Objective: To explore the regulatory role of iron regulatory protein 2 (IRP2) in iron metabolism in macrophages of the iron overload model. Method: The in vivo and in vitro IRP2 knockout and knockdown models were respectively constructed and then fed a high-iron diet (2% carbonyl iron) or treated with ammonium ferric citrate (AFC). Bone marrow-derived macrophages (BMDMs) from IRP2 knockout (Ireb2^-/-) and wild-type mice were isolated, and the expression of ferritin (FTH1) and ferroportin1 (FPN1) was observed. Prussian blue staining was used to detect iron deposition and immunohistochemistry was used to detect FPN1 expression in the spleens of Ireb2^-/- and wild-type iron-overload mice. The expression of FTH1 in IRP2-knockdown RAW 264.7 macrophages and THP1 (PMA-treated) cells with AFC treatment was detected by Western blot, and the iron content in the cell culture medium was analyzed. Results: Under AFC treatment, compared with wild-type mice, the expression of FTH1 and FPN1 in BMDMs of Ireb2^-/- mice increased significantly. Moreover, increased iron deposition was evident in the spleens of Ireb2^-/- mice, while FPN1 expression decreased. In vitro results showed that down-regulating IRP2 increased FTH1 expression in the macrophage cell lines Raw 264.7 and THP1 (PMA treated), and the iron ion content in the cell culture medium increased relatively. Conclusion: During the process of iron overload, the absence or reduction of IRP2 increased FTH1 expression in macrophages,, which aggravated the iron deposition in the spleen.

Objective: To investigate the role of the UM729 compound in the growth of hepatocellular carcinoma (HCC) and to elucidate its regulatory mechanism. Methods: (1) The half-maximal inhibitory concentration (IC₅₀) of the UM729 compound in HCC cell lines was determined using the CCK-8 assay, and the experimental concentrations were preliminarily established. (2) The effects of the UM729 compound on the proliferation of HepG2 and SMMC7721 cells were assessed via cell growth curves and colony formation assays. (3) Apoptosis in HepG2 and SMMC7721 cells induced by the UM729 compound was detected using Annexin V-FITC/PI double staining, while alterations in the expression levels of apoptosis-related proteins were examined by Western blot analysis. (4) The in vivo impact of the UM729 compound on tumor growth was evaluated using a BALB/c nude mouse xenograft model. Results: (1) The IC₅₀ values for the UM729 compound in HepG2 and SMMC7721 cells were 9.525 μmol/L and 10.41 μmol/L, respectively. (2) The UM729 compound exhibited potent anti-proliferative activity in both HepG2 and SMMC7721 cells in a dose-dependent manner. (3) The UM729 compound promoted apoptosis in HCC cells by modulating the expression of apoptosis-related proteins in a dose-dependent manner. (4) The UM729 compound was found to significantly suppress liver tumor growth in vivo. Conclusion: The UM729 compound inhibits the proliferation of HCC cells both in vitro and in vivo by inducing apoptosis through modulating apoptosis-related gene expression.

Objective: Shewanella sp. is a Gram-negative bacterium that causes diseases in aquatic animals and spoils food. In order to reduce the use of antimicrobials, this study isolated and characterized a novel Shewanella phage to determine its potential use in phage therapy. Methods: LB and TCBS media were used to isolate the Shewanella strains. Phages were isolated, purified, and counted by the double-layer plating method. Spotting was used to determine the host range of phages. A one-step growth curve was used to analyze reproductive capacity. A growth inhibition curve was used to analyze the phage killing efficiency. The blunt-end ligation method was used to determine the terminal sequences of the phage genome. High-throughput sequencing was used for genome annotation. We explored the evolutionary relationship of the isolated phage by constructing phylogenetic trees of the key viral proteins. Results: The Shewanella Y2 bacterium and its corresponding phage, phiY2, were isolated from the intestinal contents of a farmed Silurus asotus in a freshwater environment. An acid-base stability analysis was performed, revealing that phage phiY2 showed the survival percentages of 84.76% and 67.96% in environments with pH levels of 4.0 and pH 12.0, respectively. After treatment at 50℃ for 1 h, phiY2 showed a survival percentage of 23.13%. The phage phiY2 was relatively tolerant of chloroform treatment with a survival percentage of 49.33%. A one-step growth experiment was conducted, revealing that phiY2 has a latent phase of 12 min and a burst size of 95 pfu/infected cell. The growth inhibition assay showed that the phage phiY2 significantly inhibited the bacterial growth at the indicated multiplicity of infection (MOI). High-throughput sequencing and genome structure analysis were conducted, revealing that the phiY2 virion has a linear, double-stranded DNA genome of 45 066 bp. The genome has identical direct repeat sequences of 315 bp at each end. A total of 50 protein-coding genes were predicted. A comparative genomics analysis revealed that the phiY2 phage had very low homology with the currently known phage genome sequences. Further analysis of the evolutionary relationship of the key viral proteins showed that they were highly homologous to the counterparts of the Vibrio sp. and Marinomonas sp. phages, respectively. Conclusion: In this study, a novel Shewanella phage, phiY2, was isolated, which can effectively lyse the host bacteria and maintain high survival percentages under a variety of environmental stressors. Our study indicated that the bacteriophage phiY2 may be used as a new antimicrobial agent to control and prevent Shewanella contamination.

Acetamiprid (ACE) is a widely used neonicotinoid insecticide. Klebsiella variicola, which was isolated from ACE-contaminated soil, demonstrates remarkable ACE degradation capabilities. Recombinant expression of Klebsiella variicola nitrilase (Kv-NASE) was achieved, and its enzymatic and degradation properties were investigated. Gas chromatography results indicated that the recombinant Kv-NASE (rKv-NASE) exhibits an activity level of 41.2 U/mg, with an optimal temperature range from 37℃ to 40℃, and its optimal pH level of 7.0. Additionally, the presence of Ni²⁺ enhances rKv-NASE activity by 23%. Analysis by HPLC-MS/MS indicated that rKv-NASE can degrade 81% of ACE, yielding N'-[(6-chloropyridin-3-yl)methyl]-N-methylacetamide, a product that is less toxic than ACE. Molecular docking analysis revealed that the catalytic triad (E48-C127-K163) of the nitrilase (Kv-NASE) plays a crucial role in the degradation process. These findings provide a theoretical foundation for developing rKv-NASE-based enzymatic agents that could be used to clean up ACE-contaminated environments and treat ACE poisoning in emergencies.

Objective: To obtain novel AsCas12a mutant proteins that bind to target double-stranded DNA (dsDNA) with high specificity. Methods: The structure of the AsCas12a protein was predicted and molecular docking was performed using Phyre2 and Schrödinger software. This identified the following mutation sites preliminarily: R174, A282, R542 and R548. Site-directed mutagenesis of Cas12a was conducted via overlap extension PCR. The recombinant plasmid pET28b-four-Mut-AsCas12a, which is used for prokaryotic expression, was constructed and subjected to prokaryotic induction expression. The expressed product was purified using nickel-affinity chromatography. The trans-cleavage activity of the purified target protein was subsequently validated using a full-wavelength fluorescence analyzer. Results: A quadruple mutant protein of AsCas12a was obtained with a purity exceeding 95% and a concentration of 4.844 mg/mL, Its trans cleavage activity is 1.45 times that of the wild-type protein (WT). Finally, fluorescence polarization (FP) assays were employed to analyze its binding characteristics with target dsDNA and non-target strand (NTS)-complementary single-stranded DNA (ssDNA). The results demonstrated that the affinity of this AsCas12a quadruple mutant for target dsDNA was significantly higher (by up to fourfold or more) than its affinity for NTS-complementary ssDNA, showing a 3.1-fold difference in binding affinity with the same substrates compared to the wild-type protein. This suggests that it has a specific activity for binding to target dsDNA rather than ssDNA. Conclusion: The novel AsCas12a mutant protein has established a foundation for advancing the research and the application of new CRISPR-Cas12a-based biosensor technologies in the future.

Objective: To enhance the supply level of the cofactor NADPH and alleviate the limitations imposed by global carbon and nitrogen transcription factors on the uptake pathways of carbon and nitrogen sources. Methods: The pentose phosphate pathway was optimized, and the expression of the isocitrate dehydrogenase and NAD kinase genes was increased to boost the availability of NADPH. Additionally, the global transcription regulators RamA, RamB, and AmtR were knocked out to investigate their impact on carbon and nitrogen metabolism. Results: Compared with the initial strain, intracellular NADPH levels increased by 80.2%, and L-arginine production improved by 35.2%. Conclusion: Metabolic engineering strategies that boost NADPH supply and modify transcriptional regulators effectively redirect both the cellular carbon and nitrogen fluxes toward the accumulation of L-arginine.

Objective: To improve the yield of sequencing data from low-quality nucleic acid samples under field conditions using the portable, high-throughput MinION MK1C sequencing platform. Methods: Using standardized microbial samples to simulate low-quality field conditions, we evaluated three strategies to enhance sequencing output: (1) exogenous DNA-assisted library preparation by adding lambda bacteriophage genomic DNA at different ratios; (2) targeted enrichment using multi-pathogen probes; and (3) artificial polyadenylation of non-polyadenylated RNA samples. Sequencing data were analyzed using fastp, NanoStat, minimap2, and SAMtools to assess the read output, coverage, and quality. Results: Spiking exogenous nucleic acid at a ratio of 1∶2 ratio significantly increased the yield of sequencing data and produced the best results of all the tested ratios. Targeted amplification produced 150.17 k mapped reads, achieving a consistent mapping rate of 79.74%. The key pathogens Pseudomonas aeruginosa, Listeria monocytogenes, Escherichia coli, and Salmonella enterica, were reliably detected. Artificial polyadenylation increased the output of total RNA sequencing from 245 Mb to 637 Mb, improving the median read length and uniformity of genome coverage. Mapping accuracy exceeded 99.99%. Conclusion: The three strategies-exogenous DNA supplementation, targeted enrichment, and Poly(A) tailing-can each enhance sequencing performance in different scenarios. For RNA samples with sufficient concentration, Poly(A) tailing is efficient and convenient. For low-input DNA samples with known pathogens, targeted enrichment improves sensitivity. For unknown or complex cases, it is recommended that targeted enrichment be combined with the addition of exogenous DNA to balance detection breadth and sequencing yield. These approaches facilitate the rapid identification of pathogens in emergency and resource-limited settings.

Cell therapies, such as immune cell and stem cell therapies, have been widely used in preclinical and clinical settings to treat various diseases. Nevertheless, their clinical application is limited due to challenges related to efficacy and safety. Drug delivery systems operating at the nano-, micro-, and macro-scales can enhance therapeutic efficacy by optimizing pharmacokinetics, augmenting cellular functionality and viability, preventing cell exhaustion, and reducing immunogenicity. This review summarizes engineering strategies for multi-scale drug delivery systems that aim to improve the biological functions of therapeutic cells, modulate tissue microenvironments to enhance cell survival and potency, achieve targeted drug delivery in transplanted cells, and provide protective barriers for in vivo cellular therapeutics. Additionally, it summarizes key domestic and international advances in the clinical translation of cell therapies that incorporate drug delivery systems. With representative examples, this article provides a focused analysis of the core challenges of their scalable production.

Peptide-drug conjugates (PDCs) are a novel targeted drug delivery system that integrates the biological activity of peptides with the diagnostic and therapeutic capabilities of drugs. This method provides an excellent reference for developing new drug delivery system. Homing peptides, linkers and payloads are three necessary components of PDCs. The peptides’ properties enhance the excellent cell permeability of drugs, allowing them to reach the tumor site more accurately, and improve the therapeutic effect. Moreover, the linkers enable the selective release of drugs and significantly reduce off-target effects, thereby dramatically preventing tissue and cell damage caused by traditional chemotherapy drugs. At present, PDCs have extensive applications in bio-medicine, bio-sensing, and imaging probes. Therefore, this review not only elaborates on the applications of PDCs in drug delivery systems, but also the development of PDC technology. The practical applications and limitations of PDCs for clinical use are summarized and described.

Natural killer (NK) cells, a subset of innate lymphocytes, play a pivotal role in the immune system. NK-cell-mediated immunotherapy has emerged as a promising approach for cancer treatment. Among the various strategies, natural killer cell engagers (NKCEs) represent one of the most promising avenues in contemporary cancer immunotherapy. NKCEs direct NK cells towards tumor cells by targeting surface receptors on the NK cells, such as CD16a, NKG2D, NKG2C, NKp30, and NKp46, as well as tumor-associated antigens. This targeting promotes NK cell activation, thereby enhancing these cells’ tumor-killing capabilities. Notwithstanding the significant development potential of NKCEs, certain challenges stemming from the tumor microenvironment and the inherent structure of NKCEs themselves severely impact their anti-tumor efficacy in practical applications. This article provides a comprehensive review of the latest research advancements regarding NKCEs in development, organized by their NK-cell targets. It also delves into the pre-clinical and clinical progress of these products, elaborates on the challenges that NKCEs face in clinical treatment, and explores treatment modalities that can overcome these limitations. The article also underscores the significance of optimizing the therapeutic efficacy of NKCEs and offers insights for their development and application within the realm of cancer immunotherapy.

Inflammatory bowel disease (IBD), characterized as a chronic and recurrent immune-metabolic disorder involving intestinal inflammation, mainly includes Crohn’s disease (CD) and ulcerative colitis (UC). Its onset and progression are closely associated with an imbalance in the intestinal microecology, specifically the disruption of the structure and function of microbes, which is currently a therapeutic challenge. In recent years, studies have found that the total amount of bacteriophages in the intestines of IBD patients is significantly higher, by two to three times. There is also an imbalance in the ratio of temperate to lytic phages, which correlates positively with disease activity. This suggests that the “virome-bacteriome-host” interaction could be a new target for intervention. Bacteriophages (BPs) can precisely eliminate pathogenic bacteria through highly specific host recognition and lytic action, and regulate microbial structure and function via mechanisms such as horizontal gene transfer, promoting the synthesis of beneficial metabolites, like short-chain fatty acids, and enhancing intestinal barrier integrity. Furthermore, bacteriophages can modulate host immune responses via immune pathways, such as the TLR9/IFN-γ pathway, thereby alleviating intestinal inflammation. Multiple animal studies have confirmed that phage cocktails targeting IBD-related pathogens, such as adherent-invasive E. coli (AIEC) and Klebsiella pneumoniae, can significantly reduce colonic inflammation and restore intestinal barrier integrity. Early clinical trials have also demonstrated the favorable safety profile and stable intestinal colonization of oral phages. Phage therapy possesses unique advantages, such as high efficiency, specificity, and a low likelihood of inducing resistance, but it still faces challenges, including standardized preparation, control of immunogenicity, and addressing individual variability. In the future, enhanced multidisciplinary collaboration will be required to promote the clinical translation of phage therapy for IBD treatment. This review provides a systematic summary of the mechanisms and potential of bacteriophages in regulating the gut microbiota and immune microenvironment in the pathogenesis and treatment of IBD.

Microbial cell factories (MCFs) are pivotal to green biomanufacturing, yet their industrial application is often hindered by a “metabolic trade-off”-the inherent competition for resources between cell growth and target product synthesis. To address this issue, this review systematically summarizes existing literature and categorizes three types of balancing strategies for metabolic trade-offs. The “rigid” strategy employs irreversible approaches, such as gene knockout and overexpression, to reconstruct metabolic networks and forcibly redirect metabolic flux. While straightforward, it often leads to metabolic burden and reduced robustness. The “flexible” strategy adopts spatiotemporal regulation of gene expression based on transcriptional dynamics to decouple growth from production. It offers greater adaptability but relies heavily on regulatory elements and requires high specificity in target selection. The “rigid-flexible hybrid” strategy capitalizes on the strengths of both: rigid engineering provides a high-yield framework, while flexible regulation enables precise optimization, thereby balancing synthesis efficiency and cellular fitness. Finally, this review anticipates that AI-assisted component design, multi-omics-driven metabolic network analysis, and intelligent control system implementation will accelerate the resolution of metabolic trade-offs. This will ultimately enable the precise, efficient, and intelligent construction of advanced microbial cell factories.

Due to the growing global challenge of antibiotic resistance, developing novel antibacterial technologies has become a crucial focus of current medical research. Metal nanomaterials demonstrate significant potential in anti-infective therapy due to their unique physicochemical properties and multifaceted antimicrobial mechanisms. Recent advancements in omics technologies have provided new insights into the antibacterial mechanisms of metal nanomaterials. These technologies enable researchers to conduct systematic analyses at multiple levels including transcriptomics, proteomics, and metabolomics. Current research indicates that omics approaches play a pivotal role in elucidating key processes, such as the responses to oxidative stress induced by metal nanomaterials, the dysregulation of metabolic networks, the disruption of cell membrane integrity, and the inhibition of biofilm formation. Although notable progress has been made in this field, there are still several challenges that persist. Future research will focus on integrating multi-omics data, incorporating artificial intelligence technologies, and developing strategies for the precise design of nanomaterials. These innovative approaches are expected to advance metal-based antimicrobial nanomaterials toward enhanced efficacy and safety, laying a solid foundation for their clinical applications.

The “dual carbon” strategy has driven the emergence of biosynthesis as a transformative force in green manufacturing, initiating a paradigm shift in industrial production. With advantages such as mild reaction conditions and strong environmental compatibility, biosynthetic technology offers a promising pathway for the low-carbon transition of the chemical and pharmaceutical industries. However, industrial enzymes, the core functional units of biocatalysis, are still primarily derived from natural sources. Due to the inherent limitations of natural enzymes-such as insufficient thermal stability and low catalytic activity in industrial settings-they often fail to meet the demands of large-scale production. Consequently, enzyme engineering has emerged as a critical strategy to overcome these performance limitations and accelerate the industrial application of biosynthesis. This review outlines the technical framework of enzyme engineering in a systematic manner, integrating various computational tools used in common strategies, such as directed evolution, rational design, and semi-rational design. The innovative applications of artificial intelligence (AI) in high-throughput screening and performance prediction are also explored. In addition, the denovo enzyme design strategy is introduced, which constructs catalytically active artificial enzymes from scratch, based on the target reaction mechanisms and structural features, without relying on natural enzyme templates. Notably, the deep integration of systems biology and computational biology has significantly improved catalytic activity through the use of deep learning techniques, which elucidate the structure-function relationships of enzymes. The combination of multi-dimensional technologies for guiding enzyme engineering aims to provide both theoretical and methodological references for the intelligent design of industrial biocatalysts.

Feruloyl esterase (FAE) belongs to a subclass of carboxylate hydrolases that catalyze the hydrolysis of the ester bonds between phenolic acids and lignin or cellulose, in the cell walls of plants. This process produces high-value phenolic acids, such as ferulic acid and p-coumaric acid, which have diverse applications in food processing, cosmetics, papermaking, and medicine. At present, large quantities of feruloyl esterase have been found in microorganisms and plants, but their weak catalytic activity, low expression level, and poor thermal stability seriously limit their potential applications. Therefore, based on the molecular modification of the active pocket of feruloyl esterase, the exploration of the relationship between structure and function, and the analysis of the catalytic mechanism will be of great significance for promoting the efficient release of ferulic acid from feruloyl esterase enzymes and sustainable wood resource utilization. This review summarizes the recent research progress on the classification, spatial structure, catalytic mechanism, and molecular modification of feruloyl esterase. It further elaborates on the modification strategy and development direction of feruloyl esterase, providing a reference for exploring and applying feruloyl esterase with high catalytic activity.

Two key challenges in environmental microbiology detection are inhibitor interference and low target abundance. Digital PCR (dPCR) addresses these challenges by partitioning reactions into single molecules. This mechanism reduces inhibition effectively, improves the detection of rare targets, and enables absolute quantification without the need for standard curves. Over the past decade, dPCR has emerged as an indispensable tool in environmental microbiology research due to its advantages, such as high sensitivity, accuracy, and robust tolerance to inhibitors. This review systematically outlined the technological evolution of dPCR, summarized recent advances in its main applications-such as monitoring environmental pathogens, tracking microbial sources, and quantifying antimicrobial resistance genes (ARGs) and functional genes-and provided a critical analysis of its main advantages and future challenges.

Food security underpins national security, and achieving higher yields while minimizing environmental impacts has become a central goal of modern agriculture. Over the past five decades, yield gains have largely depended on genetic improvement, with transgenic approaches playing a major role in crops such as cotton, maize, and soybean. Three decades of practical experience demonstrate that, under appropriate regulatory oversight, transgenic technologies do not pose risks greater than those associated with conventional breeding. In 2023, the Royal Society of London published a policy briefing welcoming the UK Precision Breeding Act and advocating regulation based on product traits rather than breeding methods. This commentary aims to highlight the publication of the Chinese edition of the policy briefing and to discuss its contemporary relevance. The briefing highlights promising technologies, including stacking immune receptor genes for disease resistance, expanding Bt applications, improving stress tolerance, and optimizing photorespiration to increase yield. These advances are closely related to current agricultural challenges and innovations in China. While significant progress has been made in areas such as cross-species deployment of resistance genes, enhancement of photosynthetic efficiency, and AI-assisted protein design, a further proportionate regulatory framework is essential to support food security and sustainable agricultural development.