Objective To investigate the effects of combined application of organic and inorganic fertilizers on soil nutrient content and microbial community structures and functions in soybean fields, thus providing a scientific basis for rational fertilization and high-quality, high-yield soybean production. Methods Four fertilization treatments—control (CK: no fertilization), inorganic fertilizer (CF: compound fertilizer), organic fertilizer (OF: dry chicken manure), and combined organic-inorganic fertilizers (OCF: dry chicken manure+compound fertilizer)—were established. During the experiment, soil organic matter (SOM), alkali-hydrolyzed nitrogen (AN), available phosphorus (AP), available potassium (AK), and microbial community structures and functions were measured to investigate the relationships between microbial communities and soil nutrients. Results Different fertilization treatments influenced soil nutrients in soybean fields. Compared with CK, the OCF treatment increased the soil SOM, AN, AP, and AK by 60.67% (P<0.05), 68.09% (P<0.001), 15.18 folds (P<0.001), and 59.54% (P<0.01), respectively, and it also increased soil pH. Amplicon sequencing indicated that different fertilization measures did not alter the community composition of soil microorganisms but changed the relative abundance of different phyla and genera. Compared with CK, the OCF treatment increased the relative abundance of Basidiomycota and Mortierellomycota by 5.62 folds and 4.51%, respectively, while decreasing that of Ascomycota by 38.35%. The OCF treatment reduced fungal community richness and diversity (P<0.05). The alpha diversity analysis revealed that both bacterial and fungal diversity decreased after organic fertilizer application, with fungal alpha diversity showing the most significant reduction (P<0.05). Functional prediction indicated that amino acid metabolism exhibited the highest relative abundance among metabolic pathways in bacterial communities, suggesting that the OCF treatment promoted metabolic processes centered on nitrogen assimilation and protein synthesis, facilitating bacterial participation in soil nutrient transformation. Under the OF treatment, symbiotropic fungi exhibited the highest relative abundance, which suggested that organic fertilizer promoted the ecological functions of fungi in soil nutrient cycling. Conclusion Combined application of organic and inorganic fertilizers modulates soil pH, mitigates soil acidification, and enhances soil nutrient content. Fungal communities exhibit greater sensitivity to organic fertilizer application, which significantly reduces their diversity and stimulates the proliferation of certain pathogenic fungi. Conversely, inorganic fertilizer suppresses the relative abundance of pathogenic fungi. Thus, the combined application of organic and inorganic fertilizers demonstrates distinct advantages in balancing soil nutrient supply with microbial community structure and optimizing soil microbial functional composition. This approach provides theoretical foundations and practical guidance for achieving efficient, green, and sustainable fertilization management in soybean fields.

Objective Earthworm intestines, rich in carbohydrates and organic acids, are considered potential hotspots for the horizontal transfer of antibiotic resistance genes (ARGs). However, direct evidence is lacking regarding whether reactive oxygen species (ROS) are produced under anaerobic conditions in earthworm intestines and how ROS regulate plasmid conjugation. This study aimed to investigate the contribution of organic matter metabolism to ROS generation in earthworm intestines and how ROS affected the conjugative transfer of plasmids. Methods Pheretimaguillelmi was used as a model organism to establish the anaerobic microcosm systems simulating in-situ substrate concentrations of earthworm intestines. Four treatments with glucose, lactate, acetate, and amino acids as sole carbon sources were set up. The role of ROS was verified by adding ROS scavengers. Using the fluorescent probe technology, ion chromatography, and qPCR, we determined the production levels of •OH, O₂^•-, and H₂O₂, the consumption of organic substrates, and the abundance changes of the conjugation-related genes gfp, mCherry, trfA, and trbB, respectively. Results ROS was detected in all the treatments. The glucose group showed the highest •OH, O₂^•-, and H₂O₂ yields (0.684, 0.988, and 6.371 μmol/L, respectively) on day 2, which were significantly higher than those in other groups, while the acetate group showed the lowest yields. The substrate consumption rate followed the trend of glucose>lactate>amino acids>acetate, which was consistent with the ROS generation trend. Correspondingly, the glucose group exhibited the highest abundance of gfp, trfA, and trbB (3.47×10⁶, 6.73×10⁶, and 7.86×10⁶ copies/μg DNA) and conjugation frequency (8.9×10^-4), which were the lowest in the acetate group. After ROS scavenging, the conjugation frequencies in all the treatments significantly decreased by 73%‒92%. Mantel analysis revealed that hydroxyl radical showed the most significant correlation with conjugation frequency and abundance of trfA and trbB, indicating that •OH was the core ROS driving conjugative transfer. Unclassified Enterobacteriaceae and Clostridiumsensu stricto 10 were identified as the core microbial taxa coupling ROS generation and conjugation. Conclusion Organic matter metabolism in the anaerobic earthworm intestine can significantly promote ROS generation. ROS further regulates the conjugative transfer of ARGs among microbial strains by altering the abundance of conjugation-related genes.

Vulvovaginal candidiasis (VVC) is a prevalent fungal infection affecting the female reproductive tract. Although conventional therapeutic approaches for VVC are relatively well-established, they still exhibit certain limitations. Pulsatilla decoction, a classic traditional Chinese medicine formula, has demonstrated significant clinical efficacy in the treatment of VVC. However, its precise mechanism of action remains incompletely elucidated. Objective To clarify the therapeutic mechanism of the n-butanol extract of Pulsatilla decoction (BEPD) on VVC through network pharmacology and animal experiments. Methods A mouse model of VVC was established and the therapeutic effect of BEPD on VVC was evaluated. Network pharmacology was employed to screen the potential signaling pathways of BEPD on VVC. Western blotting, immunofluorescence, immunohistochemistry, and real-time fluorescence quantitative PCR were employed to measure the changes in autophagy, apoptosis, and related pathway proteins in the vaginal mucosa of mice. Results Network pharmacology analysis identified PIK3R1 and AKT1 as key targets of Pulsatilla decoction in exerting antifungal activity against VVC. KEGG pathway enrichment analysis indicated that Pulsatilla decoction exerted its therapeutic effects on VVC by regulating the PI3K-Akt signaling pathway. Animal experiments confirmed that compared with the VVC model group, the BEPD treatment down-regulated the expression of PI3K, p-Akt, and p-mTOR, significantly up-regulated the expression of autophagy-related proteins LC3B and ATG5, significantly inhibited the expression of apoptosis-related proteins Bax and Cleaved-Caspase-3, and significantly promoted the expression of anti-apoptosis-related protein Bcl-2. Conclusion BEPD may promote autophagy and inhibit apoptosis of vaginal epithelial cells by inhibiting the PI3K-Akt-mTOR signaling pathway, thereby restoring the homeostasis of the vaginal mucosal epithelial barrier and alleviating VVC.

Objective The outer membrane protein CirA serves as a specific transporter for catecholate-type siderophores and is involved in the uptake of siderophores and other nutrients, playing a crucial role in bacterial physiology. However, its impact on bacterial antibiotic resistance remains unclear. This study aimed to investigate the role of ahcirA in the antibiotic resistance of Aeromonas hydrophila ATCC 7966 under antibiotic stress, thereby providing a theoretical basis for elucidating the molecular mechanism by which ahcirA regulates bacterial resistance. Methods With A. hydrophila ATCC 7966 as the model organism, an ahcirA knockout strain (ΔahcirA) was constructed, and its susceptibility to multiple quinolones and aminoglycosides was assessed. Quantitative proteomics was further employed to compare protein expression profiles of ΔahcirA with and without antibiotic stress. Bioinformatic approaches were adopted for the functional analysis of differentially expressed proteins. Results In the media containing enrofloxacin and norfloxacin, the growth of ΔahcirA was significantly impaired compared with that of the wild-type strain. In contrast, ΔahcirA exhibited enhanced growth in the media supplemented with kanamycin and streptomycin. Proteomic and bioinformatic analyses revealed that the deletion of ahcirA may alter bacterial antibiotic resistance by affecting the expression of proteins involved in multiple biological processes, such as small molecule metabolism, and by modulating the expression of antibiotic resistance genes. Conclusion CirA plays a significant role in the antibiotic resistance of A. hydrophila. Its absence influences bacterial susceptibility to different classes of antibiotics by regulating the expression of diverse functional proteins and antibiotic resistance genes.

Objective To investigate the changes in gut microbiota, serum metabolites, and differentially expressed genes (DEGs) in the lung tissue of the mouse model of pulmonary fibrosis and explore the potential associations via multi-omics analysis. Methods A mouse model of pulmonary fibrosis was established by the dynamic inhalation exposure method and evaluated. Metagenomic sequencing was performed to analyze the microecological changes in cecal contents. Untargeted metabolomics was employed to detect serum metabolite alterations, and transcriptomic sequencing was conducted to profile DEGs in the lung tissue. Bioinformatics methods were comprehensively used to explore correlations and potential functional modules among differential microbial taxa, metabolites, and genes. Results Pathological changes of pulmonary fibrosis were successfully induced in the model mice, accompanied by the upregulated expression of transforming growth factor-beta (TGF-β), tumor necrosis factor-alpha (TNF-α), and fibrosis-related genes in the lung tissue. Omics results indicated the presence of gut microbiota dysbiosis, serum amino acid metabolic disorder, and lung transcriptome remodeling in the model mice. Correlation analysis demonstrated that the four differential bacterial species were strongly correlated with multiple serum metabolites, among which Akkermansia muciniphila and Ligilactobacillus murinus were jointly associated with 22 differential metabolites. A cross-omics network was constructed with these 22 differential metabolites and DEGs. Topological analysis identified five key subnetworks: (1) Inosine triphosphate serves as a phosphate donor and is converted to inosine diphosphate via multiple pathways; (2) Uridine triphosphate (UTP) undergoes an amination reaction to form cytidine triphosphate (CTP); (3) Serine/threonine-protein kinase 11, Fas-activated serine/threonine kinase, and cyclic GMP-dependent protein kinase act as core kinase nodes; (4) The reaction between serine and homocysteine bridges the metabolic pathways of methionine and cysteine; (5) Prostaglandin H₂ is catalytically converted into thromboxane A₂. Conclusion There are significant statistical correlations among gut microbiota, serum metabolites, and DEGs in the lung tissue in the mouse model of pulmonary fibrosis. We identify the core association network and potential functional modules, which provide references for the subsequent mechanism exploration of pulmonary fibrosis.

Objective To determine the cause of death of an adult crocodile in the Alligator sinensis Management Center in Anhui Province. Methods Bacteria were isolated from the heart, liver, lung, and spleen via the culture method, and the isolates were identified by morphological observation, biochemical tests, and molecular biological methods. Furthermore, the mucus phenotype was determined by means of the string test. Multilocus sequence typing (MLST) was conducted on the basis of seven housekeeping loci. Virulence gene analysis, pathogenicity test, drug resistance gene analysis, and antimicrobial susceptibility testing were conducted to clarify the pathogenicity and drug resistance of the isolates. Results The pathogenic bacteria isolated from the four organs were morphologically consistent Gram-negative bacilli. Through biochemical tests and 16S rRNA gene and khe sequencing, the isolates were identified as Klebsiella pneumoniae YZE01, capsular serotype K2. String test showed that the strain was hypermucinous K. pneumoniae, and MLST analysis showed that the strain belonged to sequence type 25 (ST25). The strain carried six virulence genes: fimH, entB, rmpA, rmpA2, mrkD, and wabG. Pathogenicity tests showed that some of the tested mice died within 24 h after infection with YZE01, and the same strain was isolated from the heart, liver, lung, and spleen. The lung tissue of infected mice showed hemorrhage and congestion lesions to different degrees. In addition, RT-qPCR revealed that the transcript levels of IL-1β, IL-6, IL-8, and TNF-α in the lung peaked at 12 h post-infection and then declined. The strain carried three drug resistance genes (blaSHV, armA, and ermB), and it was not sensitive to cephalexin, cefazolin, ampicillin, streptomycin, gentamicin, erythromycin, roxithromycin, and clindamycin. Conclusion The isolated strain K. pneumoniae YZE01 carries a variety of virulence genes and has strong pathogenicity and drug resistance. It is considered as a major cause of death in A. sinensis. The findings are conducive to the prevention and control of diseases in A. sinensis.

Objective To investigate the function of ring finger protein 31 (RNF31) in the replication of foot-and-mouth disease virus (FMDV) and to provide a theoretical basis for the research on the molecular mechanism by which the host protein RNF31 regulates FMDV replication. Methods CRISPR/Cas9 gene editing was employed to design two sgRNA sequences in the exon segment of RNF31, and recombinant plasmids were constructed by ligation with the pX459-puro vector. The recombinant plasmids pX459-RNF31-sgRNA were transfected into PK-15 cells, followed by screening under the action of puromycin to obtain the cell lines with RNF31 gene knockout. The effect of RNF31 gene knockout on FMDV replication was detected by Western blotting, RT-qPCR, and TCID₅₀ methods. Results Compared with wild-type cells, the knockout of RNF31 significantly increased the protein level, mRNA level, and virus titer of FMDV. Conclusion We successfully construct the cell lines with RNF31 gene knockout and prove that RNF31 plays a key role in the replication of FMDV. This result provides data support for further research on the mechanism by which RNF31 inhibits FMDV replication.

Objective To analyze the expression profile of the β-glucan-binding protein (Acβ-GBP) gene of Apis cerana cerana in response to Ascosphaera apis infection and to investigate the impacts of Acβ-GBP knockdown on the larval mortality and the incidence of chalkbrood disease following A. apis infection. These findings will provide a foundation for further functional research. Methods The sequence and structural characteristics of Acβ-GBP were analyzed via bioinformatics approaches. RT-qPCR was employed to investigate the expression profiles of Acβ-GBP in the larval midgut following A. apis infection. Furthermore, RNA interference (RNAi) was utilized to explore the impacts of Acβ-GBP on the larval mortality and the incidence of chalkbrood disease. Results The CDS length of Acβ-GBP was 1 440 bp, encoding a protein with a molecular weight of 54.68 kDa and a grand average of hydropathicity value of -0.22, which contained a typical transmembrane domain and signal peptide. Phylogenetic analysis revealed that β-GBP of A. c. cerana, Apis florea and Apis dorsata clustered into a single major clade. After A. apis infection, the expression level of Acβ-GBP in the midgut of A. c. cerana worker larvae was downregulated at 1-3 days post-infection (dpi) (P<0.05). Following RNA interference (RNAi)-mediated silencing of Acβ-GBP, its expression level was lower than that in the ds-egfp group at 2 and 3 dpi (P<0.01). The cumulative larval mortality and the incidence of chalkbrood disease both increased over the infection time, and the overall mortality was higher than that of the control group (P<0.000 1). Conclusion Acβ-GBP was capable of responding to A. apis infection, and knockdown of Acβ-GBP expression significantly impaired the resistance of honeybee larvae to A. apis. Collectively, β-GBP acts as an important immune recognition protein in A. c. cerana, and plays an important role in defending against fungal invasion.

Objective To investigate changes in ArfGAP with GTPase domain, ankyrin repeat and PH domain 2 (Agap2) expression during hepatic fibrosis progression following hepatitis E virus (HEV) infection and preliminarily explore the association between chronic HEV infection and Agap2 expression. Methods A BALB/c mouse model of HEV infection was established through inoculation in tail vein and subjected to RNA sequencing. HEV infection and Agap2 expression in the liver tissue were detected via immunohistochemistry, immunofluorescence assay, and real-time qPCR. Results Agap2 expression was upregulated following HEV infection (24 hpi group: P=0.000 3, 48 hip group: P=0.001 9). Chronic HEV infection induced hepatic fibrosis in mice, and Agap2 expression in the mouse liver was positively correlated with HEV load (r=0.797 4, P<0.000 1). Similarly, in vitro experiments demonstrated that Agap2 expression was upregulated in HEV-infected Huh 7.5.1 cells (r=0.968 3, P=0.002 4) and LX-2 cells (r=0.683 5, P=0.006 5), showing a positive correlation with HEV load. Conclusion The results demonstrate that Agap2 expression is positively correlated with HEV load during hepatic fibrosis progression after chronic HEV infection. Agap2 may serve as a potential molecular target for the treatment of HEV-associated hepatic fibrosis.

Objective The immunoinflammatory response induced by spinal cord injury is a key factor hindering the recovery of neurological functions. Recent studies have shown that gut microbiota dysbiosis can participate in the immune regulation of the central nervous system through the gut-spinal cord axis. This study aims to explore whether curcumin can exert its protective effect on spinal cord injury by reshaping the gut microbiota and thereby regulating the local Treg/Th17 balance in the spinal cord. Methods Female Sprague-Dawley rats weighing 200‒220 g were randomly assigned into the sham operation group, spinal cord injury group, curcumin group, fecal microbiota transplantation group, fecal microbiota transplantation+ curcumin group, and fecal microbiota transplantation+curcumin+GPR inhibitor group. Neurological function recovery was evaluated based on the Basso-Beattie-Bresnahan motor function score and gait analysis. Histopathological changes in the injured area were observed via hematoxylin-eosin staining, Nissl staining, and Luxol Fast Blue staining. RT-qPCR, ELISA, and Western blotting were employed to quantify the expression levels of key transcription factor forkhead box protein 3 (FOXP3) for Treg cells, anti-inflammatory cytokines interleukin (IL)-10 and transforming growth factor (TGF)-β1, as well as key transcription factor retinoic acid receptor-related orphan receptor gamma t (RORγt) for Th17 cells and pro-inflammatory cytokines IL-17 and IL-6 in the spinal cord of each group. Results Compared with the spinal cord injury group and fecal microbiota transplantation group, the curcumin group and fecal microbiota transplantation+ curcumin group showed the most significant improvement in neurological function, specifically manifested by significant increases in BBB motor function scores and gait coordination, along with a marked reduction in the scope of spinal cord injury. At the molecular level, the two groups showed significantly upregulated gene and protein levels of FOXP3, IL-10, and TGF-β1 and significantly inhibited expression of RORγt, IL-17A, and IL-6 in the spinal cord tissue. This suggests that after curcumin intervention in the gut microbiota, the immune balance shifted toward a Treg-dominated anti-inflammatory state. Notably, the aforementioned beneficial effects of curcumin-modified gut microbiota were reversed after combined use of the GPR inhibitor. Conclusion This study indicates that curcumin can act on the gut microbiota to promote the recovery of motor function after spinal cord injury. Curcumin may exert the effect by activating the GPR signaling pathway, thereby upregulating Treg viability, inhibiting Th17 differentiation, and ultimately correcting the Treg/Th17 imbalance. This provides new experimental evidence and application value for using curcumin and its modified gut microbiota as an adjuvant therapeutic strategy for spinal cord injury.