Background: The midgut undergoes histolysis and remodeling during the larval to adult transition in holometabolous insects, but the molecular mechanisms underlying this process are not well understood. Results: Using Suppression Subtractive Hybridization (SSH), we identified a 531 bp cDNA predicted to encode a 176 amino acid protein, which we call hmg176. Northern and western blot analysis suggested that high levels of hmg176 are expressed in the midgut during molting, but not during metamorphosis. HMG176 protein was detected by immunofluorescence within the membrane of fat bodies and the basement membrane of the midgut of both molting and feeding larvae, but not in metamorphically committed larvae. In situ hybridization revealed that hmg176 transcripts mainly localized to the columnar cells of the midgut. Interestingly, a non-steroidal ecdysone agonist, RH-2485, significantly upregulated expression of hmg176. Conclusion: These observations suggest that hmg176 encodes a larval-specific protein that may participate in sustaining larval midgut during larval development, possibly in response to ecdysteroid in vivo. This study will enlighten our understanding of the molecular mechanisms of tissue histolysis during metamorphosis.

Licorice (Gan-Cao, licorice) is a natural antioxidant and roasted licorice is the most common processing specification used in traditional Chinese medicine prescriptions. Traditional Chinese medicine theory deems that the honey-roasting process can promote the efficacy of licorice, including tonifying the spleen and augmenting “Qi” (energy). The antioxidant activity and mechanisms underlying roasted licorice have not yet been reported. In this study, we found that roasted licorice could relieve the oxidative stress injury induced by metronidazole (MTZ) and could restrain the production of excessive reactive oxygen species (ROS) induced by 2,2′-azobis (2-methylpropionamidine) dihydrochloride (AAPH) in a zebrafish model. It was further found that roasted licorice could exert its oxidative activity by upregulating the expression of key genes such as heme oxygenase 1 (HO-1), NAD(P)H quinone dehydrogenase 1 (NQO1), glutamate–cysteine ligase modifier subunit (GCLM), and glutamate–cysteine ligase catalytic subunit (GCLC) in the nuclear factor erythroid 2-related factor 2 (NRF2) signaling pathway both in vivo and in vitro. Furthermore, consistent results were obtained showing that rat serum containing roasted licorice was estimated to reduce cell apoptosis induced by H₂O₂. Then, the UHPLC-Q-Exactive Orbitrap MS analysis results elucidated the chemical composition of rat plasma containing roasted licorice extracts, including ten prototype chemical components and five metabolic components. Among them, six compounds were found to have binding activity with Kelch-like ECH-associated protein 1 (KEAP1), which plays a crucial role in the transcriptional activity of NRF2, using a molecular docking simulation. The results also showed that liquiritigenin had the strongest binding ability with KEAP1. Immunofluorescence further confirmed that liquiritigenin could induce the nuclear translocation of NRF2. In summary, this study provides a better understanding of the antioxidant effect and mechanisms of roasted licorice, and lays a theoretical foundation for the development of a potential antioxidant for use in clinical practice.

Background: The fecal microbiota is closely coupled to host metabolism and nutritional physiology in ruminants, but longitudinal biochemical variation across defined sika deer developmental sampling stages remains incompletely characterized. We therefore integrated microbiome, metabolomic, short-chain fatty acid (SCFA), and Raman spectroscopic measurements to describe stage-associated fecal changes. Methods: Fecal specimens were collected from male sika deer at two predefined antler-cycle stages, the rapid antler-growth stage (G; 1 March 2024) and the mature/ossification stage (M; 28 June 2024), 119 days apart. Eight animals were followed longitudinally and contributed matched specimens at both stages; two additional single-time-point specimens per stage were included, so that 16S rRNA gene sequencing, untargeted metabolomics, and SCFA quantification comprised 10 specimens per stage (n = 20), whereas Raman spectroscopy comprised the eight matched pairs (n = 16). The animal was treated as the biological unit: 16S, metabolomic, and SCFA comparisons used independent-sample tests on the 10 specimens per stage, and Raman comparisons used paired procedures on the eight matched animals, each with multiplicity correction. For Raman classification, technical spectra were averaged within each specimen and validation was structured by deer so that both stage specimens from the held-out animal were excluded from model training. Results: The two sampling stages differed in fecal microbial composition and biochemical profiles. Alpha-diversity indices did not differ significantly (all FDR-adjusted q ≥ 0.220), but genus-level analysis with FDR correction identified three differentially abundant genera (q < 0.05): UCG-005 and UCG-010 were more abundant at the G stage, and Treponema was more abundant at the M stage. Fecal butyrate was higher at the M stage (p = 0.0065, Bonferroni-adjusted p = 0.019), whereas valerate and caproate were higher at the G stage (adjusted p = 0.029 and 0.0002, respectively). Untargeted metabolomics identified 5621 differential features across the two electrospray ionization modes (|log2 fold change| ≥ 2 and p < 0.01; positive-ion features additionally selected at PLS-DA VIP ≥ 1.0), of which 2311 were increased and 3310 were decreased at the M stage relative to G, with no evidence of PLS-DA overfitting (permutation R²Y-intercept = 0.318, Q²-intercept = −0.237). Raman spectra showed stage-associated variation, with seven candidate bands showing FDR-adjusted differences (q = 0.007–0.028). A leakage-controlled classifier evaluated by grouped leave-one-deer-out cross-validation achieved an AUC of 0.875 (deer-level 95% CI 0.69–0.98; paired-label permutation p = 0.023). Conclusions: This exploratory longitudinal study identifies stage-associated variation in fecal microbial, metabolomic, SCFA, and Raman profiles in sika deer. The findings are associative rather than mechanistic because the cohort was small and developmental stage was not separated from season. Raman spectroscopy may provide complementary non-invasive biochemical phenotyping, but the classification result requires confirmation in a larger independent cohort before practical use.