Biotechnology advances最新文献

Advances and challenges in preparing membrane proteins for native mass spectrometry 为原生质谱法制备膜蛋白的进展与挑战

IF 16 1区工程技术

Biotechnology advances Pub Date : 2024-11-20 DOI: 10.1016/j.biotechadv.2024.108483

Di Cheng, Yi Guo, Jixing Lyu, Yang Liu, Wenhao Xu, Weiyi Zheng, Yuchen Wang, Pei Qiao

{"title":"Advances and challenges in preparing membrane proteins for native mass spectrometry","authors":"Di Cheng, Yi Guo, Jixing Lyu, Yang Liu, Wenhao Xu, Weiyi Zheng, Yuchen Wang, Pei Qiao","doi":"10.1016/j.biotechadv.2024.108483","DOIUrl":"https://doi.org/10.1016/j.biotechadv.2024.108483","url":null,"abstract":"Native mass spectrometry (nMS) is becoming a crucial tool for analyzing membrane proteins (MPs), yet challenges remain in solubilizing and stabilizing their native conformations while resolving and characterizing the heterogeneity introduced by post-translational modifications and ligand binding. This review highlights recent advancements and persistent challenges in preparing MPs for nMS. Optimizing detergents and additives can significantly reduce sample heterogeneity and surface charge, enhancing MP signal quality and structural preservation in nMS. A strategic workflow incorporating affinity capture, stabilization agents, and size-exclusion chromatography to remove unfolded species demonstrates success in improving nMS characterization. Continued development of customized detergents and reagents tailored for specific MPs may further minimize heterogeneity and boost signals. Instrumental advances are also needed to elucidate more dynamically complex and labile MPs. Effective sample preparation workflows may provide insights into MP structures, dynamics, and interactions underpinning membrane biology. With ongoing methodological innovation, nMS shows promise to complement biophysical studies and facilitate drug discovery targeting this clinically important yet technically demanding protein class.","PeriodicalId":8946,"journal":{"name":"Biotechnology advances","volume":"16 1","pages":""},"PeriodicalIF":16.0,"publicationDate":"2024-11-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142684169","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Leveraging independent component analysis to unravel transcriptional regulatory networks: A critical review and future directions. 利用独立成分分析揭示转录调控网络：重要综述与未来方向。

IF 12.1 1区工程技术

Biotechnology advances Pub Date : 2024-11-20 DOI: 10.1016/j.biotechadv.2024.108479

Yuhan Zhang, Jianxiao Zhao, Xi Sun, Yangyang Zheng, Tao Chen, Zhiwen Wang

{"title":"Leveraging independent component analysis to unravel transcriptional regulatory networks: A critical review and future directions.","authors":"Yuhan Zhang, Jianxiao Zhao, Xi Sun, Yangyang Zheng, Tao Chen, Zhiwen Wang","doi":"10.1016/j.biotechadv.2024.108479","DOIUrl":"https://doi.org/10.1016/j.biotechadv.2024.108479","url":null,"abstract":"<p><p>Transcriptional regulatory networks (TRNs) play a crucial role in exploring microbial life activities and complex regulatory mechanisms. The comprehensive reconstruction of TRNs requires the integration of large-scale experimental data, which poses significant challenges due to the complexity of regulatory relationships. The application of machine learning tools, such as clustering analysis, has been employed to investigate TRNs, but these methods have limitations in capturing both global and local co-expression effects. In contrast, Independent Component Analysis (ICA) has emerged as a powerful analysis algorithm for modularizing independently regulated gene sets in TRNs, allowing it to account for both global and local co-expression effects. In this review, we comprehensively summarize the application of ICA in unraveling TRNs and highlight the research progress in three key aspects: (1) extending TRNs with iModulon analysis; (2) elucidating the regulatory mechanisms triggered by environmental perturbation; and (3) exploring the mechanisms of transcriptional regulation triggered by changes in microbial physiological state. At the end of this review, we also address the challenges facing ICA in TRN analysis and outline future research directions to promote the advancement of ICA-based transcriptomics analysis in biotechnology and related fields.</p>","PeriodicalId":8946,"journal":{"name":"Biotechnology advances","volume":" ","pages":"108479"},"PeriodicalIF":12.1,"publicationDate":"2024-11-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142692671","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Recent advances in culture medium design for enhanced production of monoclonal antibodies in CHO cells: A comparative study of machine learning and systems biology approaches. 用于提高 CHO 细胞单克隆抗体产量的培养基设计的最新进展：机器学习与系统生物学方法的比较研究。

IF 12.1 1区工程技术

Biotechnology advances Pub Date : 2024-11-19 DOI: 10.1016/j.biotechadv.2024.108480

Hossein Kavoni, Iman Shahidi Pour Savizi, Nathan E Lewis, Seyed Abbas Shojaosadati

{"title":"Recent advances in culture medium design for enhanced production of monoclonal antibodies in CHO cells: A comparative study of machine learning and systems biology approaches.","authors":"Hossein Kavoni, Iman Shahidi Pour Savizi, Nathan E Lewis, Seyed Abbas Shojaosadati","doi":"10.1016/j.biotechadv.2024.108480","DOIUrl":"https://doi.org/10.1016/j.biotechadv.2024.108480","url":null,"abstract":"<p><p>The production of monoclonal antibodies (mAbs) using Chinese Hamster Ovary (CHO) cells has revolutionized the treatment of numerous diseases, solidifying their position as a cornerstone of the biopharmaceutical industry. However, achieving maximum mAb production while upholding strict product quality standards remains a significant hurdle. Optimizing cell culture media emerges as a critical factor in this endeavor, requiring a nuanced understanding of the complex interplay of nutrients, growth factors, and other components that profoundly influence cellular growth, productivity, and product quality. Significant strides have been made in media optimization, including techniques such as media blending, one factor at a time, and statistical design of experiments approaches. The present review provides a comprehensive analysis of the recent advancements in culture media design strategies, focusing on the comparative application of systems biology (SB) and machine learning (ML) approaches. The applications of SB and ML in optimizing CHO cell culture medium and successful examples of their use are summarized. Finally, we highlight the immense potential of integrating SB and ML, emphasizing the development of hybrid models that leverage the strengths of both approaches for robust, efficient, and scalable optimization of mAb production in CHO cells. This review provides a roadmap for researchers and industry professionals to navigate the complex landscape of mAb production optimization, paving the way for developing next-generation CHO cell culture media that drive significant improvements in yield and productivity.</p>","PeriodicalId":8946,"journal":{"name":"Biotechnology advances","volume":" ","pages":"108480"},"PeriodicalIF":12.1,"publicationDate":"2024-11-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142685908","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Bacterial 5' UTR: A treasure-trove for post-transcriptional regulation. 细菌 5' UTR：转录后调控的宝库。

IF 12.1 1区工程技术

Biotechnology advances Pub Date : 2024-11-17 DOI: 10.1016/j.biotechadv.2024.108478

Ya-Jun Liu, Xiaoqing Wang, Yuman Sun, Yingang Feng

引用次数: 0

Synthetic biology approaches to improve tolerance of inhibitors in lignocellulosic hydrolysates. 提高木质纤维素水解物对抑制剂耐受性的合成生物学方法。

IF 12.1 1区工程技术

Biotechnology advances Pub Date : 2024-11-15 DOI: 10.1016/j.biotechadv.2024.108477

Linyue Tian, Tianqi Qi, Fenghui Zhang, Vinh G Tran, Jifeng Yuan, Yuanpeng Wang, Ning He, Mingfeng Cao

{"title":"Synthetic biology approaches to improve tolerance of inhibitors in lignocellulosic hydrolysates.","authors":"Linyue Tian, Tianqi Qi, Fenghui Zhang, Vinh G Tran, Jifeng Yuan, Yuanpeng Wang, Ning He, Mingfeng Cao","doi":"10.1016/j.biotechadv.2024.108477","DOIUrl":"https://doi.org/10.1016/j.biotechadv.2024.108477","url":null,"abstract":"<p><p>Increasing attention is being focused on using lignocellulose for valuable products. Microbial decomposition can convert lignocellulose into renewable biofuels and other high-value bioproducts, contributing to sustainable development. However, the presence of inhibitors in lignocellulosic hydrolysates can negatively affect microorganisms during fermentation. Improving microbial tolerance to these hydrolysates is a major focus in metabolic engineering. Traditional detoxification methods increase costs, so there is a need for cheap and efficient cell-based detoxification strategies. Synthetic biology approaches offer several strategies for improving microbial tolerance, including redox balancing, membrane engineering, omics-guided technologies, expression of protectants and transcription factors, irrational engineering, cell flocculation, and other novel technologies. Advances in molecular biology, high-throughput sequencing, and artificial intelligence (AI) allow for precise strain modification and efficient industrial production. Developing AI-based computational models to guide synthetic biology efforts and creating large-scale heterologous libraries with automation and high-throughput technologies will be important for future research.</p>","PeriodicalId":8946,"journal":{"name":"Biotechnology advances","volume":" ","pages":"108477"},"PeriodicalIF":12.1,"publicationDate":"2024-11-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142646932","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Fusarium as a promising fungal genus with potential application in bioremediation for pollutants mitigation: A review 镰刀菌是一种很有潜力的真菌属，有可能应用于减轻污染物的生物修复：综述。

IF 12.1 1区工程技术

Biotechnology advances Pub Date : 2024-11-12 DOI: 10.1016/j.biotechadv.2024.108476

Carmen Sánchez

{"title":"Fusarium as a promising fungal genus with potential application in bioremediation for pollutants mitigation: A review","authors":"Carmen Sánchez","doi":"10.1016/j.biotechadv.2024.108476","DOIUrl":"10.1016/j.biotechadv.2024.108476","url":null,"abstract":"<div><div><em>Fusarium</em> is genetically diverse and widely distributed geographically. It is one of the genera with more endophytes (which cause no damage to the host plants). This review highlights the capability of <em>Fusarium</em> species to degrade environmental pollutants and describes the biodegradation pathways of some of the emerging environmental contaminants. Some <em>Fusarium</em> species use metabolic strategies enabling them to efficiently mineralize high concentrations of toxic environmental pollutants. These fungi can degrade hydrocarbons, pesticides, herbicides, dyes, pharmaceutical compounds, explosives, plastics, and plastic additives, among other pollutants, and possess high metal biosorption capabilities. According to data from consulted reports, <em>Fusarium</em> strains showed a percentage of biodegradation of a variety of contaminants ranging between 30 % and 100 % for different tested concentrations (from 1 mg to 10 g/L) in a time range between 10 h and 90 d. Enzymes such as esterase, cutinase, laccase, lignin peroxidase, manganese peroxidase, dehydrogenase, lipase, dioxygenase, and phosphoesterase were detected during the pollutant biodegradation process. <em>Fusarium oxysporum</em>, <em>Fusarium solani</em>, and <em>Fusarium culmorum</em> are the most studied species of this genus. Owing to their metabolic versatility, these fungal species and their enzymes represent promising tools for bioremediation applications to mitigate the adverse effects of environmental pollution.</div></div>","PeriodicalId":8946,"journal":{"name":"Biotechnology advances","volume":"77 ","pages":"Article 108476"},"PeriodicalIF":12.1,"publicationDate":"2024-11-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142614093","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Metabolic engineering of Corynebacterium glutamicum: Unlocking its potential as a key cell factory platform for organic acid production 谷氨酸棒状杆菌的代谢工程：挖掘其作为生产有机酸的关键细胞工厂平台的潜力。

IF 12.1 1区工程技术

Biotechnology advances Pub Date : 2024-11-07 DOI: 10.1016/j.biotechadv.2024.108475

Ming-Hou Li , Han Li , Xue Zhang , Yu-Chen Liang , Cheng Li , Meng-Lin Sun , Kai Li , Chen-Guang Liu , Anthony J. Sinskey

{"title":"Metabolic engineering of Corynebacterium glutamicum: Unlocking its potential as a key cell factory platform for organic acid production","authors":"Ming-Hou Li , Han Li , Xue Zhang , Yu-Chen Liang , Cheng Li , Meng-Lin Sun , Kai Li , Chen-Guang Liu , Anthony J. Sinskey","doi":"10.1016/j.biotechadv.2024.108475","DOIUrl":"10.1016/j.biotechadv.2024.108475","url":null,"abstract":"<div><div><em>Corynebacterium glutamicum</em>, a well-studied industrial model microorganism, has garnered widespread attention due to its ability for producing amino acids with a long history. In recent years, research efforts have been increasingly focused on exploring its potential for producing various organic acids beyond amino acids. Organic acids, which are characterized by their acidic functional groups, have diverse applications across industries such as food, agriculture, pharmaceuticals, and biobased materials. Leveraging advancements in metabolic engineering and synthetic biology, the metabolic pathways of <em>C. glutamicum</em> have been broadened to facilitate the production of numerous high-value organic acids. This review summarizes the recent progress in metabolic engineering for the production of both amino acids and other organic acids by <em>C. glutamicum</em>. Notably, these acids include, amino acids (lysine, isoleucine, and phenylalanine), TCA cycle-derived organic acids (succinic acid, α-ketoglutaric acid), aromatic organic acids (protocatechuate, 4-amino-3-hydroxybenzoic acid, anthranilate, and para-coumaric acid), and other organic acids (itaconic acid and cis, cis-muconic acid).</div></div>","PeriodicalId":8946,"journal":{"name":"Biotechnology advances","volume":"77 ","pages":"Article 108475"},"PeriodicalIF":12.1,"publicationDate":"2024-11-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142614098","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Microbial electrosynthesis technology for CO2 mitigation, biomethane production, and ex-situ biogas upgrading 用于二氧化碳减排、生物甲烷生产和沼气异地提纯的微生物电合成技术。

IF 12.1 1区工程技术

Biotechnology advances Pub Date : 2024-11-07 DOI: 10.1016/j.biotechadv.2024.108474

Tae Hyun Chung , Simran Kaur Dhillon , Chungheon Shin , Deepak Pant , Bipro Ranjan Dhar

{"title":"Microbial electrosynthesis technology for CO2 mitigation, biomethane production, and ex-situ biogas upgrading","authors":"Tae Hyun Chung , Simran Kaur Dhillon , Chungheon Shin , Deepak Pant , Bipro Ranjan Dhar","doi":"10.1016/j.biotechadv.2024.108474","DOIUrl":"10.1016/j.biotechadv.2024.108474","url":null,"abstract":"<div><div>Currently, global annual CO<sub>2</sub> emissions from fossil fuel consumption are extremely high, surpassing tens of billions of tons, yet our capacity to capture and utilize CO<sub>2</sub> remains below a small fraction of the amount generated. Microbial electrosynthesis (MES) systems, an integration of microbial metabolism with electrochemistry, have emerged as a highly efficient and promising bio-based carbon-capture-and-utilization technology over other conventional techniques. MES is a unique technology for lowering the atmospheric CO<sub>2</sub> as well as CO<sub>2</sub> in the biogas, and also simultaneously convert them to renewable bioenergy, such as biomethane. As such, MES techniques could be applied for biogas upgrading to generate high purity biomethane, which has the potential to meet natural gas standards. This article offers a detailed overview and assessment of the latest advancements in MES for biomethane production and biogas upgrading, in terms of selecting optimal methane production pathways and associated electron transfer processes, different electrode materials and types, inoculum sources and microbial communities, ion-exchange membrane, externally applied energy level, operating temperature and pH, mode of operation, CO<sub>2</sub> delivery method, selection of inorganic carbon source and its concentration, start-up time, and system pressure. It also highlights the current MES challenges associated with upscaling, design and configuration, long-term stability, energy demand, techno-economics, achieving net negative carbon emission, and other operational issues. Moreover, we provide a summary of current and future opportunities to integrate MES with other unique biosystems, such as methanotrophic bioreactors, and incorporate quorum sensing, 3D printing, and machine learning to further develop MES as a better biomethane-producer and biogas upgrading technique.</div></div>","PeriodicalId":8946,"journal":{"name":"Biotechnology advances","volume":"77 ","pages":"Article 108474"},"PeriodicalIF":12.1,"publicationDate":"2024-11-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142614109","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Biotechnological applications of purine and pyrimidine deaminases 嘌呤和嘧啶脱氨酶的生物技术应用。

IF 12.1 1区工程技术

Biotechnology advances Pub Date : 2024-11-05 DOI: 10.1016/j.biotechadv.2024.108473

Jon Del Arco , Javier Acosta , Jesús Fernández-Lucas

{"title":"Biotechnological applications of purine and pyrimidine deaminases","authors":"Jon Del Arco , Javier Acosta , Jesús Fernández-Lucas","doi":"10.1016/j.biotechadv.2024.108473","DOIUrl":"10.1016/j.biotechadv.2024.108473","url":null,"abstract":"<div><div>Deaminases, ubiquitous enzymes found in all living organisms from bacteria to humans, serve diverse and crucial functions. Notably, purine and pyrimidine deaminases, while biologically essential for regulating nucleotide pools, exhibit exceptional versatility in biotechnology. This review systematically consolidates current knowledge on deaminases, showcasing their potential uses and relevance in the field of biotechnology. Thus, their transformative impact on pharmaceutical manufacturing is highlighted as catalysts for the synthesis of nucleic acid derivatives. Additionally, the role of deaminases in food bioprocessing and production is also explored, particularly in purine content reduction and caffeine production, showcasing their versatility in this field. The review also delves into most promising biomedical applications including deaminase-based GDEPT and genome and transcriptome editing by deaminase-based systems. All in all, illustrated with practical examples, we underscore the role of purine and pyrimidine deaminases in advancing sustainable and efficient biotechnological practices. Finally, the review highlights future challenges and prospects in deaminase-based biotechnological processes, encompassing both industrial and medical perspectives.</div></div>","PeriodicalId":8946,"journal":{"name":"Biotechnology advances","volume":"77 ","pages":"Article 108473"},"PeriodicalIF":12.1,"publicationDate":"2024-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142590090","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Harnessing Raman spectroscopy for cell therapy bioprocessing 利用拉曼光谱进行细胞治疗生物处理。

IF 12.1 1区工程技术

Biotechnology advances Pub Date : 2024-10-28 DOI: 10.1016/j.biotechadv.2024.108472

Marta H.G. Costa , Inês Carrondo , Inês A. Isidro , Margarida Serra

{"title":"Harnessing Raman spectroscopy for cell therapy bioprocessing","authors":"Marta H.G. Costa , Inês Carrondo , Inês A. Isidro , Margarida Serra","doi":"10.1016/j.biotechadv.2024.108472","DOIUrl":"10.1016/j.biotechadv.2024.108472","url":null,"abstract":"<div><div>Cell therapy manufacturing requires precise monitoring of critical parameters to ensure product quality, consistency and to facilitate the implementation of cost-effective processes. While conventional analytical methods offer limited real-time insights, integration of process analytical technology tools such as Raman spectroscopy in bioprocessing has the potential to drive efficiency and reliability during the manufacture of cell-based therapies while meeting stringent regulatory requirements. The non-destructive nature of Raman spectroscopy, combined with its ability to be integrated on-line with scalable platforms, allows for continuous data acquisition, enabling real-time correlations between process parameters and critical quality attributes.</div><div>Herein, we review the role of Raman spectroscopy in cell therapy bioprocessing and discuss how simultaneous measurement of distinct parameters and attributes, such as cell density, viability, metabolites and cell identity biomarkers can streamline on-line monitoring and facilitate adaptive process control. This, in turn, enhances productivity and mitigates process-related risks. We focus on recent advances integrating Raman spectroscopy across various manufacturing stages, from optimizing culture media feeds to monitoring bioprocess dynamics, covering downstream applications such as detection of co-isolated contaminating cells, cryopreservation, and quality control of the drug product. Finally, we discuss the potential of Raman spectroscopy to revolutionize current practices and accelerate the development of advanced therapy medicinal products.</div></div>","PeriodicalId":8946,"journal":{"name":"Biotechnology advances","volume":"77 ","pages":"Article 108472"},"PeriodicalIF":12.1,"publicationDate":"2024-10-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142567634","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0