Lipases are a group of biocatalysts that efficiently catalyze the hydrolysis, alcoholysis, acidolysis, ester exchange, and synthesis of esters at the oil-water interface. These enzymes play crucial roles in various fields, including pharmaceuticals and chemical engineering. Microbial lipases are more readily available and hold greater value for research and application than animal and plant lipases. [Objective] To isolate lipase-producing microorganisms from natural environments, optimize their culture conditions, and characterize the structure of the produced lipase. [Methods] We used bromocresol purple as an indicator to screen lipase-producing strains from oil-contaminated soil and identified the strains by morphological observation and 18S rRNA gene sequencing. We optimized the culture conditions of the strains by single-factor experiments and response surface methodology. We performed proteomic sequencing on the active proteins to identify the potential lipase. We amplified and sequenced the gene of the potential lipase by PCR and analyzed its multi-level structure. [Results] A lipase-producing fungal strain FA3 was isolated from oil-contaminated soil and identified as Aspergillus sp. The strain was cultured with olive oil as the sole carbon source, and the p-nitrophenol colorimetric method revealed that the intracellular enzyme activity of strain FA3 was 263.75 U/g. The optimized culture conditions were as follows: emulsified olive oil at 4 mL/L, peptone at 18 g/L, K₂HPO₄ at 1 g/L, NaCl at 10 g/L, MgSO₄ at 0.5 mmol/L, and pH 5.3. After culture at 30 ℃ for 77.5 h, the specific activity reached 2 120.27 U/g, which was about 8.04 times higher than that before optimization. The proteomic analysis and structural prediction revealed the conserved Gly-Asp-Ser motif, confirming the lipase as a typical GDSL lipase. The enzyme contained strictly conserved Ser, Gly, Asn, and His residues and it was thus classified as a member of the N-sulphoglucosamine sulphohydrolase (SGNH) hydrolase family. [Conclusion] After optimization of the culture conditions, strain FA3 demonstrated significantly enhanced lipase production, showing great potential for application. We clarify the functions and structural characteristics of the lipase, providing a basis for the engineering of related proteins.

[Objective] Rotavirus (RV) is a major pathogen causing acute dehydrating gastroenteritis in infants and young children. Currently, no specific therapeutic drugs are available, making preventive vaccination the most effective strategy for controlling RV infection. We targeted the RV receptor-binding domain viral protein 8^* (VP8^*) and selected its functional region ΔVP8^* (amino acids 65-223) to construct a single-chain dimer ΔVP8^*-sc-dimer. We expressed and purified this recombinant protein in a prokaryotic system using the pET-30a(+) vector and evaluated its immunogenicity and neutralizing antibody induction capacity to provide scientific evidence for developing safe and effective RV subunit vaccines. [Methods] The ΔVP8^*-sc-dimer sequence was synthesized and cloned into the prokaryotic expression vector pET-30a(+) via homologous recombination. The purified recombinant protein was formulated with AddaVax adjuvant and administered to 6 to 7-week-old BALB/c mice via intramuscular injection. ΔVP8^*-specific IgG antibody titers in sera were determined by enzyme-linked immunosorbent assay (ELISA), and neutralization activity of immune sera was assessed through virus neutralization assays. [Results] The recombinant protein ΔVP8^*-sc-dimer was successfully expressed with 90% purity. ELISA results showed that both ΔVP8^* and ΔVP8^*-sc-dimer induced specific anti-ΔVP8^* IgG antibodies following immunization, with the ΔVP8^*-sc-dimer group exhibiting significantly higher antibody titers. Virus neutralization assays revealed that immune sera from both groups neutralized the RV Wa strain, with the ΔVP8^*-sc-dimer group showing significantly superior neutralizing antibody titers. [Conclusion] The ΔVP8^*-sc-dimer subunit vaccine effectively stimulates high-level antibody production against RV Wa strain, demonstrating significantly enhanced immune responses compared with ΔVP8^*. With its excellent immunogenicity, ΔVP8^*-sc-dimer represents a promising candidate antigen for developing novel RV vaccines with substantial clinical application potential.

[Objective] To develop a recombinant influenza A virus (IAV) expressing a reporter (enhanced green fluorescent protein, EGFP), study its biological characteristics in vitro, and explore its application in antiviral drug screening and neutralizing antibody (nAb) detection. [Methods] The EGFP gene was inserted into the C-terminus of NA (derived from H1N1-PR8) by reverse genetics of influenza virus, and the recombinant IAV was successfully constructed, rescued, and named PR8NAEGFP/WSN. The EGFP insertion position and genetic stability were analyzed by RT-PCR and sequencing. PR8NAEGFP/WSN and the control virus (PR8NA/WSN, without EGFP insertion) were identified based on EGFP reporter gene expression, replication kinetics, and plaque morphology. The in vitro antiviral efficacy of favipiravir, a positive drug for influenza virus, was evaluated by focus-forming assay (FFA), qPCR, and EGFP fluorescence detection. The focus reduction neutralization test (FRNT) and reporter gene activity detection were conducted for IAV nAb detection. [Results] PR8NAEGFP/WSN remained genetically stable after five passages in chicken embryos. Compared with PR8NA/WSN, PR8NAEGFP/WSN showed a slightly declined titer at the time point of 48 h and similar crystal violet plaque morphology. The EC₅₀ of favipiravir measured with the recombinant virus is consistent with that with the control virus, and the EC₅₀ values obtained through various detection methods, including FFA, qPCR, and EGFP fluorescence, show good correlations. The correlation coefficient r of the nAb titer determined by FRNT and EGFP fluorescence was 0.930 4, which indicated good consistency. [Conclusion] PR8NAEGFP/WSN, a recombinant IAV carrying the reporter gene, might be used as a real-time visualization tool for the basic research on IAV and the evaluation of antivirals and vaccines for IAV.

Colorectal cancer (CRC) remains a focal point of research due to its consistently high incidence and mortality, as well as the substantial economic and healthcare burdens it imposes. In recent years, the pivotal role of the gut microbiota in the prevention, diagnosis, and treatment of CRC has garnered increasing attention, offering promising avenues for the management of advanced stages of the disease. This article comprehensively reviews the involvement of the gut microbiota in the development and progression of CRC, elucidating the underlying mechanisms. Furthermore, it examines the potential applications of the gut microbiota in the systemic treatment of advanced CRC, with the aim of proposing novel strategies and insights to enhance the diagnosis and treatment of advanced CRC.

[Objective] To explore the effects of plant diversity and soil improvement on the soil microbial community in abandoned lead-zinc mine wasteland. [Methods] A pot experiment was conducted with nine commonly used plants for ecological restoration in mining areas. Different plant diversity levels (S1 to S5) were set up, and for each level, a control group (Y: without soil amendment) and an improvement group (G: with organic fertilizer and polyacrylamide) were established. [Results] The plant height of the improvement group was generally higher than that of the control group. After soil improvement, the content of available nitrogen, available phosphorus, and available potassium all significantly increased, while that of heavy metals Cd and Pb decreased the most when the species richness was 9 (S5), dropping by 37.20% and 14.85% respectively. The diversity of soil microorganisms increased with the increase in plant diversity. The application of soil amendments reduced the richness and diversity of the fungal community, while enhancing the richness and diversity of the bacterial community. In the improvement group, the soil fungal abundance reached the highest level when GS4 configuration was adopted, with the Observed index and Chao1 index being 110.50 and 169.23, respectively. The soil bacterial abundance reached the highest level when GS2 was adopted, with the Observed index and Chao1 index being 1 081.59 and 1 116.79, respectively. In the fungal community, the abundance ofascomycetesat both the phylum and genus levels increased to varying degrees in the improvement group compared with the control group, and the abundance of Ascomycota increased with the increase in plant diversity. Soil improvement reduced the abundance of Mucoromycota and Rhizopus, but increased the abundance of Sordaria. Moreover, the abundance of Mucoromycota and Rhizopus decreased with the increase in plant diversity. In the bacterial community, soil improvement increased the abundance of Sphingomonas and Gemmatimonas. [Conclusion] Soil microbial diversity increases as the plant diversity increases. The application of soil amendments reduces the richness and diversity of soil fungi and improves the richness and diversity of soil bacteria. As the plant diversity increases, the abundance of Ascomycota increases, while that of Mucoromycota and Rhizopus decreases.

[Objective] To elucidate the virulence-associated biological characteristics of a transposon insertion mutant with impaired biofilm formation of Avibacterium paragallinarum. [Methods] The wild-type strain JZIC-005 and a biofilm-deficient mutant Tn-1504 (from a pre-established transposon mutant library) were selected for determination of insertion sites biological characterization. [Results] PCR results demonstrated site-specific transposon integration into TonB of A. paragallinarum. While mutant Tn-1504 exhibited no growth defects compared with the wild-type strain (P>0.05), it demonstrated reductions of 46.57% in adhesion to DF-1 cells and 77.61% in cellular invasion. Cytotoxicity assays revealed a 34.97% reduction of Tn-1504 in inducing host cell damage. All of the above phenotypic differences were statistically significant (P<0.01). [Conclusion] We demonstrate that inactivation of TonB specifically impairs the adhesion, invasion, and virulence characteristics of A. paragallinarum, providing a key target for analyzing the pathogenic mechanism and developing anti-virulence strategies for this pathogen.

[Objective] To investigate the effects of fertilization regime alterations on microbial communities in paddy soils. [Methods] We conducted a long-term field experiment with three treatments: chemical fertilizer (H) and H converted to a conventional amount of organic fertilizer (HC), a conventional amount of organic fertilizer (C) and C converted to chemical fertilizer (CH), and a high amount of organic fertilizer (G) and G converted to chemical fertilizer (GH). Metagenomic sequencing was combined with bioinformatics analysis to assess the structures, diversity, and co-occurrence networks of microbial communities in paddy soils. [Results] Compared with the H treatment, the HC treatment increased the soil organic carbon, dissolved organic carbon, total nitrogen, and alkali-hydrolyzable nitrogen (P<0.05). The CH and GH treatments exhibited lower soil carbon and nitrogen levels than C and G treatments, respectively. The HC treatment markedly altered the relative abundance of Acidobacteriota, Nitrospirota, Candidatus Rokubacteria, Mucoromycota, and Thaumarchaeota. The CH treatment showed no significant changes in microbial composition at the phylum level, whereas the GH treatment significantly modified the relative abundance of Nitrospirota and the archaeal community structure. Although fertilization regime alterations showed no significant effect on microbial alpha diversity (P>0.05), the beta diversity differed across treatments (P<0.05). Co-occurrence network analysis demonstrated that the HC treatment enhanced the network complexity relative to the H treatment, with increased nodes (181), edges (2 935), average degree (16.215), modularity (0.757), and clustering coefficient (0.495). The CH treatment showed more edges (3 894) and higher average degree (21.514) but lower modularity (0.599) than the C treatment (0.751). Conversely, the GH treatment diminished all network topology parameters relative to G. Redundancy analysis identified dissolved organic carbon (22.1%), soil pH (16.8%), total nitrogen (15.6%), ammonium nitrogen (14.6%), available potassium (11.8%), and available phosphorus (10.6%) as the primary drivers of microbial community variations. [Conclusion] The findings indicate that fertilization regime alterations influence the microbial community structure and network characteristics in paddy soils by modifying soil physicochemical properties. Transitioning from chemical to organic fertilization enhances microbial community stability and soil ecological functions, while replacing organic with chemical fertilization reduces soil carbon and nitrogen availability, potentially compromising microbial network complexity and resilience.

[Objective] We systematically analyzed the growth and compared the ectoine accumulation of Halomonas campaniensis XH26 cultured with nine different amino acids, aiming to clarify the optimal amino acid for ectoine accumulation of strain XH26. [Methods] Under the optimal salt concentration of 1.5 mol/L, nine amino acids (l-monosodium glutamate, l-glutamine, l-aspartic acid, l-asparagine, l-histidine, l-tryptophan, l-glycine, l-serine, and l-lysine) were selected as the single carbon/nitrogen source of the culture medium and added within the concentration range of 20-50 mmol/L (interval of 5 mmol/L), on the basis of which the optimal concentration and optimal amino acid for ectoine accumulation were screened. l-aspartic acid was selected to culture the cells at low (L, 20 mmol/L), medium (M, 35 mmol/L), and high (H, 50 mmol/L) concentrations for targeted metabolomics sequencing and analysis. [Results] The amount of ectoine synthesis first increased and then decreased as the amino acid concentration increased and reached the highest at optimum concentration (30/35 mmol/L). Metabolomics analysis screened out 28 (L vs. M), 27 (L vs. H), and 26 (H vs. M) significantly differential metabolites, such as glyceric acid, lactose, adenosine 5′-monophosphate, α-ketoglutaric acid, glucose-1-phosphate, fumaric acid, and citric acid. KEGG metabolic pathway enrichment analysis showed that l-alanine, l-aspartic acid, and l-glutamate metabolic pathways were the most significantly enriched pathways. [Conclusion] Targeted metabolomics of differential metabolites of bacteria discovers that the strain achieves a rebalance between nitrogen homeostasis and energy supply through the aspartate-alanine axis and the arginine-proline metabolic axis.

The thioredoxin family plays crucial roles in bacterial oxidative stress defenses and virulence regulation, while the function of its member YbbN in Vibrio parahaemolyticus remains unclear. [Objective] To elucidate the regulatory role of YbbN in the biological characteristics and pathogenicity of Vibrio parahaemolyticus, providing potential targets for developing novel anti-infection strategies. [Methods] The ybbN knockout strain (ΔybbN) and complementary strain (CΔybbN) of Vibrio parahaemolyticus SH112 were constructed by homologous recombination. The strains were compared regarding the growth characteristics, motility, biofilm formation, bacterial competition, cell adhesion, cytotoxicity, and pathogenicity in mice. [Results] Although the knockout of ybbN showed no significant effects on bacterial growth, motility, cell adhesion, or colonization, it markedly attenuated key pathogenic traits. Specifically, it decreased the biofilm formation (by 19%-30%), killing efficiency against competitive bacteria (*: P<0.05; ****: P<0.000 1), and cytotoxicity in HeLa cells (by 27%), while increasing the survival rate of mice by 87.5%. [Conclusion] This study demonstrates for the first time that YbbN specifically regulates critical aspects involved in biofilm formation, bacterial competition, and cytotoxicity in host cells, significantly influencing the biological characteristics and pathogenicity of Vibrio parahaemolyticus. These findings not only expand the understanding about the functional diversity of the thioredoxin family proteins but also provide new molecular targets and a theoretical basis for preventing Vibrio parahaemolyticus infections.

[Objective] To compare the bacterial growth, oxidative resistance, and bacterial infection in cells and host among Listeria monocytogenes EGD-e, lmo0175 (LPXTG motif-anchored protein)-deleted and complementary strains, so as to investigate the roles of Lmo0175 in anti-oxidative resistance and bacterial infection. [Methods] The lmo0175-deleted and complementary strains were constructed to compare the difference in bacterial growth, oxidative resistance, adhesion, invasion, intracellular proliferation, survival of infected mice, and bacterial loads in organs of Listeria monocytogenes. [Results] The deletion of lmo0175 remarkably decreased oxidative resistance, cell proliferation, colonization in the liver and spleen, and pathogenicity in mice. However, it had no significant impact on bacterial growth, adhesion or invasion. [Conclusion] The LPXTG motif-anchored protein Lmo0175 contributes to the anti-oxidative resistance, proliferation, and colonization in specific organs of Listeria monocytogenes.