Adsorption of Polylactic-co-Glycolic Acid on Zinc Oxide Systems: A Computational Approach to Describe Surface Phenomena.

Zinc oxide and polylactic-co-glycolic acid (ZnO-PLGA) nanocomposites are known to exhibit different biomedical applications and antibacterial activity, which could be beneficial for adding to wound dressings after different surgeries. However, possible cytotoxic effects along with various unexpected activities could reduce the use of these prominent systems. This is correlated to the property of ZnO, which exhibits different polymeric forms, in particular, wurtzite, zinc-blende, and rocksalt. In this study, we propose a computational approach based on the density functional theory to investigate the properties of ZnO-PLGA systems in detail. First, three different stable polymorphs of ZnO were considered. Subsequently, the abilities of each system to absorb the PLGA copolymer were thoroughly investigated, taking into account the modulation of electrical, optical, and mechanical properties. Significant differences between ZnO and PLGA systems have been found; in this study, we remark on the potential use of these models and the necessity to describe crucial surface aspects that might be challenging to observe with experimental approaches but which can modulate the performance of nanocomposites.

Chemical Scaffolds for the Clinical Development of Mutant-Selective and Reversible Fourth-Generation EGFR-TKIs in NSCLC.

In nonsmall cell lung cancer (NSCLC), as well as in other tumors, the targeted therapy is mainly represented by tyrosine kinase inhibitors (TKIs), small molecules able to target oncogenic driver alterations affecting the gene encoding the epidermal growth factor receptor (EGFR). Up to now, several different TKIs have been developed. However, cancer cells showed an incredible adaptive tumor response to the inhibition of the sequentially mutated EGFR (EGFRM+), triggering the need to explore novel pharmacochemical strategies. This Review summarizes the recent efforts in the development of new reversible next-generation EGFR TKIs to fight the resistance against T790M and C797S mutations. Specifically, after giving an overview of the role of the EGFR's signaling pathways in cancer progression, we are going to discuss the most relevant approved drugs and drug candidates in terms of chemical structure, binding modalities, and their potency and selectivity against the mutated EGFR over the wild-type form. This could provide important guidelines and rationale for the discovery and iterative development of new drugs.

Inhibition of polymorphic MexXY-OprM efflux system in Pseudomonas aeruginosa clinical isolates by Berberine derivatives.

The MexXY-OprM multidrug efflux pump (EP) in aminoglycosides resistant Pseudomonas aeruginosa is one of the major resistance mechanisms, which is often overexpressed in strains isolated from pulmonary chronic disease such as cystic fibrosis.[1-3] In this research, we focused on the design of potential efflux pumps inhibitors, targeting MexY, the inner membrane component, in an allosteric site. Berberine[4] has been considered as lead molecule since we previously demonstrated its effectiveness in targeting MexY in laboratory reference strains.[5,6] Since this protein is often present in polymorphic variants in clinical strains, we sequenced and modeled all the mutated forms and we synthesized and evaluated by computational techniques, some berberine derivatives carrying an aromatic functionalization in its 13-C ring position. These compounds were tested in vitro against clinical P. aeruginosa strains for antimicrobial and antibiofilm activity. In conclusion, the results demonstrated the importance of the aromatic moiety functionalization in exerting the EP inhibitory activity in synergy with the aminoglycoside tobramycin. More, we found that aminoacidic composition of MexY in different strains must be considered for predicting potential binding site and affects the different activity of berberine derivatives. Finally, the antibiofilm effect of these new EPIs is promising, particularly for o-CH3-berberine derivative.

Integration of Lipid-Functionalized Epigallocatechin-3-gallate into PLGA Matrix as a Novel Polyphenol-Based Nanoantioxidant.

The search for polyphenol-based materials with antioxidant activity is a growing research area in the biomedical field. To obtain an efficient and stable nanoantioxidant, a novel biosystem was designed by integrating a lipophilic derivative of epigallocatechin-3-gallate (named EGCG-C18) on the surface of poly(lactic-co-glycolic acid) (PLGA). Poly(vinyl alcohol) (PVA) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (DSPE-PEG2000) were selected as polymeric and lipidic stabilizers, respectively, and their influence on both physical properties and the antioxidant activity of nanoantioxidant was investigated by a combined in silico and experimental approach. Full-atom molecular dynamics (MD) simulations were carried out to describe the different self-assembly processes of all components and the interactions that guided the EGCG-C18 insertion inside the PLGA matrix. Together with infrared spectroscopy results, the formation of an antioxidant lipid shell on the PLGA surface was clear. Dynamic light scattering and transmission electron microscopy showed that in the presence of DSPE-PEG2000, NPs were smaller than those treated with PVA. In addition, the different stabilizers used strongly influenced the ROS-scavenging ability of nanomaterials and this effect was strictly related to the molecular organization of EGCG-C18. MD showed that the apolar interaction between the alkyl chains of DSPE-PEG2000 and EGCG-C18 oriented the phenolic groups of the polyphenol toward the solvent, providing an ability of NP to scavenge hydroxyl radicals over to free EGCG-C18 and PLGA/PVA NPs. Finally, the ability of nanoantioxidants to protect human dermal fibroblasts from cell death induced by oxidative stress has been tested, revealing the high potential of these novel NPs as polyphenol-based materials.

Synthesis and Characterization of Edaravone Analogues as Remyelinating Agents and Putative Mechanistic Probes.

Edaravone (EDA), an antioxidant drug approved for the treatment of ischemic stroke and amyotrophic lateral sclerosis, was recently proposed as a remyelinating candidate for the treatment of multiple sclerosis. Here, we synthesized twelve EDA analogues 2b-4c showing three substitution patterns A-C, searching for improved remyelinating agents and putative molecular targets responsible for their regenerative activity. We profiled them in three primary assays to determine their stimulation of oligodendrocyte progenitor cell metabolism (tetrazolium MTT assay), their antioxidant potential (2,2-diphenyl-1-picrylhydrazyl-DPPH assay) and to predict their bioavailability (virtual ADME profile). Active 4'-carboxylate 2b, 4'-ester 2c and N1-carbamate-4'-ester 4a were further characterized, justifying their in vitro effects and selecting 4a as a putative EDA 1 prodrug suitable for in vivo testing.

Identification of Flavone Derivative Displaying a 4'-Aminophenoxy Moiety as Potential Selective Anticancer Agent in NSCLC Tumor Cells.

Five heterocyclic derivatives were synthesized by functionalization of a flavone nucleus with an aminophenoxy moiety. Their cytotoxicity was investigated in vitro in two models of human non-small cell lung cancer (NSCLC) cells (A549 and NCI-H1975) by using MTT assay and the results compared to those obtained in healthy fibroblasts as a non-malignant cell model. One of the aminophenoxy flavone derivatives (APF-1) was found to be effective at low micromolar concentrations in both lung cancer cell lines with a higher selective index (SI). Flow cytometric analyses showed that APF-1 induced apoptosis and cell cycle arrest in the G2/M phase through the up-regulation of p21 expression. Therefore, the aminophenoxy flavone-based compounds may be promising cancer-selective agents and could serve as a base for further research into the design of flavone-based anticancer drugs.

Meldonium Inhibits Cell Motility and Wound-Healing in Trabecular Meshwork Cells and Scleral Fibroblasts: Possible Applications in Glaucoma.

Meldonium (MID) is a synthetic drug designed to decrease the availability of L-carnitine-a main player in mitochondrial energy generation-thus modulating the cell pathways of energy metabolism. Its clinical effects are mostly evident in blood vessels during ischemic events, when the hyperproduction of endogenous carnitine enhances cell metabolic activities, leading to increased oxidative stress and apoptosis. MID has shown vaso-protective effects in model systems of endothelial dysfunction induced by high glucose or by hypertension. By stimulating the endothelial nitric oxide synthetase (eNOS) via PI3 and Akt kinase, it has shown beneficial effects on the microcirculation and blood perfusion. Elevated intraocular pressure (IOP) and endothelial dysfunction are major risk factors for glaucoma development and progression, and IOP remains the main target for its pharmacological treatment. IOP is maintained through the filtration efficiency of the trabecular meshwork (TM), a porous tissue derived from the neuroectoderm. Therefore, given the effects of MID on blood vessels and endothelial cells, we investigated the effects of the topical instillation of MID eye drops on the IOP of normotensive rats and on the cell metabolism and motility of human TM cells in vitro. Results show a significant dose-dependent decrease in the IOP upon topic treatment and a decrease in TM cell motility in the wound-healing assay, correlating with an enhanced expression of vinculin localized in focal adhesion plaques. Motility inhibition was also evident on scleral fibroblasts in vitro. These results may encourage a further exploration of MID eye drops in glaucoma treatment.

Structural Basis for Agonistic Activity and Selectivity toward Melatonin Receptors hMT1 and hMT2.

Glaucoma, a major ocular neuropathy originating from a progressive degeneration of retinal ganglion cells, is often associated with increased intraocular pressure (IOP). Daily IOP fluctuations are physiologically influenced by the antioxidant and signaling activities of melatonin. This endogenous modulator has limited employment in treating altered IOP disorders due to its low stability and bioavailability. The search for low-toxic compounds as potential melatonin agonists with higher stability and bioavailability than melatonin itself could start only from knowing the molecular basis of melatonergic activity. Thus, using a computational approach, we studied the melatonin binding toward its natural macromolecular targets, namely melatonin receptors 1 (MT1) and 2 (MT2), both involved in IOP signaling regulation. Besides, agomelatine, a melatonin-derivative agonist and, at the same time, an atypical antidepressant, was also included in the study due to its powerful IOP-lowering effects. For both ligands, we evaluated both stability and ligand positioning inside the orthosteric site of MTs, mapping the main molecular interactions responsible for receptor activation. Affinity values in terms of free binding energy (ΔGbind) were calculated for the selected poses of the chosen compounds after stabilization through a dynamic molecular docking protocol. The results were compared with experimental in vivo effects, showing a higher potency and more durable effect for agomelatine with respect to melatonin, which could be ascribed both to its higher affinity for hMT2 and to its additional activity as an antagonist for the serotonin receptor 5-HT2c, in agreement with the in silico results.

Mechanistic insight toward EGFR activation induced by ATP: role of mutations and water in ATP binding patterns.

The discovery of mutations within the kinase domain of the epidermal growth factor receptor (EGFR) gene has enabled a new era of targeted therapy in non-small cell lung cancer (NSCLC). Drugs belonging to the family of tyrosine kinase inhibitors (TKIs) are designed to bind ATP binding cleft, anyway, the occurrence of aminoacidic mutations decreases the effectiveness of the antitumoral treatment. Despite many efforts has been already made, the impact of the mutations on conformation and stability of EGFR-ATP complexes is still not fully understood. Therefore, we investigated the effect of mutations that leads to changes in Michaelis-Menten constant (Km) using dynamic docking simulations. We focused on six different EGFR forms in relation to different mutation states, then we found a good correlation between the calculated ATP affinities and Km values. Moreover, since dynamic switching of TK-EGFR from the inactive towards the active state is known to regulate the kinase activity, we observed that ATP induces the inwards movement of the αC-helix with the Lys745 close to Glu762 in all cases. This means that ATP binding should be the first step in promoting the conformational shift to the active state. Finally, we highlighted for the first time the key contribution of water hydrogen bond and water-bridge networks in the modulation of ATP affinity. The identified mutant-specific ATP binding patterns and conformational features could be much useful to guide cancer therapy and develop more personalized medicine. Communicated by Ramaswamy H. Sarma.

Glyoxalase 2: Towards a Broader View of the Second Player of the Glyoxalase System.

Glyoxalase 2 is a mitochondrial and cytoplasmic protein belonging to the metallo-ß-lactamase family encoded by the hydroxyacylglutathione hydrolase (HAGH) gene. This enzyme is the second enzyme of the glyoxalase system that is responsible for detoxification of the α-ketothaldehyde methylglyoxal in cells. The two enzymes glyoxalase 1 (Glo1) and glyoxalase 2 (Glo2) form the complete glyoxalase pathway, which utilizes glutathione as cofactor in eukaryotic cells. The importance of Glo2 is highlighted by its ubiquitous distribution in prokaryotic and eukaryotic organisms. Its function in the system has been well defined, but in recent years, additional roles are emerging, especially those related to oxidative stress. This review focuses on Glo2 by considering its genetics, molecular and structural properties, its involvement in post-translational modifications and its interaction with specific metabolic pathways. The purpose of this review is to focus attention on an enzyme that, from the most recent studies, appears to play a role in multiple regulatory pathways that may be important in certain diseases such as cancer or oxidative stress-related diseases.

Salt effects on mixed composition membranes containing an antioxidant lipophilic edaravone derivative: a computational-experimental study.

The protection of lipid membranes against oxidation avoids diseases associated with oxidative stress. As a strategy to contrast it, functionalized lipids with antioxidant activity are used to become part of membranes thus protecting them. For this purpose, a lipophilic edaravone derivative has been synthesized, adding a C18 saturated chain to the original structure. The antioxidant activity of C18-Edv has been demonstrated in our previous work. In this study, molecular dynamics simulations have been performed to define the effects of NaCl, MgCl2, KCl, and CaCl2 salts on a palmitoyl-oleoyl-sn-glycero-phosphocholine (POPC) lipid bilayer encapsulating C18-Edv. The results showed how different salts influence POPC lateral diffusion, and the movements of C18-Edv heads, which are antioxidant moieties, were correlated to the ability of C18-Edv molecules to protect membranes. MgCl2 showed a negative impact leading to C18-Edv clusterization and membrane stretching, while KCl and NaCl showed a moderate influence on the mixed lipid membrane structure. CaCl2 increased the exposure of the C18-Edv heads to the lipid-water interface, resulting in the salt with a higher propensity to guarantee protection against radicals in the aqueous phase. Finally, C18-Edv-POPC liposomes have been prepared following the simulation conditions, and then an experimental Oxygen Radical Absorbance Capacity (ORAC) assay has been performed to confirm the in silico predicted results.

Tailoring the Barrier Properties of PLA: A State-of-the-Art Review for Food Packaging Applications.

It is now well recognized that the production of petroleum-based packaging materials has created serious ecological problems for the environment due to their resistance to biodegradation. In this context, substantial research efforts have been made to promote the use of biodegradable films as sustainable alternatives to conventionally used packaging materials. Among several biopolymers, poly(lactide) (PLA) has found early application in the food industry thanks to its promising properties and is currently one of the most industrially produced bioplastics. However, more efforts are needed to enhance its performance and expand its applicability in this field, as packaging materials need to meet precise functional requirements such as suitable thermal, mechanical, and gas barrier properties. In particular, improving the mass transfer properties of materials to water vapor, oxygen, and/or carbon dioxide plays a very important role in maintaining food quality and safety, as the rate of typical food degradation reactions (i.e., oxidation, microbial development, and physical reactions) can be greatly reduced. Since most reviews dealing with the properties of PLA have mainly focused on strategies to improve its thermal and mechanical properties, this work aims to review relevant strategies to tailor the barrier properties of PLA-based materials, with the ultimate goal of providing a general guide for the design of PLA-based packaging materials with the desired mass transfer properties.

Tuning curvature and phase behavior of monoolein bilayers by epigallocatechin-3-gallate: Structural insight and cytotoxicity.

The use of glyceryl monooleate (GMO)-based nanoparticles has not yet been explored in overcoming the low bioavailability of Epigallocatechin-3-gallate (EGCG), a green tea polyphenol with a known anticancer activity. Since the inclusion of a guest molecule can affect the curvature and the supramolecular structure of fully hydrated GMO-based phase, the phase behavior of bulk and dispersed liquid crystalline systems containing EGCG were explored by Small Angle Neutron Scattering and X-Ray Diffraction experiments. Molecular Dynamic Simulations showed how the interaction of EGCG with polar heads of GMO strongly affects the curvature and packing of GMO phase. The EGCG encapsulation efficiency was determined in the nanodispersions and their size studied by Dynamic Light Scattering and Atomic Force Microscopy. A nanodispersed formulation has been optimized with a cytotoxic effect more than additive of GMO and EGCG.

Influence of a lipophilic edaravone on physical state and activity of antioxidant liposomes: An experimental and in silico study.

The influence of a lipophilic derivative of Edaravone (C18Edv) on a POPC liposomal bilayer has been investigated by a combined computational-experimental approach. The order and hydration degree of three different systems composed by 10%, 20% and 40% in w/w percentage of C18Edv respect to POPC were investigated through Molecular Dynamics (MD) simulations and fluorescence spectroscopy experiments. Dynamic Light Scattering measurements showed how the presence of different amounts of C18EdV determines differences on liposome size and stability. The survey revealed that the content of lipophilic antioxidant tunes liposome rigidity and influences cellular uptake and antioxidant activity which are maximized for formulation containing 20% of C18Edv.

Berberine Derivatives as Pseudomonas aeruginosa MexXY-OprM Inhibitors: Activity and In Silico Insights.

The natural alkaloid berberine has been demonstrated to inhibit the Pseudomonas aeruginosa multidrug efflux system MexXY-OprM, which is responsible for tobramycin extrusion by binding the inner membrane transporter MexY. To find a structure with improved inhibitory activity, we compared by molecular dynamics investigations the binding affinity of berberine and three aromatic substituents towards the three polymorphic sequences of MexY found in P. aeruginosa (PAO1, PA7, and PA14). The synergy of the combinations of berberine or berberine derivatives/tobramycin against the same strains was then evaluated by checkerboard and time-kill assays. The in silico analysis evidenced different binding modes depending on both the structure of the berberine derivative and the specific MexY polymorphism. In vitro assays showed an evident MIC reduction (32-fold and 16-fold, respectively) and a 2-3 log greater killing effect after 2 h of exposure to the combinations of 13-(2-methylbenzyl)- and 13-(4-methylbenzyl)-berberine with tobramycin against the tobramycin-resistant strain PA7, a milder synergy (a 4-fold MIC reduction) against PAO1 and PA14, and no synergy against the ΔmexXY strain K1525, confirming the MexY-specific binding and the computational results. These berberine derivatives could thus be considered new hit compounds to select more effective berberine substitutions and their common path of interaction with MexY as the starting point for the rational design of novel MexXY-OprM inhibitors.

Effect of Epigallocatechin-3-Gallate on EGFR Signaling and Migration in Non-Small Cell Lung Cancer.

The epidermal growth factor receptor (EGFR) is one of the most well-studied molecular targets in non-small cell lung cancer (NSCLC) and tyrosine kinase inhibitors have been shown to be effective in the treatment of advanced NSCLC. Nevertheless, the efficacy of tyrosine kinase inhibitors could be compromised by additional mutations in EGFR and compensatory activations of other pathways. Epigallocatechin-3-gallate (EGCG), the main bioactive molecule in green tea, acts as a tyrosine kinase inhibitor toward cancer cells overexpressing EGFR (wild-type). However, little information has been reported on the effect of EGCG on EGFR with activating mutations. In this study, we evaluated the ability of EGCG to inhibit EGFR signaling activation in three different NSCLC cell lines containing wild-type EGFR or EGFR with additional mutations. The effect on proliferation, apoptosis, migration, and vinculin expression was then studied. Overall, our results demonstrate that EGCG polyphenol inhibits cell proliferation and migration in NSCLC cell lines, although with different efficacy and mechanisms. These data may be of interest for an evaluation of the use of EGCG as an adjunct to NSCLC therapies.

Amidinoquinoxaline-Based Nitrones as Lipophilic Antioxidants.

The potential of nitrones (N-oxides) as therapeutic antioxidants is due to their ability to counteract oxidative stress, mainly attributed to their action as radical scavengers toward C- and O-centered radicals. Among them, nitrones from the amidinoquinoxaline series resulted in interesting derivatives, due to the ease with which it is possible to introduce proper substituents within their structure in order to modulate their lipophilicity. The goal is to obtain lipophilic antioxidants that are able to interact with cell membranes and, at the same time, enough hydrophilic to neutralize those radicals present in a water compartment. In this work, the antioxidant efficacy of a series of amidinoquinoxaline nitrones has been evaluated regarding the oxidation of 2-deoxyribose and lipid peroxidation. The results have been rationalized on the basis of the different possible mechanisms involved, depending on some of their properties, such as lipophilicity, the ability to scavenge free radicals, and to undergo single electron transfer (SET) reactions.

Insights into the Antioxidant Mechanism of Newly Synthesized Benzoxazinic Nitrones: In Vitro and In Silico Studies with DPPH Model Radical.

Synthetic nitrone spin-traps are being explored as therapeutic agents for the treatment of a wide range of oxidative stress-related pathologies, including but not limited to stroke, cancer, cardiovascular, and neurodegenerative diseases. In this context, increasing efforts are currently being made to the design and synthesis of new nitrone-based compounds with enhanced efficacy. The most researched nitrones are surely the ones related to α-phenyl-tert-butylnitrone (PBN) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO) derivatives, which have shown to possess potent biological activity in many experimental animal models. However, more recently, nitrones with a benzoxazinic structure (3-aryl-2H-benzo[1,4]oxazin-N-oxides) have been demonstrated to have superior antioxidant activity compared to PBN. In this study, two new benzoxazinic nitrones bearing an electron-withdrawing methoxycarbonyl group on the benzo moiety (in para and meta positions respect to the nitronyl function) were synthesized. Their in vitro antioxidant activity was evaluated by two cellular-based assays (inhibition of AAPH-induced human erythrocyte hemolysis and cell death in human retinal pigmented epithelium (ARPE-19) cells) and a chemical approach by means of the α,α-diphenyl-ß-picrylhydrazyl (DPPH) scavenging assay, using both electron paramagnetic resonance (EPR) spectroscopy and UV spectrophotometry. A computational approach was also used to investigate their potential primary mechanism of antioxidant action, as well as to rationalize the effect of functionalization on the nitrones reactivity toward DPPH, chosen as model radical in this study. Further insights were also gathered by exploring the nitrone electrochemical properties via cyclic voltammetry and by studying their kinetic behavior by means of EPR spectroscopy. Results showed that the introduction of an electron-withdrawing group in the phenyl moiety in the para position significantly increased the antioxidant capacity of benzoxazinic nitrones both in cell and cell-free systems. From the mechanistic point of view, the calculated results closely matched the experimental findings, strongly suggesting that the H-atom transfer (HAT) is likely to be the primary mechanism in the DPPH quenching.

Molecular dynamics simulations of quinine encapsulation into biodegradable nanoparticles: A possible new strategy against Sars-CoV-2.

A new coronavirus disease, SARS-CoV-2, has spread into a global pandemic in December 2019. Since no specific therapeutic drugs for treating COVID-19 have been approved by FDA, recent studies suggest that the known antimalarial quinine and its derivatives (chloroquine and hydroxychloroquine) inhibit receptor binding of the viral particles and inhibits the strong "cytokine storm", which is the main cause of death among infected patients. In particular, the natural alkaloid quinine has shown to possess a better safety profile and greater tolerability compared to its derivatives. Dosage optimization of quinine is still necessary as the currently available dosage forms have controversial pharmacokinetics and safety profiles. Therefore, repurposing quinine dosage forms to improve its pharmacokinetics and safety profile may be necessary to support its use against SARS-CoV-2. In this context, biodegradable/biocompatible polymeric nanoparticles may provide a safe site-specific and controlled quinine delivery, reducing the frequency of drug administration and the dose. In this study, a full atomistic molecular dynamics simulation approach has been used to investigate the use of poly-(glycolic acid) and poly-(lactic acid) and their copolymer poly-(lactic-co-glycolic acid) as potential delivery systems for lipophilic quinine to get insights into the mechanism of quinine encapsulation and release at the atomic/molecular level.

The Natural Alkaloid Berberine Can Reduce the Number of Pseudomonas aeruginosa Tolerant Cells.

The eradication of recurrent Pseudomonas aeruginosa (PA) lung infection in cystic fibrosis (CF) patients may be hampered by the development of persistent bacterial forms, which can tolerate antibiotics through efflux pump overexpression. After demonstrating the efflux pump inhibitory effect of the alkaloid berberine on the PA MexXY-OprM efflux pump, in this study, we tested its ability (80/320 µg/mL) to enhance tobramycin (20xMIC/1000xMIC) activity against PA planktonic/biofilm cultures. Preliminary investigations of the involvement of MexY in PA tolerance to tobramycin treatment, performed on the isogenic pair PA K767 (wild type)/K1525 (ΔmexY) growing in planktonic and biofilm cultures, demonstrated that the ΔmexY mutant K1525 produced a lower (100 and 10 000 times, respectively) amount of tolerant cells than that of the wild type. Next, we grew broth cultures of PAO1, PA14, and 20 PA clinical isolates (of which 13 were from CF patients) in the presence of 20xMIC tobramycin with and without berberine 80 µg/mL. Accordingly, most strains showed a greater (from 10- to 1000-fold) tolerance reduction in the presence of berberine. These findings highlight the involvement of the MexXY-OprM system in the tobramycin tolerance of PA and suggest that berberine may be used in new valuable therapeutic combinations to counteract persister survival.