Molecular Mechanisms Underlying Medium-Chain Free Fatty Acid-Regulated Activity of the Phospholipase PlaF from Pseudomonas aeruginosa.

PlaF is a membrane-bound phospholipase A1 from Pseudomonas aeruginosa that is involved in remodeling membrane glycerophospholipids (GPLs) and modulating virulence-associated signaling and metabolic pathways. Previously, we identified the role of medium-chain free fatty acids (FFAs) in inhibiting PlaF activity and promoting homodimerization, yet the underlying molecular mechanism remained elusive. Here, we used unbiased and biased molecular dynamics simulations and free energy computations to assess how PlaF interacts with FFAs localized in the water milieu surrounding the bilayer or within the bilayer and how these interactions regulate PlaF activity. Medium-chain FFAs localized in the upper bilayer leaflet can stabilize inactive dimeric PlaF, likely through interactions with charged surface residues, as has been experimentally validated. Potential of mean force (PMF) computations indicate that membrane-bound FFAs may facilitate the activation of monomeric PlaF by lowering the activation barrier for changing into a tilted, active configuration. We estimated that the coupled equilibria of PlaF monomerization-dimerization and tilting at the physiological concentration of PlaF lead to the majority of PlaF forming inactive dimers when in a cell membrane loaded with decanoic acid (C10). This is in agreement with a suggested in vivo product feedback loop and gas chromatography-mass spectrometry profiling results, indicating that PlaF catalyzes the release of C10 from P. aeruginosa membranes. Additionally, we found that C10 in the water milieu can access the catalytic site of active monomeric PlaF, contributing to the competitive component of C10-mediated PlaF inhibition. Our study provides mechanistic insights into how medium-chain FFAs may regulate the activity of PlaF, a potential bacterial drug target.

Combined in†vitro/in silico approaches, molecular dynamics simulations and safety assessment of the multifunctional properties of thymol and carvacrol: A comparative insight.

Bioactive compounds derived from medicinal plants have acquired immense attentiveness in drug discovery and development. The present study investigated in vitro and predicted in silico the antibacterial, antifungal, and antiviral properties of thymol and carvacrol, and assessed their safety. The performed microbiological assays against Pseudomonas aeruginosa, Escherichia coli, Salmonella enterica Typhimurium revealed that the minimal inhibitory concentration values ranged from (0.078 to 0.312 mg/mL) and the minimal fungicidal concentration against Candida albicans was 0.625 mg/mL. Molecular docking simulations, stipulated that these compounds could inhibit bacterial replication and transcription functions by targeting DNA and RNA polymerases receptors with docking scores varying between (-5.1 to -6.9 kcal/mol). Studied hydroxylated monoterpenes could hinder C. albicans growth by impeding lanosterol 14α-demethylase enzyme and showed a (ΔG=-6.2 and -6.3 kcal/mol). Computational studies revealed that thymol and carvacrol could target the SARS-Cov-2 spike protein of the Omicron variant RBD domain. Molecular dynamics simulations disclosed that these compounds have a stable dynamic behavior over 100 ns as compared to remdesivir. Chemo-computational toxicity prediction using Protox II webserver indicated that thymol and carvacrol could be safely and effectively used as drug candidates to tackle bacterial, fungal, and viral infections as compared to chemical medication.

Increased malondialdehyde concentration and reduced total antioxidant capacity in aqueous humor and blood samples from patients with glaucoma.

PURPOSE: To evaluate the levels of malondialdehyde (MDA) and total antioxidant capacity (TAC) in the blood and aqueous humor of glaucomatous and nonglaucomatous patients. To measure the adenosine triphosphate/adenosine diphosphate/adenosine monophosphate (ATP/ADP/AMP) concentration as a biomarker of the blood energy charge potential. METHODS: We examined 40 consecutive patients with primary open-angle glaucoma scheduled for cataract surgery. Twenty-six age-matched subjects scheduled for cataract surgery were enrolled as a control group. Blood and aqueous humor samples were collected at the time of surgery. MDA concentrations and blood nucleotides were measured with high-performance liquid chromatography. The TAC of the samples was estimated with the oxygen-radical absorbance capacity method. RESULTS: Blood and aqueous humor MDA levels in glaucoma patients (respectively, 0.976±0.370 and 0.145±0.065 µmol/ml) were significantly increased (p<0.001 for both) over those of the control group (respectively, 0.454±0.395 and 0.060±0.039 µmol/ml). In contrast, the control group presented significantly higher TACs than did the glaucoma group in both the blood (control: 2.681±1.101 and glaucoma: 1.617±0.674 µmol Trolox Equi/g; p<0.001) and aqueous humor (control: 0.963±0.302 and glaucoma: 0.788±0.346 µmol Trolox Equi/g; p=0.039). The control group (0.869±0.037) exhibited statistically significant (p<0.001) higher values of blood adenosine triphosphate/adenosine diphosphate (ATP-ADP) levels than did the glaucoma group (0.791±0.037). CONCLUSIONS: Our data further support the hypothesis that oxidative stress and decreased antioxidant defenses are involved in glaucoma. High-performance liquid chromatography appears to be an effective and sensitive method to detect altered levels of oxidative stress markers in glaucoma patients.

Modulation of RAGE isoforms expression in the brain and plasma of rats exposed to transient focal cerebral ischemia.

Activation of RAGE (receptor for advanced glycation endproducts) and of its subtypes may play a role in neuronal damage and neuroinflammation associated with brain ischemia, though the underlying mechanisms remain unclear. In this study, we have examined by Western blotting the expression of RAGE isoforms in the cerebral cortex and striatum of Wistar rats subjected to transient (1 or 2 h) middle cerebral artery occlusion (tMCAo). The findings show that the full-length RAGE (~50 kDa) and its isoforms in the 26-43 kDa range are significantly decreased in the ischemic cortex, but not in the striatum, after 1 and 2 h tMCAo when compared to the sham group. By contrast, in the striatum, ischemia-reperfusion injury caused a significant increase of full-length RAGE and its isoforms in the 72-100 kDa range. We also investigated the soluble form of RAGE, which was significantly decreased in the plasma of rats subjected to transient or permanent MCAo. In conclusion, the present data demonstrate that regional brain expression of RAGE is differentially affected by tMCAo in rat. These modifications are accompanied by a decrease in the plasma levels of soluble RAGE, thereby suggesting a potential role for soluble RAGE as a peripheral biomarker of focal ischemia.