Methylglyoxal is a cytotoxic reactive carbonyl compound produced by central metabolism. Dedicated glyoxalases convert methylglyoxal to d-lactate using multiple catalytic strategies. In this study, the DJ-1 superfamily member ORF 19.251/GLX3 from Candida albicans is shown to possess glyoxalase activity, making this the first demonstrated glutathione-independent glyoxalase in fungi. The crystal structure of Glx3p indicates that the protein is a monomer containing the catalytic triad Cys(136)-His(137)-Glu(168). Purified Glx3p has an in vitro methylglyoxalase activity (Km = 5.5 mM and kcat = 7.8 s(-1)) that is significantly greater than that of more distantly related members of the DJ-1 superfamily. A close Glx3p homolog from Saccharomyces cerevisiae (YDR533C/Hsp31) also has glyoxalase activity, suggesting that fungal members of the Hsp31 clade of the DJ-1 superfamily are all probable glutathione-independent glyoxalases. A homozygous glx3 null mutant in C. albicans strain SC5314 displays greater sensitivity to millimolar levels of exogenous methylglyoxal, elevated levels of intracellular methylglyoxal, and carbon source-dependent growth defects, especially when grown on glycerol. These phenotypic defects are complemented by restoration of the wild-type GLX3 locus. The growth defect of Glx3-deficient cells in glycerol is also partially complemented by added inorganic phosphate, which is not observed for wild-type or glucose-grown cells. Therefore, C. albicans Glx3 and its fungal homologs are physiologically relevant glutathione-independent glyoxalases that are not redundant with the previously characterized glutathione-dependent GLO1/GLO2 system. In addition to its role in detoxifying glyoxals, Glx3 and its close homologs may have other important roles in stress response.

Heart failure and arrhythmias occur at 3 to 5 times higher rates among individuals with diabetes mellitus, compared with age-matched, healthy individuals. Studies attribute these defects in part to alterations in the function of cardiac type 2 ryanodine receptors (RyR2s), the principal Ca²⁺-release channels on the internal sarcoplasmic reticulum (SR). To date, mechanisms underlying RyR2 dysregulation in diabetes remain poorly defined. A rat model of type 1 diabetes, in combination with echocardiography, in vivo and ex vivo hemodynamic studies, confocal microscopy, Western blotting, mass spectrometry, site-directed mutagenesis, and [³H]ryanodine binding, lipid bilayer, and transfection assays, was used to determine whether post-translational modification by reactive carbonyl species (RCS) represented a contributing cause. After 8 weeks of diabetes, spontaneous Ca²⁺ release in ventricular myocytes increased ∼5-fold. Evoked Ca²⁺ release from the SR was nonuniform (dyssynchronous). Total RyR2 protein levels remained unchanged, but the ability to bind the Ca²⁺-dependent ligand [³H]ryanodine was significantly reduced. Western blotting and mass spectrometry revealed RCS adducts on select basic residues. Mutation of residues to delineate the physiochemical impact of carbonylation yielded channels with enhanced or reduced cytoplasmic Ca²⁺ responsiveness. The prototype RCS methylglyoxal increased and then decreased the RyR2 open probability. Methylglyoxal also increased spontaneous Ca²⁺ release and induced Ca²⁺ waves in healthy myocytes. Treatment of diabetic rats with RCS scavengers normalized spontaneous and evoked Ca²⁺ release from the SR, reduced carbonylation of RyR2s, and increased binding of [³H]ryanodine to RyR2s. From these data, we conclude that post-translational modification by RCS contributes to the heterogeneity in RyR2 activity that is seen in experimental diabetes.

A rapid, sensitive, and reliable quality by design (QbD) based bioanalytical HPLC method was developed and validated for quantification of dapagliflozin (DFG) in rat plasma. The method was optimized using three levels and three-factor Box–Behnken statistical design (BBD). The mobile phase composition (acetonitrile % as A), flow rate (mL/min as B) and UV-wavelength (nm as C) selected as independent variable whereas area (AU as Y₁), retention time (min. as Y₂) and tailing factor (% as Y₃) selected as dependent variables. The developed method validated for linearity, sensitivity (LOD & LOQ), precision, accuracy, extraction recovery, matrix effect, and stability. Further the developed bioanalytical used to assess the pharmacokinetic study on a rat model. The developed method showed excellent linearity between 10 and 1200 ng/mL with r² = 0.997, LOD (2.15 ng/mL) and LOQ (6.52 ng/mL). The validation results of the tested parameters were found within the acceptable limit. Further, DFG loaded S-SNEDDS showed the droplet size (75.34 ± 5.34 nm), PDI (0.426) and zeta-potential (−25.6 mV). The pharmacokinetic study results revealed the C_max of S-SNEDDS was 1.42 fold higher than DFG dispersion. The significantly high AUC value observed with S-SNEDDS indicated increased bioavailability of DFG as compared to DFG dispersion. From the study, it was concluded that QbD approach based bioanalytical method is suitable for the in-vitro and in-vivo estimation of DFG from bulk as well as developed formulations.