Tibetan pigs, a typical high-altitude indigenous breed in China, have developed unique physiological and metabolic characteristics through long-term natural selection under extreme plateau environmental conditions such as low oxygen and low temperature. However, stage-specific differences in their gut microbiota composition remain to be investigated. [Objective] To compare the rectal fecal microbiota composition and short-chain fatty acids (SCFAs) in Tibetan pigs at different growth stages from birth to the fattening stage. [Methods] Ten pregnant Tibetan sows with similar parity (2-3) and good body condition were selected and allowed to farrow synchronously. The offspring were reared from birth to 140 days old. A cross-sectional sampling design was adopted to collect rectal fecal samples from 10 pigs at 1, 7, 21, 70, and 140 days old for 16S rRNA gene sequencing and determination of SCFA concentrations. [Results] Microbial diversity in rectal fecal samples of Tibetan pigs gradually increased with age and stabilized during the later growth stage. Bacillota and Bacteroidota remained dominant throughout development. At the genus level, the gut microbiota of neonatal piglets was primarily colonized by Clostridium, Escherichia-Shigella, Streptococcus, and Fusobacterium. Notably, dominant genera in the gut during the suckling stage, such as Lactobacillus and Rikenellaceae RC9 gut group, remained abundant after weaning. Christensenellaceae R-7 group and Treponema showed low relative abundance before weaning but were significantly enriched after weaning and were among the dominant genera at 70 and 140 days old. PICRUSt2-based functional prediction analysis showed that the rectal fecal microbiota of Tibetan pigs at 21 days old was mainly enriched in pathways related to the citrate cycle and oxidative phosphorylation, whereas that at 70 and 140 days old was mainly enriched in amino sugar and nucleotide sugar metabolism, glycolysis/gluconeogenesis, and starch and sucrose metabolism. The concentrations of acetate, propionate, butyrate, and total SCFAs in rectal fecal samples at 70 and 140 days old were significantly higher than those at 21 days old. Correlation analysis revealed that Treponema and Rikenellaceae RC9 gut group had significantly positive correlations with acetate, propionate, butyrate, and total SCFA concentrations. [Conclusion] The gut microbiota composition differed markedly among Tibetan pigs at different growth stages. Beneficial bacteria such as Lactobacillus maintained relatively high abundance throughout the growth stages, while Christensenellaceae R-7 group remained abundant during the fattening stage. Moreover, the significant enrichment of complex carbohydrate-fermenting bacteria, represented by Treponema and Rikenellaceae RC9 gut group, during the growing-finishing stage suggests their key role in the hindgut fermentation of Tibetan pigs.

[Objective] To evaluate the inhibitory effects of polyunsaturated fatty acids (PUFAs) on oral microorganisms and to elucidate the response patterns of the oral microbiota to docosahexaenoic acid (DHA) treatment, as well as the genomic features associated with the sensitivity or tolerance. [Methods] The minimum inhibitory concentrations of four PUFAs were determined for five common species of oral bacteria. The effects of PUFAs at different concentrations on planktonic growth and biofilm formation were assessed in both single-species and mixed-species culture systems. Human intervention and metagenomic sequencing were employed to analyze the impact of DHA on oral microbiota composition and the abundance of specific bacterial taxa. The DHA sensitivity of Fusobacterium pseudoperiodonticum isolates from saliva was compared, followed by whole-genome analysis. [Results] The inhibitory effects of the four PUFAs varied among bacterial species. In single-species cultures, Streptococcus oralis, Streptococcus mutans, and Porphyromonas gingivalis were sensitive, whereas Fusobacterium nucleatum and Prevotella intermedia showed tolerance. In mixed-species cultures, the overall antibacterial effects were reduced. In the human intervention study, an 8-day mouth rinse with DHA did not significantly alter oral microbiota at the phylum level, while selectively modulating the oral microbiota at the genus and species levels, with increased relative abundance of Neisseria and Porphyromonas and decreased relative abundance of Prevotella. Dynamic monitoring indicated that DHA selectively influenced certain taxa over time, inducing a downward trend in F. nucleatum abundance. Strain-level analysis revealed differential DHA sensitivity among F. pseudoperiodonticum isolates, which was closely associated with variations in genes related to cell envelope and lipid metabolism (e.g., lapB, pilE, and ftsZ). [Conclusion] The inhibitory effects of PUFAs on oral pathogens exhibit species- and system-dependent heterogeneity. DHA selectively suppresses specific bacteria while maintaining the overall stability of the oral microbiota, and the strain-dependent variation in sensitivity may be associated with genomic characteristics. These findings provide new evidence for understanding the role of PUFAs, particularly DHA, in oral microbiota modulation and support their potential applications in this field.

[Objective] To investigate the effects of Shouwu Pill (SWW) on physical performance, gut microbiota composition, and bile acid metabolism in the mouse model of doxorubicin (DOXO)-induced premature aging, and to preliminarily explore its potential mechanisms. [Methods] A mouse model of premature aging was established by intraperitoneal injection of DOXO. Mice were randomized into Control, DOXO, DOXO+Shouwu Pill low-dose (SWW-L), and DOXO+Shouwu Pill high-dose (SWW-H) groups. Grip strength test, hanging endurance test, and treadmill exercise test were performed to evaluate physical performance. Gut microbiota composition and diversity were analyzed by 16S rRNA gene sequencing, followed by PICRUSt functional prediction. Fecal bile acid profiling was performed via UHPLC-QQQ-MS. Spearman correlation analysis was further conducted to explore the association between differentially abundant gut microbial taxa and bile acid metabolism. [Results] Compared with the Control group, DOXO-treated mice exhibited increased systemic inflammatory cytokine levels and significantly decreased forelimb grip strength, hanging endurance, and maximal running speed, indicating successful establishment of the premature aging model. SWW treatment significantly reduced systemic inflammation and improved physical performance, with the DOXO+SWW-H group demonstrating greater efficacy than the DOXO+SWW-L group. The results of 16S rRNA gene sequencing revealed that DOXO induced gut microbiota dysbiosis characterized by altered microbial diversity and disrupted microbiota structure, including decreased abundance of Muribaculaceae and increased abundance of opportunistic pathogenic genera such as Desulfovibrio and Helicobacter. SWW treatment partially restored the microbiota structure by increasing the abundance of potentially beneficial bacteria, including Akkermansia, Lactobacillus, Bifidobacterium, and Roseburia. PICRUSt functional prediction indicated significant alterations in bile acid metabolism-related pathways following SWW intervention. Fecal bile acid profiling further demonstrated that DOXO treatment led to the accumulation of several conjugated bile acids, including T-α-MCA and T-β-MCA, accompanied by reduced levels of unconjugated bile acids. SWW intervention significantly lowered the levels of conjugated bile acids and partially restored the levels of unconjugated bile acids. Correlation analysis showed that several beneficial bacteria were positively correlated with unconjugated bile acids but negatively correlated with conjugated bile acids. [Conclusion] SWW ameliorates physical decline and gut microbiota dysbiosis in the mouse model of DOXO-induced premature aging. Its anti-aging effects may be associated with modulation of gut microbiota-related bile acid metabolism and restoration of bile acid homeostasis.

[Objective] To achieve efficient expression and comprehensive functional characterization of chitosanase (Csn, EC 3.2.1.132). [Methods] The chitosanase gene (CsnBh) was cloned from Bacillus halotolerans GXK615 and heterologously expressed in Escherichia coli BL21(DE3). [Results] Following purification via nickel-affinity chromatography, the recombinant CsnBh exhibited a specific activity of 400.47 U/mg, which represented 3.03-fold purification. Enzymatic characterization revealed that CsnBh demonstrated high specificity toward colloidal chitosan, with optimal activity observed at 40 ℃ and pH 6.0. The enzyme demonstrated remarkable thermal stability across a broad temperature range (30-70 ℃) and pH stability over pH 5.0-9.0. Among the metal ions tested, Mn²⁺ significantly enhanced enzymatic activity, whereas Cu²⁺, Fe²⁺, and Fe³⁺ completely inhibited the activity. Hydrolysis pattern analysis confirmed an endo-acting cleavage mechanism, with chitotriose to chitopentaose identified as the predominant oligosaccharide products. Further investigation showed that the chitooligosaccharide (COS) mixture generated by CsnBh-mediated hydrolysis exerted potent inhibitory effects against E. coli, Staphylococcus aureus, and Phytophthora nicotianae. Moreover, COS treatment significantly upregulated the activities of key plant defense-related enzymes, including superoxide dismutase, peroxidase, and phenylalanine ammonia-lyase, in tobacco leaves. [Conclusion] This study establishes both a robust theoretical foundation and a highly promising enzyme candidate for the efficient production of chitosanases and the green synthesis and application of bioactive COSs.

[Objective] Animal sera have the potential to induce predatory organ formation in nematode-trapping fungi, while the specific mechanism remains unclear. This study systematically compared the inductive effects of different animal sera on predatory organ formation in nematode-trapping fungi, determined the optimal serum type and concentration, and identified and validated key synergistic active metabolites, aiming to provide a basis for elucidating the chemical signaling mechanisms underlying predatory organ formation and for applications in agricultural biocontrol. [Methods] D. dactyloides capable of producing constricting rings was used as the model strain. Horse, pig, sheep, and bovine sera at different concentrations were applied in induction cultures to determine the optimal induction conditions. Constricting ring development was monitored over time. Ring inflation was assessed by stimulation with 55 ℃ hot water, and trapping and digestion activities against Caenorhabditis elegans were further evaluated. Untargeted metabolomic profiling was performed for horse, pig, and bovine sera with different inductive effects, followed by differential metabolite screening and clustering analysis. Lyso-platelet-activating factor C-16(lyso-PAF C-16), linoleic acid (LA), and glycochenodeoxycholic acid (GCDCA) were subsequently tested in synergistic induction assays. [Results] All the sera were able to induce constricting ring formation, although their induction efficiencies differed markedly. Horse serum showed the strongest induction activity at concentrations of 0.5%-1%. A small number of constricting rings appeared at 48 h, and ring formation peaked at 72-96 h. Rings formed at 48 h could not be stimulated to inflate, whereas those formed at 96 h exhibited efficient inflation and effective nematode trapping and digestion. Metabolomic analysis revealed clear separation among serum groups and identified several candidate metabolites enriched in horse serum. Functional validation showed that GCDCA significantly increased constricting ring formation when combined with 1% horse serum, producing the strongest effect at 0.04 μmol/L. [Conclusion] This study establishes a robust induction system for constricting ring formation in D. dactyloides, clarifies the optimal induction conditions and time, and identifies GCDCA as a key synergistic factor that promotes constricting ring formation.

[Objective] Mycoplasmoides pneumoniae is a major pathogen responsible for community-acquired pneumonia worldwide, and the aggravating antibiotic resistance underscores the urgent need to develop an effective vaccine. [Methods] According to the critical pathogenic mechanisms of M. pneumoniae, 13 key adhesins and the community-acquired respiratory distress syndrome (CARDS) toxin were selected as candidate antigens. Reverse vaccinology and immunoinformatics approaches were adopted to predict and screen cytotoxic T lymphocyte (CTL) epitopes, helper T lymphocyte (HTL) epitopes, and linear B-cell epitopes. The screened epitopes were then connected via specific linkers. Two adjuvants, human beta-defensin 2 (HBD-2) and 50S ribosomal protein L7/L12, were introduced at the N-terminus to assemble the complete vaccine sequence. Furthermore, the physicochemical and biological properties of the constructed vaccine sequence were comprehensively analyzed. [Results] A total of 15 CTL epitopes, 10 HTL epitopes, and 14 linear B-cell epitopes were obtained after screening. All the epitopes and adjuvants were ligated by specific linkers, and a final multi-epitope vaccine sequence consisting of 746 amino acid residues was successfully constructed. The computational analysis indicated that the vaccine construct exhibited favorable physicochemical properties, antigenicity, solubility, and a safety profile. Molecular docking results showed that this construct could bind to Toll-like receptor 2 (TLR2) and TLR4 stably, which was further confirmed with the results of molecular dynamics simulations. The immune simulation and epitope coverage analysis indicated that the vaccine could elicit potent humoral and cellular immune responses and provide potential protection for up to 99.74% of the global population. [Conclusion] Through systematic computational design and evaluation, this study develops a multi-epitope mRNA candidate vaccine with promising application prospects, laying a theoretical foundation for subsequent experimental validation and development.

[Objective] To address the current issue that pathogen detection methods rely on specialized personnel and precision instruments and are thus unsuitable for on-site rapid testing, this study established a portable and rapid detection method for Staphylococcus aureus (SA) based on the recombinase polymerase amplification (RPA)-CRISPR/dCas9 system and NanoLuc binary interaction technology (NanoBiT). [Methods] Firstly, targeting the highly conserved thermostable nuclease-encoding gene (nuc) of SA, RPA primers and single guide RNA (sgRNA) with high sensitivity were selected. Secondly, reaction parameters including the concentration ratio of sgRNA to dCas9 protein and the detection time were optimized, and detection signals were read by a self-developed portable photoelectric biosensor. Finally, with the optimized reaction system and conditions, this method was evaluated in terms of the sensitivity, specificity, adaptability under different environmental temperatures, and practical sample detection performance. [Results] The optimized method can complete the detection within 1 h. With the mean signal of the negative control plus three times the standard deviation (mean+3SD) as the threshold, the limit of detection (LOD) reached 10² copies/µL. The method was specifically sensitive only to the target bacterium SA, demonstrating high specificity, sensitivity, and environmental adaptability. [Conclusion] A highly sensitive, specific, portable, and low-cost detection method for SA was successfully established based on RPA-CRISPR/dCas9 and NanoBiT. This method is particularly suitable for on-site rapid testing in resource-limited settings and holds broad application prospects.

In the context of global carbon emission reduction and carbon neutrality strategies, microbial carbon-fixing enzymes have emerged as a research frontier in biological carbon fixation technologies due to their unique advantages, including high efficiency, low energy consumption, and the ability to produce value-added chemicals. This review systematically summarizes the classification, structural characteristics, catalytic mechanisms, and regulatory networks of both natural and engineered carbon-fixing enzymes. It focuses on the roles and kinetic regulation of key enzymes such as ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), carbonic anhydrase (CA), formolase (FLS), and carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) in CO₂ fixation. From a structural biology perspective, the review discusses how static structures and dynamic conformational changes determine catalytic efficiency. Furthermore, this paper reviews the recent advances in protein engineering strategies including rational design, directed evolution, high-throughput screening, and artificial intelligence (AI)-assisted prediction for carbon-fixing enzymes, along with the enabling role of specialized databases such as Greenase in enzyme mining and pathway reconstruction. Representative case studies, including RuBisCO, CA, FLS, and multi-enzyme cascade systems, are adopted to illustrate engineering pathways from single-enzyme optimization to integrated systems. Finally, future directions are discussed, including dynamic catalytic mechanism simulation, in vivo fitness engineering, de novo enzyme design, and electro-/photo-enzyme coupling systems. This review provides a theoretical reference for the development of efficient, stable, and industrially adaptable novel biological carbon fixation technologies.

The efficient conversion of CO₂ into fuels and chemicals represents a key route toward sustainable biomanufacturing, and microbial cell factories offer significant potential for this purpose. Despite substantial advances in carbon fixation pathway design and carbon flux rewiring, the performance of microbial CO₂-fixing systems remains largely constrained by the efficiency of energy supply and utilization. At the core of this limitation is a systemic mismatch between energy metabolism and carbon fixation, involving discrepancies in energy carrier types [ATP, NAD(P)H, and low-potential electrons], supply ratios, and redox potential, which ultimately restrict carbon flux and energy utilization efficiency. From the perspective of energy-carbon flux coupling, this review proposes an energy conversion chain framework that conceptualizes carbon fixation as a cascade network comprising energy input, electron transfer, and carbon assimilation. On the basis of this framework, a quantitative evaluation system is established, encompassing energy input efficiency (EE), redox/electron utilization efficiency (RE), and energy-to-carbon conversion efficiency (ECE). By integrating experimental measurements with metabolic modeling, we systematically analyze the distribution of energy carriers and electron fluxes and identify key bottlenecks, including limitations in energy input flux, electron transfer losses, cofactor mismatches, and reaction network efficiency. At the engineering level, we further summarize strategies for improving energy-carbon compatibility, including chassis selection under energy constraints, construction of exogenous energy-supplying modules, and rewiring of endogenous energy networks. Building upon these advances, a multi-scale energy-carbon co-design framework is proposed to achieve precise matching between energy supply and carbon fixation demand. Overall, this review redefines energy metabolism as a central design dimension in carbon fixation engineering and provides both a theoretical foundation and engineering strategies for the development of efficient and robust CO₂ bioconversion systems.