NCMN (N-(3-carboxy propyl)-4-methoxy-1,8-naphthalimide), a newly developed ratiometric two-photon fluorescent probe for human Cytochrome P450 1A (CYP1A), shows the best combination of specificity and reactivity for real-time detection of the enzymatic activities of CYP1A in complex biological systems. This study aimed to investigate the interspecies variation in NCMN-O-demethylation in commercially available liver microsomes from human, mouse, rat, beagle dog, minipig and cynomolgus monkey. Metabolite profiling demonstrated that NCMN could be O-demethylated in liver microsomes from all species but the reaction rate varied considerably. CYP1A was the major isoform involved in NCMN-O-demethylation in all examined liver microsomes based on the chemical inhibition assays. Furafylline, a specific inhibitor of mammalian CYP1A, displayed differential inhibitory effects on NCMN-O-demethylation in all tested species. Kinetic analyses demonstrated that NCMN-O-demethylation in liver microsomes form rat, minipig and cynomolgus monkey followed biphasic kinetics, while in liver microsomes form human, mouse and beagle dog obeyed Michaelis-Menten kinetics, the kinetic parameters from various species are much varied, while NCMN-O-demethylation in MLM exhibited the highest similarity of specificity, kinetic behavior and intrinsic clearance as that in HLM. These findings will be very helpful for the rational use of NCMN as a practical tool to decipher the functions of mammalian CYP1A or to study CYP1A associated drug-drug interactions in vivo.

(1) Objectives: Understanding the genetic basis of muscle development and meat quality traits in divergent pig breeds is crucial for advancing precision breeding strategies. (2) Methods: This study investigated transcriptome differences in the longissimus dorsi muscle between Chinese Congjiang Xiang (CX) and Landrace (LAN) pigs. RNA sequencing was performed on muscle tissues from ten individuals of each breed, generating 874.5 million raw reads with an average mapping rate of 89.3% to the pig reference genome. (3) Results: Transcriptional profiling revealed distinct expression patterns with 785 genes exclusively expressed in CX pigs and 457 genes unique to LAN pigs, while 7099 co-expressed genes were shared by both breeds. Differential expression analysis identified 2459 significantly different genes (|log2FC| ≥ 1, adjusted p-value < 0.05), with 1745 up-regulated and 714 down-regulated in CX pigs. Among the most significantly up-regulated genes in CX pigs were flavor-associated genes (ELOVL5/6, FASN, DGAT2, ALDH1A3, PPAR-γ) with log2FC values ranging from 1.21 to 3.88. GO and KEGG pathway analyses revealed that up-regulated genes in CX pigs were significantly enriched in immune response pathways (adjusted p-value < 0.01), while down-regulated genes were primarily associated with myosin complex formation and PPAR signaling pathway. PPI network analysis identified PPAR-γ as a central hub gene with 16 direct interactions to other flavor-related genes. (4) Conclusions: These findings demonstrate that the superior meat flavor characteristics of indigenous Chinese pigs are driven by enhanced expression of lipid metabolism genes and distinctive immune-related pathways, providing specific molecular targets for breeding programs aimed at improving meat quality while maintaining production efficiency in commercial breeds.

Understanding the genetic basis of muscle development and meat quality traits in divergent pig breeds is crucial for advancing precision breeding strategies. This study aimed to investigate transcriptome differences in the longissimus dorsi muscle between Chinese Congjiang Xiang (CX) and Landrace (LAN) pigs. RNA sequencing was performed on muscle tissues from ten individuals of each breed, with an average mapping rate of 89.3% to the pig reference genome. Transcriptional profiling revealed 785 genes exclusively expressed in CX pigs and 457 genes unique to LAN pigs, with 7,099 co-expressed genes shared by both breeds. Principal component analysis demonstrated clear separation between the breeds. Differential expression analysis identified 2,459 differentially expressed genes, with 1,745 up-regulated and 714 down-regulated in CX pigs. Notably, genes associated with meat flavor formation (ELOVL5/6, FASN, DGAT2, ALDH1A3, PPAR-γ) were significantly up-regulated in CX pigs. Functional enrichment analysis showed that up-regulated genes in CX pigs were primarily enriched in immune responses, while down-regulated genes were associated with myosin complex and PPAR signaling pathway. These findings provide novel insights into the molecular mechanisms underlying the distinctive meat characteristics of indigenous Chinese and commercial European pig breeds, offering valuable genetic resources for future breeding programs aimed at enhancing pork quality.

Meat flavor significantly influences consumer preference and market value, particularly for indigenous pig breeds renowned for distinctive sensory characteristics. While traditional research has focused on genetic factors and feeding regimens, emerging evidence suggests that gut microbiota plays a crucial role in meat quality attributes. However, the specific contribution of bacterial–fungal co-occurrence to meat flavor formation remains largely unexplored. This study aimed to characterize the associations between intestinal bacterial–fungal co-occurrence networks and the muscle flavor-related metabolite profiles of CX pigs, using an integrated multi-omics approach. Twenty male pigs (10 CX and 10 LAN, 12 months old) were subjected to comprehensive analyses, including meat quality evaluation, electronic nose analysis, 16S and 18S rRNA sequencing, and untargeted metabolomics. CX pigs exhibited significantly superior meat quality characteristics, including higher moisture content (p < 0.001), fat content (p = 0.008), and meat color scores (p < 0.001). Electronic nose analysis revealed significantly higher response values across all ten aroma sensors in CX pigs (p < 0.001), with the most pronounced differences observed in sensors detecting sulfur compounds and organic compounds. Untargeted metabolomics identified 40 differential metabolites, with 27 up-regulated in CX pigs, including key flavor compounds such as glycocholic acid, isorhamnetin, and pantothenic acid. Microbiome analysis demonstrated significantly higher bacterial alpha diversity in CX pigs (p < 0.05), with enrichment of beneficial bacteria, including Rikenellaceae_RC9_gut_group, Prevotellaceae_UCG_003, and Phascolarctobacterium, while fungal communities showed enrichment of Candida_Lodderomyces_clade. Correlation network analysis revealed that Rikenellaceae_RC9_gut_group demonstrated strong positive correlations with flavor compounds (r = 0.575 for isorhamnetin, r = 0.535 for pantothenic acid, p < 0.001) and all electronic nose responses (r = 0.434–0.691, p < 0.001). Bacterial–fungal co-occurrence networks showed synergistic relationships, with Rikenellaceae_RC9_gut_group positively correlated with Candida_Lodderomyces_clade (r = 0.711, p < 0.001) while exhibiting antagonistic relationships with Piromyces (r = −0.714, p < 0.001). These findings offer novel insights for developing microbiome-targeted strategies to enhance meat quality in pig production systems.