Current Biotechnology

Research Progress on the CRISPR/Cas System in Zebrafish
Ali WANG, Jiangdong LIU
2023, 13(4): 485-491. DOI: 10.19586/j.2095-2341.2023.0033
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The zebrafish is an important model organism in biology and a useful tool for gene function analysis， human disease pathology research， and new drug development. It has advantages of easy operation and close evolutionary relationship with humans. Currently， various zebrafish models have been developed for the study of human-related diseases. The emergence of clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins （CRISPR/Cas） technology with regularly spaced short palindromic repeats and their associated proteins has greatly reduced the complexity of zebrafish gene editing. This article mainly described the basic principles and technical innovations of the CRISPR/Cas system， and summarized the important role of the CRISPR/Cas system in zebrafish gene knockout， knock-in， live-cell imaging， transcriptional regulation， multiple targeting， and disease model establishment， with a view to providing a certain idea for exploring the application of the CRISPR/Cas system in zebrafish genomics research.

Advances on Research and Application of mRNA Vaccines
Dan YU, Yunlong MA, Fang WAN, Jianqiang WU
2023, 13(4): 492-498. DOI: 10.19586/j.2095-2341.2023.0015
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Compared with traditional vaccines， mRNA vaccines have the advantages of high efficiency， safety and low cost， but its application has been limited by technical problems such as unstable in vivo delivery and low translation efficiency. The prevalence of SARS-CoV-2 virus and their vaccine development have accelerated the development and approval of mRNA vaccines， especially breakthroughs in mRNA structural modification and construction of lipid nanoparticles， for example， the stability of mRNA vaccines using optimized nucleoside and cap structures is greatly improved， and the replacement of codons with low frequency of use can improve their translation efficiency. The commonly used mRNA delivery systems are lipid nanoparticles， polymer vectors， virus-like vectors， etc. The research and application progress of development history， mechanism of action， technological breakthroughs in modification， and delivery systems of mRNA vaccines were reviewed in order to promote the in-depth research and application of mRNA vaccines.

Research Progress on Heme Protein Targets of Gas Signaling Molecules
Jun LI, Yifei SONG, Yang YI, Chen MA, Ziyi ZHANG⁃HUANG, Linlin DU, Junyu LI, Fei XIE, Xuemei MA
2023, 13(4): 499-508. DOI: 10.19586/j.2095-2341.2023.0044
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Gas signaling molecules， including nitric oxide， carbon monoxide， and hydrogen sulfide， play crucial roles in regulating physiological processes such as vasodilation， neurotransmission， platelet aggregation， immunity， cell proliferation， and mitochondrial respiration. Studies have shown that heme protein in cells is one of the main receptors of gas signal molecules， and they all have the same cofactor—heme. Heme is a ring composed of four pyrrole subunits， with a center chelating a ferrous ion group. It is a co-group of proteins such as hemoglobin， myoglobin， peroxidase and cytochrome. The intermediate ferrous ion can coordinate and bind with gas signaling molecules， widely participating in various physiological activities of organisms. In this paper， the types of heme proteins and downstream pathways involved in the action of gas signaling molecules were reviewed， with a view to providing reference for further study of their mechanisms.

Research Progress on Experimental Models of Autism Spectrum Disorders
Jingjing FANG, Kunlun HUANG, Tao TONG
2023, 13(4): 509-523. DOI: 10.19586/j.2095-2341.2023.0014
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Autism spectrum disorder （ASD） is a typical heterogeneous neurodevelopmental disorder induced by genetic or/and environmental factors. The main characteristics of ASD patients are social communication deficits， verbal communication disorders， and stereotyped behaviors. Currently， the pathogenesis of ASD is not clear， and there is a lack of effective treatment methods. This review focused on cell models and animal models used for ASD research in recent years， including PC12 cells， SH-SY5Y cells， human induced pluripotent stem cells， lymphoblastoid cell lines， and primary neurons， as well as non-mammalian models， prenatal valproic acid exposure models， BTBR idiopathic mouse models， maternal immune activation models， rodent models based on ASD susceptibility genes， and non-human primate models. We consolidated the key information on the available cell models and animal models of ASD and highlighted the advantages， limitations， construction methods， and important caveats of each model， which was expected to lay a foundation for exploring the pathogenesis and developing new diagnosis and treatment methods for ASD.

Research Progress of Oxidative Stress and Disuse Muscular Atrophy
Shiming LI, Peng ZHANG, Pengxiang ZHAO, Fei XIE, Xiaoping CHEN, Mengyu LIU
2023, 13(4): 524-533. DOI: 10.19586/j.2095-2341.2023.0025
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Skeletal muscle has a high degree of plasticity， which can be changed according to the functional needs. Decreased or discontinued activity usually leads to muscle atrophy and metabolic dysfunction. The loss of skeletal muscle mass due to a dramatic decrease in muscle load and inhibition of nerve activation is commonly referred as disuse muscle atrophy. The contractile activity， high oxygen consumption and metabolism of skeletal muscle enable it to continuously produce appropriate amounts of oxidizing substances， such as reactive oxygen species（ROS）. When the oxidation products exceed the antioxidant defense ability， the body will enter a state of oxidative stress. During long-term skeletal muscle discontinuation， oxidative stress promotes skeletal muscle atrophy by increasing the degradation of protein in at least three ways， or by inhibiting mRNA translation and inhibiting protein synthesis at the initial level. With the rapid development of space industry in China， disuse muscle atrophy in weightlessness is also an urgent problem to be solved in space medicine. The specific purpose of this study was to outline disuse muscle atrophy and the role of oxidative stress in disuse muscle atrophy， in order to provide valuable reference for the clinical treatment and practice of disuse muscle atrophy.

Progress on Moist Healing Research
Ziyi LIU, Xujuan ZHANG, Zisong CAI, Chen MA, Mengyu LIU, Pengxiang ZHAO
2023, 13(4): 534-541. DOI: 10.19586/j.2095-2341.2023.0010
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Wound healing is the process of restoring tissue damage to its original morphological function， including coagulation， inflammation， proliferation， and remodeling. Conventional wound healing is mostly dry healing， characterized by platelet agglutination forming a blood scab covering the wound surface， and prone to scarring. If the wound is too humid， it will cause a chronic and difficult wound. Moist healing therapy is widely used in clinical treatment by maintaining suitable humidity to improve the wound microenvironment， promote the synergistic effect of various cells and factors， improve the wound healing speed and effect， avoid wound scabs and reduce the formation of ulcer surfaces. This paper reviewed the development of moist healing and the therapeutic mechanism of moist therapy， summarized the clinical application of medical dressings based on moist healing theory， and looked forward to the future development direction of moist healing therapy.

The Effects of Reactive Oxygen Species Mediated by Monoamine Oxidase A in Cardiovascular Diseases
Dilin DAI, Yuan WU, Mingwei BAO
2023, 13(4): 542-546. DOI: 10.19586/j.2095-2341.2023.0004
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Cardiovascular disease is one of the most common causes of death in the world， and its incidence rate is increasing annually. Therefore， it is becoming increasingly urgent to find effective methods for prevention， diagnosis and treatment of cardiovascular disease. Monoamine oxidase A （MAO-A）， a flavoenzyme located in the outer membrane of mitochondria， is responsible for the oxidative deamination of 5-hydroxytryptamine （5-HT） and norepinephrine （NE）， where accompanied by the accumulation of reactive oxygen species （ROS）， such as hydrogen peroxide （H₂O₂）， aldehyde and ammonia. Excessively produced ROS leads to mitochondrial dysfunction by targeting mitochondrial DNA， thereby accelerating ROS production， which is the main cause of cardiac oxidative stress and cardiac injury. Previously， MAO-A inhibitors were mostly used for the treatment of mental and nervous system diseases， but the roles of MAO-A in cardiovascular diseases have not been concerned until recently. Based on the above research findings， the role of ROS produced by MAO-A degradation of amines in cardiovascular diseases and its potential as a treatment for cardiovascular diseases were systematically reviewed， which was expected to provide reference for cardiovascular diseases treatment.

Reasearch Progress of Cryopreservation Technology in Biological Products
Ronglin LIU, Ning WANG, Yanyi LI, Weiting ZHANG
2023, 13(4): 547-555. DOI: 10.19586/j.2095-2341.2023.0045
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Biological products are prone to various physical and chemical degradation during storage， so long-term storage technology for biological products is necessary to slow down degradation， maintain biological activity and extend storage life. At present， low-temperature preservation technology is the most commonly used and effective method for long-term preservation of biological products， which can greatly reduce the activity decrease resulted from various physical and chemical degradation caused by liquid water during the storage of biological products. However， there are still some damages in the preservation process of biological products using low-temperature preservation technology， such as ice crystal damage， adsorption denaturation of ice water interface， and osmotic damage. The use of appropriate freezing processes and cryoprotectant agents （CPAs） plays an important role in reducing the negative impact of low-temperature preservation process. This article reviewed the research progress of low-temperature preservation technology in the preservation of biological products， including the influencing factors of low-temperature preservation （cryoprotectants， freezing processes and other influencing factors）， as well as the latest low-temperature preservation technologies， in order to provide reference for overcoming damage and maintaining activity of biological products during long-term preservation.

Cotton Drought Resistance Index Screening and Comprehensive Evaluation of Drought Resistance of Germplasm Resources During Germination Period
Jinping CHEN, Quanjia CHEN, Kai ZHENG, Yuanchun PU, Jianglin XU, Ting ZHOU, Yejun YANG, Guoqing SUN
2023, 13(4): 556-564. DOI: 10.19586/j.2095-2341.2022.0087
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Cotton is an important cash crop and oil crop. In order to study the relationship between morphological indicators and drought resistance at the germination stage of cotton seeds， the suitable drought resistance indexes in the germination stage of cotton were screened. Took 40 cotton varieties as materials， polyethylene glycol-6000 (PEG-6000) solution simulating drought stress， and clean water as control， seven indicators （relative seed water absorption， relative germination potential， relative germination rate， relative germination exponent， radicle/hypocotyl index， relative radicle stem/fresh weight， and relative germ stem/fresh weight） related to drought resistance in the germination stage of cotton were compared by correlation analysis， and the comprehensive drought resistance evaluation value was obtained according to the membership function， and the correlation analysis was carried out with seven indicators to obtain the most suitable drought resistance index. Results showed that the relative germination potential， relative germination rate and relative germination index were screened out as important indicators for the identification of drought resistance during the germination stage of 40 cotton varieties. Under drought stress， the drought tolerance of different varieties of cotton in the germination stage can be quickly and accurately identified and evaluated by measuring these three indicators. Among the 40 cotton materials tested， ‘Zhong 85271’ had the strongest drought resistance and ‘CF75 drop 6’ had the weakest drought resistance.

Physiological Response of Black Pepper (Piper nigrum L.) Cutting Seedlings to Waterlogging Stress
Huageng YANG, Bin LI, Na LI, Yimin YANG, Yougen WU
2023, 13(4): 565-574. DOI: 10.19586/j.2095-2341.2023.0053
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The aim of this study is to explore the physiological response mechanism of black pepper in response to waterlogging stress and provide scientific basis for preventing waterlogging stress injury of black pepper. Black pepper cutting seedlings which is used commonly in black pepper production were treated with pot waterlogging to investigate the effects of waterlogging stress on the production rate of superoxide anion， the content of hydrogen peroxide and malondialdehyde， the activity of antioxidant enzymes， the content of osmoregulation substances and photosynthetic pigments， and root activity at the 1st， 3rd， 6th， 9th day of the 9-day waterlogging treatment， respectively. Maintain 70%±5% of the field water capacity was used as the control group. The results indicated that when the plant was waterlogged for 1 day， the proline content of black pepper cutting seedlings increased by 27.24% compared to the control group， and there was no significant difference compared with the control group， while the superoxide anion production rate， the content of chlorophyll b（Chl b）， hydrogen peroxide and malondialdehyde increased significantly， and the difference were significant. And other measured physiological parameter value was lower than the control group to varying degrees. Meanwhile， the root tip of black pepper cutting seedlings began to brown and necrosis， while plants above ground part in black pepper cutting seedlings showing no symptoms of waterlogging injury were observed， and the leaves were still dark green. When the plant was waterlogged for 3 days， the content of soluble protein， proline and total chlorophyll of black pepper cutting seedlings increased slightly， and there was no significant difference compared with the control group. The activities of SOD and POD increased significantly and were significantly higher than that of the control group. Although Chl b content decreased， it was still higher than that of the control group. The superoxide anion production rate， the content of hydrogen peroxide and malondialdehyde decreased significantly， but still higher than the control group. However， superoxide anion production rate， malondialdehyde content had no significant difference compared with the control group. And other measured physiological parameter value was lower than the control group to varying degrees. Meanwhile， a few roots of black pepper cutting seedlings had rotted out， a small number of leaves turned from green to yellow， and there was no obvious wilting phenomenon. For 6 days after waterlogging， the activities of SOD， POD， CAT and APX，the content of soluble protein and soluble sugar， the ratio of Chl a to Chl b （Chl a/b） and carotenoid to total chlorophyll in black pepper cutting seedlings leaves decreased to varying degrees and were significantly lower than that of the control group， while the superoxide anion production rate， proline content， the content of Chl a and Chl b， the content of total chlorophyll and carotenoid， the content of hydrogen peroxide and malondialdehyde were increased to varying degrees and were significantly higher than that of the control group. Meanwhile， all roots of black pepper cutting seedlings had rotted away， and most or almost all the leaves wither， turn yellow， fall off and finally died. The results showed that there are sensitive to waterlogging stress and susceptible to waterlogging damage. The physiological response mechanism of black pepper cutting seedlings afraid of waterlogging was preliminarily revealed.

Analysis of Nutritional Ingredient and Active Contents from Abelmoschus moschatus Medicus
Qiaoyue ZHANG, Yuchao GUO, Hongchen JI, Houlin MAO, Dandan GU, Guanghua MAO, Ting ZHAO, Liuqing YANG
2023, 13(4): 575-579. DOI: 10.19586/j.2095-2341.2023.0023
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Abelmoschus moschatus Medicus was used as the research object to analyze its nutrition and active ingredients， which was expect to provide a theoretical basis for its deep processing and development of functional food resources. Nutritional contents and active contents from Abelmoschus moschatus Medicus were determined by national standard and colorimetric method. The results showed that okra was rich in total sugar （8 478.12 mg·100 g^-1 FW）， crude fibre （2 433.57 mg·100 g^-1 FW）， protein （386.32 mg·100 g^-1 FW） and essential amino acids. The crude fat content in okra was lower （110.84 mg·100 g^-1 FW） than that in other vegetables， and the crude fat was mainly composed of unsaturated fatty acids such as linoleic acid and so on， which accounted for about 48.95 % of the total fatty acids content. Okra was also particularly rich in potassium （1 309.21 mg·100 g^-1 DW）， which was a high K/Na ratio vegetable. Polysaccharide， polyphenols and flavonoids were main active substances in okra， their contents were 2 362.52， 65.64 and 12.01 mg·100 g^-1 FW. It can be seen that okra is a kind of green and healthy vegetable with rich nutrition， deserving further investigation on its application value.

Effect of tp53 Knockout on High Temperature Tolerance and Swimming Capacity in Zebrafish
Qianting YANG, Xiaoying TIAN, Junfang ZHANG, Bingshe HAN
2023, 13(4): 580-587. DOI: 10.19586/j.2095-2341.2023.0039
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The aim of the study was to investigate the effect of tp53 knockout on high temperature tolerance and swimming capacity in zebrafish. Through RT-qPCR， Western blot and other technologies， it was found that the expression of tp53 in wild-type zebrafish at high temperature was upregulated， suggesting that tp53 may play a role at high temperature， while zebrafish with the tp53 gene knockout had a reduced survival time at high temperature. Further studies found that tp53^-/- zebrafish had increased ROS levels （P<0.05）， decreased ATP levels （P<0.001）， and significantly increased levels of γH2A.X protein at high temperatures. It was found by hematoxylin-eosin（HE） staining that the liver tissue hyperemia of tp53^-/- zebrafish was more severe at high temperatures. Danio Vision behavior trajectory analysis showed that tp53^-/- zebrafish exercise capacity was weakened at 34 ℃. The results showed that tp53 has a positive effect on the high temperature tolerance and swimming capacity of zebrafish， which provided a new idea for the study of high temperature tolerance mechanism and exercise behavior in zebrafish.

The Effects of hdac11 Knockout of Zebrafish on Lipid Metabolism
Maolan XIONG, Siyan WEI, Juntao LUO, Bingshe HAN, Junfang ZHANG
2023, 13(4): 588-595. DOI: 10.19586/j.2095-2341.2023.0042
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To explore the function of histone deacetylase 11 （HDAC11） of zebrafish in lipid metabolism， we generated a hdac11^-/- zebrafish mutant line with CRISPR/Cas9 technology. We fed WT zebrafish and hdac11^-/- zebrafish with standard diet and high fat diet from 5 days post fertilization （dpf） to 90 dpf， measured their body weights and body lengths， observed histological sections and detected hepatic triglyceride content and lipid metabolism related gene expression of zebrafish. The results showed that there was no significant difference in body weight and body length betweeen hdac11^-/- mutants and WT zebrafish. Triglyceride content was significantly decreased in hdac11^-/- zebrafish compared to WT （P<0.001） at larval stage. The hepatic triglyceride content of adult zebrafish was significantly reduced under standard diet and high fat diet， and the liver lipid droplets were smaller. The expression levels of pnpla2 and lipeb were significantly increased， while the expression level of gpam was significantly decreased in liver. The results indicated that hdac11 participates in the lipid metabolic process in zebrafish and affects the regulation of lipid metabolism by changing the expression levels of genes involved in triglyceride synthesis and decomposition， which provides a new research direction for the epigenetic regulation mechanism to lipid metabolism in fish.

Characterization and Efficacy Evaluation of Recombinant Mussel Adhesive Protein
Min LI, Wenpei WEI, Sha QIAO, Dong HAO, Hao ZHOU, Shuowen ZHAO, Lifeng ZHANG, Zengmiao HOU
2023, 13(4): 596-603. DOI: 10.19586/j.2095-2341.2023.0021
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In order to promote the application of recombinant mussel adhesive protein in the medical and cosmetics field， the recombinant mussel adhesive protein obtained from scale fermentation and purification of Escherichia coli was characterized and its efficacy was evaluated. Amino acid N-terminal sequencing， relative molecular weight analysis， amino acid composition analysis， protein purity analysis， endotoxin content， dihydroxyphenylalanine（DOPA） content of recombinant mussel adhesive protein were determined by the following methods： Edman degradation， matrix-assisted laser desorption ionization time-of-flight mass spectrometry （MALDI-TOF-MS）， phenyl-isothiocyanate （PITC）， nonreductive SDS-polyacrylamide gel electrophoresis （SDS-PAGE）， gel method， modified Arnow. The efficacy of recombinant mussel adhesive protein was evaluated by cell migration and repairing effect of zebrafish tail fin. Results showed that the obtained recombinant mussel adhesive protein was confirmed to be consistent with the theoretical primary structure， protein purity of more than 95%， endotoxin <10 EU·mg^-1， DOPA content above 5%. When the recombinant mussel adhesive protein concentration was 60 μg·mL^-1， the effect of promoting cell proliferation was the most obvious， and it had very significant activity （P<0.01）. The caudal fin area of zebrafish in sample group was significantly increased compared with model control group （P<0.001）. The results indicated that recombinant mussel adhesive protein can promote cell migration and repair healing and has the potential to be used as biomedical materials.

Study of the Terpenoids from Stilbella sp. CGMCC 40422
Kainan SONG, Yutong AI, Yuquan XU
2023, 13(4): 604-611. DOI: 10.19586/j.2095-2341.2023.0030
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The compounds from Stilbella sp. CGMCC 40422 were isolated by silica gel column， ODS column and semi-preparative high performance liquid chromatography. And their structures were elucidated by nuclear magnetic resonance （NMR） and mass spectrum （MS） spectral analysis. The results showed that two prototerpene-type tetracyclic triterpenoids， one sesquiterpenoid and five meroterpenoids were isolated from the rice fermentation products of the fungus， and their structures were identified as helvolic acid， helvolinic acid， tricho-acorenol， ascofuranone， ilicicolin C， LL-Z1272?， deacetylchloronectrin ascochlorin and N-acetylglucosamine. The content of helvolic acid under different fermentation time was tested by HPLC chromatography. The yeild of helvolic acid reached a maximum of 3 g·kg^-1 after 15 days of fermentation， showing that Stilbella sp. CGMCC 40422 have strong production capacity of terpenoids. The results could lay the foundation for further development of the chassis cells of terpenoid cell factories.

SELEX of Vibrio parahaemolyticus Specific Aptamer
Jie LI, Xiaoyu YUE, Liwei CUI, Wentao XU, Xiangyang LI
2023, 13(4): 612-618. DOI: 10.19586/j.2095-2341.2023.0041
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As a common foodborne pathogenic bacterium in aquatic products， and a variety of rapid detection methods of Vibrio parahaemolyticus have been gradually developed. Aptamers have received widespread attention as an emerging specific identification element. In this study， 12 rounds of cell-systematic evolution of ligands by exponential enrichment (cell-SELEX) were performed with Vibrio parahaemolyticus as the target， including 9 rounds of positive selection and 3 rounds of negative selection， and the number of PCR rounds and the dosage of Lambada exonuclease were optimized in each round of positive selection. Nucleic acid sequencing was performed after SELEX， and the obtained sequence performance was verified from multiple angles by qPCR and flow cytometry. The results showed that F-28-10 showed better binding ability and specificity. And the development of aptamers can be achieved by optimizing the number of PCR amplification rounds and the amount of Lambda exonuclease in cell-SELEX. This method provided a new usable identification element for the development of rapid detection technology of foodborne pathogenic microorganisms.

Visual Analysis of the Research Status and Hotpots of Microplastic Biotoxicity
Qing XIAO, Guozeng WANG, Jing HAN, Xiaolan LIU
2023, 13(4): 619-627. DOI: 10.19586/j.2095-2341.2022.0175
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In order to explore the current status， hotspots and trends of microplastic biotoxicity， we analyzed the trend of article generation， authorship， journal and citation and keyword clustering analysis of microplastic biotoxicity-related literature since 2011 based on VOS viewer， a bibliometric visualization software taking Web of Science core collection and China National Knowledge Infrastructure （www.cnki.net） as data sources. Results showed that， the number of annual articles on microplastic biotoxicity has been increased exponentially. The total number of articles and citations published by Chinese scholars is ranked among the top in the world. Journals Science of the Total Environment， Journal of Hazardous Materials， Environmental Pollution and Environmental Science & Technology，etc. have high influence on the research of microplastic biotoxicity. The hot topics of microplastic biotoxicity research were mainly focused on four aspects： “the manifestation of microplastic biotoxicity” “the source of microplastic biotoxicity”“the combined biotoxicity of microplastic and other harmful substances” and “the intake route of microplastic biotoxicity”. In the future， researches need to focus on the establishment of rapid detection technology for microplastic， clarification of the absorption and transfer mechanisms of microplastics under different exposure pathways， and exploration of endogenous microplastics prevention and control measures as key research and development fields.

Preliminary Study on the Application of Graphene-coated Iron Nitride Magnetic Beads to Capture Lung Cancer Circulating Tumor Cells
Wenbo MA, Yiqun PAN, Qun WANG, Zhuang MA, Minglian WANG, Yishu YANG
2023, 13(4): 628-636. DOI: 10.19586/j.2095-2341.2023.0037
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Circulating tumor cells （CTCs） present in the blood play important roles in cancer recurrence and metastasis， so the capture of CTCs has become a key issue in current cancer research. The method of using antibody-dependent immunomagnetic beads to capture CTCs has the characteristics of simple operation and rapidity， but there are problems such as large sample size requirement and high preparation cost. This study intended to use a new type of magnetic bead-plasma-modified graphene-coated iron nitride magnetic beads （G@FeN-MB） to carry out a preliminary study on the capture of circulating tumor cells in lung cancer. G@FeN-MB is covered by multi-layer graphene， and the core is iron nitride， which has stronger magnetic response than the commonly used iron oxide core. Graphene used as a magnetic bead coating shell has lower mass， higher stability and better biocompatibility than the SiO₂ shell commonly used for magnetic beads on the market. In this study， two kinds of streptavidin magnetic beads were prepared by direct adsorption method and carbodiimide method respectively， and coupled with CTCs surface markers epithelial cell adhesion molecule （EpCAM） antibody preparation amino immunomagnetic beads （AIMB） and carboxylic immunomagnetic beads （CIMB）. The results showed that both kinds of immunomagnetic beads could capture A549 cells， and the capture efficiency of AIMB was slightly higher than that of CIMB； in order to simulate the CTCs capture environment， healthy human blood was mixed with A549 cells to prepare cell mixtures of different concentrations， and the capture efficiency was higher by using AIMB captures A549 in human blood， and calculated the captured A549 and capture efficiency. The results showed that the capture efficiency of AIMB ranged from 66.25% to 81.50%. The feasibility of the method of preparing immunomagnetic beads based on G@FeN-MB was proved， and the obtained AIMB can be used for the sorting of lung cancer CTCs， showing the prospect of its application in clinical research.

Effect of Erastin on the Ultrastructure of Granular Cells by High Pressure Freezing-freezing Substitution Technology
Zhen WANG, Kerang HUANG, Lei CHEN, Min ZHOU, Yuanxia XUE
2023, 13(4): 637-644. DOI: 10.19586/j.2095-2341.2023.0057
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In order to investigate the effect of ferroptosis on the ultrastructure of granulosa cells， the high pressure freezing-freezing substitution method （HPF-FS） and conventional chemical fixation method （CF） were used to study the ultrastructures of bovine and porcine granulosa cells induced by Erastin. The results showed that， after induction by Erastin for 48 h， the proliferation of both bovine granulosa cells and porcine granulosa cells were significantly inhibited， the ATP contents were significantly decreased， and the ROS levels were significantly increased when compared with the control group， all of these indicated ferroptosis in granulosa cells. Electron microscopy observation indicated that vacuoles appeared in the cells， mitochondria size were significantly decreased， cristae broken or even disappeared of granulosa cells in both treatment groups； the endoplasmic reticulum of the control porcine granulosa cells were more abundant than the treatment group. The maintenance of the ultrastructure of the cells by HPF-FS were better than CF in both cell morphology and membrane boundary. Compared with CF， the structures of mitochondria， golgi apparatus， autophagy and nucleus were clear and intact， which were closer to the transient physiological state of cells. Therefore， the technology of HPF-FS can more realistically and comprehensively display the ultrastructure of cells， which can providing more ideas and references for further research on the apoptosis regulation mechanism of granulosa cells or the related research on follicle development.

Content Estimating of Microbial Dissolved Organic Carbon Based on Machine Learning
Yunpeng MA, Jing ZHU, Xinghua CUI
2023, 13(4): 645-653. DOI: 10.19586/j.2095-2341.2022.0007
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The microbial communities has an important impact on the macro nature of the environment. However， the characteristics of high-dimensional， complex and sparse microbial data also pose new challenges for understanding the relationship between microorganisms and ecological environment. The development of machine learning and the popularization of the application of the second generation DNA sequencing technology provided a new solution to this problem. In this study， soil microbiome and dissolved organic carbon （DOC） data of 308 samples from plant litter decomposition experiments for 44 days were used， and 1 709 operational taxonomic units （OTU） of bacteria and microorganisms were used as features to build 12 commonly used machine learning models. Embedding method， packaging method and embedd-packaging fusion method were used for feature selection， and gradient boosting decision tree （GBDT） was selected as the optimal model for parameter optimization. The model adopted root mean square error， mean absolute error and linear goodness of fit was used as evaluation indexes. The results showed that， the feature selection reduced the data dimension and improved the model accuracy. In the simulation experiment， the embedding-packaging fusion method performs was the best in the application model. The prediction model of dissolved organic carbon was constructed by combining the embedding and packaging fusion method with gradient boosting decision tree， and the validity of the model was verified by experiments. The results provided a new way to estimate dissolved organic carbon using machine learning method based on bacterial and microbial data.

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