N-acetylcysteine prevents catheter occlusion and inflammation in catheter associated-urinary tract infections by suppressing urease activity.

Introduction: Proteus mirabilis is a key pathobiont in catheter-associated urinary tract infections (CA-UTIs), which is well known to form crystalline biofilms that occlude catheters. Urease activity alkylates urine through the release of ammonia, consequentially resulting in higher levels of Mg2+ and Ca2+ and formation of crystals. In this study, we showed that N-acetyl cysteine (NAC), a thiol antioxidant, is a potent urease inhibitor that prevents crystalline biofilm formation. Methods: To quantify urease activity, Berthelot's method was done on bacterial extracts treated with NAC. We also used an in vitro catheterised glass bladder model to study the effect of NAC treatment on catheter occlusion and biofilm encrustation in P. mirabilis infections. Inductively-coupled plasma mass spectrometry (ICP-MS) was performed on catheter samples to decipher elemental profiles. Results: NAC inhibits urease activity of clinical P. mirabilis isolates at concentrations as low as 1 mM, independent of bacterial killing. The study also showed that NAC is bacteriostatic on P. mirabilis, and inhibited biofilm formation and catheter occlusion in an in vitro. A significant 4-8log10 reduction in viable bacteria was observed in catheters infected in this model. Additionally, biofilms in NAC treated catheters displayed a depletion of calcium, magnesium, or phosphates (>10 fold reduction), thus confirming the absence of any urease activity in the presence of NAC. Interestingly, we also showed that not only is NAC anti-inflammatory in bladder epithelial cells (BECs), but that it mutes its inflammatory response to urease and P. mirabilis infection by reducing the production of IL-6, IL-8 and IL-1b. Discussion: Using biochemical, microbiological and immunological techniques, this study displays the functionality of NAC in preventing catheter occlusion by inhibiting urease activity. The study also highlights NAC as a strong anti-inflammatory antibiofilm agent that can target both bacterial and host factors in the treatment of CA-UTIs.

Pharmacological inhibition of TBK1/IKKε blunts immunopathology in a murine model of SARS-CoV-2 infection.

TANK-binding kinase 1 (TBK1) is a key signalling component in the production of type-I interferons, which have essential antiviral activities, including against SARS-CoV-2. TBK1, and its homologue IκB kinase-ε (IKKε), can also induce pro-inflammatory responses that contribute to pathogen clearance. While initially protective, sustained engagement of type-I interferons is associated with damaging hyper-inflammation found in severe COVID-19 patients. The contribution of TBK1/IKKε signalling to these responses is unknown. Here we find that the small molecule idronoxil inhibits TBK1/IKKε signalling through destabilisation of TBK1/IKKε protein complexes. Treatment with idronoxil, or the small molecule inhibitor MRT67307, suppresses TBK1/IKKε signalling and attenuates cellular and molecular lung inflammation in SARS-CoV-2-challenged mice. Our findings additionally demonstrate that engagement of STING is not the major driver of these inflammatory responses and establish a critical role for TBK1/IKKε signalling in SARS-CoV-2 hyper-inflammation.

Ascorbic acid modulates the structure of the Pseudomonas aeruginosa virulence factor pyocyanin and ascorbic acid-furanone-30 combination facilitate biofilm disruption.

The production of pyocyanin by Pseudomonas aeruginosa increases its virulence, fitness and biofilm formation. Pyocyanin is also a redox molecule and we hypothesize that ascorbic acid being an antioxidant will interact with pyocyanin. The main objective of this study was to investigate the potential interaction of ascorbic acid with pyocyanin, and also to investigate the impact of ascorbic acid in combination with Furanone-30 on quorum sensing and biofilm formation of P. aeruginosa. When incubated with ascorbic acid, hyperchromic and hypsochromic shifts in pyocyanin absorbance peaks at 385 nm and 695 nm were observed. In the presence of dehydroascorbic acid and citric acid, these shifts were absent, indicating that the intrinsic antioxidant property of ascorbic acid was probably essential in binding to pyocyanin. NMR spectroscopy showed shifts in 1H NMR pyocyanin peaks between 8.2 to 5.8 ppm when incubated in the presence of ascorbic acid. Density Functional Theory (DFT) supported potential interactions between the -CH2OH or -OH moieties of ascorbic acid with the -C=O moiety of pyocyanin. The pyocyanin-ascorbic acid complex impaired pyocyanin binding to DNA. Ascorbic acid combined with furanone-30 elevated quorum-sensing inhibition in P. aeruginosa, which was directly associated with significantly reduced P. aeruginosa virulence, adhesion, aggregation and biofilm formation and enhanced antibiotic-mediated bacterial killing. This study demonstrated that the antioxidant ascorbic acid directly binds to pyocyanin, modulates its structure and results in disruption of biofilm formation and associated tolerance to antibiotics.

Hydrogels with intrinsic antibacterial activity prepared from naphthyl anthranilamide (NaA) capped peptide mimics.

In this study, we prepared antibacterial hydrogels through the self-assembly of naphthyl anthranilamide (NaA) capped amino acid based cationic peptide mimics. These ultra-short cationic peptide mimics were rationally designed with NaA as a capping group, L-phenylalanine, a short aliphatic linker, and a cationic group. The synthesized peptide mimics efficiently formed hydrogels with minimum gel concentrations between 0.1 and 0.3%w/v. The resulting hydrogels exhibited desirable viscoelastic properties which can be tuned by varying the cationic group, electronegative substituent, or counter anion. Importantly, nanofibers from the NaA-capped cationic hydrogels were found to be the source of hydrogels' potent bacteriacidal actvity against both Gram-positive and Gram-negative bacteria while remaining non-cytotoxic. These intrinsically antibacterial hydrogels are ideal candidates for further development in applications where bacterial contamination is problematic.

Synthetic Bilayers on Mica from Self-Assembly of Hydrogen-Bonded Triazines.

This study describes organic thin films prepared under a range of conditions from a model series of bis-N-alkyl chloro-triazines functionalized with short alkyl chains from ethyl to hexyl. The pure films were characterized using atomic force microscopy (AFM), X-ray diffraction (XRD), Raman spectroscopy, and X-ray photoelectron spectroscopy (XPS). When cast on mica, these compounds assemble as crystalline sheets made up of a synthetic bilayer along the crystallographic ab-plane with an internal hydrogen-bonded domain between external alkyl chains. These micron-scale surfaces stack along the c-axis, and increasing the alkyl chain length results in changes to the crystal morphology from needles to nanoscale plates. Thicker films produce nanoscale, pyramidal stacks of bilayers. Compared to atomically flat mica, a rougher, unetched silicon substrate produced irregular domains in the secondary bilayer. Films of mixtures comprising the ethyl derivative with butyl, pentyl, or hexyl derivative were imaged using time-of-flight secondary-ion mass spectrometry (ToF-SIMS) that indicated a trend toward a constant stoichiometry with increasing alkyl chain length. AFM of mixed films on mica showed single bilayers of height <2 nm, with an acceptable correlation to the XRD measurements, supporting a constant stoichiometry. These materials permit easy modification of mica to a micron-scale, atomically flat hydrophobic surface, and the use of mixtures with different alkyl chain lengths suggests a method to improve the quality of functional organic thin films.

Anthranilamide-based Short Peptides Self-Assembled Hydrogels as Antibacterial Agents.

In this study, we describe the synthesis and molecular properties of anthranilamide-based short peptides which were synthesised via ring opening of isatoic anhydride in excellent yields. These short peptides were incorporated as low molecular weight gelators (LMWG), bola amphiphile, and C3-symmetric molecules to form hydrogels in low concentrations (0.07-0.30% (w/v)). The critical gel concentration (CGC), viscoelastic properties, secondary structure, and fibre morphology of these short peptides were influenced by the aromaticity of the capping group or by the presence of electronegative substituent (namely fluoro) and hydrophobic substituent (such as methyl) in the short peptides. In addition, the hydrogels showed antibacterial activity against S. aureus 38 and moderate toxicity against HEK cells in vitro.

Synthesis of Bis-Glyoxylamide Peptidomimetics Derived from Bis-N-acetylisatins Linked at C5 by a Methylene or Oxygen Bridge.

The bis-glyoxylamide peptidomimetics have been synthesized from bis-N-acetylisatins linked at C5 by ring-opening with alcohols, amines, and amino acid methyl ester hydrochlorides. X-ray images of single crystals of bis-glyoxylamide peptidomimetics have been obtained.

Glyoxylamide-based self-assembly hydrogels for sustained ciprofloxacin delivery.

In this study, we report the synthesis of glyoxylamide peptide-mimics as self-assembled gels with well-defined molecular structures for topical delivery of ciprofloxacin (CIP). The glyoxylamide peptide mimics successfully formed hydrogels with critical gel concentrations of 0.02-0.08% (w/v). The mechanical strength, secondary structure, and fiber morphology of these hydrogels can be modulated by varying the N-substituent of the ring-opened isatins. The synthesised hydrogel exhibited a high loading capacity of CIP (40% (w/w)) and a sustained release profile. The CIP-loaded hydrogels were able to release CIP for more than 15 days and the released solution was shown to retain activity against Gram-positive and Gram-negative bacteria. In addition, the hydrogels formed showed low toxicity against Cos7 cells.