Galdieria sulphuraria is a thermoacidophilic red alga that thrives in extreme environments and encodes numerous hypothetical proteins with uncharacterized functions. In this study, we describe the recombinant production, purification, and isotopic labeling of a truncated construct (residues 1-130) of the hypothetical protein Gasu_38600 for NMR-based structural analysis. The construct was expressed in Escherichia coli using a maltose-binding protein fusion system and purified via immobilized metal affinity chromatography followed by TEV protease cleavage. Uniform ¹⁵N and [¹³C,¹⁵N] isotopic labeling was achieved using minimal media supplemented with ¹⁵NH₄Cl and ¹³C-glucose. High-quality ¹H¹⁵N HSQC spectra indicated that the protein adopts a folded conformation in solution, supporting its suitability for further structural investigation. These results lay the groundwork for future studies to elucidate the function of this conserved hypothetical protein from an extremophilic organism.

Fluoxetine, a common antidepressant and an environmental contaminant, was investigated for its effects on zebrafish larvae and adults using NMR-based metabolomics. Adult zebrafish were exposed to fluoxetine concentrations of 70 and 700 ㎍/L for 72 hours, while larvae were exposed to 7 and 70 ㎍/L from 1 to 72 hours post-fertilization. Our analysis revealed alterations in 77 metabolites in adults and 58 in larvae, with both groups showing changes in citrate, serine, and glutathione levels, and decrease in succinate and fumarate, highlighting some conserved metabolic responses to fluoxetine. Pathway analysis indicated significant effects on the TCA cycle, as well as glycine, serine, and threonine metabolism across both developmental stages. Notably, larvae exhibited distinct shifts with increased glutamine and decrease in glutamate and glycine, suggesting heightened sensitivity during early development. In adults, specific changes included increases in ATP, N,N-dimethylglycine, and alanine, with decreases in phenylalanine, tyrosine, betaine, NADP⁺ and 3-hydroxyisovalerate, pointing to possible metabolic disruptions. These alterations affected key pathways, notably the glycerolipid pathway in larvae and tyrosine and β-alanine metabolism in adults, indicating stage-specific susceptibility to fluoxetine. Furthermore, the application of HR-MAS NMR has proven instrumental in elucidating the complex metabolic responses to fluoxetine exposure, underscoring its utility in biotoxicity assessments.