Objective: Effective therapeutic agents against multidrug-resistant Klebsiella pneumoniae (K. pneumoniae) are urgently needed in clinical practice. We developed a mouse model of pneumonia induced by multidrug-resistant K. pneumoniae to evaluate the efficacy of phage nebulization as a treatment.Methods: A K. pneumoniae-specific phage was isolated from hospital wastewater. The phage one-step growth curve, host spectrum, and complete genome sequencing were used to evaluate the bactericidal efficacy of the phage as a potential antimicrobial agent. Mice were challenged by nasal inhalation with highly virulent and multi-drug resistant K. pneumoniae. Purified phage was obtained by density gradient centrifugation for nebulization therapy in mice. After nebulization, mouse lung tissu was examined for bacterial load, phage titers, and immune-related gene expression levels. Additionally, changes in histopathologic sections of the lungs were compared, and fluorescence-labeled albumin was used to evaluate the integrity of the lung barrier function in mice. Results: We isolated a K. pneumoniae phage, SY201, which belongs to the Podoviridae family. The phage entered the lysis phase within 20 mins of infection and effectively lysed the highly virulent, drug-resistant K. pneumoniae strain HG-KP-3, suggesting its potential as a therapeutic agent. Whole genome sequencing confirmed the phage’s safety profile, showing no virulence genes or integrase loci. Nebulization therapy significantly reduced lung bacterial load by approximately 30-fold compared to nasal drip therapy. Histopathologic analysis indicated restored lung tissue structure and barrier function, with no inflammation or lesions observed. Expression levels of immune-related genes IL-6,IL-1β and TNF-α decreased, indicating effective treatment of pneumonia in mice.Conclusion: The combination of phage therapy and nebulization technique holds promise for clinical application, and offers a superior treatment option for pulmonary infections.

Objective: This study aims to investigate the anti-fibrotic effects of hepcidin during the progression of liver fibrosis and to identify the primary cellular targets involved.Methods: To ensure stable, high levels of hepcidin expression in vivo, a recombinant adeno-associated virus carrying the hepcidin gene (AAV-hepcidin) was constructed and administered to mice by tail vein injection. The transduction efficiency of AAV-hepcidin was verified by detecting green fluorescence in frozen liver tissue sections, while serum hepcidin levels were quantified by enzyme-linked immunosorbent assay (ELISA) six weeks post-injection. To evaluate the anti-fibrotic potential of hepcidin, liver fibrosis was induced in mice by repeated intraperitoneal injections of carbon tetrachloride (CCl₄), a known hepatotoxin. Fibrosis progression was assessed by Masson’s trichrome staining, which reveals collagen deposition in liver sections, and quantitative real-time PCR (qPCR) analysis of liver tissue to measure fibrosis-related gene expression levels. Differential expression of the membrane iron transporter ferroportin (FPN) in primary hepatic stellate cells (p-HSCs) and RAW 264.7 macrophages was assessed using qPCR, Western blotting, and immunofluorescence staining to determine the target-specific effects of hepcidin. Additionally, the influence of hepcidin-conditioned medium on p-HSCs activation under transforming growth factor-β1 (TGF-β1) stimulation and the effects of ferric ammonium citrate (FAC) on markers of ferroptosis and p-HSCs activation were investigated. To evaluate hepcidin’s impact on immune modulation, the effect of hepcidin-conditioned medium on RAW 264.7 macrophage polarization under lipopolysaccharide (LPS) stimulation was examined by analyzing the mRNA and protein expression levels of inflammation-related markers by qPCR and Western blotting. Results: Frozen sections and ELISA results confirmed stable and high hepcidin expression in mouse liver. Compared to controls, hepcidin overexpression was associated with reduced liver fibrosis as evidenced by Masson trichrome staining, and qPCR results indicated lower mRNA levels of fibrosis-related genes in mouse liver tissue. Furthermore, qPCR, Western blotting, and immunofluorescence staining all showed that FPN expression levels were higher in RAW 264.7 macrophages than in p-HSCs. After treatment with hepcidin-conditioned medium and TGF-β1, qPCR and Western blotting results showed no significant differences in the mRNA levels of fibrosis-related genes or the protein expression of fibrosis markers between the hepcidin and control groups. Therefore, hepcidin does not have a significant inhibitory effect on p-HSCs activation. Additionally, after stimulation with FAC, qPCR and Western blotting results indicated that, except for higher mRNA expression of Fth1 and Ftl1 in the FAC group-suggesting that FAC effectively increased intracellular iron levels-there were no significant differences in the mRNA and protein expression levels of other fibrosis-related marker genes and ferroptosis-related marker genes between the FAC and control groups. These results suggest that FAC treatment does not significantly affect p-HSCs activation or ferroptosis markers. After treatment with hepcidin-conditioned medium and LPS, RAW 264.7 cells exhibited decreased expression of inflammation-related genes and proteins compared to controls, indicating that hepcidin effectively suppresses M1 macrophage polarization. Conclusion: Hepcidin exerts a protective effect during the progression of liver fibrosis primarily by modulating the inflammatory microenvironment. This occurs through inhibition of M1 macrophage polarization rather than direct suppression of HSCs activation, suggesting that the hepcidin’s anti-fibrotic role is largely mediated through macrophage modulation, ultimately contributing to a reduction in liver fibrogenesis.

Human lysozyme is a typical representative of c-type lysozymes, and its derived peptides have shown a variety of physiological activities in vitro, such as antimicrobial and antiviral activities. In this study, the structural gene of a “helix-loop-helix” (HLH) antimicrobial peptide derived from the C-terminus of human lysozyme was integrated into an episomal vector, pPICZαA-HLH-pARS, and then transformed into a host strain, Pichia pastoris X33. The positive P. pastoris clone expressing the antimicrobial peptide HLH was successfully obtained. After induction by methanol for 72 h, the yield of HLH peptide reached 58.6 mg/L and the antibacterial activity was 260 U/mL. The secreted recombinant HLH peptide in the supernatant had a molecular weight of 4 kDa, consistent with the theoretical value. Moreover, to further investigate the structure-function relationship of this HLH antimicrobial peptide, the site-saturation mutagenesis libraries of 5 amino acid residues located in the loop region in the middle of two helices of the HLH peptide were constructed, respectively, and the mutants with higher bactericidal activity were screened by the enzyme-cascade fluorescence high-throughput screening method. Three positive antimicrobial peptide mutants 2-1 A10, 2-1 C10, and 2-2 A2 were obtained. This study provides new insights and mutation strategies of the loop region of HLH antimicrobial peptides to enhance their bactericidal efficacy against Gram-negative bacteria.

Objective: To express Tenebrio molitor antimicrobial peptide TmAMP1m in prokaryotic cells, to study its antibacterial activity when used alone or in combination with traditional antibiotics, and to explore the mechanism of synergistic effect between Tenebrio molitor and conventional antibiotics. Methods: The prokaryotic expression vector pET-30a-TmAMP1m was constructed and transformed into Escherichia coli BL21 for heterologous expression. The recombinant protein was identified by SDS-PAGE, the recombinant protein was purified and quantified, the bacteriostatic effect of its single and combined use was determined, and the mechanism of synergistic effect was explored. Results: The target protein TmAMP1m was obtained by prokaryotic expression, and the antimicrobial peptide TmAMP1m with a concentration of 57 μg/mL was obtained after protein purification. The antibacterial spectrum of TmAMP1m was determined, and it was found that it has bacteriostatic effect on Escherichia coli, Staphylococcus aureus, Candida albicans, Pseudomonas aeruginosa, Bacillus subtilis, Propionibacterium acne and other common bacteria, and the minimum inhibitory concentration and minimum bactericidal concentration of TmAMP1m on some bacteria were determined. The combined effect of antimicrobial peptide TmAMP1m with traditional antibiotics such as ampicillin, gentamicin, ciprofloxacin, cefepime, minocycline and polymyxin B was tested, and it was found that it has synergistic effect against some bacteria when combined with some antibiotics. The bacteriostatic combination had an obvious destructive effect on the bacterial cell membrane, resulting in leakage of cell contents and an obvious inhibitory effect on the respiratory chain dehydrogenase. Conclusion: The combination of TmAMP1m and antibiotics has a good bacteriostatic effect, is not easy to produce drug resistance, and strengthens the destruction of bacterial cell membrane and the effect of bacterial respiratory chain.

To quantify the concentration of thrombin-antithrombin III complexes (TAT), we developed and evaluated a chemiluminescent immunoassay (CLIA) for TAT detection. Initially, thrombin (T) and antithrombin (AT) were extracted from plasma and reacted in vitro to form TAT, which served as the immunogen. Through screening, we identified three hybridoma cell lines, 1E9, 16G4, and 1D7, that specifically recognize TAT. After further screening and pairing, biotin-conjugated monoclonal antibody 16G4 was used as the capture antibody and horseradish peroxidase-labled 1D7 was used as the detection antibody, thus establishing a double-antibody sandwich detection system. The optimal procedure for this method is a two-step process with an incubation time of 10 mins per step and a detection limit of 0.6 ng/mL, yielding results in less than 25 mins. The strong correlation between our measurements and those obtained using the Sysmex kit (R² = 0.976 9) showed that the reagents were equivalent for the detection of TAT. In this study, all reagent components were integrated into a single cartridge, and quantitative detection was performed using a fully automated chemiluminescence analyzer for convenience and speed.

Lipid nanoparticles (LNPs), nanoscale delivery vehicles composed of lipid molecules, are distinguished by four major components: ionizable lipids, phospholipids, cholesterol, and lipids conjugated with polyethylene glycol (PEG). These components work in synergy to encapsulate and protect mRNA molecules, ensuring their integrity and facilitating their effective transport throughout the body. The LNP-mRNA delivery mechanism, which utilizes the encapsulation of messenger RNA by lipid nanoparticles, has emerged as a promising therapeutic modality for pulmonary diseases. The emergence and success of COVID-19 vaccines has driven the development and recognition of this technology. This review discusses advances in the application of LNP-mRNA system to treat a spectrum of pulmonary diseases. It includes the underlying design rationale, refinement tactics, experimental results from both in vitro and in vivo studies, and the translational potential into clinical practice. One focus of the discussion is the precision targeting of LNPs, particularly in the context of pulmonary genetic disorders, infectious diseases, and pulmonary malignancies. The review also addresses the existing challenges and future directions for the field, with the goal of providing a scientific foundation and strategic insights for the use of LNP-mRNA technology in pulmonary therapeutics.

Virus-like particles (VLPs) are self-assembled nanoscale particles containing one or more structural proteins of a specific virus, which are highly immunogenic. They resemble natural viruses in structure and conformation, but do not contain viral genomes, making VLPs a safe and effective biological tool. In recent years, VLPs have made remarkable progress in the fields of vaccine development, drug delivery, and gene editing. The article outlines the structural properties of VLPs, the advantages and disadvantages of different VLP expression systems, the purification strategies of VLPs, and the applications of VLPs in vaccines, drug delivery, gene editing, and tumor immunotherapy, with the aim of providing references for the research and application of VLP technology and making it more useful in the biomedical environment.

African swine fever (ASF) is an acute and highly contagious animal disease caused by the African swine fever virus (ASFV), which is extremely lethal, causing huge economic losses to the global swine industry and seriously hindering its healthy development. ASFV is a DNA arbovirus belonging to the Asfarviridae family, and an in-depth study of its antigenic epitopes is essential for the development of detection methods and vaccines. With the continuing discovery of antigenic epitopes, serologic assays based on ELISA and immunochromatographic techniques have been widely used to detect antibodies in swine sera, aiming to assess the status of pigs. By screening and characterizing proteins such as p30, p54, p72, and CD2v that induce specific antibody responses during ASFV infection, studies have defined numerous linear and conformational epitopes using monoclonal antibodies and bioinformatics tools, laying the foundation for the development of diagnostic assays and vaccines. However, the complexity of the viral genome poses a significant challenge to the development of vaccines and antiviral drugs, and no commercialized ASFV vaccines or therapeutic agents are available. Although, researchers have continued to explore multiple vaccine strategies, including subunit vaccines, and have developed some promising vaccine candidates using gene editing technology, more in-depth studies are needed to improve the safety and efficacy of vaccines. Therefore, recent advances in ASFV antigenic epitopes, related antibody detection technologies, and anti-ASF vaccine development are reviewed with the aim of providing references and insights for the development of novel and efficient detection technologies and viral vaccines.

Cancer is a serious threat to human health, and although traditional treatment methods such as chemotherapy and radiotherapy have good therapeutic effects, they also show obvious side effects in clinical practice. Since the publication of American scientist William Coley’s study using Streptococcus pyogenes to alleviate sarcoma, bacteria have been reused as a means of treating tumors because natural bacteria have the ability to self-propagate and quorum sensing, the oxygen concentration gradient within tumor cells ensures the directional migration of bacteria, and the enrichment of nutrients also ensures that bacteria proliferate without being destroyed by the immune system. Bacteria can cause tumor cell death, but at the same time, they can inhibit the host’s immune response and interfere with the body’s immune effect against tumors, so bacteria used for anti-tumor therapy must be genetically engineered, such as deleting virulence factor genes or adding promoters to the bacterial cell walls to ensure that they can only reach lethal conditions under hypoxic conditions. Bacteria can also be used as drug delivery vehicles to target tumors for efficient and precise drug delivery. This article reviews bacteria and their application as delivery vectors in anti-tumor therapy, introduces in detail the protocol of bacterial genetic engineering to reduce autotoxicity and the combination application of bacterial vectors with other anti-tumor therapies, and provides an optimized, safer and more efficient treatment option for possible future research.

Metabolic reprogramming, an emerging hallmark of malignant tumors, is the mechanism by which tumor cells obtain energy to promote their initiation, development, and metastasis by altering their metabolic pattern. Exosomes are small extracellular vesicles with a lipid bilayer membrane structure that are secreted by many types of cells. They can transmit information between cells through their contents, including proteins, nucleic acids and other bioactive molecules, involved in regulating the biological functions of cells. MicroRNAs (miRNAs) are short non-coding RNAs of approximately 19 to 25 nucleotides in length. miRNAs have been shown to mediate cell-to-cell communication via exosome delivery and play an important regulatory role in cellular metabolic reprogramming. In this paper, we have summarized the effects of exosomal miRNAs on the metabolic reprogramming of tumor cells and other cells in their microenvironment, hoping to provide new ideas for the study of exosomal miRNAs in regulating tumor metabolism.

African swine fever (ASF), an acute, hemorrhagic, and fulminating infectious disease caused by the African swine fever virus (ASFV), causes an extremely high mortality rate, as high as 100%, and thus has a severe impact on the livestock industry. Currently, ASFV has spread to several regions, including Africa, Europe, and Asia. However, vaccine research and development has not yet achieved a significant breakthrough, mainly due to its complicated genome structure and special immune evasion mechanism. In this paper, recent progress in ASFV research has been comprehensively reviewed. Research into the mechanism of ASFV immune evasion has further elucidated the specific ways in which ASFV regulates the host immune system through multiple proteins. The mechanism, by which ASFV inhibits interferon production by interfering with key signaling pathways such as cGAS-STING, RIG-I-MAVS, and JAK-STAT, ultimately weakening the host’s antiviral ability, has been elucidated. Furthermore, the interaction mechanisms associated with various immune processes,, including inflammatory response regulation, apoptosis, pyroptosis, and autophagy by various proteins encoded by ASFV are being studied in detail. This research will contribute to a better understanding of the interaction between ASFV and host immunity. In terms of future research directions, there are some points that need more attention. On the one hand, the function of unknown ASFV genes and their interaction mechanisms in immune evasion should be continuously explored with the aid of continuously updated biotechnologies in order to find out more potential targets for broad-spectrum vaccines and drugs. On the other hand, it is crucial to significantly increase efforts in vaccine research and development to develop more precise and effective vaccines. Moreover, it is indispensable to strengthen the comprehensive prevention and control measures for ASF, such as implementing on prevention and control regionalization for ASF, to realize the effective control to ASF in the end.

There are more than 200 different HMOs, of which 6 HMOs are well studied, including 2'-fucosyllactose (2' -FL), 3-fucosyllactose (3-FL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), α-2,6-sialyllactose (6'-SL) and α-2,3-sialyllactose (3' -SL), which have been approved as food safety ingredients in Europe and America, and their nutritional necessity and safety are widely recognized. However, most HMOs are unique to breast milk and are difficult to separate. The limited availability of HMOs hinders their use in the food and medical applications. So far, there have been many methods to extract or synthesize HMOs, among which the chemical method has many steps and low yield, and needs to use various organic reagents, causing environmental pollution. Whole-cell synthesis method also faces many challenges related to cost, production efficiency and safety. This paper summarizes the in vitro enzymatic synthesis of HMOs, which are clearly recognized as safe components added to food, points out the advantages and disadvantages of biosynthetic means, and discusses the future direction of its development. Currently, there is still a gap between laboratory-scale synthesis of HMOs and cost-effective industrial-scale production. Despite the challenges in this field, with the rapid development of computer technology, molecular biology, chemistry and other fields, the biological enzyme synthesis of HMOs has promising prospects.

Melatonin, as an important hormone in mammals, not only regulates circadian rhythms and promotes sleep, but also plays a significant role in antioxidation and anti-inflammation. With the increasing demand for melatonin, traditional production methods are no longer sufficient to meet people’s needs. Microbial heterologous synthesis of melatonin has received widespread attention due to its low cost and sustainability. Although the synthetic pathways of melatonin vary in different organisms, fruitful results have been achieved in the microbial synthesis of melatonin through metabolic engineering optimization, as well as enzyme research and characterization. A review of the strategies and synthetic pathways for microbial synthesis of melatonin in recent years has been provided, highlighting the existing bottlenecks and providing effective references for increasing subsequent yields and achieving industrial-scale production.

Esterases are important enzymes commercialized worldwide, and one of the most interesting and unexplored sources is archaeal esterases. Archaeal esterases typically exhibit a wide range of pH, temperature, and substrate specificities, varying degrees of tolerance to metal ions, solvents, and salts, as well as specific regioselectivities, chemoselectivities and stereoselectivities, which make them important industrial biocatalysts with a great potential for applications in the fields of pharmaceuticals, chemicals, environmental bioremediation, and food processing. However, only a small fraction of archaeal esterases have been characterized to date, and their further exploration and applications have been hampered by low yields and poor enzyme stability. Here, we systematically summarize the distribution, enzymatic characterization and application prospects of two major archaeal esterases, namely, halophilic archaeal esterases and hyperthermophilic archaeal esterases, review the related technologies that can significantly enhance the yield and performance of archaeal esterases including solid-state fermentation, genetic engineering strategy, immobilization technology, and nanotechnology, and discusss the prospects for further development and applications of archaeal esterases.

Biosafety laboratories are critical infrastructure and important platforms for conducting biosafety research. From the perspective of the history of science and technology, this study systematically reviews and analyzes the development of China’s biosafety laboratories and their role in overall national security. The article reviews the construction and development of biosafety laboratories at different historical stages, emphasizing their key role in scientific research and technological progress. In addition, the article examines the construction process of relevant laws, regulations, systems, and standards. By studying the development of China’s biosafety laboratories, this paper clarifies their importance to national security, infectious disease prevention and control, and other fields, and provides important scientific support for promoting the development of China’s biosafety laboratories.

Bioplastics have attracted worldwide attention as an effective way to address the problem of plastic pollution. This study examines the development trend of global bioplastics from multiple perspectives, including national strategy, research & development status, competitive landscape, and application scenarios. The study found that the number of global bioplastics patents is growing steadily, with China becoming the leading country in terms of bioplastics patents, while the United States and Japan are leading in terms of high-value patents. Foreign patent applications are mainly filed by companies, while Chinese patent applications are mainly filed by research institutes and universities. Overall, the bioplastics industry is in a period of rapid development, with huge market potential and expanding applications due to technological advances and policy support. However, there is still a significant gap in production compared with traditional plastics, and future development needs breakthroughs to increase production, reduce costs, and enhance market competitiveness.