Universidad Autonoma Del Estado De Morelos

Pompe disease (PD) is an autosomal recessive disorder caused by a deficiency of lysosomal acid alpha-1,4-glucosidase (GAA; EC 3.2.1.20), encoded by the GAA gene, leading to progressive neuromuscular deterioration. The mutational spectrum of GAA is expanding, particularly with recognition of splicing-impacting synonymous variants. Here, we report the molecular characterization and reclassification of a novel synonymous GAA variant in a female Mexican patient with enzymatically confirmed infantile-onset PD (IOPD).

DNA and RNA were extracted from peripheral blood. Next-generation sequencing of a clinical exome panel identified variants in the GAA gene. The whole coding sequence of the GAA was amplified from cDNA, subcloned, and analyzed by Sanger sequencing to assess splicing alterations.

The pathogenic variant c.1979G > A (p.Arg660His) and a novel VUS c.2799G > A (p.Lys933=) were detected. The variant c.2799G > A is predicted to affect splicing. mRNA analysis revealed three isoforms, one corresponding to c.1979G > A and two others caused by alternative splicing of c.2799G > A (isoforms 1 and 2). The isoform-1 showed c.2799G > A, followed by 61 bp retention of intron 19, predicted to cause a frameshift p.(A934fs). Isoform 2 has a 90 bp deletion in exon 19, resulting in a p.V904_K933del in-frame deletion.

This study demonstrates that the synonymous variant c.2799G > A is pathogenic due to its impact on RNA splicing. Our findings highlight the importance of transcript analysis for VUS reclassification and stress the clinical relevance of evaluating synonymous variants in genetic diagnostics and patient management.

Lipases from Pseudomonas species are valuable biocatalysts, but their heterologous expression is often complicated by the need for different protocols to ensure proper folding and activity. In this study, we successfully produced a soluble and active form of LipGoM, a lipase derived from Pseudomonas sp., that employs a refolding strategy after solubilization in urea. Our findings indicate that refolding LipGoM in the presence of its native foldase, LifGoM, is essential for restoring its enzymatic activity. Furthermore, we identified that optimizing the refolding conditions, specifically pH, ionic strength, protein concentration, and the addition of glycerol, reducing agents, and ions, was critical for success. The lipase-lipid GoM mixture, Lip-Lif GoM, exhibited maximal hydrolytic activity toward p-nitrophenyl-octanoate at 55 °C and pH 8.0, demonstrating notable stability in the presence of detergents and organic solvents. These results demonstrate the indispensable role of its cognate foldase, LifGoM, in activating LipGoM, as well as the significance of buffer composition, particularly glycerol, for the effective refolding of the enzyme and its subsequent activity.

This study aimed to remove a mixture of the EPA's 16 priority pollutant polycyclic aromatic hydrocarbons (PAHs) using sugarcane bagasse as an adsorbent. Initially, the bagasse fibers were characterized by scanning electron microscopy and energy-dispersive spectroscopy for elemental analysis, Fourier transform infrared spectroscopy, and Brunauer-Emmett-Teller (BET). The highest loading capacity was observed for PAHs with two to three aromatic rings. Meanwhile, the highest removal percentages occurred at the highest concentration level in a contact time of approximately 40 min. Adsorption equilibrium data fitted well to the Temkin and Elovich isotherm models, suggesting a multilayer chemical adsorption process. Additionally, Density Functional Theory (DFT) revealed that the primary adsorption mechanisms of PAHs onto bagasse were driven by van der Waals forces, π-π stacking, and hydrogen bond interactions, with lignin facilitating adsorption through its encapsulating behavior.