Objective: To investigate the effects of a non-enzymatic LDHA protein on the proliferation, migration, and invasion of breast cancer cells. Methods: (1) An LDHA enzyme-deficient mutant was constructed by site-directed mutagenesis, in which key residues——arginine 105 (Arg105), asparagine 138 (Asn138), arginine 168 (Arg168), and threonine 247 (Thr247)——were substituted with alanine (Ala). (2) The protein expression of the LDHA enzyme activity-deficient mutant was detected via Western blot analysis. (3) Lactate production, ATP generation, and glucose uptake were measured using commercial assay kits to verify the loss of enzymatic activity in the LDHA mutant. (4) The effect of the catalytically inactive LDHA mutant on the proliferation of human breast cancer cells was assessed using CCK-8 and colony formation assays. (5) Cell migration was evaluated by a wound healing assay. (6) Cell invasion was measured using a Transwell invasion assay. (7) The subcellular localization of the catalytically inactive LDHA mutant was determined using cellular immunofluorescence. Results: (1) The LDHA enzyme-deficient mutant was successfully generated. (2) The mutant was expressed in breast cancer cells and exhibited a loss of the classic LDHA enzyme activity. (3) The LDHA enzyme-deficient mutant promoted the proliferation, migration, and invasion of breast cancer cells. (4) The mutation of the enzymatic active site did not affect the subcellular localization of the LDHA protein. Conclusion: The catalytically inactive LDHA mutant promotes the proliferation, migration, and invasion of human breast cancer cells, independently of its canonical enzymatic activity. These findings enhance our understanding of the role of LDHA in breast cancer progression, offering new molecular targets and perspectives for future research.
Rabies, canine distemper, canine parvovirus disease, canine parainfluenza, and canine adenovirus infections are widely prevalent viral diseases in pet dogs. The currently available inactivated and live-attenuated vaccines still have limitations regarding safety and the breadth of protection they provide. To develop a broad-spectrum candidate vaccine targeting multiple pathogens, this study adopted an artificial intelligence (AI)-assisted immunoinformatics strategy to predict and screen dominant B-cell and T-cell epitopes from the rabies virus glycoprotein, the canine distemper virus H protein, the canine parvovirus VP2 protein, the canine parainfluenza virus HN protein and the canine adenovirus hexon protein. Based on AI-assisted predictions of antigenicity, toxicity and allergenicity, 10 epitopes were prioritized and assembled in tandem to design the multi-epitope antigens BCH and its double-copy counterpart, 2BCH. pVAX1 vaccine plasmids that encode BCH/2BCH were constructed. These plasmids incorporated the SV40 enhancer and CpG motifs, and were formulated with the cell-penetrating peptide HNR for targeted delivery to generate candidate DNA vaccines. In vitro cellular assays demonstrated that the AI-designed, multi-epitope antigens BCH/2BCH were efficiently expressed and promoted the maturation of murine bone marrow-derived dendritic cells. Furthermore, they significantly up-regulated the expression of dendritic cell co-stimulatory molecules. Using BALB/c mice as a model, in vivo studies revealed that multi-epitope DNA vaccines significantly modulated serum IgG levels, splenocyte proliferation, cytokine secretion and the CD4+/CD8+ T-cell ratio.Vaccine formulations containing 2BCH and CpG/HNR induced stronger humoral and cellular immune responses. No obvious histopathological toxicity was observed in major organs. Therefore, the AI-assisted, multi-epitope antigens that target five common viral pathogens in pet dogs exhibit high antigenicity and preliminary safety in mice. This AI-assisted, multi-epitope DNA vaccine design and construction strategy lays the groundwork for designing, optimizing and evaluating the protective efficacy of other multi-epitope vaccines.
To investigate the cross-species regulatory mechanism of outer membrane vesicles (OMVs) released by multidrug-resistant Proteus vulgaris PV14 under stress from temperate phage vB_BPM-V3 on the virulence phenotypes and pathogenicity of Proteus mirabilis CMCC 49005. Methods: OMVs secreted by PV14 under phage stress were prepared via ultracentrifugation. Phenotypic assays (motility, urease activity, biofilm formation, and environmental tolerance) combined with RT-qPCR were employed to detect the expression of the following virulence genes in the recipient bacteria: flhDC, pmfA, scsA, and sodA. A RAW264.7 macrophage infection model was established to evaluate the bacterial anti-phagocytic capacity and changes in the expression of host inflammatory cytokines (IL-6, TNF-α, and IL-10). Results: The results indicated that OMVs induced by phage stress did not affect the normal growth kinetics of the recipient bacteria. However, OMV treatment significantly inhibited the swarming motility of the recipient P. mirabilis and downregulated flhDC expression, while significantly enhancing its urease activity, biofilm formation capacity (upregulation of pmfA), and tolerance to high temperatures, strong alkalis, and oxidative stress (upregulation of scsA). Cell experiments revealed that after OMV pretreatment, the anti-phagocytic capacity of the bacteria against macrophages was significantly enhanced. Furthermore, OMVs caused an imbalance in the response of macrophages, which was characterized by high pro-inflammatory responses (high expression of IL-6 and TNF-α) and low anti-inflammatory responses (low expression of IL-10). Conclusion: OMVs secreted by Proteus vulgaris serve as interspecies signal carriers under temperate phage stress. They improve the environmental adaptability and pathogenicity of Proteus mirabilis by switching to a motility-defense phenotype and employing enhanced immune evasion strategies.
25-Hydroxyvitamin D3 (25-OH-VD3) is the biologically active form of vitamin D3 (VD3), which has physiological functions such as maintaining calcium and phosphorus metabolism, regulating immune responses, and maintaining cell osmotic pressure in the human body. It is widely used in clinical practice. Previous studies have shown that CciUPO, a peroxygenase from Coprinopsis cinerea, can selectively hydroxylate VD3 to produce 25-OH-VD3 in a single step. In this study, Pichia pastoris X33 was used as the host to optimize the heterologous expression of CciUPO through signal peptide screening and promoter engineering in order to increase the expression level and catalytic efficiency of CciUPO. An in vitro multi-enzyme cascade catalytic system was constructed to efficiently convert VD3 to 25-OH-VD3. The results showed that using the SPα factor signal peptide increased the yield of 25-OH-VD3 by 3.23 times (42.85 mg/L) compared to the previous optimized level. On this basis, the expression of CciUPO was co-regulated by the dual promoters PAOX1 and PDAS2, which increased the product concentration to 62.21 mg/L. To improve the conversion efficiency of the catalytic system, the conditions of the multi-enzyme cascade reaction were further optimized. The optimal conditions were determined by regulating the concentrations of glucose, glucose oxidase, and the cosolvent 2-hydroxypropyl-β-cyclodextrin, as well as the reaction time. The optimal conditions were found to be 25 mmol/L, 20 U/mL, 45 mg/mL, and 24 h, respectively. The final product concentration of 25-OH-VD3 was 101.50 mg/L. This study achieved efficient secretory expression of CciUPO in Pichia pastoris, and significantly improved the efficiency of VD3’s C25 hydroxylation through system optimization. This provides a novel technical route for the green biomanufacturing of 25-OH-VD3.
Objective: To identify the halophilic bacterium MM7, which was isolated from a mixture of water and mud from Gahai Salt Lake, and to screen its antibacterial activity. Then, the antibacterial substances will be isolated and identified. Methods: The strain was identified through analysis of the 16S rRNA gene sequence. Optimal growth conditions were determined by single-factor experiments, while physiological and biochemical characteristics were assessed using paper tests or reagent methods. Using Pseudomonas aeruginosa, Klebsiella pneumoniae, Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Candida albicans as indicator bacteria, the antibacterial activity of the fermentation supernatant from strain MM7 was tested using the agar well diffusion method. The antibacterial substance was primarily isolated and purified using ethyl acetate extraction, acid precipitation, thin-layer chromatography, silica gel column chromatography, semi-preparative liquid chromatography, high-performance liquid chromatography, and gel column chromatography. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the extract were determined using the micro-broth dilution method in a 96-well plate. The chemical structures of the active products were analyzed and identified using high-resolution mass spectrometry (HRMS) and nuclear magnetic resonance spectroscopy (NMR). Results: Identification confirmed that strain MM7 was Bacillus licheniformis. Its fermentation broth exhibited significant antibacterial activity against all six indicator bacteria. The strain grew within the following ranges: temperature, 5-55℃; pH, 5-14; and NaCl concentration, 0-25%. The optimal growth conditions were a temperature of 37℃, a pH of 8.0, and a concentration of 5% NaCl. The strain produced positive results in the Methyl Red test and negative results in the Indole test. The ethyl acetate and acid precipitate extracts from the fermentation broth of strain MM7 both demonstrated excellent antibacterial activity, with MIC and MBC values against six indicator bacteria ranging from 0.75 to 3 mg/mL and 1.5 to 12 mg/mL, respectively. These extracts also exhibited tolerance to high temperatures and acidic or alkaline environments. Among these, Fraction A4 from the ethyl acetate extract exhibited the strongest antibacterial activity, while Fraction B6 from the acid precipitate extract demonstrated the greatest antibacterial potency. A comprehensive analysis using HRMS and NMR identified L-malic acid as the primary antibacterial component in A4, and surfactin-C15 as the key antibacterial compound in B6. Conclusion: This study successfully isolated and identified L-malic acid from B. licheniformis for the first time. Basic antibacterial data and characteristics of L-malic acid and surfactin-C15 were obtained against opportunistic pathogens, including P. aeruginosa. This work provides significant references for developing new antimicrobial agents and advances the resource mining and application of halophilic microorganisms in biomedicine.
Bone morphogenetic protein 2 (BMP2) is a member of the transforming growth factor β (TGF-β) family and it plays an important role in the formation of bone and cartilage, as well as the maintenance of adult bone homeostasis. BMP2 is crucial for osteogenesis and is currently the most widely used factor for bone defect repair. However, its clinical application is limited by its short half-life and rapid diffusion, as well as the potential for side effects with prolonged treatment. This study designed BMP2-ColG, which has a collagen-binding domain that enhances its affinity for type I collagen and improves its targeted retention capacity. Moreover, the His-SUMO dual tag not only facilitates purification via Ni-affinity chromatography but also increases the expression level of BMP2-ColG by 2.04-fold compared to native BMP2 alone. The collected expression inclusion bodies were dissolved and refolded using the freeze-thaw method. We remove the affinity tags via protease digestion directly on a Ni-affinity chromatography column to obtain unlabeled, high-purity protein. Our in vitro osteogenic differentiation assays showed that BMP2-ColG exhibits collagen-binding activity and significantly increases calcium deposition in human bone marrow mesenchymal stem cells (hBMSCs). Furthermore, it upregulates the expression of the key osteogenic markers Runx2 and Ocn. Taken together, our findings suggest that the BMP2-ColG fusion protein was successfully expressed in Escherichia coli, the freeze-thaw method effectively improved the refolding efficiency of its inclusion bodies, and the refolded fusion protein exhibited collagen-binding ability and promoted the expression of key osteogenic markers during osteogenic differentiation. This study presents a new strategy for obtaining targeted, functional BMP2 with a simplified purification process for potential clinical applications.
Core fucosylation is an important form of N-glycosylation modification. Its aberrant expression is closely associated with the occurrence and progression of various cancers, making it a potential target for discovering biomarkers and drugs. Mass spectrometry-based glycoproteomics offers an effective approach for the comprehensive profiling of core fucosylation. However, analyzing core fucosylated peptides remains challenging due to their relatively low abundance, the different fragmentation behaviors between glycan chains and peptide backbones, and the difficulty of interpreting glycopeptide spectra. This review focuses on enrichment methods for core fucosylated peptides, including lectin affinity-based and chemoenzymatic strategies. It provides a detailed comparison of various mass spectrometry fragmentation modes, and systematically introduces data analysis strategies and related software tools for both data-dependent and data-independent acquisition. Furthermore, it discusses challenges and recent advancements in glycopeptide quantification, as well as the potential value of core fucosylation in disease biomarker research. Finally, future directions in the field are outlined, including developments in enrichment techniques, data analysis algorithms and tools.
Chinese hamster ovary (CHO) cells are the core chassis cells for the production of biotechnological drugs. The construction of traditional engineered cell lines is primarily reliant on random integration (RI) technology, a method that confronts limitations such as uncontrollable integration sites, pronounced expression heterogeneity, compromised genetic stability, protracted screening cycles, and low success rates. The recent breakthrough development of CRISPR/Cas9 and other gene-editing technologies has brought the practical application of site-specific integration (SSI) in CHO cells to a new level. By precisely inserting transgenes into pre-validated genomic safe harbors (GSHs), SSI enables sustained, high-yield recombinant protein expression, significantly enhancing predictability, genetic stability, and process efficiency. This review systematically summarizes recent advances in CRISPR/Cas9, recombinase, and transposon systems, and hybrid integration strategies for constructing SSI platforms in CHO cells. It offers the theoretical foundation and practical framework necessary for developing next-generation CHO cell lines with enhanced productivity and stability for industrial biologics manufacturing.
The tumor microenvironment (TME) exhibits unique pathological and physiological characteristics, such as weak acidity, high levels of reactive oxygen species (ROS), high concentrations of specific enzymes, and hypoxia. These characteristics provide intrinsic targets for the development of intelligent drug delivery systems. Due to their high specific surface area, tunable pore size, and excellent biomimetic extracellular matrix structure, electrospun nanofibers have become an outstanding drug carrier platform. In recent years, by combining the “intelligent response” property with nanofibers, intelligent responsive nanofibers that can sense and respond to specific signals of the tumor microenvironment have been developed. These nanofibers achieve precise and controllable drug release, and demonstrate great potential in regulating immunosuppressive microenvironments, reversing hypoxia, and providing synergistic treatment. This article reviews the construction strategies, drug release mechanisms, and application progress of intelligent nanofibers that respond to different stimuli (e.g., pH, ROS, enzymes, hypoxia, light and heat). It also discusses their role in regulating and treating the tumor microenvironment, and discusses the current challenges and future development directions in this field.
The growing prevalence of age-related disorders and trauma-induced injuries has increased the clinical need for effective repair of hard tissues such as bone and cartilage. However, regenerating these tissues is is particularly challenging due to their intrinsic complexity involving coordinated biochemical signaling, mechanical support, and dynamic remodeling. Within the native extracellular matrix, glycoproteins and proteoglycans represent essential structural and functional constituents. They regulate cell recognition, adhesion, proliferation, and lineage specification by means of highly orchestrated glycan-receptor interactions and localized growth factor modulation. Despite their central roles, the application of natural glycosylated biomolecules in tissue engineering is severely constrained by their pronounced structural heterogeneity, low abundance, and the difficulty of purifying well-defined molecular species from biological sources. Glycopeptides have recently emerged as a promising class of biomimetic molecules, capable of recapitulating many of the functional attributes of native glycoproteins and proteoglycans, while offering marked advantages in structural precision and synthetic accessibility. By covalently linking carbohydrate moieties to sequence-defined peptide backbones, glycopeptides integrate the tunability of peptide chemistry with the biological specificity of glycan structures. This modularity enables the rational design of materials that both reproduce extracellular matrix cues and deliver targeted biological functions, which makes glycopeptides highly attractive candidates for next-generation hard tissue regenerative platforms. Significant progress has been made in developing glycopeptide synthesis strategies. Advances in solid-phase peptide synthesis, including linear and convergent assembly, have expanded the range of accessible glycopeptide architectures. Strategies for radical-mediated glycosylation, particularly those based on photochemical or metal-catalyzed activation, enable site-selective glycan installation without extensive protecting group manipulation, thus improving synthetic efficiency. Collectively, these strategies have broadened the chemical space and functionality of synthetic glycopeptides, supporting their translation into biomaterial design. Increasing evidence demonstrates the utility of glycopeptide-based materials in hard-tissue regeneration. In cartilage repair, glycopeptide amphiphiles and supramolecular GAG-mimetic nanofibers emulate the hydration and mechanobiological functions of aggrecan and hyaluronic acid, promote chondrocyte viability, and guide stem cell chondrogenesis. In bone tissue engineering, glycopeptide hydrogels modulate immune responses by inducing macrophage M2 polarization, thereby enhancing osteogenic differentiation, vascularization, and defect healing. Moreover, glycopeptide constructs that combine antibacterial activity with osteoinductive signaling have shown promise in treating infected bone defects. Within inflammatory periodontal environments, glycopeptide-functionalized scaffolds regulate cytokine expression, inhibit osteoclast-mediated bone resorption, and support alveolar bone regeneration. Overall, the classification and functions of glycopeptides, together with chemical synthesis, chemoenzymatic synthesis, and biosynthetic strategies, are summarized, with particular emphasis on the advantages and limitations of these approaches in the construction of complex glycopeptides. Recent advances in the application of glycopeptides in the repair and regeneration of hard tissues, such as bone and cartilage, are further outlined. Glycopeptide-based materials are expected to provide new strategies and a material basis for precise and functional hard tissue regeneration.
Molybdenum disulfide has demonstrated significant potential in the field of field-effect transistor (FET) biosensors due to its unique structure and outstanding properties. This paper systematically reviews the structural characteristics and synthesis methods of molybdenum disulfide. It explores the unique properties of molybdenum disulfide as a channel material for FET biosensors, and provides a detailed analysis of the progress in biomedical sensing applications of molybdenum disulfide-based FET biosensors. Finally, the challenges and future development directions of molybdenum disulfide-based FET biosensors are outlined.
Antibiotic resistance genes (ARGs) are emerging contaminants that are characterized by environmental persistence and the potential for cross-species transmission. Due to their occurrence and widespread dissemination across diverse environmental media, ARGs pose significant challenges to global public health and ecological safety. Not only do environmental media serve as reservoirs for ARGs, they also serve as critical hubs for disseminating them among microbial communities via horizontal gene transfer (HGT). Traditional monitoring techniques face substantial hurdles in addressing the high complexity of environmental samples, the urgent need for rapid on-site screening, and the difficulty of deciphering propagation mechanisms. Biosensor technology offers high sensitivity and specificity, as well as the potential for miniaturization and integration. It is gradually becoming a frontier tool for the highly sensitive monitoring and dynamic tracking of environmental ARGs. This review systematically summarizes the latest research achievements in this field. First, it reviews the recent progress of applying biosensors to reveal the distribution patterns and dissemination dynamics of ARGs in different environmental media. Second, it provides an in-depth analysis of genotypic biosensors that target ARG nucleic acid sequences, focusing on elucidating the innovative mechanisms of various isothermal signal amplification strategies, as well as the role of advanced functional nanomaterials, such as surface-enhanced Raman scattering (SERS) substrates, upconversion nanoparticles, and MXene, in enhancing optoelectronic sensing performance and overcoming matrix interference. Subsequently, it summarizes advancements in genotypic and phenotypic biosensors for rapid antimicrobial susceptibility testing and resistance risk assessment. Lastly, it outlines the trends in point-of-care testing (POCT), which involves the deep integration of complex sample pretreatment, microfluidics, and portable terminals. It also discusses the development of intelligent platforms that integrate monitoring and remediation functions.
As biotechnology continues to be integrated into the industrial sector, industrial biotechnology has emerged as a key driver of transformation in the bio-manufacturing industry, fueling the sustainable development of the bioeconomy. During the 14th Five-Year Plan period, China has placed significant emphasis on advancing bio-manufacturing. This article provides a comprehensive analysis of China’s bio-manufacturing sector, focusing on four key areas: biopharmaceuticals and healthcare, bio-based chemicals and materials, bio-agriculture and food, and bioenergy and environmental protection. From various perspectives, it explores these domains, including the policy framework, advancements in development and research (R&D), innovation outcomes, industrial development, and financing trends. This analysis aims to highlight the emerging trends and characteristics in China’s industrial biotechnology landscape, offering valuable insights for developing future bio-manufacturing strategies.
