Objective: Mitochondria possess their own genome, and the unique post-transcriptional processing and modifications of mitochondrial RNA plays a crucial role in normal physiological functions. The m⁶A modification is the most prevalent RNA modification in organisms. However, the occurrence and regulatory patterns of m⁶A modifications in mitochondrial RNA remain largely unexplored. Methods: The novel computational method for analyzing miCLIP-seq and third-generation nanopore direct RNA sequencing data was developed to investigate m⁶A modifications in mitochondrial RNA. Additionally, SELECT technology was employed to predict and identify potential m⁶A sites on mitochondrial RNA. Results: The miCLIP-seq data identified 520 high-confidence m⁶A modification sites, and the nanopore sequencing data predicted 377 sites. The results of multi-omics data analysis and subsequent validation experiments confirmed that mitochondrial RNA, similar to genomic RNA, also undergoes m⁶A modification. However, mitochondrial RNA m⁶A modifications tend to occur at the HHACH motif, which differs from the common DRACH motif observed in genomic RNA. Conclusion: Mitochondrial-encoded RNA can undergo m⁶A modification and exhibit novel regulatory characteristics distinct from those of genomic RNA, providing new insights into the mechanisms underlying mitochondrial functional homeostasis and the catalytic mechanisms and functions of m⁶A modification.

Objective: The accumulation and characteristics of citrate in CHO fed-batch culture were investigated to provide a basis for understanding and regulating the accumulation of citrate. Methods: Using CHO cells as the research object, the flow addition culture process established in the laboratory was used to investigate the accumulation characteristics of citrate during the fed-batch culture. The effects of cultivation process parameters (temperature and glucose concentration) on citrate accumulation were investigated. The correlation between key metabolic parameters (Q_Gluc, q_Lac, q_p, and total carbon flux of the TCA cycle) and citrate accumulation was established. Results: Temperature affected the accumulation of citrate during the fed-batch culture of CHO cells, and the specific rate of citrate formation was significantly increased at lower temperature (31℃). The effect of glucose concentration on the accumulation process of citrate was limited, and the control of glucose concentration in the range of 2 ~ 55 mmol/L did not affect the accumulation process of citrate. The accumulation process of citrate under different cultivation process parameters was investigated in multiple batches. The specific production rate of citrate is positively correlated with the specific consumption rate of glucose, the specific production rate of antibodies, and the total carbon flux of the TCA cycle, while negatively correlated with the specific production rate of lactate. Conclusions: The above correlation indicates that extracellular citrate accumulation may reflect sufficient nutrient supply, vigorous intracellular TCA cycle metabolism, and enhanced antibody production capacity.

Objective: The artificial control and functionalization of the self-assembly process of light chain and heavy chain ferritin were studied to explore the effective method of artificially controlling the synthesis of ferritin hybrid cage-like nanoparticles, and to provide references for designing and customizing the functionalized protein nanoparticle drug delivery system. Methods: Recombinant human heavy chain (H) and light chain (L) ferritin nanoparticles were expressed, and their molecular weight and cage-like structure were verified by mass spectrometry and transmission electron microscopy. H and L ferritin were first dissociated under acidic conditions and mixed in different molar ratios, and then the pH of the solution was slowly raised to neutral by dialysis. The formation of hybrid ferritin nanoparticles was verified by denaturing and non-denaturing electrophoresis. L-ferritin nanoparticles containing a single cysteine mutation were constructed, the sulfhydryl group of the cysteine was coupled with Dansyl fluorescent dyes containing maleimide, and the L-ferritin nanoparticle functionalized with fluorescent dyes was co-assembled with H-ferritin. Denatured and non-denatured electrophoresis was performed to verify the formation of fluorophore functionalized ferritin hybrid cages. Results: Both the recombinant H and L ferritin nanoparticles could form cage-like structures, and a series of hybrid ferritin nanoparticles with different H and L contents could be formed by artificially controlling their self-assembly process. The L-ferritin nanoparticles modified with the fluorescent dye Dansyl can also co-assemble with H-ferritin to form hybrid ferritin nanoparticles. The molar ratios of functionalized LFt and HFt within the ferritin hybrid nanoparticles can be artificially controlled during the co-assembly process. Conclusion: H and L ferritin can form a series of hybrid cage-like structures with different concentration ratios by artificially controlling its self-assembly process. After the functional modification of ferritin nanoparticles, the assembly of ferritin hybrid nanocarriers can also be artificially controlled, which verifies the feasibility of ferritin nanoparticles as a multi-functional and customized drug delivery system.

Objective: Epoxide hydrolases (EHs, EC.3.3.2.-) catalyze the enantioselective hydrolysis of racemic epoxides to the corresponding chiral diols, offering a wide range of applications. A Rhodotorula paludigena epoxide hydrolase mutant, RpEH^L360C, was previously obtained through molecular modification. This study aimed to further investigate the catalytic properties of this mutant by analyzing its catalytic activity and regioselectivity towards different epoxide substrates. Methods: Recombinant Escherichia coli expressing rpeh^L360C (E. coli/rpeh^L360C) was used as a catalyst to investigate the catalytic activity, regioselectivity, optimal reaction conditions, and temperature stability of RpEH^L360C. Molecular dynamics simulations were employed to elucidate the mechanism behind the high regioselectivity of RpEH^L360C. Results: E. coli/rpeh^L360C exhibited a catalytic activity of 301 U/g wet cell weight when catalyzing the hydrolysis of rac-3-chloroepoxyphenylethane (4a). After complete hydrolysis, the product, (R)-1-(3-chlorophenyl)-1,2-ethanediol (4b), had an enantiomeric excess (ee) of 97.6%, demonstrating excellent regioselectivity (α_S = 98.9%, β_R = 98.3%). Optimal reaction conditions were found to be pH 7.0 and 25℃, with a half-life (t_1/2) of 4.7 h at 25℃. In a large-scale (R)-4b preparation using E. coli/rpeh^L360C, 200 mmol/L of rac-4a was hydrolyzed within 12 h, with a product ee of 96.3%, a yield of 90.1%, and a maximum space-time yield of 130.5 g/(L·h). Molecular dynamics simulation analysis revealed that the substrate-binding pocket of RpEH^L360C contains a hydrophobic region, and hydrophobic interactions between the chlorine atoms of (R)- and (S)-4a and amino acid residues in this region contribute to the high regioselectivity of the enzyme. Conclusion: RpEH^L360C demonstrates significant potential in catalyzing different epoxide substrates, providing a foundation for its application in fine chemical and pharmaceutical synthesis.

Objective: To isolate bacteriophages targeting Aeromonas hydrophila from aquaculture effluents, and analyze the biological characteristics and whole genome sequences. Methods: The double-layer plate method was used to isolate phage. The morphology of the isolated phage was analyzed by transmission electron microscope and the bactericidal effect of the phage was examined by a one-step growth curve. The classification of the isolated phage was sequenced and subsequently analyzed by comparative genomics. Results: A novel phage, phaST21, was isolated from sewage using the pathogenic bacterium A. hydrophila as a host. In addition, the biological characterization was determined and the whole genome sequence was analyzed. The results showed that phaST21 contains an icosahedral head and a non-contractile tail. The optimal MOI of phaST21 is 0.001, whereas the latent period and the burst size are 20 min and 12 PFU/cell, respectively. PhaST21 displayed a broad temperature and pH tolerance, and strong endurance to chloroform. However, phaST21 only infected its indicator host. Whole genome sequence analysis showed that the phage phaST21 is a circular double-standed DNA with a total length of 42 593 bp. A total of 51 open reading frames (ORFs) were predicted in the genome, and there were no tRNA, virulence genes, or resistance genes. Sequence alignment and phylogenetic analysis revealed that phaST21 is a new member of the order Caudovirales, family Autographiviridae, and genus Ahphunivirus. Conclusion: A novel A. hydrophila phage was isolated. This novel phage enriches the resources of the bacteriophage library and provides insight into the future development of therapeutic agents to control multidrug-resistant A. hydrophila.

Objective: Antibiotics have been widely exploited in the poultry industry, which leads to the emergence of drug-resistant bacterial strains. Phage therapy can lyse drug-resistant strains and is considered a potential alternative to antibiotics. The purpose of this study is to isolate and characterize Escherichia phages from duck feces. Methods: We used MacConkey agar plates for isolation of E. coli strains and the double-layer plating method for phage isolation and analysis. The broth dilution method was used to determine minimum inhibitory concentration (MIC). Genome sequencing and bioinformatics analyses were used for characterization of the phage genome. Sterilized milk was used as the medium for estimating phage killing efficiency. Results: An E. coli strain EK31-2 was isolated from the goose fecal sample and found to be resistant to 3 antibiotics. With strain EK31-2 as an indicator strain, a new phage, named phiE31-2, was isolated from the duck fecal sample. The tolerance of phiE31-2 to environmental stressors was analyzed. The data showed that more than 77.67% of phiE31-2 was survived after incubation in the pH range of 5.0 ~ 10.0 and about 52.8% of phiE31-2 was alive after treatment with 400 μg/mL sodium cholate, suggesting that phage phiE31-2 is very stable when exposed to the environmental stressors. Genomic analyses revealed that the phiE31-2 genome is a dsDNA molecule of 51 468 bp with 82 CDS regions, 35 of which have been annotated with putative functions. Comparative genomic analysis showed that phage phiE31-2 is an unclassified member of the genus Hanrivervirus in the subfamily Tempevirinae. In the biocontrol assay, phage phiE31-2 can kill 86.8% of the environmental strain EK31-2 in milk at 4℃. Conclusion: Phage phiE31-2 was isolated from the duck fecal sample and can effectively lyse the drug-resistant strain EK31-2. Additionally, it was highly resistant to a wide pH range and sodium cholate, suggesting that phage phiE31-2 may be a good candidate for potential phage applications in the poultry industry.

Objective: To enhance L-arginine accumulation in Corynebacterium crenatum by reducing non-essential genes and provide a reference for synthetic biology. Methods: Ten sequences from insertion sequence elements, prophage, and gene island were individually deleted using a traceable knockout technology. Among them, 8 gene fragments favorable to arginine accumulation were co-knocked out. Results: Arginine yield of the 8 gene fragments increased by 56.9% and the cell dry weight increased by 26.5% compared with the starting strain. Conclusion: This provides a reference for reducing the growth burden of microorganisms, improving the utilization rate of carbon sources, and constructing ideal chassis cells for synthetic biology services.

DNA barcoding technology as an efficient molecular biology approach to species identification, product addressing, and traceability. It exhibits high specificity in rapidly identifying plants, animals, and microorganisms, as well as in distinguishing closely related species. This technology offers an effective molecular identification means for detecting adulteration in food, pharmaceuticals, and related products, while also enabling the tracking and traceability of synthetic products. DNA barcoding techniques currently available include conventional barcoding, mini barcoding, and super barcoding. Additionally, with the advent of encapsulation technology, encapsulated barcoding has become a new technology for tracing and identification. Existing DNA barcoding technologies have been reviewed and compared in terms of their application scopes, advantages, and disadvantages. Furthermore, the potential use of DNA coding technology for information storage is anticipated. The objective is to provide guidance on the selection of appropriate barcoding technologies for the identification, recognition, and traceability of relevant species and products, thereby improving their identification and traceability accuracy, in order to facilitate the future development of DNA barcoding technology in different fields.

Protein glycosylation is one of the most important and heterogeneous post-translational modifications. Glycosylation is crucial in physiological and pathological processes such as cell growth and differentiation, signal transduction and tumor immune surveillance. Abnormal protein glycosylation is closely linked to the onset and development of disease, and many glycoproteins can be used as biomarkers for the early diagnosis and prognosis of cancer. Mass spectrometry-based glycoproteomics technology provides a powerful analytical tool for the comprehensive analysis of protein glycosylation modifications. In bottom-up glycoproteomics, glycopeptides often need to be separated and enriched due to their low relative abundance. In recent years, in order to analyze protein glycosylation more comprehensively, researchers have developed a variety of new materials and methods based on different enrichment mechanisms. The glycopeptide enrichment efficiency has been significantly improved, providing strong technical support for glycoproteomic analysis and the discovery of protein glycosylation-related disease targets and biomarkers. The new progress in glycopeptide enrichment methods is summarized and reviewed. Prospects for its future development are discussed.

S100A8/A9 proteins, also known as calprotectin, are heterodimeric proteins that belong to the S100 family of calcium-binding proteins. S100A8/A9 proteins (calprotectin) are mainly released by neutrophils and are widely involved in a variety of biological processes such as infection, inflammation and immunity. In recent years, S100A8/A9 (calprotectin) has been extensively studied as a biomarker and potential therapeutic target. This article reviews the current status of research and application of S100A8/A9 in inflammatory bowel diseases, infectious diseases, rheumatoid arthritis, cardiovascular diseases and cancer, and discusses future research directions and application scenarios.

Nucleic acid vaccines have garnered widespread attention in the field of animal infectious disease prevention due to their design flexibility, short production cycles, and ability to induce both cellular and humoral immunity. The application of delivery carriers has greatly facilitated nucleic acid vaccines as a promising preventive and therapeutic strategy. while most nucleic acid carrier development has been focused on human diseases, there is a growing demand for low-cost, safe, efficient, and non-pathogenic nucleic acid delivery carriers in the veterinary field. Some progress has been made on nucleic acid delivery carriers and vaccines for major animal infectious diseases such as porcine epidemic diarrhea, porcine reproductive and respiratory syndrome, foot-and-mouth disease, avian influenza, Newcastle disease, and rabies. This review summarizes the research progress of animal nucleic acid vaccine nano-delivery carriers at home and abroad, and briefly describes the process of nano-carrier delivery of nucleic acids and the mechanism of action of nucleic acid vaccines. It analyzes the impact of physicochemical factors of nano-delivery carriers on cellular uptake and discusses the future directions and challenges of this promising vaccine platform in the veterinary field, aiming to provide references for the development of animal nucleic acid vaccines and delivery carriers.

Innovative medicines are the core competitiveness and key area of the biopharmaceutical industry. In recent years, although significant progress has been made in the development of the innovative drug industry with policy support, there are still issues with poor coordination and inconsistent direction. There are mainly six major bottlenecks: the review and approval process is not coordinated with the innovative industry policy, the focus is concentrated on targets but enterprises are scattered, the medical insurance policy is not coordinated with the upgrading of clinical medication structure, the rights of doctors and patients to use medicines are restricted, the capital market support for innovative medicines is insufficient, and the connection with the international regulatory system is inadequate. It is necessary to further clarify the overall coordination responsibility system, fill in the policy gaps, unblock the policy transmission, strengthen project review, optimize the layout of production capacity, accelerate the link with international regulations, and carry out policy consistency assessments, in order to build a healthier and more sustainable environment for the creation of innovative medicine.