Confronting the Threat: Designing Highly Effective bis-Benzimidazolium Agents to Overcome Biofilm Persistence and Antimicrobial Resistance.

The objective of this study is to take the initial steps toward developing novel antibiotics to counteract the escalating problem of antimicrobial and bacterial persistence, particularly in relation to biofilms. Our approach involves emulating the structural characteristics of cationic antimicrobial peptides. To circumvent resistance development, we have designed a library of bis-benzimidazolium salts that selectively target the microbial membranes in a nonspecific manner. To explore their structure-activity relationship, we conducted experiments using these compounds on various pathogens known for their resistance to conventional antibiotics, including Gram-positive methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecium (VRE), and Gram-negative Escherichia coli (E. coli). Notably, two bis-benzimidazolium salts exhibited robust antimicrobial activity while maintaining a high level of selectivity compared with mammalian cells. Our investigations revealed significant antibiofilm activity, as these compounds rapidly acted against established biofilms. In addition, bis-benzimidazolium compounds exhibited consistent results in resistance development and cross-resistance studies. Consequently, amphiphilic bis-benzimidazolium salts hold promise as potential candidates to combat resistance-associated infections.

The NAMPT Inhibitor FK866 Increases Metformin Sensitivity in Pancreatic Cancer Cells.

Pancreatic cancer (pancreatic ductal adenocarcinoma: PDAC) is one of the most aggressive neoplastic diseases. Metformin use has been associated with reduced pancreatic cancer incidence and better survival in diabetics. Metformin has been shown to inhibit PDAC cells growth and survival, both in vitro and in vivo. However, clinical trials using metformin have failed to reduce pancreatic cancer progression in patients, raising important questions about molecular mechanisms that protect tumor cells from the antineoplastic activities of metformin. We confirmed that metformin acts through inhibition of mitochondrial complex I, decreasing the NAD+/NADH ratio, and that NAD+/NADH homeostasis determines metformin sensitivity in several cancer cell lines. Metabolites that can restore the NAD+/NADH ratio caused PDAC cells to be resistant to metformin. In addition, metformin treatment of PDAC cell lines induced a compensatory NAMPT expression, increasing the pool of cellular NAD+. The NAMPT inhibitor FK866 sensitized PDAC cells to the antiproliferative effects of metformin in vitro and decreased the cellular NAD+ pool. Intriguingly, FK866 combined with metformin increased survival in mice bearing KP4 cell line xenografts, but not in mice with PANC-1 cell line xenografts. Transcriptome analysis revealed that the drug combination reactivated genes in the p53 pathway and oxidative stress, providing new insights about the mechanisms leading to cancer cell death.

Phenylethynylbenzyl-modified biguanides inhibit pancreatic cancer tumor growth.

We present the design and synthesis of a small library of substituted biguanidium salts and their capacity to inhibit the growth of pancreatic cancer cells. We first present their in vitro and membrane activity, before we address their mechanism of action in living cells and in vivo activity. We show that phenylethynyl biguanidium salts possess higher ability to cross hydrophobic barriers, improve mitochondrial accumulation and anticancer activity. Mechanistically, the most active compound, 1b, like metformin, activated AMPK, decreased the NAD+/NADH ratio and mitochondrial respiration, but at 800-fold lower concentration. In vivo studies show that compound 1b significantly inhibits the growth of pancreatic cancer xenografts in mice, while biguanides currently in clinical trials had little activity.

Highly Active, Entirely Biobased Antimicrobial Pickering Emulsions.

We present the development of surfactant-free, silica-free and fully biobased oil-in-water antimicrobial Pickering emulsions, based on the self-assembly of ß-cyclodextrin and phytoantimicrobial oils (terpinen-4-ol or carvacrol). Undecylenic acid (UA), derived from castor oil, can be used as bio-based drug to treat fungal infection, but is less effective than petroleum-based drugs as azole derivatives. To maximize its antifungal potential, we have incorporated UA in fully biobased Pickering emulsions. These emulsions are effective against fungi, Gram-positive and Gram-negative bacteria. The carvacrol emulsion charged with UA is +390 % and +165 % more potent against methicillin-resistant S. aureus (MRSA), compared to UA and azole-based commercial formulations. Moreover, this emulsion is up to +480 % more efficient that UA ointment against C. albicans. Finally, remarkable eradication of E. coli and MRSA biofilms was obtained with this environmental-friendly emulsion.

Enhancing the principles of experiential marketing in ophthalmology services.

Introduction. Consumers are getting more empowered, the process of empowerment transforming them into the partners of organizations or co-producers of services. Health care services, as well as ophthalmology services, have been a sensitive topic and raised several debates that focused on the principles of Experiential Marketing and hedonic experiences. Aim. The aim of this paper was to investigate the experiences of patients in a public ophthalmology organization by using the components of Experiential Marketing: sense, feel, think, act, and relate. Materials and methods. The case study was cross-sectional, and the sampling method was non-probabilistic, following a snowball technique. The sample was made up of 100 patients of the Clinical Emergency Eye Hospital in Bucharest. The instrument for data collection was a self-administered questionnaire. The statistical analysis was conducted in SPSS version 20 and frequencies or percentages were used for describing the qualitative data. Findings. Experiential Marketing principles may successfully be applied in public ophthalmology services if a special attention is given to the Feel and Relate components.

Phytochemical- and Cyclodextrin-Based Pickering Emulsions: Natural Potentiators of Antibacterial, Antifungal, and Antibiofilm Activity.

We present self-assembled Pickering emulsions containing biocidal phytochemical oils (carvacrol and terpinen-4-ol) and ß-cyclodextrin able to potentiate the antimicrobial and antibiofilm activity of miconazoctylium bromide. The carvacrol-containing emulsion is 2-fold more sensitive against C. albicans and S. aureus and highly active against E. coli, compared to the commercial cream containing miconazole nitrate. Moreover, this emulsion shows a synergistic effect against fungi, additive responses against bacteria, and remarkable staphylococcal biofilm eradication. These results are associated with membrane permeabilization, enzymes inhibition, and the accumulation of reactive oxygen species in microorganisms.

Benzimidazolium salts prevent and disrupt methicillin-resistant Staphylococcus aureus biofilms.

Emergence of resistant bacteria encourages us to develop new antibiotics and strategies to compensate for the different mechanisms of resistance they acquire. One of the defense mechanisms of resistant bacteria is the formation of biofilms. Herein we show that benzimidazolium salts with various flexible or rigid side chains act as strong antibiotic and antibiofilm agents. We show that their antibiofilm activity is due to their capacity to destroy the biofilm matrix and the bacterial cellular membranes. These compounds are able to avoid the formation of biofilms and disperse mature biofilms showing a universal use in the treatment of biofilm-associated infections.

Antimicrobial and Antibiofilm Activity of Disubstituted Bis-benzimidazolium Salts.

The increased prevalence of antibiotic-resistant bacteria is a critical issue for human health. Developing new antibiotic agents is vital for fighting persistent infections and lowering mortality rates. In this study, we designed lutidine-disubstituted bis-benzimidazolium salts (lutidine-bis-benzimidazolium core with octyl, adamantyl, and cholesteryl lipophilic side chains), and tested their antimicrobial activity, their capacity to inhibit planktonic bacterial and fungal growth, and their ability to inhibit the formation of or disrupt mature methicillin-resistant Staphylococcus aureus (MRSA) biofilms. The antibiofilm activity of these salts was analyzed in terms of their lipophilicity, capacity to induce transmembrane ion transport, perturbation of the cellular membrane, and mechanism of action in the phospholipid bilayer. The synthesized compounds were not active against MRSA biofilms, as the formation of transmembrane channels had no effect on the integrity of the extracellular polymeric substance matrix and only octyl and adamantyl derivatives possessed the capacity to inhibit biofilm formation. The synthesized derivatives could be used as lead candidates for the development of anti-MRSA agents.

Anti-staphylococcal biofilm activity of miconazoctylium bromide.

We designed and synthesized miconazole analogues containing a substituted imidazolium moiety. The structural modification of the miconazole led to a compound with high potency to prevent the formation and disrupt bacterial biofilms, as a result of accumulation in the biofilm matrix, permeabilization of the bacterial membrane and generation of reactive oxygen species in the cytoplasm.

Synthesis and Characterization of Biguanide and Biguanidium Surfactants for Efficient and Recyclable Application in the Suzuki-Miyaura Reaction.

We report here the synthesis and thorough characterization of a new family of alkylbiguanides and alkylbiguanidium chlorides by 1H and 13C NMR and X-ray diffraction. Their critical micelle concentration was first determined by surface tension measurements. Hexylbiguanide was then studied as a surfactant in the micellar Suzuki-Miyaura cross-coupling reaction. The unexpected low reactivity of the system at high Pd/hexylbiguanide ratios was due to the change of the size and the shape of the aggregates, observed by transmission electron microscopy. The best catalytic activity was obtained for a 1:1 Pd/hexylbiguanide ratio for which the micellar conditions were conserved. Better results were obtained for several substrates, when compared to those previously obtained with metformin under the same reaction conditions. Higher yields and a better recyclability were obtained under micellar conditions with hexylbiguanide.

Correction: Ultra-low fouling alkylimidazolium modified surfaces for the detection of HER2 in breast cancer cell lysates.

Correction for 'Ultra-low fouling methylimidazolium modified surfaces for the detection of HER2 in breast cancer cell lysates' by Alexandra Aubé et al., Analyst, 2017, 142, 2343-2353.

Ultra-low fouling alkylimidazolium modified surfaces for the detection of HER2 in breast cancer cell lysates [corrected].

We synthesized novel ultra-low fouling ionic liquids and demonstrated their use with surface plasmon resonance (SPR) sensing for the analysis of HER2 in breast cancer cell lysates. Whilst biomarkers are commonly detected in serum, this remains challenging for cancer diagnosis due to their low concentrations in circulation and in some cases, there is a poor correlation between serum and tissue concentrations. Therefore, a cell lysate constitutes an interesting biosample for cancer diagnosis and typing, which has been largely unexploited for chemical biosensing of cancer biomarkers. However, high fouling of surfaces in contact with the cell lysate and the absence of effective surface chemistry to prevent fouling are currently limiting biomarker analysis in cell lysates. To address this challenge, we report the synthesis of 1-(carboxyalkyl)-3-(12-mercaptododecyl)-1H-imidazolium ionic liquids with different anions (Br-, BF4-, PF6-, ClO4-, and NTf2-) and ethyl and pentyl chains to form monolayers and analyse specific proteins from cell lysates. The most efficient ionic liquid monolayer, 1-(carboxyethyl)-3-(12-mercaptododecyl)-1H-imidazolium bromide, was able to eliminate the nonspecific adsorption (surface coverage of 2 ± 2 ng cm-2) of a concentrated cell lysate (protein concentration of ∼3.5 mg mL-1), which was significantly better than carboxy-PEG (surface coverage of 14 ± 7 ng cm-2), a benchmark monolayer commonly used to reduce nonspecific adsorption. These ionic liquid monolayers were modified with anti-HER2 and the detection of the HER2 breast cancer biomarker was carried out in crude breast cancer cell lysates, as shown with HER2-negative MCF-7 cells spiked with HER2 and with HER2 positive SK-BR-3 cells.

A field-deployed surface plasmon resonance (SPR) sensor for RDX quantification in environmental waters.

A field-deployable surface plasmon resonance (SPR) sensor is reported for the detection of the energetic material (commonly termed explosives) 1,3,5-trinitroperhydro-1,3,5-triazine (RDX) at ppb concentration in environmental samples. The SPR sensor was first validated under laboratory conditions with uncontaminated natural water samples spiked with known concentrations of RDX near the EPA limit of 2 ppb, which was then applied to monitor environmental samples collected in different downgradient wells near a grenade training range. The SPR sensor was finally tested on the field, where environmental samples were analysed on location in less than 90 minutes per well, which included the time to setup the equipment, sample the well and analyse the sample. The SPR analysis time was less than 45 minutes for equilibration, recalibration and measuring the water sample. Results obtained with the SPR sensors were cross-validated with the standard HPLC method (EPA method 8330b), and they showed good agreement with an accuracy within less than 1.6 ppb for analysis at the sampling sites, and with the relative standard deviation (RSD) better than 20% for field and laboratory measurements. The SPR sensor worked in a range of environmental conditions, including operation from about 0 °C to nearly 30 °C. The instrument was easily deployed near the sampling site using motor vehicles under summer conditions (Lab-in-a-Jeep) and using a sled under winter conditions (Lab-on-a-sled), showcasing the field deployability of the RDX SPR sensor and the possibility of continuously monitoring RDX in the environment.

Investigation of Classical Organic and Ionic Liquid Cosolvents for Early-Stage Screening in Fragment-Based Inhibitor Design with Unrelated Bacterial and Human Dihydrofolate Reductases.

Drug design by methods such as fragment screening requires effective solubilization of millimolar concentrations of small organic compounds while maintaining the properties of the biological target. We investigate four organic solvents and three 1-butyl-3-methylimidazolium (BMIm)-based ionic liquids (ILs) as cosolvents to establish conditions for screening two structurally unrelated dihydrofolate reductases (DHFRs) that are prime drug targets. Moderate concentrations (10%-15%) of cosolvents had little effect on inhibition of the microbial type II R67 DHFR and of human DHFR (hDHFR), while higher concentrations of organic cosolvents generally decreased activity of both DHFRs. In contrast, a specific IL conserved the activity of one DHFR, while severely reducing the activity of the other, and vice versa, illustrating the differing effect of ILs on distinct protein folds. Most of the cosolvents investigated preserved the fold of R67 DHFR and had little effect on binding of the cofactor NADPH, but reduced the productive affinity for its substrate. In contrast, cosolvents resulted in modest structural destabilization of hDHFR with little effect on productive affinity. We conclude that the organic cosolvents, methanol, dimethylformamide, and dimethylsulfoxide, offer the most balanced conditions for early-stage compound screening as they maintain sufficient biological activity of both DHFRs while allowing for compound dissolution in the millimolar range. However, IL cosolvents showed poor capacity to solubilize organic compounds at millimolar concentrations, mitigating their utility in early-stage screening. Nonetheless, ILs could provide an alternative to classical organic cosolvents when low concentrations of inhibitors are used, as when characterizing higher affinity inhibitors.

Metal-Organic Synthetic Transporters (MOST): Efficient Chloride and Antibiotic Transmembrane Transporters.

We present the synthesis of two functionalized 2,4,7-triphenylbenzimidazole ligands and demonstrate the formation of their respective metal assemblies in phospholipid membranes. Anion transport experiments demonstrate the formation of metal-organic synthetic transporters (MOST) directly in phospholipid membranes. The formation of MOST in phospholipid membranes results in efficient architectures for chloride transport. We also demonstrate the insertion of these ligands and the formation of their metal-organic assemblies in bacterial membranes; the use of MOST makes the membranes of resistant bacteria more permeable to antibiotics. We also demonstrate that a combination of MOST with tetracycline lowers the sensitivity of resistant bacteria to tetracycline by 60-fold.

Adaptation of a bacterial membrane permeabilization assay for quantitative evaluation of benzalkonium chloride as a membrane-disrupting agent.

We describe the use of the ortho-nitrophenyl-ß-galactoside (ONPG) assay developed by Lehrer et al. to which a new mathematical data treatment was applied. In this simplified assay, only one enzymatic assay is needed to provide direct evidence of the kinetics of Escherichia coli membrane permeabilization induced by different concentrations of benzalkonium chloride (BAC). Analysis of the data obtained from the revised ONPG assay with our adapted mathematical formula indicates that BAC induces permeabilization of the bacterial outer and inner membranes in a two-step process. The two effective concentration (EC50) values obtained in this study, combined with the results from an outer membrane permeabilization assay, suggest that the two steps observed in the permeabilization process are related to the two different bacterial membranes. We show that membrane permeabilization occurs very fast upon the addition of bacterial cells to the BAC solutions and demonstrate that sub-lethal concentrations of BAC disturb the integrity of the Gram-negative bacterial membranes. Overall, our work broadens our knowledge on the mode of action of BAC on bacterial cells and emphasizes that BAC, and quaternary ammonium compounds in general, should not be used at sub-lethal concentrations in order to lower the risk of bacterial tolerance and resistance to antibiotics.

Host-Guest Strategy to Reversibly Control a Chloride Carrier Process with Cyclodextrins.

Herein, we report a reversible modular chloride transport process based on host-guest competitive interactions between an imidazolium-based chloride carrier and beta-cyclodextrin. We report evidence for the formation of the supramolecular complex between 1,3-bis(2-(adamantan-1-yl)ethyl)imidazolium bis(trifluorometyl-sulfonyl)imide with two ß-cyclodextrins. Through fluorescence assays in liposomes and black lipid membrane experiments, we demonstrate that the formation of the supramolecular complex results in the inhibition of the chloride transport. We show that the chloride transport process can be entirely restored in the presence of competitive adamantyl-functionalized guests. This is the first example of an entirely reversible modular chloride transport process in phospholipid bilayers involving a mobile carrier transporter and cyclodextrin supramolecular complex.

Strong antibacterial properties of anion transporters: a result of depolarization and weakening of the bacterial membrane.

The development of low molecular weight anionophores is an emerging topic in chemistry, as the need for these compounds increases with the continuous discovery of pathologies involving anomalies in anion transport processes. Development of new concepts to initiate anion imbalance in living cells while fighting multidrug-resistant bacteria is a paramount topic. In this study, three series of compounds including N,N'-diphenylethynylbenzyl benzimidazolium salts (1 and 2), 1,1'-(pyridine-2,6-diyl)bis(3-(4-(phenylethynyl)benzyl)-1H-benzo[d]imidazol-3-ium) salts (3-5), and 1,1'-(pyridine-2,6-diylbis(methylene))bis(3-(4-(phenyl ethynyl)benzyl)-1H-benzo[d]imidazol-3-ium) salts (6-8) displaying high antimicrobial activity and low toxicity against human cells were designed, synthesized, and studied. The most potent compound displayed micromolar minimal inhibitory concentrations in different Gram-negative and Gram-positive bacteria, while its hemolytic activity remained around 10% or less, even after a prolonged period of exposure. The mechanism of action of these benzimidazolium salts on bacterial membrane was assessed by bioanalytical techniques including assays in model membrane liposomes, membrane depolarization studies, and scanning electron microscopy (SEM) in living bacteria.

Asymmetric mutations in the tetrameric R67 dihydrofolate reductase reveal high tolerance to active-site substitutions.

Type II R67 dihydrofolate reductase (DHFR) is a bacterial plasmid-encoded enzyme that is intrinsically resistant to the widely-administered antibiotic trimethoprim. R67 DHFR is genetically and structurally unrelated to E. coli chromosomal DHFR and has an unusual architecture, in that four identical protomers form a single symmetrical active site tunnel that allows only one substrate binding/catalytic event at any given time. As a result, substitution of an active-site residue has as many as four distinct consequences on catalysis, constituting an atypical model of enzyme evolution. Although we previously demonstrated that no single residue of the native active site is indispensable for function, library selection here revealed a strong bias toward maintenance of two native protomers per mutated tetramer. A variety of such "half-native" tetramers were shown to procure native-like catalytic activity, with similar KM values but kcat values 5- to 33-fold lower, illustrating a high tolerance for active-site substitutions. The selected variants showed a reduced thermal stability (Tm ∼12°C lower), which appears to result from looser association of the protomers, but generally showed a marked increase in resilience to heat denaturation, recovering activity to a significantly greater extent than the variant with no active-site substitutions. Our results suggest that the presence of two native protomers in the R67 DHFR tetramer is sufficient to provide native-like catalytic rate and thus ensure cellular proliferation.

Molecular parameters and transmembrane transport mechanism of imidazolium-functionalized binols.

We describe the molecular parameters governing the transmembrane activity of imidazolium-functionalized anion transporters and present a detailed mechanistic study. These ionophores adopt a mobile-carrier mechanism for short methyl and butyl chains, a combined mobile-carrier/transmembrane-pore mechanism for octyl and dodecyl chains, and form transmembrane aggregates for hexadecyl chains.