[Objective] To improve the overall quality of upper tobacco leaves. [Methods] We isolated a hemicellulase-producing bacterium from tobacco soil and identified it as Actinacidiphila bryophytorum. After optimizing the culture conditions of this strain, we prepared the crude enzyme liquid, which was sprayed on upper tobacco leaves. The physical properties and chemical composition of the treated tobacco leaves were then evaluated. [Results] The optimal fermentation conditions for the strain were an inoculation rate of 4%, pH 6.0, and incubation at 30 ℃ for 36 h. Enzyme preparations of different concentrations (50, 100, 150, 200 and 250 U/mL) were then sprayed on the surface of upper tobacco leaves, and the appearance quality, conventional chemical composition, lignocellulose content, and sensory scores of the tobacco leaves were evaluated. The results indicated that treatment of the enzyme preparation at 150 U/mL performed better than other treatments. Compared with the control group, the 150 U/mL treatment increased the overall score for the appearance quality (maturity, color, characteristics, and structure) of tobacco leaves by 5.15 points. Additionally, this treatment increased the total sugar content and reducing sugar content by 29.87% and 35.77%, respectively. Meanwhile, it decreased the content of nicotine, cellulose, hemicellulose, and lignin by 16.10%, 16.37%, 20.22%, and 17.13%, respectively. Furthermore, the aroma characteristics were enhanced, and off-flavors were reduced. [Conclusion] The enzyme preparation produced by the fermentation of A. bryophytorum S2 improves the appearance quality, increases the soluble total sugar and reducing sugar content, and reduces the lignocellulose content of tobacco leaves, thus improving the overall quality of tobacco leaves.

[Objective] Yersinia enterocolitica is the primary pathogenic agent of yersiniosis, and it is one of the major foodborne pathogens worldwide. The purpose of this study is to isolate and identify a virulent phage against Y. enterocolitica and explore its application potential. [Methods] A novel virulent phage, named Yersinia phage Yen-yong1 (abbreviated as Yen-yong1), was isolated from the sewers of Lulin Market in Beilun District, Ningbo City, Zhejiang Province, China, by the double-layer agar method with Y. enterocolitica CICC 10869 as the indicator host. The morphology, host range, genome sequence, gene functions, phylogenetic position, physical and chemical tolerance, and biofilm removal ability of Yen-yong1 were studied. Additionally, the sterilization effect of Yen-yong1 on Y. enterocolitica in raw pork slices was evaluated. [Results] Yen-yong1 clarified the turbid CICC 10869 culture within 1.5 h. Yen-yong1 was Siphovirus-like, consisting of an elliptical head with a length of (83.32±0.15) nm and a width of (59.57±2.28) nm and a long tail with a length of (168.04±3.21) nm. The double-stranded DNA genome of Yen-yong1 was 51 321 bp, with the G+C content of 47.80% and 93 open reading frames (ORFs). No known lysogeny-associated genes, resistance genes, or virulence genes detrimental to application were found in Yen-yong1. The highest nucleotide sequence similarity between Yen-yong1 and the other phages in public databases was 20.64%, which was <70.00% (the threshold to define a genus). Yen-yong1 shared 20.00% of core genes with the closest relative, which was lower than the ICTV criteria for subfamily classification (27.00%‒79.00%). The results suggested that Yen-yong1 represented a novel genus and a novel subfamily belonging to Caudoviricetes. Yen-Yong1 was species-specific and capable of lysing 3 out of 4 tested Y. enterocolitica strains. Yen-yong1 maintained high bacteriolytic activity at -80‒60 ℃ and pH 2.0‒13.0, as well as in the presence of 0‒3% NaCl. Yen-yong1 can disrupt the biofilms of Y. enterocolitica. At 25 ℃ and MOI=100, the sterilization rates of Yen-yong1 against Y. enterocolitica CICC 10869, CICC 21669, and CMCC 52202 in raw pork slices were 93.48%, 52.77%, and 43.24%, respectively. All the 3 tested Y. enterocolitica strains proliferated at 4 ℃. At 4 ℃ and MOI=100, the sterilization rates of Yen-yong1 against Y. enterocolitica CICC 10869, CICC 21669, and CMCC 52202 in raw pork slices were 98.28%, 78.35%, and 96.33%, respectively. [Conclusion] Yen-Yong1 reveals a previously unknown evolutionary lineage. Yen-Yong1 has excellent biological characteristics and potential applications. This study enriches the knowledge of Y. enterocolitica phages, phage database, and gene database, laying a foundation for the application of phages in the prevention and control of food-borne yersiniosis.

[Objective] The enzymatic depolymerization of polyethylene terephthalate (PET) involves the hydrolysis of PET catalyzed by PET hydrolases, leading to the production of terephthalic acid and ethylene glycol. In this study, we identified a new PET hydrolase through gene mining, with the goal of providing enzymes for efficient depolymerization of waste PET. [Methods] A previously uncharacterized functional enzyme, named as AePETase, was identified through gene mining. This enzyme demonstrated the ability to hydrolyze amorphous PET films. Subsequently, heterologous expression, purification, and enzymatic characterization were performed for AePETase. [Results] AePETase showed a T_m value of (53.0±0.7) ℃, and an optimal reaction temperature of 45 ℃. The optimal buffer was Na₂HPO₄-NaH₂PO₄ (100 mmol/L, pH 8.0), and the optimal enzyme concentration was 1 µmol/L. After 72 h, the hydrolysis of amorphous PET films by AePETase resulted in 13.4-fold more products than that by the reported IsPETase. [Conclusion] AePETase demonstrates significant hydrolytic activity on PET and represents a promising new enzyme for hydrolyzing PET.

[Objective] To study the cytopathic effects (CPE) and replication characteristics of HCoV-OC43 VR1558 strain in cell lines derived from different species. [Methods] Thirteen cell lines from humans (MRC-5, HRT-18, Huh7, Huh7.5, RD, and HeLa), non-human primates (LLC-MK2 and Vero), and rodents (17Cl-1, Mv.1Lu, BHK-21, BHK-21-APN, and Neuro 2a) were selected and infected with HCoV-OC43 VR1558 strain. CPE were observed for several consecutive days. Virus-infected cells and supernatants were collected daily. RT-qPCR was conducted to monitor the changes in viral RNA copy number. The load of viruses with infectious ability was determined based on the tissue culture infectious dose 50% (TCID₅₀), and the kinetic curves of viral replication in different cell lines were established. [Results] Following infection with HCoV-OC43 VR1558 strain, CPE were observed in all the 13 cell lines. CPE primarily manifested as cell aggregation, shrinkage, rounding, and detachment. CPE appeared early in MRC-5, Mv.1Lu, HeLa, and 17Cl-1 cells, being noticeable within 72 h post-infection (hpi). The virus induced CPE in other cell lines after 120 hpi, and CPE were the mildest in HRT-18 and Huh7.5 cells. RT-qPCR results indicated that the viral RNA copy number increased most significantly within 24 hpi, although the time to reach the peak and the peak copy number varied among cell lines. Specifically, the RNA copy number in Huh7.5 cells reached the peak (10⁷ copies/mL) at 24 hpi, and that in 17Cl-1, BHK-21-APN, Mv.1Lu, and BHK-21 cells reached the peaks (10⁷ to 10⁹ copies/mL) at 48 hpi. In MRC-5, LLC-MK2, Neuro 2a, and Vero cells, the replication peaks (10⁶ to 10⁹ copies/mL) occurred at 72 hpi. In HRT-18, HeLa, Huh7, and RD cells, the viral RNA copy number peaked after 96 hpi, reaching 10⁸ to 10⁹ copies/mL. TCID₅₀ assay results demonstrated rapid viral proliferation within 24 hpi, while the time to reach the peak titer and the peak titers varied. The peak titer (2.68× 10⁷ TCID₅₀/mL) in BHK-21-APN cells was observed at 48 hpi. In BHK-21 and Neuro 2a cells, the peak titers (10⁶ to 10⁷ TCID₅₀/mL) were observed at 72 hpi. In MRC-5, 17Cl-1, HeLa, Huh7, Huh7.5, Mv.1Lu, LLC-MK2, and RD cells, the peak titers (10⁶ to 10⁸ TCID₅₀/mL) were observed at 96 hpi. In Vero cells, the virus strain reached the peak titer (10⁵ TCID₅₀/mL) at 120 hpi, while the strain reached the peak titer of 10⁸ TCID₅₀/mL in HRT-18 cells at 144 hpi. [Conclusion] HCoV-OC43 VR1558 strain exhibits a wide spectrum of cell tropism, demonstrating rapid replication and proliferation within 24 hpi across 13 cell lines derived from various species. However, the time to reach the replication peak varied among different cell lines. The highest viral titer achieved was 10⁸ TCID₅₀/mL, observed in MRC-5 and HRT-18 cells. This study provides experimental reference for further investigation of the replication characteristics, infection mechanism, and pathogenicity of HCoV-OC43, as well as for the screening and evaluation of antiviral drugs.

[Objective] To understand the molecular functions and potential applications of the significantly up-regulated gene cluster chr1_2605-chr1_2604 in response to tetrabromobisphenol A (TBBPA) stress, we investigated the roles of chr1_2605 and chr1_2604 in the specific recognition and efficient degradation of TBBPA. [Methods] Synthetic biology methods were employed to construct Sphingobiumxenophagum C1 (pBBR-2605-HiBiT) and Escherichiacoli BL21(DE3, pET30b-2604) as chassis cells for biosensing and degrading, respectively. The response characteristics of Chr1_2605 in the chassis cells to different pollutants were analyzed by the luciferase activity assay. Additionally, the degradation activity of TBBPA by Chr1_2604 in the chassis cells was determined by high-performance liquid chromatography. [Results] The xenobiotic-responsive element Chr1_2605 exhibited a highly specific response to TBBPA. The Chr1_2605-based chassis cell of S. xenophagum C1 (pBBR-2605-HiBiT) demonstrated high responsivity and sensitivity to TBBPA, with a limit of detection ranging from 0.010 to 0.050 μmol/L. The 2-oxoglutarate/Fe-dependent dioxygenase Chr1_2604 in the chassis cell of E. coli BL21(DE3, pET30b-2604) displayed the degradation rate of 44.415% for 2.0 mg/L TBBPA within 3 d [0.296 mg/(L·d)], which was significantly higher than those of most reported microbial strains under non-co-metabolic conditions. [Conclusion] The chr1_2605-chr1_2604 gene cluster can accurately recognize and degrade TBBPA. Specifically, the xenobiotic-responsive element Chr1_2605 specifically recognizes TBBPA, whereas the 2-oxoglutarate/Fe-dependent dioxygenase Chr1_2604 efficiently degrades TBBPA.

Lactic acid bacteria (LAB) are the predominant probiotics with significant health-promoting potential. Xizang pigs, an invaluable indigenous breed in China, harbor a unique and largely unexplored reservoir of intestinal LAB. [Objective] To screen high-quality LAB isolated from Xizang pigs. [Methods] LAB were isolated by the streak plate method and identified by 16S rRNA gene sequencing. Two Limosilactobacillus reuteri strains with significantly different lactic acid production capacities were selected from Xizang pigs. The two strains, together with one L. reuteri strain previously preserved in our laboratory, which originated from duroc×landrace×yorkshire pigs, were assessed in terms of acid and bile salt tolerance, antioxidant activity, antibacterial properties, and antibiotic resistance. [Results] A total of 21 LAB strains were isolated and identified from the intestinal microbiota of Xizang pigs, including ten Streptococcus alactolyticus strains, six L. reuteri strains, three Lactobacillus amylovorus strains, one Leuconostoc mesenteroides strain, and one Limosilactobacillus vaginalis strain. Among the three strains screened out, L. reuteri T-B5L2 exhibited the highest lactic acid production. Moreover, this strain demonstrated strong survival under pH 3.0 and 0.1% bile salts. L. reuteri T-B5L2 exhibited the strongest inhibitory activity against enteropathogenic Escherichia coli and Salmonella Choleraesuis. There were no significant differences in antioxidant activity among the three strains. All the three strains exhibited high sensitivity to penicillins and cephalosporins but displayed resistance to tetracyclines, aminoglycosides, and glycopeptides. [Conclusion] In this study, 21 LAB strains were isolated from the feces of Xizang pigs, among which L. reuteri T-B5L2 exhibited the highest acid production capacity. Further in vitro biochemical characterization demonstrated that this strain exhibited good growth performance, acid and bile salt tolerance, antioxidant activity, and the ability to inhibit pathogenic bacteria. These findings suggest that L. reuteri T-B5L2 is a promising probiotic candidate with potential applications in improving intestinal health and mitigating pathogenic infections.

[Objective] To clarify the diversity characteristics of gut microbiota in the important wild pollinator, Xylocopa appendiculata, and provide theoretical basis for the conservation and utilization of wild pollinator resources. [Methods] Utilizing employed 16S rRNA gene high-throughput sequencing, we conducted comparative analyses of gut microbiota diversity in adults X. appendiculata from different habitats and sexes. Functional prediction of gut microbial communities was performed using PICRUSt2. [Results] The gut microbiota in adults X. appendiculata was predominantly composed of Bacillota (relative abundance of 50.17%), Pseudomonadota (relative abundance of 32.04%), Actinomycetota (relative abundance of 12.84%), and Bacteroidota (relative abundance of 3.77%). The genus Lactobacillus was identified as a core microbiota present in all sampled individuals. Significant differences in the relative abundance of gut microbiota were observed across habitats and sexes. The samples from the Sui and Tang Dynasties City Ruins Botanical Garden habitat (HXST) exhibited the highest richness, diversity, and evenness of gut microbiota. In the Sui and Tang Dynasties City Ruins Botanical Garden habitat (ST) and the Caoyao Village habitat (CY), female adults showed higher microbial richness, diversity, and evenness compared to males. In the Shuiquan Village habitat (SQ), male adults showed higher microbial richness, diversity, and evenness compared to females. The genus Gilliamella, as the dominant genus of male samples from Caoyao Village (HXCYM), accounts for a very small proportion in other samples. Functional prediction PICRUSt2 showed that the metabolic gene functions of the gut microbiota of adults X. appendiculata are the most enriched, with an abundance ratio of 74.00%. [Conclusion] The gut microbiota of adults X. appendiculata exhibits ecological adaptive features with social bees (e.g., Apis mellifera and Bombus spp.), characterized by low diversity, highly conservation, and specialized core microbial community structure. Environmental heterogeneity and host sex were identified as key drivers of gut microbiota divergence. These findings suggest that the gut microbiota of X. appendiculata may play a central role in maintaining host energy homeostasis and population adaptability.

[Objective] To explore the autophagy induced by the African swine fever virus (ASFV) protein E423R. [Methods] The RFP-LC3 fluorescent spots following the transfection of HeLa-Difluo^TM hLC3 cells with pCMV-Myc-E423R was quantified by the High-Content Analysis System, and the autophagic flux was assessed. The expression levels of key autophagy proteins, LC3-Ⅱ and SQSTM1/p62, in HeLa cells were analyzed via Western blotting. Co-localization of autophagosomes and lysosomes was examined by laser confocal microscopy. Additionally, Western blotting was employed to investigate the dose-dependent effect of E423R on LC3-Ⅱ expression and the regulatory effect of E423R on the AKT/mTOR/ULK1 signaling pathway. [Results] The High-Content Analysis System demonstrated a significant increase in RFP-LC3 fluorescence spots in the reporter cells expressing E423R, suggesting that E423R induced the activation of autophagy. Western blotting further confirmed that the expression of E423R significantly elevated the LC3-Ⅱ/β-actin ratio while decreasing the expression level of p62. Confocal microscopy results indicated that E423R enhanced the expression of GFP-LC3, promoted the co-localization of GFP-LC3 with Lyso-Tracker Red, and facilitated the fusion of autophagosomes and lysosomes. Additionally, the expression level of E423R exhibited a positive correlation with the expression level of LC3-Ⅱ in HeLa cells. Furthermore, E423R down-regulated the expression of p-AKT, p-mTOR, and p-ULK1 (Ser757) in the mTOR signaling pathway. [Conclusion] This study demonstrates that the ASFV E423R protein induces complete autophagy via the AKT/mTOR/ULK1 signaling pathway, exhibiting a dose-dependent effect on LC3-Ⅱ expression to a certain degree. These findings provide a foundation for further investigation into the infection and pathogenic mechanisms of ASFV.

[Objective] To improve the fermentation performance by optimizing the fermentation medium and conditions of Bacillus subtilis subsp. spizizenii YC25, thereby providing a theoretical basis for the subsequent industrial development and application of the strain. [Methods] With the spore yield as the indicator, the optimal fermentation conditions were screened by single factor tests. On this basis, the response surface methodology was employed to optimize the fermentation medium. Pot experiments were conducted to evaluate the control effects of the optimized fermentation broth on tobacco black shank and tobacco Fusarium root rot. [Results] The optimum fermentation conditions were as follows: 30 ℃, 180 r/min, an inoculation amount of 2%, a liquid-loading volume of 20%, and fermentation time 36 h. The optimum fermentation medium was as follows: corn starch 15.53 g/L, soybean meal 8.70 g/L, KH₂PO₄ 0.50 g/L, MgSO₄ 0.92 g/L, and CaCO₃ 0.50 g/L. Under these conditions, the spore yield reached 1.423 3×10¹⁰ CFU/mL. The results of pot experiments showed that the control effects of the fermentation broth diluted by 20 times on tobacco black shank and tobacco Fusarium root rot were 77.88% and 62.09%, respectively. [Conclusion] The optimized medium could significantly increase the spore yield of strain YC25, and the disease indexes of tobacco black shank and tobacco Fusarium root rot were significantly reduced after the application of YC25 fermentation broth. This study provides a theoretical basis for the development and application of the biocontrol strain YC25 and the prevention and control of field diseases in the future.

[Objective] To develop a new and rapid fluorescent recombinase-aided amplification (RAA) method for the detection of Enterocytozoon hepatopenaei (EHP), one of the major pathogens restricting the development of the prawn farming industry. [Methods] Multiple pairs of RAA primers were designed based on the highly conserved 18S rRNA gene of EHP, and the primers with the highest amplification efficiency were screened by basic RAA and fluorescent RAA. The reaction system and conditions of fluorescent RAA were then optimized. The optimization results showed that the developed method achieved detection at 37 ℃ within 20 min. Finally, the sensitivity, specificity, precision, stability, and actual sample application of the developed fluorescent RAA method were evaluated. [Results] The fluorescent RAA method had high specificity and was only sensitive to EHP, and it had no response to other pathogens causing mixed infection or secondary infection. Moreover, it had high sensitivity and high precision, with the limit of detection being 5 copies/μL and the coefficient of variation less than 5% for repeat test results. The lyophilized premix of primers and probe demonstrated good stability. It can be stably transported, used, and stored for 10 d at room temperature (25 °C) and 5 d at the high temperature (37 °C) in summer, with the shelf life longer than one year at -20 ℃. Additionally, the detection results of ninety clinical samples of prawn by the developed method were consistent with those obtained by the fluorescence quantitative PCR method recommended by fishery industry standard Code of Diagnosis for Enterocytozoon hepatopenaei Disease (SC/T 7232—2020), with the result coincidence rate of 97.78% (88/90). In addition, the fluorescent RAA method showed the sensitivity of 100.00% (30/30) and the specificity of 96.67% (58/60). [Conclusion] The established fluorescent RAA method has the characteristics of rapid detection, simple operation, high sensitivity, high specificity, high precision, and good stability, providing a technical reference for the field rapid detection of EHP. It has a wide market application prospect in the accurate and rapid detection of aquatic diseases.