Objective: The MDCK-STAT1-KO engineered cell line has been shown to efficiently produce H1N1 influenza virus; however, its application in influenza vaccine production is limited by the inherent characteristics of adherent culture, such as scale-up challenges and serum dependence. Single cell suspension culture is preferred for vaccine production due to its ease of scale-up and operation. Nevertheless, the viral production capabilities and stress response characteristics of the MDCK-STAT1-KO cell line under suspension culture conditions remain unclear, which restricts its further application and research. Methods: The growth characteristics of suspension MDCK-STAT1-KO cells (susMDCK-STAT1^-) were assessed using both passage and batch culture methods. The viral production capacity of these cells was assessed under conventional infection conditions (MOI = 0.001). Key infection parameters, including MOI, nutrient conditions, and viral strain type, were modified to evaluate their effects on viral yield from susMDCK-STAT1^- cells. Synchronous infection experiments were performed to investigate cell apoptosis levels and immune stress responses following viral infection. Results: The maximum hemagglutinin (HA) titer and maximum specific virus yield (Svy) produced by susMDCK-STAT1^- cells were (12.40 ± 0.11) log₂HAU/100 μL and (15.86 ± 1.34) × 10³ virions/cell, respectively, indicating a high production level for H1N1 virus. Moreover, alterations in key process parameters did not affect the cell line’s ability to efficiently produce influenza virus. Additionally, susMDCK-STAT1^- cells exhibited certain anti-apoptotic properties and an overall lower level of immune defense, compared to the wild-type MDCK suspension cell line (susMDCK), facilitating virus invasion and replication and rapid virus release. Conclusions: susMDCK-STAT1^- cells are capable of efficiently producing influenza virus, and their anti-apoptotic properties and low intracellular immune response levels are critical factors contributing to the highly efficient virus production process. This cell line shows promise for future applications in the practical production of influenza virus vaccines, enhancing the overall production levels of influenza viruses.

Objective: CD44 (cluster of differentiation 44) is a popular target for tumor targeted therapy. It, especially its variant subtype (CD44 variant forms, CD44v), has the key mediating function in promoting tumor proliferation, invasion and metastasis and drug resistance and enhancing tumor stemness. Targeting CD44v, CD44v nanobodies with high affinity to and high expression on the surface of hepatoma cells were screened, opening a new avenue for the development of CD44v-positive tumor-targeted antibody drugs. Method: Camels were immunized with the CD44v-Fc fusion protein to generate a high-quality phage display library of CD44v-specific nanobodies; Phage display technology was employed for solid-phase affinity panning to enrich CD44v-specific nanobody phages; Positive clones were screened using soluble-phase enzyme-linked immunosorbent assay (PE-ELISA) to identify nanobodies with high binding affinity; Selected nanobodies, characterized by distinct complementarity-determining region 3 (CDR3) sequences, were prokaryotically expressed and purified to high purity. Results: A high-quality CD44v nanophage library with a capacity of 2.8×10⁹ CFU was constructed. After preliminary screening and sequencing by PE-ELISA, three nanobodies targeting CD44v were obtained, and three high-purity nanobodies were obtained after prokaryotic purification in WK6. One of the three strains showed good binding activity with the CD44v antigen and hepatocellular carcinoma cells. Conclusions: This study provides promising candidate nanobodies for the targeted therapy of CD44v-positive tumors, not only broadening the development pathways for CD44v-based antibody drugs but also offering critical technical support and experimental foundations for the future screening and optimization of additional CD44v-targeting nanobodies.

Objective: To isolate and characterize Escherichia coli strains and associated phages from cattle feces. Methods: MacConkey medium was used to isolate E. coli strains. The microbroth dilution method was used to analyze antibiotic susceptibility, the double-layer plating method was used to isolate and characterize phages, and the spotting assay was used to determine host range. Stability analysis was used to evaluate the effects of the environmental stressors, sequence analysis was used for genome annotation, and comparative genomics was used for phage classification. A transposon insertion library was used to identifiy the phage receptor. Results: E. coli strain EX3-6 and its associated phage phiP4 were isolated from cattle feces. Antibiotic analysis revealed that strain EX3-6 was resistant to 4 antibiotics. Host range analysis showed that phiP4 can only lyse strains DH5α and EX3-6. Thermal stability assay showed that approximately 90.12% of phiP4 survived after treatment at 50℃. The pH stability assay showed that about 73.45% and 69.07% of phiP4 survived after treatment at pH 4.0 and 12.0, respectively. The effect of sodium cholate was also evaluated, and showed that approximately 59.35% survived when exposed to 400 μg/mL. Analysis of the genome sequence and the genomic termini revealed that the phiP4 genome is a circular dsDNA of 106 340 bp, encoding 153 predicted protein-coding regions, 65 of which have putative functions. The phage also encodes 26 tRNA genes. Comparative genomic analysis revealed that phiP4 was an unidentified member of the genus Tequintavirus in the subfamily Markadamsvirinae. Outer membrane protein BtuB was identified as the receptor of phage phiP4 for infection. Conclusions: In our work, phage phiP4 was isolated from cattle feces and is highly resistant to the environmental stressors, suggesting that the biocharacteristics of phiP4 may be helpful in improving the survival of phage phiP4 in its preparation, transportation, storage, and applications. Phage phiP4 may be a potential antimicrobial candidate for the prevention and control of E. coli infections in livestock industry.

Diphtheria toxin mutant protein (Cross-reacting material 197, CRM197) is a commonly used polysaccharide conjugated vaccine vector. however, it tends to be expressed as inclusion bodies in E. coli system. To improve the low yield and tedious operation of CRM197, we designed a strategy to enhance the solubility of the recombinant protein. The SUMO tag tandem strategy was used to improve the solubility of recombinant CRM197 in an Origami B (DE3) E. coli host, resulting in a significant increase in the percentage of soluble recombinant CRM197 protein in the supernatant, exceeding 95%. Moreover, the accessibility of the CRM197 protein was simplified, after two-step Ni affinity chromatography and one-step enzyme removal to remove the fusion tag, CRM197 protein can be efficiently obtained, with a purity of more than 98% and a yield of more than 80%. The spatial structure of the purified CRM197 was confirmed to be correctly folded through the use of circular dichroism spectroscopy and fluorescence emission spectroscopy. The results of antigenicity ELISA showed that the produced CRM197 protein was antigenically equivalent to the CRM197 standard protein. The outcomes of this research indicate that the double SUMO tag has the capacity to facilitate the efficient and soluble expression of recombinant CRM197 in Escherichia coli. Furthermore, it has established a rapid purification system to obtain CRM197 protein with better immunogenicity, which provides a new idea to improve the design of soluble expression of recombinant proteins in the prokaryotic expression system.

S-Adenosyl-L-methionine (SAM) plays a crucial role in numerous physiological processes and has a wide range of applications. Whole-cell catalytic production of SAM based on methionine adenosyltransferase (MAT) offers numerous advantages; however, the low enzyme activity of MAT is a significant limitation for the implementation of biocatalytic production of SAM. In this study, ScMAT from the yeast Saccharomyces cerevisiae was selected for heterologous expression in the bacterium Escherichia coli, and the screening of expression vectors and optimization of induction conditions were performed. On this basis, ScMAT was modelled and evaluated using AlaphFold 2 and SWISS-MODEL, respectively, and the most suitable model was selected for molecular docking of ScMAT to L-methionine (L-Met). To investigate the impact of active pocket residues, alanine scanning was employed to identify optimal mutation sites. The N371R mutant, which exhibits enhanced catalytic activity and stability, was obtained by targeted and saturation mutagenesis. The specific enzyme activity of the mutant was found to be 1.21 U/mg, representing a 384% increase over the wild-type (WT) enzyme, and the half-life at 65℃ was approximately 15.07 h, a 40% improvement over the WT. Subsequently, the whole-cell catalysis process of the mutant was optimized by selecting the optimal medium components, controlling the reaction temperature at 60℃, pH 9.0, and adding 20 mmol/L ATP, 40 mmol/L L-Met, 60 mmol/L K⁺, and 60 mmol/L Mg²⁺. Whole-cell catalysis was performed for 2 h, resulting in a SAM concentration of 2 340.87 mg/L. The present study provides a solid foundation for theoretical and applied research on SAM.

Nicotine, the primary psychoactive component of tobacco, acts as a cholinergic agonist by binding to nicotinic acetylcholine receptors (nAChRs), thereby activating the cholinergic system and affecting the central nervous system (CNS). While traditional research has focused primarily on the role of neuronal nAChRs in mediating the neurobiological effects of nicotine, recent studies have highlighted the essential role of glial cell nAChRs in neuroprotection and functional regulation. This review systematically summarizes the neuroprotective mechanisms of nicotine, including its ability to inhibit Aβ deposition, modulate inflammatory responses, and regulate apoptosis through the activation of glial cell nAChRs, emphasizing its potential therapeutic applications in Alzheimer’s disease and Parkinson’s disease. Additionally, nicotine regulates synaptic plasticity and dopamine release via glial cell nAChRs, contributing to synaptic remodeling and the development and maintenance of addictive behaviors. These findings deepen our understanding of the functional role of glial cell nAChRs in mediating the effects of nicotine and provide novel insights and potential therapeutic targets for the treatment of neurodegenerative diseases and nicotine addiction.

As an important part of the biotechnology industry, the fermentation industry occupies an important position in China’s industrial structure. Under industrial fermentation conditions, the harsh environment and toxic substrates or metabolites expose microorganisms to severe osmosis, oxidation and other stresses, which seriously affect cell growth and target product synthesis, resulting in low fermentation yield and production efficiency. Exogenous additives can improve the adaptability of microorganisms to poor conditions, and the operation is simple, the cost is low, and the effect is fast. In this paper, common exogenous additives in the fermentation process, including trehalose, fatty acids, short peptides, amino acids, antioxidants and surfactants, were summarized, and the principles and application scenarios of the above-mentioned exogenous additives for improving cell stress resistance were discussed in detail, in order to provide new ideas and technical support for improving the stress resistance in the fermentation process.

The global burden of cancer is mounting, and existing treatment modalities such as surgery, chemotherapy, and radiotherapy face significant challenges in dealing with multiple metastatic tumor sites and tumor multidrug resistance. Biomimetic mineralization, a method of synthesizing minerals or organic-inorganic composites by mimicking the mineralization process in living organisms and utilizing the biomineralization mechanism, has been demonstrated to have great potential for external stress resistance, vaccine improvement, bone regeneration, energy production, and biosensing. Biomimetic mineralized nanomaterials are widely used in tumor therapy due to their targeted drug delivery, physical barrier, excellent biocompatibility, and intrinsic therapeutic properties. This article reviews the biomedical applications of common biomimetic mineralized nanomaterials and the potential correlation between tumor mineralization and tumor growth and prognosis. It focuses on the recent progress of biomimetic mineralized nanomaterials-mediated tumor diagnosis and therapy, and discusses the challenges and future research and development directions in tumor therapy to further expand the applications of biomimetic mineralization in tumor diagnosis and therapy.

Antimicrobial peptides (AMPs) are a class of naturally occurring small molecule peptides that play a pivotal role in the innate immune defense mechanism of organisms. In addition to their potent bactericidal, fungicidal, and protozoacidal activities, some of them also exhibit remarkable cytotoxic effects against tumor cells. Given their broad spectrum of antimicrobial activity and potential medical applications, AMPs hold great promise for the research and development of novel antimicrobial drugs. Marine invertebrates have evolved diverse immune defense systems to adapt to the complex and variable marine environment in the absence of specific immune mechanisms. AMPs, as innate immune defense factors, are a crucial component of the non-specific immune defense mechanism in this group of organisms and represent one of the most important sources for the screening and development of novel antimicrobial drugs. This paper reviews the physiological functions and mechanisms of action of AMPs and summarizes the representative marine invertebrate AMPs reported to date, their functions, and methods of preparation The structure-activity relationships and evolutionary mechanisms of marine invertebrate AMPs are also presented. This paper lays the foundation for the screening, research and application of this class of compounds.

Siderophores are low molecular weight compounds produced by microorganisms that have a high affinity for iron. They help microorganisms grow under iron-limited conditions. Different microorganisms live in different environments. In order to adapt to specific environmental conditions, microorganisms produce siderophores with different structures and properties. In recent years, many kinds of siderophores from different microorganisms have been thoroughly studied, and the formation of siderophores has been analyzed from the perspective of biosynthesis mechanism, and some siderophores with unique structures from specific microorganisms have been modified and applied. Desferrioxamine is the major siderophore produced by Streptomyces. Since there are many types of siderophores, the desferrioxamine produced by Streptomyces was selected for a detailed introduction. Here, the biosynthetic pathway, classification and identification of siderophores and their applications in different fields are reviewed, and the future development prospects are discussed, hoping to provide help for the research of siderophores.

From the perspective of the history of science and technology, this paper reviews in detail how the high-level biosafety laboratory has gradually developed since the establishment of China’s first biosafety level 3 laboratory in 1987, and how to respond to national security needs and global biosafety challenges. After 2004, the national high-level biosafety laboratory construction plan promoted the integration and improvement of regulations and standards, and greatly enhanced the ability of China’s high-level biosafety laboratory to deal with the threat of major infectious diseases. This paper systematically reviews the development process of China’s high-level biosafety laboratories at different historical stages and discusses the evolution process of high-level biosafety laboratories in the construction of laws, regulations, systems, and standards. By reviewing the development of China’s high-level biosafety laboratory, its development context was clarified. The key significance of a high-level biosafety laboratory in national security, effective prevention and control of major infectious diseases, and promotion of scientific research was demonstrated, which provided an important scientific basis and support to further promote the construction of China’s high-level biosafety laboratories.