As the core saccharification agent in traditional Chinese brewing processes, Daqu plays an irreplaceable role in Baijiu production due to its complex microbial community and multifunctional enzyme system. This review begins by outlining the application scenarios and characteristics of saccharification agents in both Eastern and Western brewing industries. Subsequently, based on the research of microbiology, genomics, proteomics, etc., this review elaborates on the diversity of microorganisms and enzymes with saccharifying function in Daqu, along with the roles of Daqu in saccharification. Finally, strategic approaches are proposed, including utilizing multi-omics technologies to elucidate the functional mechanisms of Daqu during brewing and enhancing the integration of mechanism research with industrial practice, which may provide references for the innovative development of saccharification agents in the brewing industry.

[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.

[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 fluoride exposure on the gut microecosystem of the model insect Bombyx mori and elucidate the toxic mechanisms of fluoride under different conditions on insect hosts. [Methods] We measured physiological enzyme activity changes in the gut tissue of B. mori following fluoride exposure and observed histopathological changes by using hematoxylin and eosin (HE) staining. We also analyzed the dynamic changes in gut microbiota by using 16S rRNA amplicon sequencing and examined the expression changes of immune-related genes in gut tissue by qPCR. [Results] Fluoride exposure significantly increased the malondialdehyde (MDA) level and decreased the reduced glutathione (GSH) level in the gut tissue. Enzyme activities of catalase (CAT), superoxide dismutase (SOD), acetylcholinesterase (AChE), carboxylesterase (CarE), alkaline phosphatase (AKP/ALP), and lactate dehydrogenase (LDH) were also markedly suppressed. Additionally, we observed epithelial cell rupture and separation from the basement membrane. The Toll immune pathway was inhibited, which resulted in significantly reduced expression of antimicrobial peptide genes, such as attacin, cecropin, lebocin, and lysozyme. Gut microbiota analysis revealed significant declines in the relative abundances of Glutamicibacter, Staphylococcus, Acinetobacter, and Methylobacterium. The gut microbiota exhibited notable structural heterogeneity with strengthened functions related to metabolic pathways and biosynthesis of secondary metabolites. [Conclusion] Fluoride exposure significantly weakened the host insect’s antioxidant capacity, basal metabolism, and immune function, damaged the integrity of gut tissue, and caused gut microbiota dysbiosis. This disruption of the gut microecosystem might be a major factor contributing to reduced insect species diversity and biomass.

Double-crossover homologous recombination using the SacB negative selection system is commonly employed for genome editing in Gram-negative bacteria. However, its negative selection efficiency varies significantly across strains, being frequently compromised by differences in metabolic characteristics or genomic composition. [Objective] To develop a novel counterselection system based on the type VI secretion system (T6SS) effector Txe1 to enhance the efficiency of seamless genome editing. [Methods] We first modified the conventional suicide plasmid pDM4 by introducing a kanamycin resistance gene, generating the derivative plasmid pDM4K. Subsequently, we replaced sacB with an l-arabinose-inducible expression cassette encoding the C-terminal domain of Txe1 (araC-P_BAD::txe1^CTD ), constructing a novel counterselection plasmid pTL1010. Using the virulence gene tssB of Edwardsiella piscicida FC2 as the target, we systematically evaluated and compared the counterselection efficiency of the Txe1 system with that of the conventional SacB system. [Results] Under induction with l-arabinose, the Txe1-based counterselection system achieved the efficiency of 91.1% (false-positive rate of 8.9%), outperforming the SacB system which had a false-positive rate of 100% (P<0.01). [Conclusion] The newly developed Txe1-based counterselection plasmid pTL1010 significantly enhances the efficiency of seamless genome editing in E. piscicida and provides a highly effective tool for precise genetic manipulation in Gram-negative bacteria.

[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.

Endometriosis is a chronic, estrogen-dependent gynecological condition often associated with pelvic pain, dyspareunia, difficulties of defecation and urination, and infertility. The disease poses significant treatment challenges and has a high recurrence rate, causing considerable distress to patients and substantial social and economic impacts. Currently, the exact etiology of endometriosis remains unclear, and the lack of specific biomarkers complicates early diagnosis. Advances in high-throughput sequencing technologies have significantly propelled research into the human microbiome, revealing intricate connections between microbiota and diseases. Notably, the microbiota in the female oral cavity, genital tract, peritoneal cavity, and gut are closely linked to the development and progression of gynecological disorders. This article reviews the correlations between the human microbiome and endometriosis and explores the potential applications of microbiota in the diagnosis and treatment of endometriosis.

[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.

[Objective] To construct a PK15 cell line stably expressing the prolyl oligopeptidase (POP) by using the PiggyBac transposon system and systematically investigate its regulatory role in the proliferation of foot-and-mouth disease virus (FMDV). [Methods] A recombinant PiggyBac vector carrying the POP gene was constructed and transfected into PK15 cells, and the expression of the target protein was detected by Western blotting. The monoclonal cell line stably expressing POP was isolated via the limiting dilution method. The stability of mRNA and protein levels of POP was verified by RT-qPCR and Western blotting, respectively. The viability of the selected cell line was assessed by the Cell Counting Kit-8 (CCK-8) assay. FMDV replication kinetics in the stable cell line were comprehensively analyzed by Western blotting, RT-qPCR, and the 50% tissue culture infective dose (TCID₅₀) assay. [Results] A PK15 cell line stably expressing POP was established. No significant differences in cell viability were observed between the stable cell line and the control cell line. The protein level of POP remained stable in the established cell line after 30 passages. The results of the viral infection assay demonstrated that the FMDV proliferation level in the PK15 cell line stably expressing POP was significantly higher than that in the control group, with this stimulatory effect being maintained across multiple passages. [Conclusion] We successfully constructed a PK15 cell line stably expressing POP. The results reveal that POP overexpression enhances FMDV replication in a passage-independent manner. These findings provide a valuable experimental model for elucidating the molecular mechanism underlying the role of POP in FMDV replication.

[Objective] To clarify the succession patterns of Lactobacillus and their regulatory effects on the ethyl acetate-to-ethyl lactate ratio during Xiaoqu Qingxiangxing Baijiu fermentation. [Methods] High-throughput sequencing was employed to analyze the species-level succession patterns of Lactobacillus in Xiaoqu and fermented grains (Jiupei). Modified culture media and enrichment methods were employed to isolate Lactobacillus from Jiupei. The dominant Lactobacillus strains were then evaluated for tolerance, subjected to single-carbon-source fermentation experiments, sorghum hydrolysate fermentation experiments, and lab-scale simulated solid-state fermentation with Lactobacillus. The physiological and biochemical characteristics of different Lactobacillus strains, their fermentation characteristics on single carbon sources, and their impact on the ethyl acetate-to-ethyl lactate ratio in the Baijiu were investigated. [Results] A total of 15 Lactobacillus strains were isolated from Jiupei, and high-throughput data analysis identified eight dominant species: Limosilactobacillus pontis, Lactobacillus helveticus, Lentilactobacillus buchneri, Lentilactobacillus hilgardii, Levilactobacillus brevis, Limosilactobacillus fermentum, Lactiplantibacillus plantarum, and Lactobacillus acetotolerans. Further tolerance tests and single-carbon-source fermentation characterization revealed that all the eight dominant strains could withstand actual fermentation conditions (ethanol/acetic acid/lactic acid). However, L. plantarum exhibited impaired maltose utilization, while L. acetotolerans showed defective d-galactose metabolism. L. helveticus and L. pontis displayed a single-product fermentation profile in single-carbon-source cultures, whereas L. brevis, L. buchneri, and L. hilgardii exhibited multi-product fermentation patterns in both single-carbon-source and sorghum hydrolysate media. In lab-scale simulated solid-state fermentation with Lactobacillus, compared with the control, supplementation with L. hilgardii increased the ethyl lactate and ethyl acetate content by 175% and 44%, respectively, reducing the ethyl acetate-to-ethyl lactate ratio to 0.357. Supplementation with L. buchneri increased the ethyl acetate content by 50% while decreasing the ethyl lactate by 71%, raising the ethyl acetate-to-ethyl lactate ratio to 4.496. [Conclusion] The dominant Lactobacillus strains in Xiaoqu Qingxiangxing Baijiu fermentation exhibit strong overall environmental tolerance, and their metabolic profiles vary in single-carbon-source fermentation. The supplementation with different dominant Lactobacillus strains differentially modulates ester formation. These findings provide a theoretical foundation for dynamically regulating key flavor compounds during Xiaoqu Qingxiangxing Baijiu fermentation.