[Objective] To study the relieving effects and potential mechanisms of Lactiplantibacillus plantarum DY6 and the soybean meal fermented with this strain on constipation in mice and to develop non-toxic feed additives with probiotic potential. [Methods] Constipation was induced in male BALB/c mice by gavege of loperamide hydrochloride. The modeled mice underwent long-term feeding with the soybean meal fermented with DY6 or gavage with DY6 suspension. Linaclotide served as the positive control. We then systematically assessed the effects of fermented soybean meal on intestinal functions, immune factors, metabolic profiles, and gut microbiota in constipated mice. [Results] Compared with the model group, long-term intake of the soybean meal fermented with DY6 increased fecal moisture content and intestinal motility (P<0.05), ameliorated goblet cell reduction and lymphocyte aggregation in the colon tissue, lowered the serum levels of tumor necrosis factor-α, interleukin-6, and vasoactive intestinal peptide (P<0.05), and elevated the fecal levels of acetic acid and butyric acid (P<0.01). The fermented soybean meal modulated the gut microbiota structure. Specifically, the significantly enriched probiotics Odoribacter and Blautia were positively correlated with indicators of relieving constipation, while the significantly reduced pathogenic bacteria Helicobacter and Colidextribacter were negatively correlated with indicators of relieving constipation. [Conclusion] The soybean meal fermented with DY6 alleviates constipation by modulating the gut microbiota structure and metabolic profiles, restoring intestinal electrolyte homeostasis, and suppressing inflammation-associated signaling pathways. This intervention establishes novel applications and a theoretical framework for developing functional biological feed additives capable of both preventing and managing constipation in animals.

[Objective] To prepare secretory IgA (sIgA) antibody against foot-and-mouth disease virus (FMDV) and provide a new idea and basis for the comprehensive prevention and control of foot-and-mouth disease (FMD). [Methods] We constructed an eukaryotic expression plasmid harboring the heavy chain of IgA antibody by replacing the constant region gene of heavy chain of IgG with the codon-optimized corresponding region of IgA based on POA-8, a previously screened IgG neutralizing antibody capable of neutralizing both type O and type A FMDV. At the same time, the eukaryotic expression plasmids harboring the J chain and secretory component (SC) of pigs as well as the eukaryotic expression plasmid containing heavy chain, light chain, and J chain genes were constructed respectively. CHO-S cells were transiently co-transfected with 4 plasmids (heavy, light, J, and SC) or 2 plasmids (heavy+light+J and SC) for assembling and expression of sIgA antibody. sIgA antibody was purified and identified by SDS-PAGE and Western blotting. The binding activity and neutralizing activity of sIgA antibody against FMDV were evaluated by indirect ELISA and virus neutralization assay. [Results] sIgA antibody against FMDV could be successfully assembled and expressed after transfection of CHO-S cells with 4 or 2 plasmids. The binding and neutralizing abilities of sIgA against type O and type A FMDV were stronger than those of IgG. [Conclusion] This study established an efficient expression system for sIgA against FMDV, laying a foundation for the development of mucosal vaccines and antiviral drugs against FMDV in the future.

[Objective] To investigate whether Fusobacterium nucleatum promotes the development of lung cancer and find out the underlying mechanisms. [Methods] The cell experiment proved that F. nucleatum can promote the proliferation and metastasis of lung cancer cells; Animal experiments showed that F. nucleatum can promote the development of lung cancer; 16S rRNA gene sequencing of alveolar lavage fluid and transcriptome sequencing of lung tissue were performed to analyze the effects of F. nucleatum on the lung flora of mice with lung cancer and the activation of relevant pathways in lung tissue. [Results] The cell experiment showed that F. nucleatum significantly promoted the proliferation and metastasis of A549 cells. The animal experiments confirmed that F. nucleatum infection in the lungs increased the number of nodules and enhanced the fluorescence intensity in the lung tissue. The 16S rRNA gene sequencing revealed the enrichment of Muribaculum and Simplicispira in the lung of infected mice. The transcriptome sequencing results showed that the mouse model of lung cancer activated the interleukin-17 signaling pathway, cytokine-cytokine receptor interactions, Staphylococcus aureus infection pathway, tumor necrosis factor signaling pathway, and other signaling pathways related to inflammation or immunity after being infected with F. nucleatum. [Conclusion] F. nucleatum infection in mice with lung cancer disrupts the microbiota and activates the immunity- and inflammation-related signaling pathways in the lung, thereby promoting the metastasis of lung cancer in mice.

[Objective] To develop an efficient catalyst for organophosphorus pesticide degradation by immobilizing organophosphorus hydrolase (OPH) on the surface of Pichia pastorisvia the SpyCatcher/SpyTag (SpyC/SpyT) system, addressing the poor stability and low reusability of OPH in practical applications and providing a new method for the bioremediation of organophosphorus pesticide pollution. [Methods] The “bait protein” SpyCatcher (SpyC) was first displayed on the surface of P. pastoris, and the display efficiency was increased by increasing the copy number and optimizing the culture conditions. Then based on the specific interaction between SpyC and SpyT, OPH-SpyTag (OPH-SpyT) was efficiently displayed on the yeast surface. The thermal stability, pH stability, and reusability of immobilized OPH were evaluated, and the hydrolysis efficiency of immobilized OPH against methyl parathion, dimethoate, and chlorpyrifos was assessed. [Results] The display efficiency of SpyC on the P. pastoris surface reached over (97.0±0.4)%, with an optimized binding capacity of (21.4±0.7) mg green fluorescent protein for 1 g wet cells. OPH was successfully displayed on the cell surface via the SpyC/SpyT system. The immobilized OPH exhibited significantly enhanced thermal and pH stability, retaining more than 50% activity after five repeated uses. Under optimum conditions, the immobilized OPH showed the hydrolysis rates of (96.5±2.7)%, (79.5±2.3)%, and (82.6±2.8)% against 100 mg/L methyl parathion, dimethoate, and chlorpyrifos, respectively. This indicated that the method showed high hydrolysis efficiency for the organophosphorus pesticides. [Conclusion] The immobilization of OPH on P. pastoris surface via the SpyC/SpyT system effectively improves its stability and reusability, offering an efficient and environmentally friendly solution for the bioremediation of organophosphorus pesticide pollution. Meanwhile, this study provides a powerful tool and method for research in the field of P. pastoris surface display.

Sauropus androgynus has high medicinal and edible values. However, its growth is threatened by various viral diseases, which severely affect both the yield and quality of S. androgynus. Since research is limited regarding the viral diseases affecting S. androgynus in China. [Objective] To isotation, identifying the viral pathogens of S. androgynus in China. [Methods] The small RNA sequencing (sRNA-seq) data of S. androgynus leaves from our previous study were analyzed. RT-PCR was employed to detect the datura yellow vein virus (DYVV) in leaf samples of 10 different varieties of S. androgynus. With the total RNA of positive S4 leaves as a template, reverse transcription-polymerase chain reaction (RT-PCR), rapid amplification of cDNA ends (RACE), and Sanger sequencing were employed to determine the full-length genome sequence of the DYVV isolate from S. androgynus (named DYVV-sa). [Results] The analysis of the sRNA-seq data revealed the presence of DYVV in S. androgynus. RT-PCR detection of different varieties showed that only S4 and S12 tested positive for DYVV, and the full-length sequence of DYVV-sa was cloned based on S4. The genome of DYVV-sa was 13 185 nt in length and contained six open reading frames (ORFs). The DYVV-sa showed the identity as high as 95.8%-98.1% with the DYVV sequences isolated from Thunbergia alata. Moreover, the phylogenetic tree also demonstrated that DYVV-sa shared the closest genetic relationship with DYVV, clearly indicating that DYVV-sa was an isolate of DYVV. In addition, the majority of DYVV-sa virus-derived small interfering RNA (vsiRNA) were 21 nt and 22 nt, and those of 21 nt were more abundant. The first nucleotide at the 5′ termini of vsiRNAs derived from DYVV-sa preferred U and C. The proportion of vsiRNAs derived from the negative strand was higher than that from the positive strand. The distribution of vsiRNAs along the viral genome was generally even, with some hot spots formed in local regions. [Conclusion] This study found that DYVV can infect S. androgynus and successfully obtains the full-length genomic sequence of the DYVV-sa isolate. These findings expand the known natural host range of DYVV, provide crucial theoretical foundations for research on its genetic diversity and phylogenetic relationship, and offer clues for the prevention and control of viral diseases attacking S. androgynus.

Cyanobacteria, commonly known as blue-green algae, are important primary producers in aquatic ecosystems and common dominant algae causing algal blooms in freshwater. Cyanophages, especially procyanophages, are important planktonic ecological factors that affect the evolution of blue-green algae and aquatic microbial communities. Yet studies on procyanophages in cyanobacteria remain scarce. So far, few studies have reported the procyanophages in Microcystis. [Objective] To investigate the prevalence of lysogeny in Microcystis and characterize the genomic features of procyanophages in Microcystis. [Methods] All the 354 genome sequences of Microcystis spp. in GenBank were downloaded. PHASTER was used to predict procyanophage regions in the Microcystis genomes. Resistance and virulence factors in intact procyanophages and questionable procyanophages were annotated via the virulence factors of bacterial pathogens (VFDB) and comprehensive antibiotic resistance database (CARD). Bioinformatics tools were used for gene annotation and phylogenetic analysis of the procyanophages in Microcystis. Microcystis flos-aquae FACHB-1344 and M. aeruginosa FACHB-1326 each was predicted to harbor an intact procyanophage by PHASTER. To evaluate the infection activities of these two procyanophages, mitomycin C induction assays and dot-ELISA were conducted. [Results] Among all the 354 Microcystis genomes, 98.3% were predicted to harbor intact procyanophages, questionable or imcomplete procyanophages. A total of 13 intact procyanophages, 5 questionable procyanophages and 725 incomplete procyanophages were predicted by PHASTER. The 13 intact procyanophages and 5 questionable procyanophages were named as WZ1-WZ13 and YS1-YS5, respectively. No antibiotic resistance or virulence gene was detected in them. The phylogenomic tree displayed distant evolutionary relationships between the 18 procyanophages and other known viruses. Bioinformatics analysis suggested that YS5 revealed a previously unknown novel genus. WZ2, WZ3, WZ4, WZ5, WZ6, WZ7, WZ9, WZ10, WZ11, WZ12 and YS3 together revealed a novel family. WZ1 and YS1 together revealed a novel family. YS2, YS4, WZ8 and WZ13 each revealed a novel family. The procyanophages in M. flos-aquae FACHB-1344 and FACHB-1326 were verified to be activated by mitomycin C. [Conclusion] Lysogeny widely exists in Microcystis spp. The novel procyanophages in Microcystis spp. unlock novel viral evolutionary lineages previously unknown. This study enriches the understanding about cyanobacterium-virus interactions and the diversity of aquatic viruses.

[Objective] To investigate the role of dppC2 in the survival of Yersinia pestis in macrophages. [Methods] The strain (201-ΔdppC2) with traceless knockout of dppC2 was constructed with a suicide plasmid via homologous recombination based on Y. pestis biovar Microtus strain 201. Phenotypes were compared between 201-ΔdppC2 and the wild type (201-WT) by the acid survival assay, hydrogen peroxide survival assay, macrophage intracellular survival assay, reactive oxygen species (ROS) detection, and cytotoxicity and mouse challenge assays. The gene expression was compared between 201-ΔdppC2 and 201-WT by transcriptomics analysis and RT-qPCR. [Results] Compared with 201-WT, 201-ΔdppC2 exhibited multiple phenotypic alterations, including significantly increases in intracellular survival rates in RAW264.7 and THP-1 cells and under acidic and hydrogen peroxide conditions, upregulation of the acid resistance gene hdeD and the catalase-related genes katA and katG, enhancement of catalase and peroxidase activities, and declines in intracellular ROS levels in 201-ΔdppC2 and RAW264.7 cells infected with the mutant. Furthermore, 201-ΔdppC2 showed reduced cytotoxicity to HeLa cells but no change in the virulence in mice. [Conclusion] The deletion of dppC2 has been demonstrated to enhance the fitness of Y. pestis to acidic and hydrogen peroxide environments, which promote the survival and replication of Y. pestis in macrophages.

6′-sialyllactose (6′-SL) is an important component of porcine breast milk oligosaccharides, and sialic acid (SIA) is the monomer of sialylated breast milk oligosaccharides. Both can regulate the structure of human colonic microbiota. However, there are few reports regarding the effects of SIA and 6′-SL on the composition and fermentation characteristics of piglet colonic microbiota in vitro. [Objective] To investigate the effects of SIA and 6′-SL on the composition and fermentation characteristics of colonic microbiota in suckling piglets, aiming to provide a reference for using these substances to regulate gut health in piglets. [Methods] We used mixed colonic chyme from three suckling piglets as the microbial inoculum. The experiment was conducted with four groups: no carbon (NCB), lactose (LAC), SIA, and 6′-SL, each with four replicates. [Results] After 24 h of fermentation, the pH in the 6′-SL group was higher than that in the LAC group (P<0.05). After fermentation for 12 h and 24 h, the gas production in the SIA and 6′-SL groups was lower than that in the LAC group (P<0.05). At the time point of 24 h, the SIA and 6′-SL groups had higher acetic acid content and lower propionic acid and butyric acid content than the LAC group (P<0.05). Moreover, the SIA and 6′-SL groups had higher Chao1 and ACE indices than the LAC group (P<0.05). Beta diversity of the microbial community in the LAC, SIA, and 6′-SL groups changed (P<0.05). As fermentation progressed, compared with LAC, SIA increased the relative abundance of Bacillota, Anaerovibrio, and Faecalibacterium and decreased that of Pseudomonadota, Escherichia-Shigella, Mitsuokella, and Streptococcus (P<0.05); 6′-SL increased the relative abundance of Bacteroidetes, Prevotella, Bacteroides, and Roseburia and decreased that of Pseudomonadota, Bacillota, Megamonas, Escherichia-Shigella, Mitsuokella, and Prevotellaceae_NK3B31_group (P<0.05). [Conclusion] Both 6′-SL and SIA can effectively regulate the gut microbiota structure in piglets. Specifically, 6′-SL increases the relative abundance of Bacteroidetes, Prevotella, and Bacteroides, while SIA increases that of Bacillota and Anaerovibrio. Both substances promote colonic microbial fermentation in piglets to increase the production of short-chain fatty acids (SCFAs), especially acetic acid.

[Objective] To study the effect of ferulic acid on the laccase activity of Dichomitus squalens and its molecular mechanism, providing a theoretical basis for microbial degradation of aromatic compounds. [Methods] Ferulic acid was added to the synthetic medium at different concentrations to investigate its effects on the growth of D. squalens and laccase activity. Transcriptomic and proteomic analyses were performed to examine the changes in laccase transcription and protein expression levels induced by ferulic acid. The lcc3 gene was knocked down using RNAi technology, and the impact on laccase activity and the ability to degrade various aromatic compounds was assessed. A self-constructed dual-luciferase system based on Gaussia luciferase and Nano luciferase was employed to identify the core promoter region of the lcc3 gene. [Results] An appropriate concentration of ferulic acid can significantly enhance laccase activity in D. squalens. Transcriptomic and proteomic analyses revealed that the transcription and expression levels of the lcc3 gene were markedly up-regulated under ferulic acid induction. In the lcc3-gene-knocked-down strain, both laccase activity and the ability to degrade various aromatic compounds decreased significantly, confirming that lcc3 is a key gene for laccase-related activities in degrading aromatic compounds in D. squalens. Moreover, the dual-luciferase system successfully identified the core promoter region of the lcc3 gene. [Conclusion] This study first revealed that ferulic acid can induce D. squalens laccase activity and clarified the molecular mechanism. It was also proved that lcc3 is a key gene for ferulic acid-induced laccase activity and degradation of aromatic compounds in D. squalens. Identifying the core promoter region of lcc3 lays a foundation for gene expression regulation research, and these findings offer theoretical support for using microbial laccases to degrade aromatic compounds.

[Objective] To explore the protective effect of selenomethionine (Se-Met) on oxidative stress and intestinal barrier damage in mice infected with porcine deltacoronavirus (PDCoV) and the potential regulatory mechanism. [Methods] Forty female C57 mice were randomly grouped as follows: control, Se-Met (0.3 mg/kg Se), PDCoV, and Se-Met+PDCoV (0.3 mg/kg Se). After being fed with or without Se-Met for 23 days, the mice in the PDCoV group and the Se-Met+PDCoV group were administrated with 300 μL suspension of PDCoV HNZK-02-P5 strain (1×10⁶ TCID₅₀) by gavage, while those in the other two groups were administered with the same volume of Dulbecco’s Modified Eagle Medium (DMEM). All the mice were observed daily for clinical signs, food intake, and body weight changes until day 28. At five days post-inoculation (dpi), intestinal tissues were collected and PDCoV titers were determined. Hematoxylin staining and eosin staining were used to monitor pathological changes in intestinal tissues. Oxidative stress-related indicators such as malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-PX) were investigated. The level of ROS in the jejunum tissue was measured via a 2′,7′-dichlorofluorescein diacetate (DCFH-DA) probe. Immunofluorescence was used to analyze the changes of small intestinal tight junction proteins (ZO-1 and Occludin). The mRNA levels of inflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-10), intestinal tight junction proteins (ZO-1 and Occludin), and the Nrf2 signaling pathway-associated factors (Nrf2, HO-1, and NQO1) were determined by RT-qPCR. Western blotting was employed to assess the protein levels of factors related to the Nrf2 signaling pathway. [Results] The results of body weight, food intake, pathological examination, and viral RNA titers in different intestinal tissues revealed that Se-Met might increase the body weight, decrease viral titers in intestinal tissues, and attenuate PDCoV-induced structural damage of intestinal villi in PDCoV-infected mice. Se-Met attenuated PDCoV-induced inflammation by lowering the mRNA levels of major inflammatory cytokines, such as IL-1β, IL-6, and TNFα in the jejunum. Se-Met ameliorated PDCoV-induced intestinal mucosal barrier damage by up-regulating the mRNA levels of ZO-1 and Occludin in the jejunum. Se-Met ameliorated PDCoV-induced oxidative stress by decreasing the levels of ROS and MDA and increasing the levels of GSH-PX and SOD in the jejunum. Se-Met inhibited PDCoV-induced oxidative stress by activating the Nrf2 signaling pathway. [Conclusion] Se-Met may attenuate the intestinal injury in mice infected with PDCoV by activating the Nrf2 signaling pathway, which provides a theoretical basis for the prevention and treatment of PDCoV infection.