Current Biotechnology

Research Advances on the Resource Utilization of Carbon Dioxide Based on Microbial Cell Factories
Xiaoyu WANG, Lin CUI, Miao LIU, Huiying LUO, Bin YAO, Xiaolu WANG
2026, 16(3): 454-484. DOI: 10.19586/j.2095-2341.2026.0087
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The extensive consumption of fossil fuels has led to a continuous increase in atmospheric carbon dioxide （CO?） concentrations， triggering global warming and even food security crises that threaten the sustainable development and stability of human society. With the rapid advancement of synthetic biology， converting CO? into value-added products through microbial cell factories offers great potential for mitigating climate change while delivering considerable economic benefits. This review first introduced carbon fixation pathways and strategies in autotrophic and heterotrophic microorganisms， as well as chassis strains applied to CO? valorization. It then summarized recent advances in the biosynthesis of CO?-derived products. On this basis， metabolic engineering strategies and fermentation approaches for improving product yields were further discussed. Finally， future perspectives on efficient CO? bioutilization were proposed. This review was expected not only to facilitate the advancement of CO? conversion technologies based on microbial cell factories， but also to provide strategic and technical references for the industrial bioproduction of chemicals from CO?.

Advance in Genetic Modification Tools and High-value Compound Production in Methanogenic Archaea
Xiaoxuan WU, Yuqian LIANG, Tianer ZHENG, Boyan LIU, Lei CHENG, Yan HUANG
2026, 16(3): 485-495. DOI: 10.19586/j.2095-2341.2026.0108
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Methanogenic archaea are strictly anaerobic microorganisms capable of utilizing one-carbon （C₁） or two-carbon （C₂） compounds for growth and methane production. Owing to their unique membrane lipid structures and tolerance to various extreme environmental conditions， they represent promising chassis cells for the high-value conversion of carbon resources such as CO₂. To date， genetic engineering toolkits have been progressively established in model methanogens， particularly Methanosarcina and Methanococcus， including shuttle vectors， expression regulation systems， homologous recombination， and gene editing technologies. Leveraging these tools， methanogenic archaea have been metabolically engineered to produce a range of high-value non-methane compounds， including isoprenoids and bioplastics， as well as metabolic intermediates such as organic acids and organic alcohols， through metabolic pathway remodeling and the integration of heterologous metabolic pathways. This review systematically summarized the research progress of genetic engineering tools and high-value compound synthesis in methanogenic archaea， evaluated the application potential of methanogenic archaeal cell factories， and outlined future research directions， which was expected to provide reference for advance synthetic biology research in methanogens.

Microbial Cell Factory Mediated Multi-enzyme Cascades Reactions Empower High-value Utilization of Biomass
Yuqing SUN, Lige TONG, Wenxia FANG
2026, 16(3): 496-509. DOI: 10.19586/j.2095-2341.2026.0039
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Facing the dual challenges of global fossil resource depletion and worsening environmental problems， the high-value utilization of renewable biomass has become a critical pathway to achieve green and sustainable development. Microbial cell factories， with unique advantages in efficient metabolic network integration， autonomous cofactor regeneration and modular assembly， have emerged as core platforms for driving multi-enzyme cascade reactions. They provide innovative solutions to address bottlenecks including low catalytic efficiency of single enzymes， limited product diversity and complicated processing procedures. This review systematically summarized the core construction strategies of multi-enzyme cascade reactions mediated by microbial cell factories， focusing on the modification and adaptation mechanisms of typical host cells represented by Escherichia coli， Saccharomyces cerevisiae and Aspergillus niger， as well as the application features of in vivo cascade and hybrid cascade systems. Furthermore， we highlighted recent advances in the high-value conversion of complex biomass via this technology， and analyzed key challenges in depth， such as screening and modification of rate-limiting enzymes， compatibility of enzymatic reaction conditions， metabolic flux competition in host cells， and cost constraints restricting large-scale industrialization. This review aims to offer theoretical references and future perspectives for the industrial promotion and application of microbial cell factory-based multi-enzyme cascade reactions in biomass biorefinery.

Advances in the Biosynthesis of Betalains
Tianjie HAN, Jinfeng WEI, Yongliang YAN, Min LIN, Zhengfu ZHOU
2026, 16(3): 510-522. DOI: 10.19586/j.2095-2341.2025.0171
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Betalains are a class of natural pigments， mainly divided into red-purple betacyanins and yellow-orange betaxanthins. Due to their excellent coloring properties and bioactivities such as antioxidant， anti-inflammatory and anti-obesity effects， betalains show broad application prospects in food， nutraceuticals， medicine and other fields. However， natural extraction faces bottlenecks including high cost， low efficiency and poor product stability， which restrict their commercial development. In contrast， the construction of microbial cell factories via synthetic biotechnology for heterologous and high-yield synthesis of betalains has become a research focus. This article reviewed the chemical properties and natural synthesis pathways of betalains， focused on analyzing the synthesis strategies of different chassis organisms such as prokaryotes and eukaryotes， systematically expounded the functions of key synthases， metabolic pathway regulation and optimization schemes， discussed future research directions and application prospects， and was expected to provide new ideas for the industrial production of betalains.

Metabolic Engineering Strategies and Application Advances of Microbial Cell Factories for Carotenoid Production
Juan LI, Yuting HE, Xiaoxuan YE, Jiayi JIA, Yaohui ZHANG, Hongyang ZHANG, Tao WU
2026, 16(3): 523-534. DOI: 10.19586/j.2095-2341.2026.0065
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Carotenoids represent an important class of terpenoid natural pigments with wide applications in food， pharmaceutical， and cosmetic industries. Benefiting from rapid growth， facile genetic manipulation， and scalable cultivation， microbial cell factories have emerged as a promising platform for carotenoid production. This paper systematically summarized metabolic engineering strategies applied in carotenoid biosynthesis in prokaryotic microorganisms typified by Escherichia coli and eukaryotic microorganisms represented by Saccharomyces cerevisiae and Yarrowia lipolytica. Major advances were highlighted， including optimization of precursor supply pathways， engineering of key enzymes， redirection of metabolic flux， regulation of cofactors， membrane engineering， and the combined application of multiple strategies. On this basis， the article compared the differences in membrane systems between prokaryotes and eukaryotes and discussed their implications for membrane engineering strategies. The unique advantages of the oleaginous yeast Yarrowia lipolytica for carotenoid synthesis and its adaptive laboratory evolution strategies were also addressed. Finally， prospects for artificial intelligence （AI）-assisted optimization of metabolic elements and other future research directions were presented， with the aim of providing references for the green and efficient biomanufacturing and industrial application of carotenoids.

Green Biomanufacturing of Indigo: Enzymatic and Fermentation Routes-advances and Challenges
Zhihao LU, Yonghui ZHANG, Zewang GUO, Jun CHEN, Xiaoyan ZHUANG
2026, 16(3): 535-548. DOI: 10.19586/j.2095-2341.2025.0182
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Indigo， a historically significant and widely used blue pigment， faces challenges in its traditional production， such as low extraction efficiency from plants and substantial pollution from chemical synthesis. These limitations have propelled the green biomanufacturing of indigo. Currently， enzymatic strategies mainly focus on the discovery and engineering of flavin-containing monooxygenases and cytochrome P450 monooxygenases， with efforts directed toward improving catalytic efficiency through directed evolution and rational design. Meanwhile， fermentation methods emphasize the use of wild-type or genetically engineered microbial strains， coupled with metabolic engineering and fermentation process optimization to enhance yield. This paper systematically outlined the key enzymes， microbial hosts， engineering strategies， and optimization techniques associated with both routes. It also discussed major challenges hindering industrial application， including low productivity and insufficient operational stability. The review aims to provide theoretical insights and technical support for the green and efficient biomanufacturing of indigo， thereby facilitating its sustainable development and application.

Metabolic Regulation Strategies for Aromatic Amino Acids and Derivatives in Yeast: A Review
Yingjie LIU, Ying MA, Yide LI, Dongqing KONG, Hanyu LIU
2026, 16(3): 549-561. DOI: 10.19586/j.2095-2341.2026.0076
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Aromatic amino acids （AAA） and their derivatives possess diverse biological functions and are widely applied in food， pharmaceutical， and other industrial sectors. With advances in synthetic biology， microbial synthesis using yeast has emerged as a research hotspot in green manufacturing. However， yeast-based production systems still face problems limiting large-scale application， including imbalanced metabolic flux， insufficient regulatory precision， and poor chassis adaptability. This review summarized recent advances in metabolic regulation strategies for the synthesis of AAA and their derivatives in yeast cell factories. Firstly， it outlined the core synthetic pathways and main rate-limiting bottlenecks of AAA； then， it summarized the evolution of metabolic pathway regulation methods from multiple dimensions such as static regulation， spatiotemporal dynamic regulation， systematic rational guidance， and artificial intelligence （AI）； further， it analyzed the metabolic characteristics of model chassis （Saccharomyces cerevisiae） and unconventional yeasts in synthesis； finally， it summarized the current challenges and looked forward to future directions， with the aim of providing strategic references for the construction of high-yield yeast engineering strains.

Research Progress on Recombinant Protein Production in the Yarrowia lipolytica Cell Factory
Xuan ZHAO, Zhongxue LIU, Lingling WU, Yongming GOU, Yingjie LIU, Wenjian MA
2026, 16(3): 562-573. DOI: 10.19586/j.2095-2341.2026.0088
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High-yield recombinant protein production represents a core demand in biomanufacturing， driving the urgent need to develop genetically stable and highly productive cell factories. As a non-conventional yeast， Yarrowia lipolytica has emerged as a novel and efficient expression host due to its excellent protein secretion capacity， well-established eukaryotic post-translational modification machinery， broad substrate utilization， and favorable industrial fermentation characteristics. However， native yeast systems still face numerous challenges and often fall short of meeting the diverse requirements for recombinant protein production. The review summarized the research progress on recombinant protein production using Y. lipolytica cell factories， analyzed the characteristics of Y. lipolytica compared to common expression hosts， sorted out the core elements of its expression systems and corresponding optimization strategies， elaborated the genetic editing tools available for this host， outlined strategies and application examples for optimizing protein secretion and stability and finally， provides perspectives on future development directions， with the aim of advancing the engineering of efficient protein expression hosts.

Engineering Microbial Cell Factories: Advances in Material Systems and Biomanufacturing Technologies
Wenzhu WEI, Sen PANG, Xiaoli HUANG, Jian WANG, Xing FAN, Siyan CHEN, Yujian CHEN, Jian JIAO
2026, 16(3): 574-584. DOI: 10.19586/j.2095-2341.2026.0069
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Architectural microorganisms offer a novel pathway for the low-carbon transformation of the construction industry. This paper systematically reviewed the engineering development of architectural microbial cell factories， focusing on durable chassis cells like Sporosarcina pasteurii and Bacillus spp. adapted to extreme environments. Through metabolic engineering and synthetic biology tools， directional enhancement of mineralization efficiency and interfacial bonding stability is achieved. A factory architecture comprising modular reactors， resource-oriented supply， and closed-loop waste recycling was proposed， exploring technical routes for localized production using industrial solid waste. Finally， by analyzing applications in crack repair， geotechnical reinforcement， and bio-material preparation， the current bottlenecks were summarized， and "design-on-demand" biomanufacturing was envisioned. This study provides a theoretical basis and technical reference for green building materials and their engineering applications.

Research Satus of Microbial Biostimulant in Ariculture
Yaxiong WEN, Yan ZHOU, Dacheng LIU, Lisheng ZHANG, Rou JIANG, Cong ZENG, Shiyong TAN
2026, 16(3): 585-594. DOI: 10.19586/j.2095-2341.2025.0180
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Application of Python Program in Bioinformatics
Zeyu LIU, Mengyang ZHANG, Baobao ZHANG, Lin SU, Zhuo LI, Wen HU
2026, 16(3): 595-609. DOI: 10.19586/j.2095-2341.2025.0142
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With the rapid development of high-throughput sequencing technologies， bioinformatics has entered the era of multi-omics big data. Python， featured by its flexibility and abundant toolbox ecosystem， has become an indispensable analytical language in this field. Despite its wide adoption， Python still faced bottlenecks in processing ultra-large-scale datasets， algorithm generalizability and model interpretability， which restricted its in-depth application in bioinformatics. This review systematically summarized the applications of Python in genomics， transcriptomics， proteomics and metabolomics， focusing on its functions in data preprocessing， analytical workflow construction and data visualization. It elaborated on Python-based machine learning methods （e.g.， random forest， support vector machine） and deep learning approaches （e.g.， neural networks， graph convolutional networks） in biomarker screening， disease prediction and drug research and development. Typical research cases were analyzed to clarify current challenges and potential optimization directions. This review aimed to provide a systematic reference for researchers who adopt Python for multi-omics data analysis， help address practical issues including large-scale data processing and model optimization， and facilitate the progress of biological data analysis technologies.

Application of Non-mammalian Models in Predictive Toxicology: Progress, Challenges and Prospects
Yingxuan DU, Yutong SUN, Jinyu PENG, Jiahao ZHAO, Keran DONG, Xinyuan GAO, Haoze WU, Yunyao LUO, Xiaoping MU, Hongshu SUI
2026, 16(3): 610-617. DOI: 10.19586/j.2095-2341.2026.0002
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Traditional toxicological evaluation has long relied on mammalian models such as rodents， which suffers from multiple limitations including high costs， long experimental cycles， uncertain interspecies extrapolation and ethical concerns. Against this backdrop， the development of efficient， reliable and ethically acceptable alternative testing models has become an urgent requirement in this field. Owing to their unique biological features， high reproductive efficiency and low ethical concerns， non-mammalian models have shown great application potential in predictive toxicology research. This review elaborated on typical non-mammalian models， including Artemia， Caenorhabditis elegans， zebrafish， chick embryo chorioallantoic membrane， Drosophila and Hydra. Furthermore， it systematically summarized the current applications of promising emerging technologies such as organ-on-a-chip， computational toxicology， artificial intelligence and multi-omics in toxicity screening and assessment. It focused on analyzing the advantages and challenges of various models with respect to the correlation of toxicological responses， as well as existing issues such as interspecies metabolic differences and insufficient validation of model applicability. This review aims to provide theoretical references for researchers in selecting alternative models and optimizing research strategies， and to facilitate the innovative development of predictive toxicology models.

Construction Technology Innovation and Application Progress in the Disease of Humanized Zebrafish Model
Xiaoyue SUN, Ranran ZHU, Kechun LIU, Xuehui JIAN, Xue WANG, Yun ZHANG, Qing XIA, Yuanshuai YU, Hairong HOU, Chen SUN
2026, 16(3): 618-625. DOI: 10.19586/j.2095-2341.2026.0019
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Humanized zebrafish models can accurately simulate human physiological and pathological processes， effectively overcome interspecies research barriers， and have become important research carriers in biomedicine. On this basis， the paper systematically elaborated on the inherent advantages of zebrafish for constructing humanized animal models， and comprehensively summarized the technical principles， characteristics and strengths of three commonly used approaches for establishing humanized zebrafish models， namely clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9（CRISPR/Cas9）， Tol2 transposon system， and xenotransplantation. It further highlighted the research progress and latest breakthroughs of humanized zebrafish models in multiple disease areas， including cardiovascular diseases， tumors， neurological disorders and liver diseases. Potential future research prospects were also proposed， including the combined application of diverse models and the integrated innovation of multiple technologies. The review aims to provide technical references for researchs in humanized zebrafish models and further advance studies on pharmaceutial research and development.

Effects of Chemical Constituents in Schisandra chinensis on Tumor Microenvironment
Shaokang WANG
2026, 16(3): 626-637. DOI: 10.19586/j.2095-2341.2026.0020
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Tumor microenvironment （TME） is a complex and dynamic system composed of tumor cells， immune cells， blood vessels， extracellular matrix， and various other components， playing a pivotal role in tumor initiation， progression， and metastasis. Schisandra chinensis， a widely used traditional Chinese medicinal herb， contains diverse bioactive constituents such as lignans， polysaccharides， volatile oils， organic acids， terpenoids， and flavonoids. Among these， lignans and polysaccharides are recognized as the primary active components， capable of modulating the TME through multiple mechanisms—including the regulation of tumor cell proliferation and apoptosis， modulation of immune cell activity， inhibition of angiogenesis， and remodeling of the extracellular matrix—ultimately contributing to antitumor effects. This article systematically reviewed the current understanding of how the active components of Schisandra chinensis influence TME， with the aim of providing a solid theoretical foundation and strategic guidance for the development of Schisandra-based antitumor agents， the design of TME-targeted therapeutic strategies in traditional Chinese medicine， and their translation into clinical practice.

Bioinformatics Analysis of GhCPK4 Protein and its Expression Pattern in Cotton
Xuan DING, Zuying ZHANG, MAYILA·Yusuyin, Jiajia WU, Xiujuan SU, Wenran HU
2026, 16(3): 638-646. DOI: 10.19586/j.2095-2341.2025.0168
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A large number of studies have shown that calcium-dependent protein kinases （CPKs） play important roles in plant responses to abiotic and biotic stresses. To further elucidate the mechanism underlying GhCPK4 gene function， bioinformatics analyses and expression pattern profiling of the GhCPK4 protein were conducted. The results showed that the GhCPK4 protein contains a protein kinase domain and EF-hand domains， with high sequence conservation. GhTCP4 lacks a signal peptide， is mainly localized in the nucleus and is characterized as an unstable hydrophilic protein. It has 62 phosphorylation and 6 glycosylation modification sites. Its secondary structure is mainly composed of random coils and α-helices， and its existence form is monomer. The GhCPK4 gene was expressed in all cotton tissues examined， however， the expression levels varied significantly， with the highest leves observed in petals compared to other tissues. This study provides a theoretical foundation for further research into the biological function of the GhCPK4 gene in cotton resistance to Verticillium wilt.

The Stability of 12 Foodborne Standard Strains Preserved by Porcelain Beads Cryopreservation Method
Bilian LIN, Peng MENG, Menghang HE, Chaohui ZHANG, Ming DAI, Fang ZHANG, Binhong HUANG, Fengling JIANG, Tianxi WANG, Yun HE, Hui XU, Yan CHEN, Zhenhua KE
2026, 16(3): 647-653. DOI: 10.19586/j.2095-2341.2026.0012
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The easy-to-operate magnetic bead cryopreservation method has become the mainstream approach for strain storage in detection laboratories. To clarify the stability of magnetic bead cryopreservation for maintaining standard foodborne strains， this study selected 12 common standard foodborne strains， including gram-positive， gram-negative， spore-forming， obligate anaerobic， and aerobic bacteria. The plate counting method was used to determine the effects of repeated freeze-thaw cycles and different storage durations on strain viability； the VITEK2 Compact automated microbial identification system and 16S rDNA sequence analysis were employed to verify biochemical and genetic stability.The results showed that after 10 repeated freeze-thaw cycles， 9 strains （including Staphylococcus aureus and Escherichia coli，etc.） exhibited minimal fluctuations in viable counts and strong stability. In contrast， the viable count of Pseudomonas aeruginosa continued to decline， while Clostridium perfringens and Vibrio parahaemolyticus were completely inactivated after 6 freeze-thaw cycles. Under unopened storage at -70°C for 15 years， 10 strains （including S. aureus， E. coli， Bacillus cereus， etc.） maintained high viability， whereas C. perfringens and V. parahaemolyticus were almost completely inactivated. Both the VITEK 2 Compact identification and 16S rDNA sequence analysis confirmed that all strains remained stable in biochemical characteristics and genetic genotype without obvious variation after treatment. In conclusion， the magnetic bead cryopreservation method is generally suitable for the long-term preservation of common foodborne pathogenic bacteria. However， C. perfringens and V. parahaemolyticus showed poor tolerance to repeated freeze-thaw cycles， indicating the need for optimized preservation conditions. This study provides scientific data references for applications such as standardized management of strains and interim verification of strains， filling the gap in strain stability research in the domestic strain preservation field.

Screening and Safety Evaluation of Probiotics Promoting GLP-1 Secretion
Xiaona DU, Fengjie MA, Fengzhen FENG, Linhao SU, Lishan ZHANG, Wei XU, Kai QIAN, Xiaofeng SONG
2026, 16(3): 654-664. DOI: 10.19586/j.2095-2341.2025.0158
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Type 2 diabetes mellitus can lead to a variety of severe complications and affects more than 400 million people worldwide. Studies have found that probiotics can stimulate intestinal L cells to secrete glucagon-like peptide-1 （GLP-1）， thereby achieving a significant blood glucose-lowering effect. This study aimed to screen bacterial strains that significantly promote GLP-1 secretion， with the goal of providing a new therapeutic strategy for type 2 diabetes mellitus. The enzyme-linked immunosorbent assay （ELISA） was used to compare the GLP-1 secretion levels of 5 candidate bacterial strains in stimulating the mouse small intestine endocrine cell line （STC-1）， so as to screen out the strain with the highest GLP-1 secretion. The inhibitory activity of this strain against glucose-related metabolic enzymes （α-amylase） was evaluated. Physiological and biochemical experiments were conducted to assess the safety of the strain， providing data support for its application. Quantitative real-time PCR （qPCR） was used to detect the relative expression levels of G protein-coupled receptors （Gpr41 and Gpr43）， so as to conduct a preliminary exploration of its blood glucose-lowering mechanism. The fermentation supernatant of Limosilactobacillus fermentum TH02105 could promote GLP-1 secretion in STC-1 cells， with a secretion level of 140.340 ng·mL^-1， which was significantly higher than that of other strains （P0.001）. Its inhibition rate against α-amylase reached 66.30%. The logarithmic growth phase of L. fermentum TH02105 was 2~8 h， with an optimal temperature of 37 ℃ and an optimal pH of 6. After being treated with artificial simulated intestinal fluid and gastric fluid for 2.5 h， the survival rate of this strain was more than 50%， and the number of its adhesion to human colonic adenocarcinoma cells Caco-2 was 334.02 CFU·cell^-1. Meanwhile， the antibiotics nitrofurantoin， minocycline， polymyxin B， ciprofloxacin， chloramphenicol， norfloxacin， and levofloxacin could all inhibit the activity of this strain. In addition， this strain significantly upregulated the relative expression levels of Gpr41/43 genes （P0.01）. A strain of L.fermentum TH02105 was screened out， which can significantly promote GLP-1 secretion and exert a blood glucose-lowering effect. At the same time， this strain has good tolerance to gastrointestinal fluids and good adhesion to Caco-2 cells， which is conducive to its colonization. The study provides a potential bacterial strain for the development of drugs and health products for the prevention and treatment of diabetes.

Establishment and Optimization of RNA Interference Technology in Platelets
Yuting WU, Wei LIANG, Hongtao WANG, Cuicui LIU, Jiaxi ZHOU
2026, 16(3): 665-676. DOI: 10.19586/j.2095-2341.2026.0036
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Platelets are the smallest anucleate blood cells in circulation， their rich and diverse RNA and protein contents constitute the molecular basis underlying their hemostatic， immune， and inflammatory functions. RNA interference represents a powerful tool for investigating platelet gene function， however， current studies remain at an early exploratory stage， and efficient and stable RNA interference approaches for platelets are still lacking. To establish a robust and effective platelet RNA interference platform， murine peripheral blood platelets were used as the experimental model， and key parameters—including transfection reagents and RNA modalities—were systematically optimized. Transfection efficiency， intracellular retention duration of nucleic acids， platelet viability， and gene-silencing efficacy were comprehensively evaluated. Through this optimization， we developed a highly efficient RNA interference platform by replacing the conventional cationic liposomal transfection reagent Lipofectamine 2000 with Mate Plus lipid nanoparticles， and substituting traditional double-stranded small interfering RNA （siRNA） with single-stranded antisense oligonucleotides. This strategy increased oligonucleotide transfection efficiency from 10% to 70% and sustained stable intracellular delivery for more than 48 h. Using the highly abundant platelet transcript thymosin β4 （Tmsb4x） as a proof-of-concept target， we successfully achieved effective knockdown of Tmsb4x mRNA via the ASO/Mate-Plus platform， reducing Tmsb4x mRNA levels by 30%. Notably， suppression of Tmsb4x RNA did not alter its corresponding protein expression， indicating that this system enables selective interrogation of platelet RNA function independent of protein abundance. Moreover， knockdown of Tmsb4x RNA alone significantly impaired adhesion and spreading of activated platelets， suggesting a potential protein-independent regulatory role of Tmsb4x RNA in platelet cytoskeletal dynamics. In summary， this study established and optimized an efficient platelet RNA interference strategy， providing a powerful experimental tool for dissecting platelet-specific RNA functions and elucidating the molecular basis of rapid platelet functional responses.

FUS and PTBP1 Cooperatively Promote Chronic Myeloid Leukemia Progression via Transcriptional and Post-transcriptional Regulation
Lexuan LIN, Xu JIN, Fang HE, Lihong SHI
2026, 16(3): 677-685. DOI: 10.19586/j.2095-2341.2026.0010
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The heterogeneous nuclear ribonucleoproteins （hnRNPs） family is involved in the development of various tumors， but its role in chronic myeloid leukemia （CML） remains unclear. Previous studies have confirmed that fused in sarcoma （FUS） is a key gene maintaining CML cell proliferation， and its high expression is associated with poor patient prognosis. The study integrated clustered regularly interspaced short palindromic repeats （CRISPR） screening， RNA interference， and cell function experiments to systematically verify the key role of FUS in CML. Knockdown of FUS significantly inhibited the proliferation of K562 cells， leading to quiescent phase （G0 phase） arrest and promoting apoptosis. Nuclear FUS binds to promoter regions and shows overlapping target genes with the transcriptional co-activator bromodomain protein 4 （BRD4） and the chromatin remodeler brahma-related gene 1 （BRG1）， suggesting potential collaborative regulation of chromatin structure and transcriptional activity. Additionally， FUS can interact with polypyrimidine tract binding protein 1 （PTBP1）， and the two may cooperatively regulate gene expression and post-transcriptional processing， jointly promoting CML cell survival. In summary， FUS may maintain oncogenic transcriptional programs by regulating chromatin spatial architecture and forms a functional cooperative network with PTBP1 at both transcriptional and post-transcriptional levels， collectively driving CML progression.

Comparative Analysis of Erythroblastic Island in Bone Marrow Versus Spleen Under Steady-state and Stress Conditions
Qimei CHENG, Jingwei WANG, Lingyue ZHENG, Xinyu LI, Chengjie GAO, Xu JIN, Jie GAO, Lihong SHI, Jingyuan TONG
2026, 16(3): 686-699. DOI: 10.19586/j.2095-2341.2026.0026
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To investigate the morphology and cellular composition of erythroblastic island （EBI） in the bone marrow and spleen of mice under physiological and stress conditions， and to elucidate the microstructural basis of extramedullary hematopoiesis （EMH）. Healthy adult C57BL/6J mice were assigned to a saline-treated control group and a phenylhydrazine （PHZ）-induced acute hemolytic anemia group. EBI from the bone marrow and spleen were enriched via density gradient sedimentation. Their cellular composition was analyzed by flow cytometry， and their morphology and abundance were assessed using imaging flow cytometry. The results showed that EBI primarily consisted of erythroid precursor cells， macrophages， and granulocytic/monocytic cells， with occasional lymphocyte association. No significant differences were observed in bone marrow EBI morphology or in the proportions of erythroid cells at different stages between physiological and hemolytic conditions. Under physiological conditions， the spleen of adult mice contained only a small number of EBI； following acute hemolytic stress， spleen weight increased by more than twofold， the number of EBIs increased markedly， and densely distributed "rosette-like" structures were observed， yet the proportions of erythroid cells at different stages within the islands remained stable， with proerythroblasts， basophilic erythroblasts， polychromatic erythroblasts， and reticulocytes exhibiting distribution patterns consistent with those in the bone marrow. These findings demonstrated that the spleen compensates for increased erythrocyte production through quantitative expansion of EBI， while erythroid precursors within the islands maintain their intrinsic differentiation program， thereby providing experimental evidence for understanding the cellular mechanisms of EMH.

Evaluation of the Inhibitory Effect of Antibodies on Tumor Cell Migration Using Hybridoma Supernatant
Haijiao JI, Wenlei LI, Ruijing HUANG, Xiaodan CAO, Hai TANG, Junjun DAI
2026, 16(3): 700-705. DOI: 10.19586/j.2095-2341.2025.0167
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The study aims to evaluate the inhibitory effects on tumor cell migration using early screened hybridoma supernatant. Firstly， anti-αvβ3 hybridoma positive clones were screened by enzyme-linked immunosorbent assay （ELISA）. After accumulating the fermentation broth， Biacore was used to detect the affinity， and 9 mixed clone supernatants with different affinity gradients were randomly selected. Using genetically engineered H-αvβ3-A375 cells （stably highly expressing αvβ3 receptor） as the model， the inhibitory effect of 9 mixed clone supernatants on tumor cell migration was detected by Transwell method， and 5 clones with strong-medium-weak gradient inhibitory effects were screened out. These 5 clones were subcloned， and the migration inhibitory activity of the subclone supernatants was also detected by Transwell method. Chimeric antibodies were synthesized based on the sequencing results of the subclones， and 4 concentration gradients （160， 80， 40， 20 μg·mL^-1） and a negative control （no antibody treatment group） were set to verify the activity consistency between the chimeric antibodies and the supernatants. The results showed that there were significant differences in the inhibition rates of the 9 mixed clone supernatants on the migration of H-αvβ3-A375 cells （P0.05）， ranging from 26.77% to 99.89%. There was no statistical difference in the inhibitory effect between the 5 selected subclone supernatants and the corresponding mixed clone supernatants （P0.05）， while the difference in inhibition rates between groups was significant （P0.05）. The migration inhibition rates of the chimeric antibodies at each concentration gradient were consistent with those of the mixed clone and subclone supernatants （P0.05）. The "hybridoma supernatant-Transwell" combined evaluation method established in this study can directly realize the early screening of antibody migration inhibition function at the mixed clone stage without antibody purification steps， with an accuracy rate close to 100%. The method significantly shortens the antibody research and development cycle and reduces the early research cost， providing a reliable technical means for the efficient development of anti-metastatic antibodies.

Transcriptomic Analysis of the Role of NFIA in HER2-positive Breast Cancer Brain Metastasis
Mengjie DONG, Peiwen FAN, Ruozheng WANG
2026, 16(3): 706-718. DOI: 10.19586/j.2095-2341.2026.0016
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This study systematically investigated the expression characteristics of NFIA in HER2-positive breast cancer brain metastasis （BCBM） and its association with patient prognosis， as well as its potential roles in the tumor microenvironment （TME）， aiming to identify novel therapeutic targets. NFIA expression patterns and their prognostic relevance were analyzed using multiple public databases， and immunohistochemistry （IHC） validation was performed on paired clinical samples. Furthermore， by integrating public datasets with our single-cell transcriptomic data， we systematically analyzed the functional states of NFIA-high cell subpopulations and their interactions within the TME. The results demonstrated that NFIA was significantly upregulated in HER2-positive BCBM and that its high expression was closely associated with poor patient prognosis. Single-cell analyses further revealed that NFIA-high tumor epithelial cells exhibited pronounced genomic instability and highly malignant features， accompanied by activation of pathways related to invasion and proliferation. In addition， NFIA-high cells acted as key modulators within the TME by regulating angiogenesis， immune suppression， and extracellular matrix remodeling. Collectively， NFIA high expression defines a highly malignant tumor epithelial cell subpopulation in HER2-positive BCBM and promotes tumor progression through TME remodeling， providing a potential molecular target for precision therapy in HER2-positive breast cancer brain metastasis.

Exploring the Mechanism of Wogonin in Cerebral Ischemia-reperfusion Injury Using Network Pharmacology
Xiying LIU, Qiangrui LIU
2026, 16(3): 719-728. DOI: 10.19586/j.2095-2341.2025.0177
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Using network pharmacology combined with cellular experiments， this study investigated the neuroprotective effects of wogonin （WOG） on cerebral ischemia-reperfusion injury （CIRI） and its underlying molecular mechanisms. Potential targets of wogonin were predicted using SwissTargetPrediction， and CIRI-related genes were collected from GeneCards databases. Common targets were identified for GO and KEGG enrichment analyses and for constructing a protein-protein interaction network. Molecular docking was performed to evaluate the binding affinity between wogonin and key targets. PC12 cells were used for in vitro experiments and divided into a control group， an oxygen-glucose deprivation （OGD） group， and an OGD+WOG group. Colony formation assays were conducted to assess cell proliferation. FerroOrange and C11 BODIPY 581/591 probes were used to detect Fe2⁺ levels and lipid peroxidation， respectively. Immunofluorescence was used to measure the levels of glutathione peroxidase 4 （GPX4） and solute carrier family 7 member 11 （SLC7A11）. A GSH/GSSG assay kit was used to quantify glutathione （GSH） and Western blotting was performed to evaluate arachidonate 15-lipoxygenase （ALOX15） protein expression. The results showed that a total of 51 common targets of WOG and CIRI were identified. GO and KEGG analyses revealed significant enrichment in arachidonic acid metabolism. Molecular docking showed strong binding between WOG and ALOX 15 （binding energy -8.6 kcal·mol^-1）. Cell experiments demonstrated that WOG significantly alleviated OGD-induced suppression of colony formation， reduced Fe2⁺ accumulation and lipid peroxidation， increased GSH levels， upregulated GPX4 and SLC7A11， and downregulated ALOX15. In conclusion， wogonin may alleviate CIRI by inhibiting OGD-induced ferroptosis in PC12 cells via targeting ALOX15.

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