Pseudorabies virus (PRV) is a member of the genus Varicellovirus in the Herpesviridae family. It primarily causes pseudorabies characterized by reproductive failure in sows, and neurological and respiratory symptoms in piglets, posing a significant threat to pig production. Vaccination is the most important measure to prevent PRV in pigs. However, due to variations of the virus and its latent infection characteristics, the effectiveness of traditional vaccines is compromised. Consequently, there is an urgent need for new drug preparations to assist vaccine immunization. It has been found that natural plant polysaccharides and small molecules such as flavonoids, phenols, and acids can inhibit PRV infection either by directly blocking the viral infection process or by regulating the immune response. In addition, host antiviral protein type Ⅰ interferon and its downstream interferon-stimulated genes have significant inhibitory effects on PRV infection. Host defense peptides, including antimicrobial peptides and defensins, also show good inhibitory effects on PRV infection. Interestingly, researchers have recently found that extracts and metabolites from bacteria and fungi also exhibit anti-PRV effects, and it is expected that these bacteria and fungi and their products could be applied for the prevention and treatment of viral diseases in the future. This study focused on the recent research progress of natural bioactive molecules against PRV infection, aiming to provide important references for the research and development of anti-PRV infection drugs.

The widely used trypsin is mainly extracted from animal pancreas, which has the disadvantages of limited raw materials, high costs, and low purity. In addition, the autolysis of trypsin affects its stability in storage and application. [Objective] To obtain an anti-autolysis recombinant trypsin by heterologous expression and methylation. [Methods] We employed gene recombination to realize the heterologous expression of porcine trypsinogen in Pichia pastoris. Furthermore, we conducted single factor experiments to investigate and optimize the temperature, pH, and time of enzyme activation and improved the anti-autolysis performance of the recombinant trypsin by methylation. [Results] The engineered strain of P. pastoris expressing trypsinogen was successfully constructed. Under the trypsin concentration of 10 mg/mL, methylation reagent addition of 30 μL, and reaction time of 3 h, the methylated trypsin showed the activity loss of only 22% and the relative activity of 79% after autolysis for 6 h, which was about 3.4 times higher than that of the control, suggesting that the anti-autolysis performance of the recombinant trypsin was greatly improved. [Conclusion] This study successfully produced a novel anti-autolysis recombinant trypsin by heterologous expression and methylation, which can improve the production and application of trypsin in China.

[Objective] Human norovirus (HuNoV) is one of the most common pathogens causing acute gastroenteritis in humans worldwide. Currently, there are no approved vaccines to prevent this disease. This study aimed to prepare virus-like particles (VLPs) of HuNoV in the flashBAC baculovirus expression system, laying a foundation for the development of vaccines against HuNoV. [Methods] After codon optimization, the full-length gene sequence of the VP1 protein of HuNoV GⅡ.4 was synthesized and cloned into the baculovirus pBacPAK9 transfer vector to obtain the recombinant plasmid pBacPAK9-8his-VP1. After enzyme digestion and sequencing, the recombinant plasmid was co-transfected with the linear baculovirus plasmid (Bacmid) into SF9 cells to obtain a recombinant baculovirus carrying the VP1 gene. Hi-Five (HF) cells were infected by the recombinant baculovirus for protein expression, and the expression of VP1 was analyzed by SDS-PAGE and Western blotting. VP1 was purified by Ni-NTA affinity chromatography and identified by SDS-PAGE and Western blotting. The purity of VP1 was examined by high performance liquid chromatography (HPLC). The VLPs were observed by transmission electron microscopy. [Results] A transfer plasmid pBacPAK9-8his-VP1 was constructed, and the VP1 protein, with a molecular weight of approximately 58 kDa, was mainly expressed in the cytoplasm of HF cells. The HPLC results showed that the purity of VP1 was over 99%. The VLPs with a regular shape, uniform sizes, and diameters of 30–40 nm were observed by transmission electron microscopy. [Conclusion] The VLPs of HuNoV GⅡ.4 were prepared with a baculovirus expression system, laying a foundation for the development of HuNoV vaccines.

The aquaculture industry has rapidly expanded in recent years in China, whereas it faces the challenge brought by bacterial diseases. Antibacterial agents have been the primary tools to combat these diseases. However, prolonged and haphazard usage of antibacterial agents in aquaculture has exacerbated antimicrobial resistance and led to severe antimicrobial residues. Considering these challenges, scholars worldwide have been exploring natural alternatives, such as Chinese herbal medicines. Among them, Pithecellobium clypearia stands out due to its antibacterial, antiviral, and anti-inflammatory properties, coupled with its safety and lack of antimicrobial resistance. Nonetheless, the potential of P. clypearia in the prevention and control of aquatic diseases remains underexplored. [Objective] This study evaluated the in vitro inhibitory activity of P. clypearia aqueous extract against aquatic pathogenic bacteria, including an artificially induced antimicrobial-resistant strain. Additionally, we investigated changes in bacterial cell membrane permeability and observed cellular alterations by transmission electron microscopy to elucidate the mechanism of the extract. Our findings are expected to pave the way for developing P. clypearia as an environmentally friendly antibacterial agent, reducing antibacterial agent dependency, and mitigating pathogen resistance in aquaculture. [Methods] We employed the microbroth method to assess the antimicrobial resistance of 107 pathogen strains attacking aquatic animals and analyzed the inhibitory activity of P. clypearia aqueous extract against aquatic pathogenic bacteria, including an artificially induced antimicrobial-resistant bacterial strain. Furthermore, we determined the extracellular K⁺ content and ultrastructural changes in Streptococcus agalactiae and Vibrio parahaemolyticus after treatment with the extract. [Results] The resistance rate of 107 pathogen strains to sulfonamides was as high as 67.29%, and 46.73% of the strains showed multidrug resistance, among which Aeromonas sp. showed the most severe resistance. The aqueous extract (12.50 mg/mL) of P. clypearia exerted inhibitory effects on all the pathogenic bacteria, especially on Aeromonas sp. with the minimum inhibitory concentration (MIC) as low as 0.39 mg/mL. The MICs of the extract were similar for the strains belonging to the same genus but with different antimicrobial resistance characteristics. The aqueous extract of P. clypearia showed stronger inhibitory effect on the artificially induced enrofloxacin-resistant strain of A. schubertii than on the original strain, with the MICs of 0.78 mg/mL on the original strain and 0.20 mg/mL on the resistant strain. In addition, the treatment with P. clypearia aqueous extract significantly increased the extracellular K⁺ concentration, leading to damage to the bacterial cell membrane structure, leakage of intracellular contents, and vacuolation of cytoplasm, which suggested that P. clypearia exerted the antibacterial effect by destroying the bacterial membrane structure. [Conclusion] P. clypearia exerts in vitro inhibitory effects on aquatic pathogenic bacteria and demonstrates great potential for further research and development in the prevention and treatment of bacterial diseases in aquatic animals. The antibacterial mechanism of P. clypearia appears to involve disrupting bacterial cell membranes. The application of P. clypearia in aquaculture promises to reduce antibiotic dependency and pathogen resistance, paving the way for a healthy and sustainable aquaculture industry.

Anaerobic digestion (AD) of biomass waste combined with waste activated sludge (WAS), which is characterized by great stability, low energy consumption, and biogas production, can effectively reduce the volume and improve the dehydration of WAS and thus has been widely applied in methane production. However, the AD process is susceptible to external factors such as microplastics (MPs) or nanoplastics (NPs), which can lead to reduced efficiency or even collapse of AD. The AD system needs the interdependence and interaction of the microbial community to keep stable operation in a dynamic equilibrium state, in which phages play a key role. Phages can not only regulate the structure of the microbial community in the sludge and direct the energy flow but also attach to MPs and NPs with bacteria and archaea for transmission. Nevertheless, the effects of MPs and NPs on such a process were underestimated in previous studies. In this paper, we summarize the research progress in the effects of different types and sizes of MPs and NPs on AD systems, with focus on the ecological connections among microbial communities, especially bacteria, archaea, and phages, in anaerobic systems. Furthermore, we put forward novel viewpoints about the effects of MPs and NPs on microbial communities and make an outlook on the future research directions in this field.

In recent years, antimicrobial peptides (AMPs) are considered alternatives to antibiotics and have received increasing attention. AMPs have a broad antibacterial spectrum and extensive sources and are not prone to drug resistance. At present, AMPs are mainly produced with three methods: extraction from natural sources, chemical synthesis, and microbial expression via genetic engineering. The application of the former two methods is limited due to their complicated processes, high costs, and low yields. Microbial expression via genetic engineering is more economical, scientific, and effective than the above two methods. This article introduces and compares the various expression systems and summarizes the strategies for increasing the heterologous expression levels, with a view to providing theoretical support for large-scale production of AMPs with low costs.

[Objective] To study the role of napF3 in Thermoanaerobacter tengcongensis at different temperatures. [Methods] We constructed ΔnapF3 from T. tengcongensis by homologous recombination and observed the growth of ΔnapF3 at 50 ℃, 60 ℃, 75 ℃, and 80 ℃. Transcriptome sequencing was employed to identify the differentially expressed genes (DEGs) between ΔnapF3 and the wild type (WT) at 75 ℃. real-time PCR was conducted to measure the transcriptional levels of 13 genes and 3 sRNAs in WT and ΔnapF3 at 50 ℃, 60 ℃, 75 ℃, and 80 ℃. [Results] ΔnapF3 was successfully constructed, and it showcased suspended growth at 50 ℃ and 80 ℃ and slow growth at 60 ℃ and 75 ℃. A total of 899 DEGs between WT and ΔnapF3 at 75 ℃ were identified, including 363 genes with up-regulated expression and 536 genes with down-regulated expression. These DEGs were mainly involved in the biosynthesis of valine, leucine and isoleucine, ABC transporters, two-component system, fatty acid synthesis, thiamine metabolism and other pathways. The transcriptional levels of 13 genes and 3 sRNAs related to the thermophilic mechanism changed under specific temperatures. [Conclusion] napF3 plays a role in the thermophilic adaptation of T. tengcongensis.

[Objective] To mine the key enzyme genes associated with spinosad synthesis and the biosynthetic gene clusters (BGCs) in Saccharopolyspora spinosa at different developmental stages by transcriptomics, thus laying the groundwork for the construction of high-yield strains. [Methods] The transcriptomes of S. spinosa during the logarithmic phase (T2-48 h) and the stationary phase (T6-144 h) were compared. The results from qRT-PCR and transcriptome sequencing were mutually validated. Gene ontology (GO) annotation and Kyoto encyclopedia of genes and genomes (KEGG) enrichment were performed for the differentially expressed genes (DEGs). Central carbon metabolism analysis was performed. [Results] The transcriptome sequencing of S. spinosa revealed 2 542 DEGs, including 1 188 genes with significantly up-regulated expression and 1 354 genes with significantly down-regulated expression. GO annotation indicated that the DEGs were primarily involved in carboxylic acid metabolic process, oxoacid metabolic process, organic acid metabolic process, and amino acid metabolic process. KEGG enrichment analysis demonstrated DEGs were mainly involved in pathways such as glycine, serine, and threonine metabolism, oxidative phosphorylation, and arginine biosynthesis. Further analysis identified seven genes related to spinosad biosynthesis. Among them, accB, Pfk, G6PD, and dsdA showed significantly up-regulated expression, while GAPDH, aceE, DLAT involved in the consumption of spinosad precursors, as well as genes in the TCA cycle and arginine biosynthesis, exhibited significantly down-regulated expression. The results of qRT-PCR were consistent with the trends observed in transcriptome sequencing, which revealed 12 upregulated BGCs: BGC2 (43 846 bp), BGC4 (18 330 bp), BGC9 (20 501 bp), BGC18 (62 621 bp), BGC22 (19 626 bp), BGC25 (42 896 bp), BGC26 (40 086 bp), BGC28 (39 392 bp), BGC30 (20 282 bp), BGC31 (53 657 bp), BGC34 (20 787 bp), and BGC35 (40 232 bp). [Conclusion] This study elucidated DEGs in S. spinosa at different developmental stages through transcriptome analysis, and analyzed the biosynthetic pathways and BGCs of spinosad. These findings pave the way for optimizing the spinosad biosynthetic pathways and genetically modifying S. spinosa to enhance the spinosad production in subsequent studies.

Bacterial biofilms are aggregates surrounded by extracellular polymeric substances produced by bacteria, exhibiting significant resistance to antimicrobials and host immune defense mechanisms. As a result, they become a crucial factor in the recalcitrance of bacterial infections. Phages, as a class of viruses capable of specifically infecting and lysing bacteria, have demonstrated immense potential in the prevention and treatment of biofilm-associated infections. This review summarizes the efficacy, in vitro and in vivo research methods, and mechanisms of phages, phage endolysins, and phage-antibiotic combinations in combating biofilm-associated infections. Furthermore, it discusses the prospects and potential obstacles of phages in this field, aiming to give insights into the development of more effective therapeutic agents.

Pullulan is an exopolysaccharide produced by Aureobasidium spp. Despite its widespread biotechnological applications, the mechanisms underlying the biosynthesis and regulation of pullulan remain to be studied. In recent years, researchers have employed molecular biological techniques to elucidate the molecular mechanisms of pullulan synthesis and regulation. The transmembrane protein AmAgs2 is identified as a key enzyme for the synthesis of pullulan, and the cAMP-protein kinase A (cAMP-PKA), target of rapamycin 1 (TORC1), high osmotic glycerol 1 (HOG1), and sucrose nonfermentable 1 (Snf1) signaling pathways are involved in the regulation of pullulan synthesis. We reviewed the research progress in this field, aiming to give insights into the research on the synthesis and regulation mechanisms of fungal extracellular polysaccharide and provide theoretical support for building cell factories with high yields of pullulan.