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.

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.

Root-knot nematode, among the most destructive plant-parasitic nematodes, poses a severe threat to global agricultural production. The plant root microbiome is considered as the “second genome” of host plant and plays an indispensable role in plant growth, development, and stress response. Parasitism by root-knot nematode significantly disrupts the community structure and function of root-associated microbial communities in plants. The disturbance of host microbiome not only exacerbates plant pathological processes but also may induce cascade effects through tripartite interactions among microorganism, plant, and nematode. This review comprehensively elucidates the multifaceted impacts of root-knot nematode parasitism on the root micro-ecosystem of host plant, particularly focusing on the variation in the structural and functional characteristics of both the rhizosphere and endophytic microbiome, as well as their roles in the occurrence of nematode diseases and maintaining plant health. Investigating the interaction between pathogenic nematodes and plant microbiome on community level will not only advance our understanding the intricate network among plant, nematode, and microorganism, but also provide theoretical and practical insights for developing innovative strategies for controlling plant nematode diseases.

Alcoholic liver disease (ALD) is a liver disease caused by long-term excessive alcohol consumption and is one of the most common chronic liver diseases worldwide. At present, no effective approach is available for preventing or reversing this disease, causing a serious social burden and increasing the pressure for prevention and treatment. In recent years, researchers have found that long-term alcohol abuse can lead to significant changes in the structure and function of the gut microbiota, thereby disrupting the balance of the gut microbiota, which can promote the progression of ALD. Therefore, maintaining gut microbiota balance can provide new targets for the prevention and treatment of ALD. This article reviews the research and intervention progress of the gut microbiota and its metabolites in ALD in recent years, providing reference for future studies on the pathogenesis and treatment of this disease.

Currently, the sustainable development of global agriculture is facing multiple challenges, including soil degradation, resource constraints, and environmental pollution. With the continuous growth of the population and the increasing demand for food quality, improving soil health has become a crucial foundation for ensuring food security. Although chemical fertilizers play an important role in maintaining the high yields and high quality of plants, their excessive or unreasonable use can cause environmental problems, such as soil acidification and water eutrophication. Rhizosphere microbial communities play an essential role in plant nutrient acquisition, tolerance to environmental stress, and adaptation to environmental changes. Among them, synthetic microbial communities (SynComs) are designed via the targeted assembly of multiple microorganisms with well-defined functions and clear genetic backgrounds, enabling the achievement of complex functionalities that cannot be accomplished by single strains. They are powerful tools for deciphering the key interface interaction mechanisms among plants, soil, and microorganisms and play a vital role in promoting efficient utilization of plant nutrients, enhancing plant stress resistance, and increasing the efficiency and reducing the application of fertilizers. This study reviews the conceptual evolution, current research trends, and construction principles, methods, and tools of SynComs, and summarizes the role of SynComs in the sustainable development of agriculture from the aspects of promoting plant growth, inhibiting biotic and abiotic stresses, and improving and restoring soil health. Furthermore, this paper makes an outlook on the future research directions and emphasizes the research and development of targeted microbial agents, the application of artificial intelligence (AI) in community assembly, and the performance improvement of SynComs in field applications, aiming to support the coordinated and multi-objective development of food security, efficient resource utilization, and environmental protection through near-natural microbial means, thereby facilitating the green agricultural development of China.

Loop-mediated isothermal amplification (LAMP), a rapid and sensitive method of nucleic acid isothermal amplification, is characterized by high specificity and sensitivity and holds broad application prospects in nucleic acid detection. However, the development of detection schemes based on LAMP encounters issues, such as non-specific and non-template amplification during the amplification process, which can affect the accuracy and specificity of the detection results. This article elaborates on the recent research progress in the strategies avoiding false positives and enhancing detection efficacy of LAMP, which include primer design, optimization of conditions, and introduction of special chemical substances. Finally, this article explores the breakthroughs and innovations of LAMP in the field of detection.

SnoaL family proteins belong to a class of the nuclear transport factor 2 (NTF2)-like superfamily, which are characterized by a distinctive α+β barrel fold structure. To date, proteins of this family have been demonstrated to catalyze a variety of reactions, such as aldol reaction, hydroxylation, decarboxylation, epoxide hydrolysis, and cycloaddition reaction, playing crucial roles in the biosynthesis of complex bioactive natural products. These proteins are widely distributed in various organisms including bacteria and fungi. However, research on proteins of this family is relatively limited at present. We reviewed the research progress on proteins of SnoaL family. The review not only provides beneficial guidance for the subsequent functional identification and mechanism research of related proteins but also helps to discover novel biocatalytic tools, laying a foundation for the research of bioactive molecule biosynthesis.

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] 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] Porcine circovirus type 3 (PCV3) is associated with the development of reproductive disorders in sows, piglet dermatitis, and myocarditis. However, due to the difficulty in stably passaging PCV3 in vitro, the animal infection model construction and specific pathogenicity of PCV3 remain to be investigated. This study aims to solve the above problems. [Methods] A PCV3 strain (PCV3-HK) was successfully isolated by inoculating PK-15 cells with lymph node samples from PCV3-positive weaned piglets. The strain was identified using PCR, indirect immunofluorescence assay, and transmission electron microscopy, and its complete genome was sequenced. The whole genome of the strain was sequenced. The pathogenicity of the strain was evaluated based on the clinical symptoms, body weight changes, virus excretion, and pathological changes of 21-day-old non-weaned piglets after inoculation with the 15th-passage viral cell culture at a dose of 10^6.4 TCID₅₀/mL. [Results] PCV3-HK had a genome length of 2 000 bp and belonged to PCV3c subtype. The viral particles were spherical, non-enveloped, about 15-20 nm in diameter, and capable of reacting specifically with the monoclonal antibody against PCV3 Cap protein. The results of infection in piglets showed that the PCV3-HK infected pigs developed obvious symptoms such as dermatitis, slow weight gain and even wasting, as well as anatomical changes such as lymph node edema and interstitial pneumonia. The viral nucleic acid was detected in oral swabs and anal swabs on day 7-21 post infection, and viremia was observed. The virus infected multiple organs, including the heart, lungs, kidneys, and lymph nodes. Notably, inflammatory cell infiltration and aggregation of necrotic cells were observed in the kidneys and lymph nodes. [Conclusion] We isolate a PCV3 strain with classical circovirus morphology and establish a piglet infection model, which provides key materials for pathogenic mechanism study and vaccine development regarding PCV3.

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 identify and develop a phage-derived lyase that can be heterologously expressed with high activity and stability and determine its optimal working conditions. [Methods] We employed the turbidity reduction assay to evaluate the bacteriolytic activity and identify the optimal parameters. [Results] Genome annotation and protein prediction of the Vibrio alginolyticus phage phiV208 showed that ORF30 encoded a lyase, named lysV208. This enzyme demonstrated soluble expression in Escherichia coli BL21(DE3), reaching a purified concentration of 204 μg/mL after 16 h induction with 0.25 mmol/L IPTG. Its bacteriolytic activity (turbidity reduction rate) increased from 24.2% to 68.0% in the presence of 0.5 mmol/L EDTA. Enzymatic characterization revealed that lysV208 exhibited the maximum bacteriolytic activity (75.6%) at 45 ℃ while maintaining high activity (52.8%-71.9%) within the temperature range of 25-37 ℃, which is typical for bacterial disease outbreaks in aquatic and terrestrial animals. The enzyme showed the maximum activity at pH 7.0 and retained substantial bacteriolytic activity (44.0%-63.2%) under alkalescence conditions (pH 7.0-9.0), demonstrating adaptability to marine and freshwater aquaculture environments. Divalent metal ions including Zn²⁺, Mg²⁺, Mn²⁺, and Fe²⁺ at 0.1-1.0 mmol/L moderately enhanced the bacteriolytic activity of lysV208, whereas those at 10.0 mmol/L reduced the activity (P<0.01). In addition, lysV208 displayed broad-spectrum lytic effects, showing the bacteriolytic activity of 59.7% against V. alginolyticus V039, 68.9% against Vibrio vulnificus H1, 65.8% against Vibrio parahaemolyticus GH32, and 38.0% and 65.6% against Vibrio harveyi TY13 and G1, respectively. [Conclusion] The recombinant lyase lysV208 demonstrates robust and stable in vitro bacteriolytic activity and a broader spectrum than its source phage. These findings highlight its potential for the control of bacterial infections and the development of phage-lyase synergistic agents.

[Objective] Microorganisms play a critical role in the treatment of food waste. Elucidating their community structure and functional characteristics is essential for optimizing treatment processes and improving operational efficiency. [Methods] We employed high-throughput 16S rRNA gene amplicon sequencing to investigate the microbial community structures and potential functions in three systems: the anaerobic digestion system, the wastewater treatment system, and the air purification system (including biofilters and deodorization towers), in a food waste treatment facility. [Results] The microbial communities differed significantly in diversity and composition across systems. The anaerobic digestion system exhibited lower microbial diversity and richness, primarily influenced by environmental factors such as temperature, pH, and biochemical oxygen demand (BOD). Functional analysis indicated that organic matter degradation was the dominant microbial function across the facility. Specifically, the anaerobic system was enriched with genera such as Defluviitoga, Methanothermobacter, and Lentimicrobium, which were involved in hydrolysis, fermentation, and methanogenesis. The wastewater treatment system was dominated by Candidatus Anammoximicrobium, Nitrolancea, and Truepera, which drove organic matter degradation and nitrogen transformation processes, including anaerobic ammonium oxidation (anammox), nitrification, and denitrification. The air purification system was enriched with Chryseobacterium and Paracoccus, which were associated with biofilm formation and the degradation of volatile organic compounds and sulfur-containing compounds. [Conclusion] The microbial community characteristics in each treatment system was closely associated with system-specific operational conditions. The enrichment of functionally distinct microbial taxa reflected their ecological roles. In the anaerobic digestion system, methanogens and syntrophic bacteria cooperatively facilitated organic matter degradation and methane production. In the wastewater treatment system, nitrogen-cycling bacteria ensured efficient nitrogen removal. In the air purification system, the enrichment of heterotrophic degraders and sulfur compound-metabolizing genera (e.g., Sphingobium and Flavobacterium) enhanced deodorization performance. These findings provide a theoretical basis for microbial community regulation and process optimization in food waste treatment facilities.

[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.

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] We isolated endophytes from the roots of Isatis indigotica Fort. and investigated the effects of endophytes on the growth, physiology, and medicinal quality of I. indigotica, aiming to provide a reference for microbial regulation of the cultivation and production of this plant. [Methods] The relevant culture media were selected to analyze the plant growth-promoting activities of the endophytes. Three endophyte strains (Bacillus sp. BC00, Bacillus sp. BV11, and Pseudomonas sp. PA28) were selected and applied singly to I. indigotica four times. The physiological indexes of the leaves were measured at different time points after the last treatment, and the growth and physiological indexes and the content of active ingredients of the roots and the leaves were measured at the harvesting stage. [Results] The photosynthetic pigment content and nutrient-metabolizing enzyme activities of I. indigotica leaves in each treatment group were higher than those in the control group at each time point. The three strains of endophytic bacteria significantly promoted the growth and biomass accumulation of I. indigotica. Strain BC00 had the best promoting effect on the dry weight of the above-ground part, which was increased by 74.16% compared with that of the control. Strain BV11 had the most significant promoting effect on the dry weight of the below-ground part, which was increased by 216.02% compared that of the control. The treatments with endophytic bacteria significantly affected the content of endogenous hormones and soluble substances. The three strains of endophytic bacteria had different effects on the active ingredients. Compared with the control, strain BC00 increased the content of indigo and epigoitrin by 7.83% and 5.64%, respectively, while strain BV11 slightly decreased the content of indigo and epigoitrin. The total active biomass of each index ingredient in the strain treatments was significantly higher than that of the control. [Conclusion] The endophytic bacterial strains BC00, BV11, and PA28 had significant promoting effects on the growth, physiological indexes, and active ingredients of I. indigotica, and strain BC00 demonstrated the best comprehensive promoting effect. The findings provide a basis for the later development of microbial fertilizers conducive to the growth of I. indigotica.

[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.

[Objective] To investigate the pathogenicity of Lichtheimiaramosa LYSF001 and ITS resistance to common fungicides, providing experimental evidence for the diagnosis and treatment of fungal infections in animals. [Methods] The strain was identified by morphological characterization and molecular biology techniques. PCR was employed to detect virulence genes in strain LYSF001, and an animal infection model was established to evaluate the pathogenicity of the strain. Histopathological examination, Grocott’s methenamine silver (GMS) staining, and ITS sequences amplification were employed to analyze pathological changes in the livers and kidneys of infected animals. Additionally, antimicrobial susceptibility tests were conducted to assess the sensitivity of strain LYSF001 to caspofungin, amphotericin B, and itraconazole. [Results] Strain LYSF001 was identified as L. ramosa. PCR analysis revealed that strain LYSF001 carried five virulence genes (CalA, PKP2, LaeA, Alp2, and AspF1). Animal experiments demonstrated that the strain led to the mortality of 40% and caused visceral hyperemia in mice, with the most significant pathological changes (inflammatory cell infiltration and tissue necrosis) observed in the livers and kidneys. Severe infections led to animal mortality. GMS staining revealed the presence of dark-colored hyphae in the heart and liver, and ITS sequences amplification further confirmed fungal infection. The antimicrobial susceptibility test results indicated that strain LYSF001 was resistant to caspofungin but sensitive to amphotericin B and itraconazole. [Conclusion] L. ramosa LYSF001 exhibits strong pathogenicity, capable of causing severe infections and even death in animals. Additionally, the strain showed resistance to caspofungin but sensitivity to amphotericin B and itraconazole. The findings provide important experimental evidence and technical support for the diagnosis and treatment of fungal infections in animals.

[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 molecular mechanism by which Listeria monocytogenes regulates the expression of mitochondrial calcium uptake 2 (MICU2) through the virulence factor listeriolysin O (LLO) and their interaction, thereby affecting mitochondrial calcium homeostasis and bacterial intracellular proliferation. [Methods] Western blotting was employed to analyze MICU2 expression in HeLa cells infected with L. monocytogenes EGD-e or Δhly. Gene silencing and eukaryotic overexpression approaches were used to examine how MICU2 regulated the intracellular proliferation of L. monocytogenes. AlphaFold3 was used to predict the interaction sites between LLO and MICU2, and co-immunoprecipitation (Co-IP) was performed to verify their interaction. Mitochondrial calcium fluorescence probe (Rhod-2 AM) was used to analyze the regulatory role of MICU2 in calcium homeostasis. [Results] EGD-e infection upregulated MICU2 expression at 4 h and 6 h post-infection (P<0.001), whereas Δhly showed no effect (P>0.05). The silencing of MICU2 enhanced bacterial proliferation (P<0.01) and elevated mitochondrial calcium levels (P<0.05), whereas overexpression of MICU2 reduced bacterial proliferation (P<0.01) and decreased mitochondrial calcium levels (P<0.05). AlphaFold3 predicted that alanine (Ala) at position 462 of LLO interacted with glutamate (Glu) at position 119 of MICU2 via a hydrogen bond, and Co-IP confirmed their interaction. [Conclusion] L. monocytogenes upregulates MICU2 expression via LLO, and MICU2 inhibits bacterial intracellular proliferation by reducing mitochondrial calcium levels. The interaction between LLO and MICU2 is a key molecular basis for this process. These findings provide insights into the pathogenic mechanism of L. monocytogenes.

[Objective] To construct a strain XH02Δlpxtg270 with knockout of the LPXTG motif protein-coding gene from Corynebacterium pseudotuberculosis XH02 and explore the role of lpxtg270 in the growth, biofilm formation, and infection of XH02. [Methods] CRISPR/Cas9 was employed to construct XH02Δlpxtg270. The knockout strain and the wild strain XH02 were compared in terms of biological characteristics, invasion into J774A.1 macrophages, and pathogenicity in mice. [Results] Compared with XH02, XH02Δlpxtg270 did not change significantly in the colony morphology, growth curve, adhesion to J774A.1 macrophages, or intracellular proliferation, while it demonstrated reductions in the biofilm formation and invasion into J774 A.1 cells. Moreover, the release of lactate dehydrogenase and secretion of interleukin-1β from J774 A.1 cells infected with the knockout strain decreased compared with those infected with the wild strain. Compared with XH02, XH02Δlpxtg270 showed weakened pathogenicity in mice and decreased loads in the liver, spleen, kidney, lung, and brain, causing milder pathological changes of above organs in mice. [Conclusion] LPXTG270 of C. pseudotuberculosis is related to the biofilm formation and invasion into macrophages, playing a key role in the pathogenicity of this bacterium in mice.

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] 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 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.

[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] 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 deeply understand the molecular evolution pattern, pathogenicity, and drug resistance mechanism and provide a scientific basis for the prevention and control of Streptococcus equi subsp. zooepidemicus (SEZ), we compared the pathogenicity, drug resistance, and genome sequences of two SEZ strains (YLCD588 and HT222) isolated from donkeys in Xinjiang. [Methods] Whole-genome sequences of the two strains were determined by next-generation sequencing and a phylogenetic tree was constructed based on multilocus sequence typing (MLST) of sequencing data and existing sequences in the database. The virulence factor database (VFDB) (https://www.mgc.ac.cn/VFs/) and the center for genomic epidemiology (CGE) (http://genomicepidemiology.org) were used for annotation of the virulence and drug resistance genes of the two strains. The growth curves, antimicrobial susceptibility, and biofilm formation of the two strains were examined and compared. Mice were challenged with these strains separately and the pathogenicity of the strains was observed and evaluated. Then, histopathological changes and bacterial loads in the lung and spleen tissues of the morbid mice were observed and determined. [Results] Sequencing data showed that the genome sizes of YLCD588 and HT222 were 2 090 225 bp (1 905 coding sequences) and 2 105 005 bp (1 995 coding sequences), respectively. HT222 and YLCD588 carried 214 and 212 virulence genes, respectively. HT222 and YLCD588 had 235 and 233 drug resistance genes, respectively. YLCD588 was assigned to a novel sequence type (ST) 545 by MLST. The MLST phylogenetic tree indicated that YLCD588 was closely related to the goat-derived SEZ strain, while HT222 was closely related to the canine SEZ strain. YLCD588 displayed resistance to six antibiotics and HT222 exhibited resistance to four. The crystal violet assay and confocal laser scanning microscopy results showed that YLCD588 exhibited stronger biofilm formation than HT222 (P<0.05), whereas HT222 caused higher mortality rate (P<0.05), higher bacterial load (P<0.01), and severer pathological damage in mice than YLCD588. [Conclusion] The two SEZ strains exhibit distinct genomic characteristics, sequence types, pathogenicity, and drug resistance. HT222 possesses more drug resistance genes and virulence genes and exhibits stronger pathogenicity than YLCD588, while YLCD588 showcases stronger biofilm formation and drug resistance than HT222. These findings broaden the understanding about the molecular epidemiology, pathogenicity, and drug resistance of different SEZ strains from donkey and provide references for the effective control of the infection and spread of SEZ and clinical treatment of SEZ infection.

[Objective] To investigate the effects of calcium ion-free (Ca²⁺-free) conditions on the gene expression, biofilm formation, and virulence of Vibrio parahaemolyticus. [Methods] Ethylene glycol-tetraacetic acid was used to chelate Ca²⁺ in culture media to create Ca²⁺-free conditions. Crystal violet staining was employed to evaluate the biofilm formation of V. parahaemolyticus. Swimming and swarming assays were performed to assess the motility. Additionally, the Kanagawa phenomenon test, HeLa cell adhesion assay, and cytotoxicity experiment were conducted to analyze the virulence phenotypes of V. parahaemolyticus. By comparing the expression profiles, we analyzed the effect of Ca²⁺-free conditions on the gene expression in V. parahaemolyticus. [Results] Ca²⁺-free conditions inhibited the growth and significantly reduced the biofilm formation, intracellular c-di-GMP levels, and motility of V. parahaemolyticus. Furthermore, Ca²⁺-free conditions suppressed the hemolytic activity and reduced the bacterial adhesion to HeLa cells, while enhancing the cytotoxicity of V. parahaemolyticus. Transcriptomic analysis revealed 359 differentially expressed genes (DEGs) under Ca²⁺-free conditions. These DEGs were mainly associated with biofilm formation, virulence factors, and regulators. Notably, the genes involved in lateral flagella and polar flagellum were downregulated, while most virulence genes were upregulated. The majority of putative regulator genes were downregulated. [Conclusion] Ca²⁺-free conditions significantly affect the biofilm formation, motility, virulence, and gene expression of V. parahaemolyticus.

[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.

[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.

Candida auris, as a multidrug-resistant fungus, pose a challenge to clinical treatment because of biofilm formation. Currently, effective intervention measures against its biofilm remain to be developed. [Objective] To explore the antifungal activity and biofilm inhibition mechanism of the Chinese medicine active compound matrine (MT) against C. auris. [Methods] The minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC), and sessile minimum inhibitory concentration (SMIC) of MT against C. auris were determined by the microdilution method. The time-growth curve and colony morphology of C. auris under the intervention of MT were observed by the plate method. The changes in the hydrolytic enzyme activity of the C. auris biofilm treated with MT were determined. The changes in cell surface hydrophobicity (CSH) of C. auris biofilm treated with MT were observed by the water-hydrocarbon two-phase method. The effects of MT on the metabolic activity and structure of C. auris biofilms were observed by the XTT method, crystal violet method, and confocal laser scanning microscopy (CLSM). The changes in the cell nucleus of C. auris in the biofilm treated with MT were detected by DAPI staining. The protective effect of MT on the host infected with C. auris was observed by the Galleria mellonella larvae infection model. [Results] The MIC of MT against C. auris was 128 μg/mL, while the MFC and SMIC were both 512 μg/mL. The inhibition mechanism of MT against the proliferation of C. auris mainly involved reducing the CSH, inhibiting the mature biofilm formation, significantly decreasing the metabolic activity, and inducing abnormal nuclear morphology. The experiments with G. mellonella larvae further confirmed that MT could alleviate the invasive damage caused by C. auris. [Conclusion] MT has a significant antifungal effect on C. auris and can effectively inhibit the biofilm formation, providing a new candidate drug and potential target for clinical treatment of multidrug-resistant C. auris infections.