aureus. Results indicated that blue light photoactivated Ber-Ab and Cur-Ab combinations led to substantial ROS generation, even at low concentrations, causing oxidative stress that severely impacted bacterial membrane integrity (approximately 90 % in MRSA and 40 % in MSSA). Membrane destabilization was further confirmed by elevated intercellular potassium release (approximate to 2.00 and 2.40 mu g/mL in MRSA and MSSA, respectively). Furthermore, significant DNA damage was observed in both strains (approximate to 50 %). aPDI treatment with blue light also reduced S. aureus pathogenicity by impairing motility and inhibiting key virulence factors such as proteases, lipases, and gelatinases, all of which play key roles in the infectious process. Overall, Ber-Ab combinations demonstrated the highest efficacy across all parameters tested, highlighting for the first time the multi-target therapeutic potential of this phytochemical-based aPDI strategy to combat antibiotic-resistant S. aureus infections and improve wound infection treatment outcomes.

Abstract The ability of Salmonella enterica subsp. enterica to persist and form biofilms on different surfaces can constitute a source of food contamination, being an issue of global concern. The objective of this study was to understand the biofilm formation profile of 14 S. enterica strains among different serovars and sources and to evaluate the ability of essential oil (EO) components (carveol, citronellol, and citronellal) to disinfect the biofilms formed on stainless steel and polypropylene surfaces. All the strains were able to form biofilms with counts between 5.34 to 6.78 log CFU cm(-2). Then, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of EO components were evaluated on two selected strains. All compounds inhibited the growth of Salmonella Typhimurium (strain 1; MIC = 800-1000 mu g ml(-1)) and Salmonella Enteritidis (strain 5; MIC = 400-1000 mu g ml(-1)) and only carveol showed bactericidal activity against strains 1 and 5 (MBC = 1200 mu g ml(-1)). Biofilms were exposed to the EO components at 10 x MIC for 30 min and polypropylene surfaces were more difficult to disinfect showing reductions between 0.9 and <1.2 log CFU cm(-2). In general, the S. enterica biofilms demonstrated a significant tolerance to disinfection, demonstrating their high degree of recalcitrance on food processing surfaces.

Abstract Introduction: Quorum sensing (QS) is a bacterial communication mechanism that regulates gene expression, playing a crucial role in various physiological processes. Interfering with this signalling pathway is a promising strategy to control bacterial pathogenicity and virulence. Objectives: This study evaluated the potential of two cinnamic acid derivatives, ferulic and sinapic acids, to inhibit the las and pqs systems in Pseudomonas aeruginosa. Their effects on biofilm architecture, virulence factor production and bacterial motility were also investigated. Methods: Bioreporter strains and bioluminescence-based assays were used to evaluate the modulation of QS-activity by cinnamic acid-type phenolic acids. In addition, in silico docking analysis was performed to validate the binding interactions of the cinnamic acid derivatives with QS-receptors. The biofilm architecture was analysed by optical coherence tomography, and virulence factors production (pyoverdine, pyocyanin, total proteases, lipases, gelatinases and siderophores) and motility were measured by absorbance measurement and plate agar method. Results: Ferulic and sinapic acids at 1000 µg mL−1 inhibited the las and pqs systems by 90 % and 80 %, respectively. The N-3-oxododecanoyl-homoserine lactone production was reduced by 70 % (6.25 µg mL-¹). In silico analysis demonstrated that cinnamic acid derivatives exhibited comparable interactions and higher docking scores than reference ligands and inhibitors. Biofilm thickness decreased from 96 µm to 11 µm, and virulence factors and swarming motility were significantly impaired. The comparable anti-QS activity of cinnamic acid derivatives suggests that the additional methoxy group in sinapic acid does not directly contribute to its anti-QS effect. Conclusion: Ferulic and sinapic acids compromised the biofilm architecture and virulence of P. aeruginosa through QS inhibition. © 2025

Abstract Aim This study investigates the mechanisms of action of a promising series of previously synthesized quaternary ammonium (QASs) and phosphonium (QPSs) salts, which have shown potent activity against Staphylococcus aureus, including methicillin-resistant strains (MRSA).Methods and results The effects of QASs and QPSs on S. aureus surface charge, total surface hydrophobicity, intracellular potassium release, membrane integrity, and ultrastructure were examined. QASs and QPSs significantly altered bacterial surface properties by reducing negative surface charge, disrupting membrane integrity, and inducing potassium leakage and propidium iodide uptake. Furthermore, S. aureus became less hydrophilic due to changes in surface hydrophobicity. Transmission electron microscopy revealed cytoplasmic leakage and the presence of electron-dense extracellular material around damaged bacterial cells upon exposure to high concentrations of these salts.Conclusions The antimicrobial activity of QASs and QPSs is driven by their ability to alter bacterial surface properties, destabilizing and disrupting membranes.

Abstract S-1 MitoPlates™ from Biolog enable the characterization of mitochondria’s function in live cells by measuring the rates of electron flow into and through the electron transport chain from different NADH or FADH2 producing metabolic substrates. This technology uses 96-well microplates pre-coated with triplicate repeats of a set of 31 substrates. Those 31 metabolic substrates have different routes of entry into the mitochondria, use different transporters, and are also oxidated by different dehydrogenases, producing reducing equivalents in the form of NADH or FADH2. The electrons produced upon oxidation of NADH or FADH2 at complex I or II, respectively, then travel to cytochrome c, where a tetrazolium redox dye (MC) can act as terminal acceptor, turning purple and absorbing at 590 nm. This mechanism allows the evaluation of cellular substrate preference by following the kinetics of MC reduction in the presence of selected substrates. In this chapter, we describe the step-by-step protocol to prepare an experiment using MitoPlate S-1 array and the OmniLog instrument to assess the metabolism of human dermal fibroblasts. We also give detailed information on how to analyze the raw data generated by the Biolog Data Analysis software to extract meaningful information and produce useful data visualizations, using reproducible methods based on a single structured dataset. © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2025.

Abstract Metabolic syndrome (MetS) has been associated with disruptions in tissue mechanical homeostasis and inflammatory and metabolic derangements. However, the direct correlation between metabolic alterations and changes in tissue stiffness, and whether they could play a role as upstream initiators of disease pathology remains to be investigated. This emerging concept has yet to be put into clinical practice as many questions concerning the interplay between extracellular matrix mechanical properties and regulation of metabolic pathways remain unsolved. This review will highlight key foundational studies examining mutual regulation of cell metabolism and mechanotransduction, and opening questions lying ahead for better understanding MetS pathophysiology.

Abstract Mitochondrial dysfunction and increased reactive oxygen species (ROS) generation play an import role in different human pathologies. In this context, mitochondrial targeting of potentially protective antioxidants by their coupling to the lipophilic triphenylphosphonium cation (TPP) is widely applied. Employing a six-carbon (C6) linker, we recently demonstrated that mitochondria-targeted phenolic antioxidants derived from gallic acid (AntiOxBEN2) and caffeic acid (AntiOxCIN4) counterbalance oxidative stress in primary human skin fibroblasts by activating ROS-protective mechanisms. Here we demonstrate that C6-TPP (but not AntiOxBEN2 and AntiOxCIN4) induce cell death in human skin fibroblasts. This indicates that C6-TPP cytoxocity is counterbalanced by the antioxidant moieties of AntiOxBEN2 and AntiOxCIN4. Remarkably, C6-TPP and AntiOxBEN2 (but not AntiOxCIN4) induced a glycolytic switch, as exemplified by a reduced cellular oxygen consumption rate (OCR), increased extracellular acidification rate (ECAR), elevated extracellular lactate levels, and higher protein levels of glucose transporter 1 (GLUT-1). This switch involved activation of AMP-activated protein kinase (AMPK) and fully compensated for the loss in mitochondrial ATP production by sustaining cellular ATP content. When glycolytic switch induction was prevented ( i.e. by using a glucose-free, galactose-containing medium), AntiOxBEN2 induced cell death whereas AntiOxCIN4 did not. We conclude that, despite their similar chemical structure and antioxidant capacity, AntiOxBEN2 and AntiOxCIN4 display both common (redox-adaptive) and specific (bioenergetic-adaptive) effects.