The failure of antibiotic therapies to clear Pseudomonas aeruginosa lung infection, the key mortality factor for cystic fibrosis (CF) patients, is partly attributed to the high tolerance of P. aeruginosa biofilms. Mannitol has previously been found to restore aminoglycoside sensitivity in Escherichia coli by generating a proton-motive force (PMF), suggesting a potential new strategy to improve antibiotic therapy and reduce disease progression in CF. Here, we used the commonly prescribed aminoglycoside tobramycin to select for P. aeruginosa persister cells during biofilm growth. Incubation with mannitol (10–40 mM) increased tobramycin sensitivity of persister cells up to 1,000-fold. Addition of mannitol to pre-grown biofilms was able to revert the persister phenotype and improve the efficacy of tobramycin. This effect was blocked by the addition of a PMF inhibitor or in a P. aeruginosa mutant strain unable to metabolise mannitol. Addition of glucose and NaCl at high osmolarity also improved the efficacy of tobramycin although to a lesser extent compared to mannitol. Therefore, the primary effect of mannitol in reverting biofilm associated persister cells appears to be an active, physiological response, associated with a minor contribution of osmotic stress. Mannitol was tested against clinically relevant strains, showing that biofilms containing a subpopulation of persister cells are better killed in the presence of mannitol, but a clinical strain with a high resistance to tobramycin was not affected by mannitol. Overall, these results suggest that in addition to improvements in lung function by facilitating mucus clearance in CF, mannitol also affects antibiotic sensitivity in biofilms and does so through an active, physiological response.

NR1/NR2A is a subtype of N-methyl-D-aspartate receptors (NMDARs), which are glutamate and glycine-gated Ca 2ϩ -permeable channels highly expressed in the central nervous system. A high-throughput screening (HTS) campaign using human osteosarcoma (U-2 OS) cells transiently transduced with NR1/NR2A NMDAR subunits, tested in a specifically designed fluorometric imaging plate reader (FLIPR)/Ca 2ϩ assay, identified sulfonamide derivative series, exemplified by,4-thiadiazol-2-yl}thio)acetamide (compound 13) as novel NR1/NR2A receptor antagonists. Compounds 1 and 13 displayed submicromolar and micromolar potency at NR1/NR2A receptor, respectively, although they did not show activity at NR2B-containing receptor up to 50 M concentration. Addition of 1 mM glycine, but not 1 mM L-glutamate, was able to surmount compound 1 and 13 inhibitory effects in FLIPR NR1/NR2A assay. However, compounds 1 and 13 displaced a glutamate site antagonist,519), in rat brain cortex binding assay. Results of FLIPR cell-based, electrophysiological, and biochemical binding assays suggest that compounds 1 and 13 are the prototypes of novel classes of NMDAR ligands, which to the best of our knowledge are the first selective antagonists at NR1/NR2A over NR1/NR2B receptor, and might constitute useful tools able to elucidate the relative role of the NR2A subunit in physiological and pathological conditions.

Fibrosis is characterized by the excessive deposition of extracellular matrix and crosslinked proteins, in particular collagen and elastin, leading to tissue stiffening and disrupted organ function. Lysyl oxidases are key players during this process, as they initiate collagen crosslinking through the oxidation of the ε‐amino group of lysine or hydroxylysine on collagen side‐chains, which subsequently dimerize to form immature, or trimerize to form mature, collagen crosslinks. The role of LOXL2 in fibrosis and cancer is well documented, however the specific enzymatic function of LOXL2 and LOXL3 during disease is less clear. Herein, we describe the development of PXS‐5153A, a novel mechanism based, fast‐acting, dual LOXL2/LOXL3 inhibitor, which was used to interrogate the role of these enzymes in models of collagen crosslinking and fibrosis. PXS‐5153A dose‐dependently reduced LOXL2‐mediated collagen oxidation and collagen crosslinking in vitro. In two liver fibrosis models, carbon tetrachloride or streptozotocin/high fat diet‐induced, PXS‐5153A reduced disease severity and improved liver function by diminishing collagen content and collagen crosslinks. In myocardial infarction, PXS‐5153A improved cardiac output. Taken together these results demonstrate that, due to their crucial role in collagen crosslinking, inhibition of the enzymatic activities of LOXL2/LOXL3 represents an innovative therapeutic approach for the treatment of fibrosis.

Semicarbazide-sensitive amine oxidase (SSAO), also known as vascular adhesion protein-1 (VAP-1), is a member of the copperdependent amine oxidase family that is associated with various forms of inflammation and fibrosis. To investigate the therapeutic potential of SSAO/VAP-1 inhibition, potent and selective inhibitors with drug-like properties are required. PXS-4681A [(Z)-4-(2-(aminomethyl)-3-fluoroallyloxy)benzenesulfonamide hydrochloride] is a mechanism-based inhibitor of enzyme function with a pharmacokinetic and pharmacodynamic profile that ensures complete, longlasting inhibition of the enzyme after a single low dose in vivo.PXS-4681A irreversibly inhibits the enzyme with an apparent K i of 37 nM and a k inact of 0.26 min 21 with no observed turnover in vitro. It is highly selective for SSAO/VAP-1 when profiled against related amine oxidases, ion channels, and seven-transmembrane domain receptors, and is superior to previously reported inhibitors. In mouse models of lung inflammation and localized inflammation, dosing of this molecule at 2 mg/kg attenuates neutrophil migration, tumor necrosis factor-a, and interleukin-6 levels. These results demonstrate the drug-like properties of PXS-4681A and its potential use in the treatment of inflammation.

Genes encoding fiketothiolase (phaA), acetoacetyl-CoA reductase (phaB) and PHB-synthase (phaC) from R. meliloti 41, together with a fourth gene, referred to as ORF1, presumed to be involved in PHB biosynthesis, have been cloned and sequenced. phaA, phaB and ORFl were identified by heterologous hybridization on a cosmid library, while phaC was isolated by cloning the transposon-tagged fragment from a R. meliloti PHB-Tn5 mutant. phaA and phaB were functionally expressed in Escherichia coli while phaC was able to complement a PHB-strain of R. meliloti 41. The three genes were sufficient to direct the production of polyhydroxyalkanoate in E. coli. The homology of ORFl with an ORF located near the PHB genes in two phototrophic bacteria suggests its involvement in PHB synthesis.