Objective: The SARS-CoV-2 virus is the pathogen responsible for the pandemic disease known as COVID-19. Studying its infection mechanisms is crucial to understanding the viral pathogenesis and identifying potential therapeutic targets. Research indicates that various conditions, such as receptor-binding affinity, hypoxic environments, and viral replication efficiency, may play a significant role in regulating viral infectivity. This study investigates the impact of hypoxic conditions on SARS-CoV-2 infectivity and identifies the related genes that influence viral infection. Methods: A cell model of viral infection under hypoxic conditions was established. A dual-luciferase reporter assay was used to measure luciferase intensity, determining the regulatory effect of hypoxia on viral infectivity. A549 and H1299 cells that stably express ACE2 were infected with the viral supernatant. Transcriptomic and proteomic analyses were performed, and differentially expressed genes were subjected to gene annotation and signaling pathway analysis to identify genes and pathways affecting viral infection. Further investigation was conducted into the influence of these genes on viral infection under hypoxic conditions. Results: (1) Different oxygen concentrations variably affected viral infectivity. Infectivity increased under hypoxic conditions of 10% and 5% O2, but decreased under 1% O2. (2) Under 1% O2 hypoxia, integrated transcriptomic and proteomic analysis revealed 279 differentially expressed proteins, involving the ribosome pathway and related genes, such as RPS19 and RPL28. (3) Measurements of cellular luciferase activity under normoxia and 1% O2 hypoxia revealed that knockdown of ribosome pathway-related genes inhibited viral infectivity under hypoxic conditions, whereas overexpression enhanced it. Conclusion: Under 1% O2 hypoxia, viral infectivity can be inhibited by regulating ribosome-related proteins and gene expression.
Human mesenchymal stem cells (hMSCs) are a type of adult stem cells with multipotent differentiation potential and robust secretory capabilities, which have been widely used in tissue repair and regenerative medicine research. Enhancing the pro-angiogenic capacity of hMSCs has become a key research focus for their application in treating ischemic diseases. This study aims to develop a novel fusion protein substrate based on neural cadherin (N-cadherin) for the routine culture of hMSCs, and to investigate its effects on hMSC adhesion and proliferation, with the goal of further studying its regulatory role in the pro-angiogenic function of hMSCs. The recombinant fusion protein N-cad1-2-Fc was produced by genetically fusing the N-cadherin extracellular domains EC1-EC2 to the Fc fragment of immunoglobulin G (IgG). The adhesion mechanisms of hMSCs on the N-cad1-2-Fc substrate were analyzed using calcium ion chelation and antibody blocking assays. The effects of N-cad1-2-Fc on hMSC adhesion and proliferation were evaluated via cell adhesion and cell proliferation assays, respectively. qPCR and Western blot analysis were employed to examine the regulatory effect of N-cad1-2-Fc on endogenous N-cadherin expression in hMSCs. Transcriptome sequencing was performed to screen for differentially expressed genes (DEGs) and enriched pathways in hMSCs cultured on N-cad1-2-Fc fusion protein-coated substrates. The pro-angiogenic potential of N-cad1-2-Fc-treated hMSCs was evaluated using endothelial cell wound healing and tube formation assays. The results demonstrated that N-cad1-2-Fc could be stably immobilized on culture plate surfaces and promoted hMSC adhesion and proliferation by activating N-cadherin-mediated downstream signaling pathways. As a novel culture matrix, N-cad1-2-Fc not only mediated hMSC adhesion and proliferation but also significantly upregulated endogenous N-cadherin expression and activated signaling pathways associated with cell proliferation and hypoxic adaptation. This study establishes an experimental basis for the directional regulation of hMSC proliferation and their subsequent applications in regenerative medicine.
Objective: To improve the targeted delivery of chemotherapeutic drug paclitaxel (PTX) to tumors, cancer cell membrane-biomimetic poly (lactic-co-glycolic acid) (PLGA) nanorods were developed. The study aims to evaluate how membrane camouflage and nanoparticle shape influence tumor targeting and in vivo anti-tumor performance, thereby providing experimental evidence for the design and optimization of biomimetic nanodrug delivery systems. Methods: Two types of PLGA nanorods with different aspect ratios (AR2 and AR4) were prepared. Then, CT26 cancer cell membranes were used to coat the nanorods to construct membrane-coated nanorods (MN-AR2 and MN-AR4). The optimal membrane coating ratio was determined by Zeta potential measurement, and particle morphology was observed using transmission electron microscopy (TEM). SDS-PAGE and Western blot analysis were used to analyze the retention of membrane proteins on the coated nanoparticles. Cellular uptake efficiency was assessed in CT26 cells using confocal laser scanning microscopy (CLSM) and flow cytometry. The internalization mechanism of membrane-coated nanorods by tumor cells was also investigated. The tumor-targeting ability of nanoparticles was investigated in mice by in vivo fluorescence imaging. The in vivo anti-tumor activity and biosafety of the nanoparticles were evaluated in CT26 tumor-bearing mice. Results: PTX-loaded nanorods with aspect ratios of 2 and 4 (AR2 and AR4) were successfully fabricated. SDS-PAGE and Western blot analysis confirmed the retention of various membrane proteins, including E-cadherin, a specific membrane protein that mediates effective homotypic tumor targeting. Consequently, CLSM and flow cytometry revealed significantly enhanced CT26 cellular uptake of membrane-coated nanoparticles compared to uncoated ones. Further in vivo experiments demonstrated the superior tumor-targeting and anti-tumor effects of MN-AR4. These effects were mediated by the synergistic effects of membrane camouflage and shape. These biomimetic nanorods, which are constructed from cancer cell membranes, also exhibited good biosafety. Conclusion: This study systematically investigated the impact of cancer cell membrane biomimetic coating and shape regulation with aspect ratio on the tumor-targeting efficiency and anti-tumor performance of PLGA nanorods. The MN-AR4 biomimetic nanoplatform shows promising potential in tumor-targeted drug delivery and provides theoretical and experimental support for the development of non-spherical biomimetic nanodrug delivery systems.
Objective: The prevalence of multidrug-resistant Acinetobacter baumannii (A. baumannii) poses a serious public health challenge. This study aims to isolate and characterize a lytic bacteriophage targeting drug-resistant A. baumannii, providing a reserve resource for phage therapy. Methods: The bacteriophage HHU4 was isolated from hospital sewage using clinically drug-resistant A. baumannii Ab3 as its host. The morphology, growth characteristics, stability, genetic functions, phylogenetic relationships, lytic spectrum, and in vivo therapeutic efficacy were investigated. Results: Transmission electron microscopy revealed that HHU4 has an icosahedral head measuring (52.89 ± 2.30) nm in diameter and a tail measuring (159.42 ± 2.40) nm. The optimal multiplicity of infection (MOI) for HHU4 was determined to be 0.01. It exhibited a latent period of 20 min, a lytic period of 60 min, and a burst size of (185±23)PFU/cell. HHU4 maintained stable infectivity without a reduction in titer after incubation for 2 h across temperatures ranging from 4℃ to 50℃ and pH values from 6.0 to 10.0. The genome of HHU4 is 51,384 base pairs (bp) long with a 38.3% GC content, encoding 66 open reading frames (ORFs) without any virulence or antibiotic resistance-related genes. A phylogenetic analysis revealed a close relationship between HHU4 and the Acinetobacter phage MD-2021a. Treating Ab3-infected Galleria mellonella larvae with HHU4 (MOI = 1) increased the survival rate of the larvae by 90% compared to the untreated control group. Conclusion: The bacteriophage HHU4, isolated from hospital sewage, demonstrates therapeutic potential against infections caused by drug-resistant A. baumannii, which enriches the phage library specific to this pathogen.
Objective: Addressing the dual challenges of the high production cost of bacterial cellulose (BC) and the significant environmental impact of discarded tobacco stems, we explored the feasibility of producing BC through fermentation with Acetobacter xylinum using tobacco stem extract as a substrate. We also examined the antibacterial activity of the resulting silver-loaded nanomaterials. Methods: The Plackett-Burman design and orthogonal experiments were employed to establish a “low-nitrogen, elevated-pH” regulatory strategy involving 1.5 g/L of citric acid, a pH of 5.5, and 0.5 g/L of (NH4)2SO4. This strategy effectively alleviated the salt stress and acid inhibition that the tobacco waste extract imposed on bacterial growth. Results: Under optimized conditions, the BC yield reached 2.40 g/L, and the product fully retained the natural type I crystalline cellulose structure and three-dimensional nanonetwork. Furthermore, the BC-AgNPs composites were formed through in situ chemical reduction, resulting in an 8.84 wt% silver loading. This composite exhibited significant antibacterial activity against both Staphylococcus aureus and Escherichia coli, demonstrating excellent long-term, sustained-release characteristics. Conclusion Waste tobacco stem extract serves as an efficient, cost-effective alternative substrate for BC fermentation. Precise regulation of nitrogen source concentration and initial pH can effectively overcome substrate biotoxicity. The prepared BC-AgNPs composites possess superior structural stability and long-term antibacterial functionality, providing technical support for the valorization of tobacco waste.
Objective: Hirudin (HN) is a natural anticoagulant peptide derived from the salivary glands of medicinal leeches. In order to obtain a recombinant hirudin that more closely resembles the physiological processing pathway and to establish a reproducible, scalable process for evaluating the expression and function of mammalian cells, the pcDNA3.1-HN-3FLAG hirudin mammalian expression vector was constructed for transient expression and systematic identification in HEK293T cells. Methods: RT-qPCR was used to detect the transcription levels, and Western blot analysis was used to detect protein expression in cell lysates and concentrated culture supernatants. Immunological ELISA was used to determine the amount of recombinant hirudin in the culture supernatant, functional ELISA was used to assess its functional potency, and the inhibitory effect on thrombin was further evaluated using the Chromozym TH chromogenic substrate assay. Meanwhile, the half-maximal inhibitory concentration (IC50) was calculated based on the dose-response relationship of the residual enzyme activity. Results: The constructed expression vector efficiently transcribed the hirudin gene in HEK293T cells, increasing mRNA expression by about 5×104 times compared with the control group. Specific bands were detected by Western blot analysis in both cell lysates and concentrated culture supernatants. Immunological ELISA showed that the concentration of recombinant hirudin in the culture supernatant was 84.6 ng/mL, while functional ELISA indicated that its potency in the culture supernatant was 148.3 U/mL. The results of the chromogenic substrate method showed that the culture supernatant had a clear and quantifiable inhibitory effect on thrombin, with a translated potency of about 47.85 ATU/mL and a half inhibitory concentration of about 49.52 ATU/mL. These results suggest that the system can quantitatively characterize recombinant hirudin products based on their functional activity. Conclusion: pcDNA3.1-HN-3FLAG can achieve effective expression of recombinant hirudin in HEK293T cells and produce functional products with thrombin inhibitory activity. The established evaluation process realizes the continuous quantification of recombinant hirudin from molecular expression to functional strength, which provides a technical basis for subsequent optimization of the expression system and further functional evaluation.
Cell-free protein synthesis (CFPS) is a technology that achieves efficient synthesis of target proteins by reconstituting the transcription and translation machinery in an in vitro environment. Compared with conventional cell-based expression systems, CFPS bypasses cell membrane barriers and cellular physiological constraints, and features an open reaction architecture, programmable control, short development cycles, high-throughput compatibility, and integration with automated platforms. This review summarizes the fundamental characteristics and application scope of CFPS systems derived from diverse biological sources. We compare prokaryotic and eukaryotic chassis in terms of productivity, cost, post-translational modification capacity, and suitability for complex protein expression. Particular emphasis is placed on Escherichia coli-based systems, including energy regeneration, extract preparation, template protection, protein maturation, and reaction stability. We discuss frontier optimization strategies involving inorganic and organic factors, physical regulation, and material-based immobilization in more detail. In the context of intelligent biomanufacturing, we emphasize the integration of CFPS with microfluidics, automation, machine learning, and AI-enabled protein design for directed evolution, pathway prototyping, point-of-care diagnostics, genetic circuit engineering, and synthetic cell construction. By connecting basic principles, process optimization, and data-driven applications, this review provides a reference for the continued improvement and intelligent deployment of CFPS technologies.
Probiotics provide humans with health benefits by modulating the gut microbiota, enhancing intestinal barrier function, and producing beneficial metabolites. In recent years, probiotic-based functional preparations have shown great potential in the biomedical field. However, delivering probiotics faces significant challenges due to the extreme pH levels, digestive enzymes, and bile acids found in the gastrointestinal tract. Traditional encapsulation techniques fail to provide precise protection for individual probiotic cells, limiting the efficacy of probiotic therapies. Currently, engineering modifications of probiotics, encompassing techniques such as layer-by-layer self-assembly, biomineralization, electrostatic adsorption, and genetic engineering, can protect and functionalize probiotics, enhancing their viability in complex environments both inside and outside the human body. Bacterial engineering modifications introduce new functions to probiotics, which substantially improves their environmental resistance in the digestive tract and promotes effective colonization at target sites. This review summarizes the challenges that probiotics face during delivery and colonization, and describes probiotic functionalization techniques and encapsulation strategies. It also discusses the benefits and limitations of probiotic engineering and suggests future research directions. The goal is to provide novel insights and ideas for developimg multifunctional engineered probiotics.
Virus-like particles (VLPs), which are non-infectious nanoparticles that self-assemble from viral structural proteins, have emerged as a pivotal platform for developing next-generation vaccines. Their capacity to effectively mimic native viral conformations and elicit robust immune responses underscores their broad prospects, particularly in the prevention and control of emerging infectious diseases and malignancies. Mammalian cell expression systems, capable of ensuring precise protein folding and authentic post-translational modifications, represent an ideal platform for the production of high-quality VLPs. Nevertheless, this system encounters significant engineering challenges, including low efficiency in multi-protein assembly, complexities arising from glycosylation, limited budding efficiency, and difficulties in separating VLPs from host-derived exosomes. This review systematically summarizes the core technical issues associated with VLP production in mammalian cells. Spanning the spectrum from basic research to engineering applications, it explores strategies to optimize VLP structure, assembly, and production via molecular engineering. The objective is to provide a reference for preparing VLPs that are characterized by a high yield, quality, and consistency, thereby facilitating their translational application in vaccines and drug delivery.
Respiratory viruses pose a continuous threat to global public health due to their rapid transmission, swift mutation, and strong immune evasion. Live attenuated vaccines (LAVs) represent a significant strategy for the prevention and control of respiratory viruses, given their capacity to emulate natural infections and elicit mucosal, cell-mediated, and humoral immune responses. However, traditional empirical passage-based attenuation methods face limitations, including unclear mechanisms, poor genetic stability, and a risk of virulence reversion. Recent breakthroughs in synthetic biology have created a new path for the rational design of LAVs. Synthetic biology enables precise regulation of viral replication, tissue tropism, and immunogenicity by integrating reverse genetics, genome synthesis, and the design-build-test-learn (DBTL) engineering cycle with rational attenuation strategies such as temperature-sensitive mutations, microRNA-mediated replication restriction, codon/codon pair deoptimization, and proteolysis-targeting. Meanwhile, artificial intelligence (AI)-assisted attenuation design, automated high-throughput virus construction, biofoundries and organ-on-chip evaluation models are transforming vaccine development from “empirical screening” to “rational engineering”. This review provides a comprehensive summary of engineering platforms and rational attenuation strategies enabled by synthetic biology, as well as their applications in representative respiratory viruses, including the influenza virus, respiratory syncytial virus, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We discuss key challenges related to genetic stability, biosafety, regulatory considerations, and clinical translation. We also highlight future perspectives in AI-enabled vaccine design, intelligent biomanufacturing, and precision attenuation, with the aim of providing insights into developing next-generation LAVs against respiratory viruses.
Oral probiotics have demonstrated significant therapeutic potential for various diseases; however, their efficacy is severely constrained by multiple gastrointestinal barriers, including strong acidity, bile salts, enzymatic degradation, and mechanical stress, which compromise bacterial viability and intestinal colonization. To address these challenges, researchers have developed various intestinal delivery systems. This review systematically summarizes recent advances in probiotic delivery technologies, focusing on the design principles, advantages, and limitations of micro/nanoencapsulation, hydrogel-based carriers, metal-phenolic network coatings, and probiotic and algae symbiotic systems. Furthermore, the application prospects of emerging technologies such as microfluidics, 3D printing, and genetic engineering for precision probiotic delivery are discussed. Studies indicate that multi-level optimization of material selection, structural design, and functional modification can significantly enhance probiotic survival and targeted accumulation under complex physiological conditions. Nevertheless, challenges regarding safety, batch-to-batch consistency, and quality control during large-scale production remain significant obstacles to clinical translation. Future developments will likely emphasize the integration of multiple strategies, intelligent functionality, and a design oriented toward clinical translation.
Iron is an essential cofactor in bacterial metabolism. The scarcity of bioavailable iron in the environment, coupled with the oxidative toxicity of intracellular free iron, poses a dual challenge to bacterial survival. This article, combining bacterial metabolic mechanisms with advances in environmental biotechnology, focuses on the synergistic mechanism of bacterial iron uptake and storage systems and its application value. It systematically elucidates two mechanisms. The first is the molecular mechanism, which explains how a siderophore acts as a bacterial “tentacle” to overcome environmental iron deficiency limitations through its high affinity. The second is the biological basis of how bacterioferritin acts as an intracellular “reservoir” that uses a nanocage structure to rapidly mineralize and sequester iron, releasse it on demand, and buffers iron concentration fluctuations. The closed-loop network of “uptake-transport-sequestration-utilization” formed by these two mechanisms under the coordination of global regulatory factors such as the iron uptake regulator protein Fur, is also outlined. This synergistic mechanism serves as both a survival strategy, allowing bacteria to adapt to their environment. It also provides new insights into the bioremediation of heavy metals. The review discusses progress in the application of siderophores and bacterioferritins in various fields. Considering unresolved scientific questions, such as the precise distribution of intracellular iron, this review provides a prospective outlook on future research directions in bacterial iron homeostasis.
In recent years, immobilized metal ion affinity chromatography (IMAC) has been widely applied in bioseparation and purification due to its advantages of high selectivity, ease of scaling up, and low cost. Its working principle involves immobilizing transition metal ions on the matrix surface via ligands. Then, stable complexes form through the specific interaction between the metal ions and the target molecules. Finally, competitive elution occurs to enrich and purify the target molecules. This paper reviews the research progress of the IMAC technology based on various matrices, including cotton fibers, nanomaterials, microspheres, monolithic materials, membranes, and composite materials. It also elaborates on the applications of this technology in purifying metalloproteins and antibodies, enriching phosphopeptides, and purifying recombinant proteins, mainly His-tagged proteins. This paper analyzes the problems encountered when applying IMAC, such as metal ion leakage and a relatively low protein adsorption capacity. Finally, the future development directions of the IMAC technology are discussed.
