Current Biotechnology

Research Advances in Transgenic Detection Technologies and Screening Strategies for Major Crop Seeds
Qingchun YUE, Siyuan MENG, Guohua LIU
2026, 16(1): 1-9. DOI: 10.19586/j.2095-2341.2025.0132
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Transgenic technology has played a pivotal role in improving key agronomic traits in crops， such as high yield， resistance to pests and diseases， and herbicide tolerance， and has been widely adopted in agricultural production. However， with the continuous expansion of genetically modified （GM） crop cultivation and the emergence of new breeding technologies like gene editing， the regulatory oversight of transgenic seeds is facing increasingly serious challenges. This article systematically reviewed the current application of detection technologies for transgenic events in major crop seeds， elucidated their underlying principles and mechanisms， and proposed a screening framework tailored to different crop species. It provided a comprehensive analysis of critical technical limitations， including low detection sensitivity， insufficient capability for simultaneous multi-target detection， and lagging development of portable devices for on-site rapid screening. Building on this assessment， future research directions were outlined， aiming to offer a theoretical foundation for strengthening the biosafety regulation of transgenic crops in seeds.

Research Progress on Screening of High-yield Xylanase-producing Strains
Jiazeng GAO, Yunshu XIAO, Wenzheng YAN, Yongli LI, Zhanying LIU, Jianhua HU
2026, 16(1): 10-17. DOI: 10.19586/j.2095-2341.2025.0070
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Xylan， the main component of plant hemicellulose， is a complex pentosan polysaccharide characterized by abundant content， low cost and renewability. However， it suffers from problems such as poor degradability， low utilization rate， and easy causes of resource waste and environmental pollution. Xylanase is the primary enzyme for degrading xylan and boasts great development potential， being widely applied in the industrial， agricultural， pharmaceutical， food and other fields. Microorganisms are the major source of xylanase， and xylanases from different sources vary greatly， yet the common drawback of low enzyme activity exists universally. Therefore， screening high-yield xylanase-producing strains is of great significance for resource recycling. This paper briefly elaborated on the classification and structure of xylanase， the mutagenesis breeding methods of xylanase-producing strains， as well as the cloning and heterologous expression of xylanase genes， and revealed the hydrolysis mechanism of xylanase and its expression in bacterial and fungal expression systems， aiming to provide a reference for the research on xylanase production.

The Thioredoxin System: Recent Progress and Developments
Ruping ZHAO, Xue JIANG, Yue JING, Xinyuan DUAN, Fei XIE
2026, 16(1): 18-28. DOI: 10.19586/j.2095-2341.2025.0127
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The thioredoxin system represents a major antioxidant and redox regulatory system in biological systems， primarily composed of thioredoxin， thioredoxin reductase， and nicotinamide adenine dinucleotide phosphate （NADPH）. This system plays a critical role in maintaining intracellular redox homeostasis， regulating signal transduction， and modulating cell proliferation and apoptosis. In recent years， extensive researches have elucidated the involvement of the thioredoxin system in the pathogenesis of various diseases， thereby offering novel therapeutic targets and strategies for related conditions. This review comprehensively examined the thioredoxin system-from its molecular architecture and mechanistic principles to its physiological and pathological functions- and evaluates its potential as a drug target. By also outlining future research avenues， it aims to deepen the mechanistic understanding of its pathophysiological roles and provide a foundation for innovating targeted therapies.

Deep Learning Strategies for Intelligent Development of Antibody Drugs
Xin ZENG, Chen WANG, Zhengjun WANG, Hechu LIANG, Zhengping ZHANG
2026, 16(1): 29-37. DOI: 10.19586/j.2095-2341.2025.0107
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The development of antibody drugs faces significant industrial challenges， including extended timelines （>3 years）， high costs （>$200 million） and difficulties in collaborative optimization of multiple attributes. Traditional methods such as hybridoma technology are limited by low throughput and inadequate global optimization capabilities. In recent years， deep learning （DL） has provided breakthrough solutions for the intelligent development of antibody drugs. This review systematically summarized the research progress of DL in antibody drug development， with a focus on exploring representative methods and technical challenges in core aspects such as antibody sequence design， structure prediction， affinity prediction and maturation， and multi-objective optimization. It also provides an outlook on future development， aiming to provide a reference for the transformation of antibody drug research and development towards intelligence and globalization.

The Role of MicroRNAs in Acrylamide Toxicity
Zekai YANG, Shihao MA, Ke DOU, Yinghao JIANG, Yilin HAO, Zhizheng ZHOU, Jiahang LYU, Xiuwen HUA, Zihao FAN, Hongshu SUI
2026, 16(1): 38-43. DOI: 10.19586/j.2095-2341.2025.0101
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Acrylamide （AA） as a white photosensitive crystal， was classified by the International Agency for Research on Cancer as a possible human carcinogen of grade 2A in 1994. AA has been proved to have definite mutagenicity and carcinogenicity. MicroRNAs （miRNAs）， a class of single-stranded non-coding RNAs with a length of 20~25 nucleotides， are widely involved in the regulation of human gene expression， which are closely related to the toxicity mechanism induced by AA and the screening and identification of biomarkers. This review aimed to clarify the in vivo process of AA， explored the mechanism of miRNAs in AA-induced cytotoxicity， and provide reference basis for intervention strategies of AA-induced related diseases.

Progress in Exosome Engineering Modification and its Application in Disease Therapy
Xinbo LIU, Haochen SUN, Yixuan WU, Huan LI, Wenjie WU, Lishuo JIAO, Limeng GUO, Caiyue XUE, Hongshu SUI, Lixin XIONG, Liwei REN
2026, 16(1): 44-52. DOI: 10.19586/j.2095-2341.2025.0103
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Exosomes， natural extracellular vesicles measuring 30~150 nm in diameter， serve as critical carriers for intercellular communication. The bioactive molecules they carry， including proteins， nucleic acids and lipids， have made them a research hotspot in biomedicine. With the development of biotechnology， exosomes have shown great potential in the fields of disease diagnosis， drug delivery and tissue regeneration. This review systematically elaborated on the basic biological characteristics of exosomes， the latest breakthroughs in their engineering modification technologies （including genetic engineering， microfluidic technology and chemical modification）， and their innovative applications in cancer therapy， tissue repair and neurodegenerative diseases. Furthermore， we conducted an in-depth analysis of the key challenges in clinical translation： large-scale production， targeting optimization and standardization. Finally， we looked forward to future directions such as artificial intelligence （AI）-assisted design， personalized medicine， and other future directions. Thanks to their unique biocompatibility and programmability， engineered exosomes are expected to serve as a "next-generation drug delivery platform"， providing a new paradigm for precision medicine and holding broad clinical application prospects.

Advances in Point-of-care Testing Technologies for Pathogen Detection
Shuo CAO, Zhengkun ZHANG, Chenchen LI
2026, 16(1): 53-60. DOI: 10.19586/j.2095-2341.2025.0091
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Pathogens are microorganisms that can cause infectious diseases， posing a serious threat to human health and imposing economic and social burdens. Therefore， developing highly sensitive， specific and portable real-time pathogen detection methods is of vital importance for preventing the spread of infectious diseases. Point-of-care testing （POCT） is an in vitro diagnostic method that uses portable devices and reagents at the sampling point for rapid detection and immediate acquisition of results. Portable biosensors， as an important implementation form， have advantages such as rapid detection， low cost， and easy large-scale and rapid application， and have attracted much attention in pathogen detection. The latest progress of portable biosensors in the real-time detection of bacteria and viruses is systematically reviewed. Based on the signal sensing mechanism， the detection methods such as electrochemical， colorimetric， fluorescence， surface-enhanced Raman and surface plasmon resonance were mainly discussed. Meanwhile， the portable sensor platform constructed by integrating lateral flow assay （LFA） and microfluidic technology was summarized， and its future development was prospected， aiming to provide references for further research and technological development in this field.

Pathological Correlation Mechanism of MicroRNA in Alzheimer′s Disease and its Potential for Early Diagnosis
Xiaocong GUO, Xiaoya XU, Ziling XU, Honglin LUO, Xiaoqian MAO, Yongcan GUO
2026, 16(1): 61-67. DOI: 10.19586/j.2095-2341.2025.0130
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Alzheimer's disease （AD） is the most common neurodegenerative disease. MicroRNAs （miRNAs）， as important molecules regulating gene expression， play a key role in neurodegenerative diseases. This review summarized the research progress on the pathological association mechanisms between miRNAs and AD， as well as their relevance to early diagnosis. It focused on exploring the critical regulatory roles of miRNAs in the core pathological processes of AD， such as regulating the production of β-amyloid （Aβ） by targeting genes like β-site amyloid precursor protein cleaving enzyme 1 （BACE1）， affecting the phosphorylation process of Tau protein by regulating kinases like microtubule affinity regulating kinase 1 （MARK1） and cyclin-dependent kinase 5 （CDK5）， and extensively participating in the regulation of neuroinflammation （e.g.， miR-155， miR-146a） and oxidative stress （e.g.， miR-34a）. Meanwhile， this review summarized the great potential of miRNAs as AD biomarkers and discussed the current challenges and future directions， aiming to provide references for the research and clinical treatment of Alzheimer's disease.

Phthalate Exposure and Female Reproductive Health
Jingshu ZHAO, Huan LI, Yingxuan DU, Jingya CUI, Dongyu ZHANG, Yunyao LUO, Haoke SONG, Hongshu SUI, Wenlong MA, Yutong SUN, Yan YANG
2026, 16(1): 68-75. DOI: 10.19586/j.2095-2341.2025.0143
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Phthalate esters （PAEs） are a class of widely distributed environmental endocrine disruptors that can lead to widespread human exposure through multiple pathways， posing significant threats to female reproductive health. Current epidemiological findings remain inconsistent， and the toxic mechanisms of PAEs on the ovaries， uterus， and pregnancy outcomes have not yet been fully elucidated， which limits accurate risk assessment and the development of prevention and control strategies. This review systematically summarized the characteristics of human exposure to PAEs， with a focus on their multifaceted reproductive toxicity mediated by disrupting various hormonal balances and signaling pathways. Key manifestations and mechanisms include inhibiting folliculogenesis， reducing anti-Müllerian hormone levels， disrupting steroidogenesis， increasing the risk of polycystic ovary syndrome， promoting the proliferation of uterine leiomyomas and the invasion of endometriosis， as well as significantly elevating the risks of adverse outcomes such as embryo implantation failure， abnormal placental development， pregnancy loss， and preterm birth. By integrating multidisciplinary evidence， this study systematically deciphered the mechanisms of action， providing a scientific basis for comprehensively assessing the female reproductive risks associated with PAEs and formulating targeted intervention measures.

Effects of Nitrogen Application on Yield-quality Formation and Aroma Compounds Accumulation in Flue-cured Tobacco ‘Yunyan 87’
Xiaochun REN, Xin LIU, Ying HAN, Kai DUAN, Feng LIU, Ruili HAO, Huan SI, Yingqun HUANG, Jianjun XIA
2026, 16(1): 76-85. DOI: 10.19586/j.2095-2341.2025.0123
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Nitrogen application rate is a critical factor affecting both the yield and quality of tobacco leaves； however， excessive nitrogen application is prevalent in practical production， resulting in a contradiction between tobacco leaf yield and quality. To clarify the appropriate nitrogen application range and improve tobacco leaf production efficiency， field experiments were conducted using ‘Yunyan 87’ as the test material， with three nitrogen application gradients set： low nitrogen （pure nitrogen 135 kg·hm^-2）， medium nitrogen （150 kg·hm^-2）， and high nitrogen （165 kg·hm^-2）. The effects of different nitrogen levels on agronomic traits， yield， output value， as well as appearance quality， sensory quality， physical characteristics and aroma components of flue-cured tobacco were systematically evaluated. The results showed that the high nitrogen treatment group significantly promoted plant growth and development： compared with medium and low nitrogen treatments， plant height increased by 6.0% and 11.7% respectively， and leaf width increased by 9.3%~13.3%； the yield of flue-cured tobacco after curing increased by 7.4% and 18.4% respectively， and the appearance quality score was also improved. Nevertheless， high nitrogen treatment significantly reduced the accumulation of aroma-forming components： the total content of aroma-forming components in middle leaves was 86.6% of that in the low nitrogen group， and the scores of several sensory evaluation indices were lower than those in the low nitrogen group. In contrast， low nitrogen treatment led to a more than 10% decrease in tobacco leaf yield， but significantly promoted the accumulation of aroma-forming components such as terpenoids， and the sensory quality performance was also better. These findings indicate that optimizing nitrogen application is a key approach to achieving the coordinated unification of tobacco leaf yield and quality. Although excessive nitrogen application can improve yield and appearance quality， it reduces the accumulation of aroma-forming components in tobacco leaves， thereby impairing their intrinsic quality. Therefore， the current nitrogen application strategy should be optimized according to actual production conditions to ensure the high-quality and efficient production of tobacco leaves and the sustainable development of the industry.

Effects of Drought Stress on Physiological Characteristics of Licorice Seedlings
Dong ZHANG, Xiaoyan ZHANG, Jiale LI, Yuanxin ZHANG, Fenglan ZHANG, Zhongren YANG, Jianwen LIU, Hua XU
2026, 16(1): 86-93. DOI: 10.19586/j.2095-2341.2025.0140
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To explore the effects of drought stress on the physiological characteristics and drought resistance adaptation mechanism of the seedlings of the medicinal plant Glycyrrhiza uralensis， one-year-old licorice seedlings were used as materials. Using potted plants with controlled water supply to simulate drought stress， we setted up normal water supply control （CK）， light stress （LS）， moderate stress （MS） and severe stress （SS） gradients， and measured key physiological parameters such as the antioxidant system and osmotic regulatory substances. The results showed that with the intensification of drought stress， the contents of malondialdehyde （MDA） and H₂O₂ in the leaves and roots of licorice generally showed an increasing trend， while the content of $O 2 -$ in the roots of licorice in the MS group and the SS group was lower than that of CK. The total antioxidant capacity and peroxidase （POD） of roots both showed a trend of first increasing and then decreasing， while the POD and catalase （CAT） activities of leaves first decreased and then increased， and the superoxide dismutase （SOD） activities of leaves and roots both showed a trend of first increasing and then decreasing. After drought stress， the ascorbic acid （ASA） content， glutathione peroxidase （GPX） and GSSG activities in licorice leaves and roots were all higher than those in CK. In conclusion， studies have shown that licorice seedlings can resist mild to moderate drought stress by enhancing the antioxidant enzyme system in leaves and roots and accumulating osmotic regulatory substances. Severe drought exceeds their physiological tolerance threshold， and the drought resistance of leaves is stronger than that of roots. This result provides a physiological basis for water management and drought-resistant germplasm screening of artificial cultivation of licorice in arid areas.

Comprehensive Evaluation of Salt Stress Tolerance of Seedling Stage of Mustard Germplasm Based on Membership Function Method
Xuming XIE, Qinwen LEI, Jingzhou WANG, Li CHEN, Huachuan HE, Zhengjie WAN, Heping WAN
2026, 16(1): 94-104. DOI: 10.19586/j.2095-2341.2025.0131
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As one of China's distinctive vegetable crops， mustard serves as an excellent experimental material for investigating the effects of salt stress—a key abiotic stress factor—on plant growth during the seedling stage. The germination performance and root morphology of 132 mustard germplasm accessions were evaluated under both normal conditions and optimal salt stress （1.0% NaCl） derived from a salt tolerance screening system tailored for the germination-to-seedling stage. Comprehensive analysis was conducted using growth status， salt tolerance indices for root traits， and membership functions to derive the D-value， a composite measure of salt tolerance. Ultimately， the 132 Brassica juncea accessions were classified into 38 salt-sensitive types， identifying 3 highly salt-tolerant accessions （D>0.6）， 22 salt-tolerant accessions （0.3<D<0.6）， and 69 salt-intolerant accessions （D<0.3）. Among them， a principal component analysis was conducted on the root salt tolerance coefficients， resulting in a composite index 1 （CI₁） and CI₂ with contribution rates of 66.886% and 26.835%， respectively. This demonstrated that ST-AD hud stronger independence when CI₂ accounted for a higher proportions. Other root coefficients exhibited linear relationships with D values， enabling regression equation construction. In conclusion， the root coefficient of rapeseed can be used to more conveniently assess salt tolerance comprehensively， adding a new approach to evaluating the salt tolerance of rapeseed.

Study on Quality Evaluation of Dandelion Based on HPLC Fingerprint and Chemometrics
Bin LI, Yumei ZHOU, Xia WANG, Zhijun SONG, Bingxiong YAN
2026, 16(1): 105-114. DOI: 10.19586/j.2095-2341.2025.0110
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To comprehensively evaluate the quality characteristics of dandelion from Huining region， this study established an high performance liquid chromatography（HPLC） fingerprint and conducted similarity evaluation. Chemical pattern recognition methods， including principal component analysis （PCA） and orthogonal partial least squares discriminant analysis（OPLS-DA）， were applied to analyze 15 batches of dandelion samples. Additionally， multi-component content determination was performed to comprehensively assess the quality of dandelion from Huining region. The results showed that the dandelion fingerprint contained 20 common peaks， with similarity values greater than 0.89 for 13 batches except S3 and S12. PCA classified the 15 batches into two categories based on their origin and OPLS-DA further identified five differential makers， namely caffeic acid， neochlorogenic acid， ferulic acid， chicoric acid， and chlorogenic acid. The significance of differences ranked as follows： caffeic acid>neochlorogenic acid>ferulic acid>chicoric acid>chlorogenic acid. The content determination results indicated that the chicoric acid content in all dandelion samples exceeded the current pharmacopoeia standards， and the levels of caffeic acid and chicoric acid in cultivated dandelion were generally higher than those in wild dandelion. The established comprehensive quality evaluation method for dandelion effectively analyzed the differences in quality among different batches， providing a scientific basis for quality assessment， cultivation， and the development of health products derived from dandelion.

Molecular Characteristics, Phylogeny and Spatiotemporal Expression Profile of the Cytochrome c Gene in Apis mellifera
Jingxian LI, Shiyu MI, Zhenzhen ZUO, Wei WANG, Zinuo LI, Haining GONG, Dafu CHEN, Rui GUO, Jianfeng QIU
2026, 16(1): 115-122. DOI: 10.19586/j.2095-2341.2025.0141
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This study focused on the cytochrome c gene （CytC） in Apis mellifera， with the aim of characterizing its molecular features， phylogenetic evolution， and spatiotemporal expression patterns. The coding sequence （CDS） of CytC was amplified by PCR and verified through Sanger sequencing alignment. Bioinformatics tools including ProtParam， Porter5， SWISS-MODEL and STRING were employed to predict the physicochemical properties， structural domains， phosphorylation sites， and protein-protein interaction networks of CytC. Phylogenetic analysis of CytC proteins from 10 species was conducted using MEGA software. RT-qPCR was used to examine the expression profiles of CytC across seven tissues of worker bees， larval developmental stages， and different ages of adult bees. The results indicated that a 327 bp CytC CDS encoding 108 residues， with predicted molecular mass of 11 kD and isoelectric point at 9.50. The CytC polypeptide lacks signal peptides and transmembrane domains but harbors 13 putative phosphorylation sites. And its secondary structure is primarily composed of random coils （48.15%） and α-helices （25.93%）. Phylogenetic analysis revealed that the CytC protein of A. mellifera， is clustered with that of A. florea and A. cerana， exhibiting marked divergence from Bombus species. Spatiotemporal expression profiling indicated that CytC expression was highest in the brain and lowest in the hypopharyngeal glands. During development， CytC expression was highest at the egg stage， significantly exceeding that at all other stages （P<0.05）， and gradually decreased throughout larval development. In adult bees，CytC expression increased with age， peaking at 18 days after eclosion and reaching the lowest level at 1 day. In summary， CytC is a hydrophilic intracellular protein with conserved molecular characteristics and functions， potentially playing a significant role in the development of western honey bees.

Enzymatic Properties of Glucose Oxidase from Aspergillus chevalieri and Optimization of its Specific Activity
Meiqi REN, Yuan WANG, Yingguo BAI, Jian TIAN, Huoqing HUANG, Haomeng YANG
2026, 16(1): 123-130. DOI: 10.19586/j.2095-2341.2025.0122
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This study introduced the AcGox gene from Aspergillus chevalieri into a Aspergillus niger strain via protoplast transformation technology， yielding the recombinant AcGox protein. Results indicated that recombinant AcGox exhibits optimal activity at pH 6.0 and 32 ℃， demonstrating excellent acid tolerance and good thermal stability. However， the specific activity of AcGox was only 70.81 U·mg^-1，to be a critical bottleneck limiting its application. To address this issue， this study further overexpressed genes encoding riboflavin kinase and FAD synthase in the AcGox-5 strain. RT-qPCR validation confirmed significantly elevated gene transcription levels. Ultimately， the specific activity of glucose oxidase produced by AcGox/FAD/FMN-3 strain increased to 111.55 U·mg^-1，with a 57.5% improvement. This study confirmed that cofactor FAD supply levels are a critical factor limiting the catalytic efficiency of recombinant AcGox. It provides direct experimental evidence for the "cofactor engineering" strategy in enzyme engineering and offers a scalable host modification approach for industrial glucose oxidase production， advancing the application of glucose oxidase.

Screening and Optimization of Fermentation Medium for Biocontrol Bacillus velezensis SD23LJ1314
Menghua ZHANG, Yuanhang QU, Peipei WANG, Lihong DONG, Jiaqi ZHANG, Yifan FU, Wei YANG, Qinggang GUO, Shezeng LI, Xuejun WEI
2026, 16(1): 131-139. DOI: 10.19586/j.2095-2341.2025.0114
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Bacillus velezensis SD23LJ1314 is a biocontrol strain that has excellent efficacy against Phytophthoracapsici， and its fermentation level directly affects the field control effect. To enhance the fermentation level of this strain， this study used the bacterial quantity as the core evaluation index. Firstly， the fermentation bacterial quantity of 8 commonly used industrial culture media was compared using the plate counting method， and the most suitable basic culture medium for SD23LJ1314 fermentation was selected. On this basis， through single-factor experiments， 11 commonly used industrial fermentation carbon sources and 9 nitrogen sources were optimized to determine the optimal carbon and nitrogen sources. Subsequently， the Plackett-Burman experimental design was further adopted， combined with the Box-Behnken design and response surface analysis method， to precisely optimize the addition concentration of the optimal carbon and nitrogen sources. The experimental results showed that fructose and yeast powder were the optimal carbon source and nitrogen source combinations， respectively. After optimization by the above methods， the final optimized culture medium formula was determined as follows， 10.75 g·L^-1 fructose， 21.48 g·L^-1 yeast extract， 5.65 g·L^-1 NaCl， and 0.71 g·L^-1 MnSO₄·7H₂O. Under this optimized condition， the fermentation bacterial quantity of this strain reached 9.32×10⁹ CFU·mL^-1， and the bacterial yield was significantly increased by 31.6% compared to the basic culture medium. This study through a series of experiments clarified the efficient fermentation process of Bacillus velezensis SD23LJ1314. The optimized culture medium formula and related technical parameters obtained have laid a solid foundation for the large-scale production of this biocontrol strain and have significant value for promoting its application in the development of biological control agents for Phytophthora capsici.

Effect of Reprocessing on the Quality of Medicinal Aromatic Vinegar and its Nanostructure Revelation
Yanhui WANG, Xinyue LAN, Yitong LIU, Shenghao LIN, Ming CHEN, Wentao XU, Longjiao ZHU
2026, 16(1): 140-148. DOI: 10.19586/j.2095-2341.2025.0135
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The development of functional vinegars represents a key direction for the diversification and value-added upgrading of the vinegar industry. To evaluate how different reprocessing methods improve the product， we systematically compared the nutritional composition and functional activity of “Huatuo medicinal aromatic vinegar” subjected to either soaking （25 ℃） or boiling， using liquid chromatography-tandem mass spectrometry （LC-MS/MS） technology， in-vitro antioxidant activity assays， and other methods. The results showed that the soaking process increased the nutritional content of the product， with a 10.43% and 30.04% increase in total organic acids and total amino acids， respectively， but did not reach a significant level. Meanwhile， soaking also markedly improved antioxidant capacity， elevating 1，1-diphenyl-2-picrylhydrazine （DPPH） and 2， 2'-azino-bis（3-ethylbenzothiazoline-6-sulfonic acid）（ABTS） radical-scavenging activities by 87.39% and 58.36% （P<0.05）. Importantly， scanning electron microscopy （SEM） revealed intrinsic nanostructures within the vinegar， and their morphology， particle size， and Zeta potential were distinctly modulated by the reprocessing method employed. Collectively， soaking is an effective strategy for enhancing the nutritional and functional properties of medicinal aromatic vinegar， and the discovery of these process-tunable nanostructures offers direct microscopic evidence for the enhanced efficacy.

Fluorescence PCR Detection Method for Shiitake-derived Components in Food
Lina SU, Fengxin LI, Jie WU, Xiandong ZENG, Jian ZHENG
2026, 16(1): 149-156. DOI: 10.19586/j.2095-2341.2025.0075
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Lentinula edodes （Shiitake mushrooms） possess significant edible and medicinal value. However， adulteration frequently occurs in the marketplace， and traditional morphological identification methods struggle to authenticate L. edodes in deeply processed products. Establishing a molecular biological detection method for L. edodes-derived components is therefore of great importance. In this study， a real-time fluorescent PCR detection method for L. edodes-derived components in food was developed， utilizing specific primers and probes designed based on the sequence of the mitochondrial NADH dehydrogenase subunit 1 （ND1） gene of L. edodes. This method specifically amplified DNA from various L. edodes varieties （including winter shiitake， thick-cap shiitake， thin-cap shiitake， and wild L. edodes）， while showing no cross-reactivity with 28 other common edible fungi. The detection limits of the method were determined to be 100 copies·μL?1 and 0.05% （mass fraction）， with excellent repeatability and verified accuracy in practical applications. This highly specific， sensitive， stable， and practical method effectively meets the requirements for detecting L. edodes-derived components in food products.

Evaluation of Detection Methods for Burkholderia cepacia Complex in Hydrosol Cosmetics
Cong LI, Zhao FU, Yakun JI, Likang AN, Cuihong WU, Liye ZHANG, Yijun ZHANG, Yuyan ZHOU, Zhenxia ZHAO
2026, 16(1): 157-163. DOI: 10.19586/j.2095-2341.2025.0099
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This study aimed to evaluate the detection method for Burkholderia cepacia complex （Bcc） in hydrosol cosmetics. A total of 20 batches of hydrosol cosmetics known to be contaminated with Bcc were subjected to Bcc detection and determination using the plate method. Additionally， matrix-assisted laser desorption/ionization time-of-flight mass spectrometry （MALDI-TOF MS）， polymerase chain reaction （PCR）， and phylogenetic analysis were employed to identify the isolated Bcc strains and analyze their genetic characteristics. The results showed that the detection results of the plate method had a good correspondence with those of MALDI-TOF MS and phylogenetic analysis based on gene sequencing， indicating that the plate method is simple， highly specific， and can be used for the detection of Bcc （an unacceptable microorganism） in hydrosol cosmetics and fot the establishment of standard method.

Establishment and Research of MRC-5 Working Cell Bank for Rabies Vaccine Production
Jing ZUO, Lu ZHAO, Chunling MA, Jing ZI, Chao CHENG, Yong LIU, Weiye ZHANG, Wei TONG
2026, 16(1): 164-169. DOI: 10.19586/j.2095-2341.2025.0112
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To establish a working cell bank of medical research council 5 （MRC-5） cells and conduct relevant research on the application of this cell bank in the production of inactivated rabies vaccine， the MRC-5 working cell bank was established in strict accordance with the Pharmacopoeia of the People's Republic of China， and all mandatory tests were performed following the pharmacopoeial specifications. Meanwhile， the growth curve， passage stability， and rabies virus susceptibility of MRC-5 cells were systematically investigated. The cells from the established MRC-5 working cell bank exhibited a typical long spindle shape with clear edges and distinct outlines， and the cell viability reached 99.03%. The results of sterility test， mycoplasma test， and exogenous factor test all conformed to the pharmacopoeial requirements. The growth curve of MRC-5 cells presented a standard sigmoidal （S） shape， with cells entering the logarithmic growth phase in 2~3 days and a cell doubling time of 21 hours. Moreover， the cell morphology remained normal even after continuous subculture to the 66th passage. After inoculation of the PM strain of rabies virus into MRC-5 cells of 36， 38， 40 and 42 passages， the mean virus titers were determined as 6.57±0.21， 6.40±0.26， 6.23±0.21 and 6.40±0.17 lgCCID??·mL^-1， with the coefficient of variation （CV） ranging from 2.70% to 4.13%. In conclusion， the established MRC-5 working cell bank meets the requirements specified in the Pharmacopoeia of the People's Republic of China. It shows excellent passage stability and high susceptibility to rabies virus， indicating that it is suitable for the industrial production of inactivated rabies vaccine.

Study on Optimizing Antibody Screening Strategy by Using pH Characteristics of Tumor Microenvironment
Jinxiang ZHANG, Xiaodan CAO, Wenlei LI, Junjun DAI, Yan YU, Hanmei XU
2026, 16(1): 170-177. DOI: 10.19586/j.2095-2341.2025.0134
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Conventional antibody screening is based on physiological pH conditions for affinity evaluation and screening， which may ignore the influence of acidic characteristics of tumor microenvironment on antibody binding. The purpose of this study is to establish a multi-pH gradient screening strategy to screen 5T4 antibody clones with high affinity and stability under acidic pH conditions by simulating tumor microenvironment conditions， so as to improve the clinical transformation success rate of antibody development. The 5T4 hybridoma clones obtained in the previous screening were selected. After the antibody concentration in the supernatant was quantified， a multi-pH gradient （7.4， 7.0， 6.6，6.0， 5.5） experimental system was constructed by Biacore T200 system， and the dynamic changes of antibody affinity were monitored in real time by surface plasmon resonance technology. The results showed that the affinity of high affinity antibody 5T4-15 screened under normal physiological conditions decreased gradually with the decrease of pH in acidic environment， and it lost its binding ability to antigen when pH was 5.5. In contrast， the antibody 5T4-45 obtained by pressure screening at pH 5.5 can maintain stable high affinity in the tumor microenvironment related range of pH 7.4 to 6.0， showing excellent adaptability to acidic environment. The introduction of acid stress test （pH 5.5） in the initial antibody screening stage could effectively identify and screen tumor microenvironment-adaptive antibodies， break through the limitation of screening high-affinity antibodies at conventional physiological pH， improve the screening efficiency of high-quality candidate molecules， and provide a new strategy for developing therapeutic antibodies for solid tumors with higher clinical transformation potential.

The Relationship Between the Expression Level of Ferroptosis-related Gene NCOA4 and Prognosis and Therapeutic Efficacy in Colon Cancer
Xiaolin LIU, Qihui WANG
2026, 16(1): 178-189. DOI: 10.19586/j.2095-2341.2025.0038
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As a ferroptosis gene， nuclear receptor coactivator 4 （NCOA4） protects cells by eliminating free iron and glutathione. To explore the value of NCOA4 in predicting the therapeutic efficacy of colon cancer， its expression in colon cancer was analyzed using data from The Cancer Genome Atlas （TCGA）， with the aim of investigating its associations with prognosis， the immune microenvironment， and treatment response. The results showed that NCOA4 expression was significantly lower in colon cancer tissues than in normal tissues （P<0.05）， and its low expression was correlated with shorter overall survival （P<0.05）. Differentially expressed genes between the high and low NCOA4 expression groups were mainly enriched in immune- and tumor-related pathways. Additionally， NCOA4 expression was associated with the immune microenvironment， immune cell infiltration， and immune checkpoints. Further analyses of drug sensitivity and immunotherapy response revealed that colon cancer patients with low NCOA4 expression may exhibit better therapeutic responses to cytotoxic T-lymphocyte-associated protein 4 （CTLA4）-targeted therapy and chemotherapeutic agents such as Raf kinase inhibitors. In conclusion， low NCOA4 expression is associated with poor prognosis and impaired immune cell infiltration in colon cancer. Combined immune checkpoint-targeted therapy and chemotherapy may effectively treat colon cancer patients with low NCOA4 expression.

Effects and Mechanisms of TWIST1 on Mitochondrial Function and Chemoresistance in Acute Myeloid Leukemia Cells
Yiwen LU, Yiyi DING, Nini GUO, Wanling HUANG, Jingwen XU-ZHANG, Peiwen ZHANG, Nan WANG, Qian REN, Xiaotong MA
2026, 16(1): 190-195. DOI: 10.19586/j.2095-2341.2025.0120
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The aim of this study was to investigate the effects of transcription factor TWIST1 on autophagy in acute myeloid leukemia （AML） cells， as well as whether it regulates mitochondrial function and tolerance to the chemotherapeutic agent cytarabine via autophagy. Lentiviral vectors carrying TWIST1 shRNA and scramble control were constructed and transduced into AML cells. Western blotting was performed to detect the expression levels of LC3-Ⅱ and p62 in TWIST1-knockdown AML cells for analyzing autophagic flux. TMRM and MitoTracker staining were used to determine the effects of TWIST1 knockdown on mitochondrial membrane potential and mass of AML cells. Annexin V/7-AAD staining was applied to assess the changes in cell apoptosis level and the sensitivity of AML cells to cytarabine. Additionally， TWIST1 shRNA-infected cells were treated with sirolimus （an autophagy activator） to evaluate its intervention effect on the aforementioned processes. The results confirmed that TWIST1 knockdown inhibited autophagy， characterized by decreased LC3-Ⅱ expression and p62 accumulation. Concomitantly， impaired mitochondrial function and a significant increase in cell apoptosis were observed （P<0.05）. Sirolimus could partially restore autophagic flux， improve mitochondrial function， and reduce the apoptosis rate. Moreover， TWIST1 knockdown enhanced the sensitivity of AML cells to cytarabine， which could be partially reversed by sirolimus. In conclusion， TWIST1 affects mitochondrial function and chemosensitivity of AML cells by regulating autophagy. These findings provide a theoretical basis for the mechanism of drug resistance in AML and autophagy-targeted therapy.

Transcriptional Group Analysis Identifies Lactylation-related Genes in Triple-negative Breast Cancer and Establishes a Prognostic Model
Haiyue ZHANG, Wenjie GUO, Xiaoyi ZHAI, Lili SUN, Zhongqi DIAO, Wenjia GUO
2026, 16(1): 196-204. DOI: 10.19586/j.2095-2341.2025.0121
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Triple-negative breast cancer exhibits metabolic reprogramming characteristics， with abnormal activation of the glycolytic pathway being one of its key features. As the end product of glycolysis， lactate functions as a signaling molecule between cells， organs， and tissues， thereby promoting tumor invasion and metastasis. Recent studies have revealed a novel biological function of lactate： it serves as a substrate for a novel post-translational modification termed "lactylation". Consequently， this study explored the role of lactylation-related genes in triple-negative breast cancer. Transcriptome data and corresponding clinical information from 151 triple-negative breast cancer samples and 113 adjacent normal samples were downloaded from The Cancer Genome Atlas （TCGA）. Differential analysis， pathway enrichment， univariate， and multivariate Cox analyses identified lactylation-associated prognostic genes. A prognostic model incorporating lactylation-related genes was constructed using the TCGA-TNBC training cohort and validation was performed using the GSE21653， GSE58812， and METABRIC-TNBC cohorts. Samples were stratified into high- and low-lactylation risk groups based on lactylation risk scores. Survival analysis， receiver operating characteristic curves， and scatter plots further validated model performance， with additional analysis exploring associations between the model and immune cell infiltration/drug sensitivity. The results showed that a prognostic model based on four lactylation-related genes has good predictive ability for triple-negative breast cancer patients. At the same time， patients in the high- and low-lactylation risk groups exhibited significant differences in tumor immune microenvironment infiltration characteristics and drug response sensitivity. This finding suggests that lactylation-related genes may have potential as novel molecular biomarkers and therapeutic targets for triple-negative breast cancer.

Research on CPEB1 Reversing Chemoresistance of Ovarian Cancer Cells by Inhibiting Epithelial-mesenchymal Transition
Lipei GUO, Qunchang ZHANG, Xin CAO, Xiuling ZHANG
2026, 16(1): 205-212. DOI: 10.19586/j.2095-2341.2025.0124
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This study aimed to investigate the role of cytoplasmic polyadenylation element-binding protein 1 （CPEB1） in cisplatin （cis-diamminedichloroplatinum Ⅱ， DDP） resistance in ovarian cancer and its underlying mechanism. The expression of CPEB1 in ovarian cancer tissues and drug-resistant cell lines was analyzed using the Gene Expression Profiling Interactive Analysis （GEPIA） and Gene Expression Omnibus （GEO） databases. Cisplatin-resistant ovarian cancer cell lines A2780/DDP and SKOV3/DDP were established by the concentration gradient increment method. Cell viability and half-maximal inhibitory concentration （IC??） were detected via the CCK-8 assay. The mRNA and protein expressions of CPEB1 were determined using RT-qPCR and Western blot analysis， respectively. CPEB1-overexpressing plasmids were transfected into A2780/DDP and SKOV3/DDP cells. Cell invasion and migration abilities were evaluated by Transwell assay and wound healing assay， respectively. The expressions of epithelial-mesenchymal transition （EMT）-related marker proteins （E-cadherin， N-cadherin， Vimentin） were detected by Western blot analysis. The results showed that CPEB1 was lowly expressed in ovarian cancer tissues and cisplatin-resistant cell lines. Overexpression of CPEB1 significantly enhanced the sensitivity of A2780/DDP and SKOV3/DDP cells to cisplatin， reduced their IC?? values， and inhibited cell invasion and migration capacities. Meanwhile， overexpression of CPEB1 upregulated the expression of the epithelial marker E-cadherin， downregulated the expressions of the mesenchymal markers N-cadherin and Vimentin， and thereby reversed the EMT process. In conclusion， CPEB1 can enhance the sensitivity of ovarian cancer cells to cisplatin and reverse their drug-resistant phenotype by inhibiting the EMT process， indicating that it may serve as a potential therapeutic target for overcoming cisplatin resistance in ovarian cancer.

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