International Journal of Molecular Sciences

Tyrosinase, encoded by Tyr, is a key rate-limiting enzyme in melanin biosynthesis. Knockout of Tyr results in a distinct albino phenotype, making it a widely used target for evaluating gene-editing efficiency. Here, we found that the tyrosinase-deficient skin melanophore lineage of Xenopus tropicalis (X. tropicalis) tadpoles shows strong autofluorescence under the GFP filter, which may interfere with in vivo fluorescence imaging. Through spectral scanning analysis, we characterized the emission spectrum of the autofluorescence under commonly used excitation wavelengths for fluorescent proteins. Based on this, we established a reference protocol for identifying and excluding such interference in Tyr-targeted knockin studies. Furthermore, knockout of the GTP cyclohydrolase 2 gene (Gch2) using CRISPR-Cas9 significantly reduced the fluorescence intensity induced by tyrosinase deficiency, indicating an essential role of the enzyme and its mediated pterine biosynthesis in the generation of the autofluorescence. This study systematically characterized these fluorescent mutant melanophores in X. tropicalis tadpoles, providing a practical basis for avoiding fluorescent interference in experimental science and a new perspective on pigment cell development and evolution.

The 2022 global mpox outbreak represented a turning point in the Monkeypox virus (MPXV) epidemiology, highlighting the incredible capability of this virus to adapt to different conditions, also in a non-endemic context. To investigate the genomic dynamics of MPXV 2022 strains, we performed whole-genome sequencing of 40 clinical samples from 16 Italian patients across multiple anatomical sites and timepoints between May and December 2022. Combining single-nucleotide analysis with detailed investigation of short tandem repeats (STRs), we explored inter- and intra-host viral dynamics. We identified 19 STR loci located near or within genes involved in immune modulation and virion morphogenesis. While most STRs remained stable across patients, a subset displayed locus- or matrix-specific variation. Among these, STR-VII—embedded within the coding sequence of OPG153, an envelope-associated protein implicated in viral attachment—showed a 12-nucleotide in-frame deletion, resulting in the loss of four aspartic acid residues (Δ4D variant). Structural modeling indicated that this deletion slightly alters a disordered acidic loop without affecting the global fold, potentially modulating surface charge and immune recognition. Integrating STR profiling into genomic surveillance may enhance resolution in outbreak reconstruction and reveal subtle adaptive processes underlying poxvirus–host interaction and immune escape.

The thermophilic bacterium Thermus thermophilus represents a crucial genetic reservoir for exploring thermostable enzymes as valuable biocatalysts for industrial and biotechnology applications. Here, we identify, clone, and characterize Ces1-ET, Est1-ET, and Plp1-ET, three lipolytic enzymes obtained from T. thermophilus strain ET-1 isolated from El Tatio Geothermal Field in Northern Chile. To enable recombinant expression, we constructed the pTGT-1 expression system, a versatile bifunctional shuttle vector compatible with both Escherichia coli and T. thermophilus. The three thermoenzymes Ces1-ET, Est1-ET, and Plp1-ET, were successfully cloned, expressed, and purified using the pTGT-1 system, with a molecular mass of 25 kDa, 36 kDa, and 28 kDa, respectively. The recombinant purified enzymes displayed optimal temperatures at 60 °C, 80 °C, and 70 °C and optimal pH of 7.5, 9.0, and 8.0 for Ces1-ET, Est1-ET, and Plp1-ET, respectively. Functional biochemical assays revealed a broad tolerance to surfactants, detergents, divalent cations, and high salinity, relevant properties for their application in an industrial setting. These thermostable esterases expand the repertoire of thermozymes from Thermus spp., introducing pTGT-1 as an innovative tool for thermophilic protein expression and highlighting T. thermophilus strain ET-1 from El Tatio Geothermal Field as a valuable source of thermostable enzymes for industrial and biotechnology applications.