Biotechnology and Bioprocess Engineering最新文献_第4页

Tn5 transposase: a key tool to decrypt random transposition Tn5 转座酶：解密随机转座的关键工具

IF 3.2 4区生物学

Biotechnology and Bioprocess Engineering Pub Date : 2024-06-03 DOI: 10.1007/s12257-024-00118-1

Ying Xu, Huimin Zhou, Fangyuan Song, Huayou Chen, Zhong Ni

引用次数: 0

Suppressive activities of lupeol on sepsis mouse model 羽扇豆醇对败血症小鼠模型的抑制作用

IF 3.2 4区生物学

Biotechnology and Bioprocess Engineering Pub Date : 2024-05-27 DOI: 10.1007/s12257-024-00112-7

Sanghee Cho, Yun Jin Park, Jinhee Lee, Jong-Sup Bae

{"title":"Suppressive activities of lupeol on sepsis mouse model","authors":"Sanghee Cho, Yun Jin Park, Jinhee Lee, Jong-Sup Bae","doi":"10.1007/s12257-024-00112-7","DOIUrl":"https://doi.org/10.1007/s12257-024-00112-7","url":null,"abstract":"Sepsis is a life-threatening condition triggered by the body’s extreme response to an infection, leading to widespread inflammation, organ dysfunction, and potentially fatal complications. While lupeol, a significant phytosterol found in various herbal plants, has been considered as a potential anti-cancer agent, its anti-septic activities and underlying molecular mechanisms remain unclear. The aim of this study is to investigate the effects of lupeol on a cecal ligation and puncture (CLP)-induced septic mouse model. Animals were categorized into six groups: control, CLP-operated, CLP plus maslinic acid, and CLP plus lupeol (0.5, 1, or 2 mg/kg). The assessment included survival rate, body weight changes, inflammatory cytokines, and histological analyses. Additionally, human endothelial cells were stimulated with high mobility group box1 (HMGB1) protein and lupeol, with cell viability determined. Inflammatory markers and gene expression were evaluated through enzymelinked immunosorbent assay and Western blot analysis, respectively. After CLP surgery, the group treated with lupeol showed improved survival rates and body weight compared to the untreated control group. Lupeol treatment also decreased levels of tumor necrosis factor (TNF)-α, interleukin-1β, nitric oxide, and cytokines associated with kidney inflammation. When administered to HMGB1-activated cells, lupeol reduced the expression of Toll-like receptor 4 and TNF-α, while simultaneously activating phosphoinositide 3-kinase/AKT signaling to enhance cell survival. In conclusion, lupeol demonstrated anti-inflammatory properties and conferred protective effects against CLP-induced sepsis, reinforcing cell survival in the face of septic responses.","PeriodicalId":8936,"journal":{"name":"Biotechnology and Bioprocess Engineering","volume":"8 1","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-05-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141166754","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Development of in vitro lycopene biosynthesis from geranyl pyrophosphate employing cell-free protein synthesis 利用无细胞蛋白质合成技术，从焦磷酸香叶酯中开发体外番茄红素生物合成技术

IF 3.2 4区生物学

Biotechnology and Bioprocess Engineering Pub Date : 2024-05-25 DOI: 10.1007/s12257-024-00111-8

Young Hwan Goh, Ye Chan Kim, Sang Hun Jeong, Sangwoo Joo, You Kyoung Kwon, Hyunseok Yoon, Seohee Jung, Taresh P. Khobragade, Pritam Giri, Seonga Lim, Subin Yun, Sunga Cho, Sang Hyun Lee, Woo-Jae Chung, Jae Eun Lim, Taek Jin Kang, Hyungdon Yun

{"title":"Development of in vitro lycopene biosynthesis from geranyl pyrophosphate employing cell-free protein synthesis","authors":"Young Hwan Goh, Ye Chan Kim, Sang Hun Jeong, Sangwoo Joo, You Kyoung Kwon, Hyunseok Yoon, Seohee Jung, Taresh P. Khobragade, Pritam Giri, Seonga Lim, Subin Yun, Sunga Cho, Sang Hyun Lee, Woo-Jae Chung, Jae Eun Lim, Taek Jin Kang, Hyungdon Yun","doi":"10.1007/s12257-024-00111-8","DOIUrl":"https://doi.org/10.1007/s12257-024-00111-8","url":null,"abstract":"Lycopene is a compound classified as carotenoid, also known as tetraterpenoids, and its high antioxidative capabilities make demand in pharmaceutical and nutrient fields. For these reasons, much research on microbial production of lycopene has been developed and reported for more than two decades. Nevertheless, a standardized in vitro biosynthesis method for lycopene synthesis has not been reported to date. The major reasons of the absence of this method lie on the poor solubility of hydrophobic intermediates (geranylgeranyl pyrophosphate [GGPP] and phytoene), and the difficulty of employing membrane-binding enzyme, phytoene desaturase (CrtI) into in vitro reactions. In this study, we developed a standard method of in vitro biosynthesis of lycopene from geranyl pyrophosphate using four enzymes, namely farnesyl pyrophosphate synthase (IspA), GGPP synthase (CrtE), phytoene synthase (CrtB), phytoene desaturase (CrtI), and liposome-the key material, which can provide both hydrophobic area and a lipid membrane for the membrane-binding enzyme CrtI. Moreover, we performed a screening of the in vitro lycopene synthetic pathway using cell-free protein synthesis system, which verifies the applicability of our system as a tool for screening the lycopene synthesis pathway.","PeriodicalId":8936,"journal":{"name":"Biotechnology and Bioprocess Engineering","volume":"43 1","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-05-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141147809","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

High-yield β-alanine production from glucose and acetate in Escherichia coli 大肠杆菌利用葡萄糖和醋酸盐高产生产 β-丙氨酸

IF 3.2 4区生物学

Biotechnology and Bioprocess Engineering Pub Date : 2024-05-18 DOI: 10.1007/s12257-024-00107-4

Toan Minh Vo, Sunghoon Park

{"title":"High-yield β-alanine production from glucose and acetate in Escherichia coli","authors":"Toan Minh Vo, Sunghoon Park","doi":"10.1007/s12257-024-00107-4","DOIUrl":"https://doi.org/10.1007/s12257-024-00107-4","url":null,"abstract":"β-Alanine is a versatile amino acid with wide-range industrial applications, but its production from glucose has been limited by a low yield. This study addresses this challenge by developing efficient Escherichia coli strains with modified carbon metabolism as microbial cell factories and implementing a two-stage fermentation strategy. The introduction of aspartate decarboxylase (PanDE56S/I88M) facilitates the conversion of aspartate to β-alanine, while the overexpression of key enzymes such as phosphoenolpyruvate carboxylase and aspartate dehydrogenase increases the carbon flow from phosphoenolpyruvate to aspartate. To mitigate oxidative stress, the glutathione cycle was enhanced by overexpressing BtuE and Gor. In a bioreactor, the optimized strain achieved β-alanine production of 71.7 g/L with a yield of 1.0 mol/mol glucose, reaching a peak of 1.29 mol/mol. Notably, the utilization of acetate as a carbon feedstock enabled the production of 50 g/L of β-alanine with a 0.33 mol/mol acetate yield, showcasing the potential for sustainable production. This research offers valuable insights into improving the carbon yield in β-alanine production, which is of great importance for industrial applications.","PeriodicalId":8936,"journal":{"name":"Biotechnology and Bioprocess Engineering","volume":"2012 1","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-05-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141063725","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Y-shaped oligonucleotides: a promising platform for enhanced therapy with siRNA and CpG Oligodeoxyribonucleotides Y 型寡核苷酸：利用 siRNA 和 CpG 寡脱氧核苷酸进行强化治疗的前景广阔的平台

IF 3.2 4区生物学

Biotechnology and Bioprocess Engineering Pub Date : 2024-05-12 DOI: 10.1007/s12257-024-00109-2

In Seop Yoon, Hye Jeong Nam, Cheol Am Hong

{"title":"Y-shaped oligonucleotides: a promising platform for enhanced therapy with siRNA and CpG Oligodeoxyribonucleotides","authors":"In Seop Yoon, Hye Jeong Nam, Cheol Am Hong","doi":"10.1007/s12257-024-00109-2","DOIUrl":"https://doi.org/10.1007/s12257-024-00109-2","url":null,"abstract":"Nucleic acids (DNA and RNA) have been recognized as promising building blocks to fabricate a variety of well-defined two- and three-dimensional architectures through the programmable molecular self-assembly of multiple oligomeric strands. Y-shaped oligonucleotides are currently among the most widely employed nanostructures in the field of nucleic acid nanotechnology due to their unique features, including high structural stability, excellent biocompatibility, simplicity and ease of synthesis, and precisely controlled sizes. To functionalize biological activity, Y-shaped oligonucleotides can be incorporated with therapeutic genes such as small interfering RNA (siRNA) for target gene-specific silencing and CpG oligonucleotides (CpG ODN) for the activation of innate immune responses. Compared to the linear structures of siRNA and CpG ODN, Y-shaped siRNA and CpG ODN structures have demonstrated significant potential in the treatment of various diseases due to improved serum stability and intracellular uptake. Here, we review a broad spectrum of related topics, including the design, construction, and characteristics of Y-shaped oligonucleotides with a specific focus on their potential as a promising platform for enhancing the therapeutic efficacy of siRNA and CpG ODN.","PeriodicalId":8936,"journal":{"name":"Biotechnology and Bioprocess Engineering","volume":"43 1","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-05-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140933336","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Reactive extraction for the separation of glyceric acid from aqueous solutions with 2-naphthaleneboronic acid and tri-octyl methyl ammonium chloride 用 2-萘硼酸和三辛基甲基氯化铵反应萃取分离水溶液中的甘油酸

IF 3.2 4区生物学

Biotechnology and Bioprocess Engineering Pub Date : 2024-05-10 DOI: 10.1007/s12257-024-00110-9

Long Hoang Dang Bui, Keitaro Aoki, Tomonari Tanaka, Yuji Aso

{"title":"Reactive extraction for the separation of glyceric acid from aqueous solutions with 2-naphthaleneboronic acid and tri-octyl methyl ammonium chloride","authors":"Long Hoang Dang Bui, Keitaro Aoki, Tomonari Tanaka, Yuji Aso","doi":"10.1007/s12257-024-00110-9","DOIUrl":"https://doi.org/10.1007/s12257-024-00110-9","url":null,"abstract":"Glyceric acid (GA), a carboxylic group-containing diol, is obtained from bioresources via microbial processes. In this study, we aimed to develop a reactive extraction method to separate GA from aqueous solutions using 2-naphthaleneboronic acid (2NB) and tri-octyl methyl ammonium chloride (TOMAC). Different feed molar amounts of 2NB (0–25 µmol), TOMAC (0–500 µmol), and NaOH (0–250 µmol) were used for GA (2.5 µmol) separation. A combination of 25 µmol 2NB, 100 µmol TOMAC, and 25 µmol NaOH was determined to be optimal for GA separation, providing 66.8 ± 3.2% GA yield at pH 11. GA was extracted by 2NB and TOMAC in a coordinated manner. Moreover, effects of various carboxylic acids (acetic, lactic, succinic, malic, tartaric, and citric acids) on GA separation from aqueous solutions were investigated. Interestingly, no significant effect on GA yeild (60.3 ± 1.2–65.2 ± 2.5%) was observed regardless of the type of carboxylic acid. The optimized protocol was subsequently applied to separate GA from crude GA solution prepared by incubating glycerol with the cells of the acetic acid bacterium, Acetobacter tropicalis NBRC 16470. GA separation was achieved at a comparable level (yield: 70.6 ± 4.6% and purity: 76.1 ± 4.1%) as that achieved using a GA reagent. This study demonstrated the efficiency of the repeated use of the organic phase for GA separation, with no significant changes in GA yield. Query Text=\"Please confirm if the author names are presented accurately and in the correct sequence (given name, middle name/initial, family name). Author 1 Given name: [specify authors given name] Last name [specify authors last name]. Also, kindly confirm the details in the metadata are correct.\"","PeriodicalId":8936,"journal":{"name":"Biotechnology and Bioprocess Engineering","volume":"246 1","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-05-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140941911","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Effect of 4-phenylbutyrate addition timing on titer of Fc-fusion protein in Chinese hamster ovary cell cultures 4-苯基丁酸盐添加时间对中国仓鼠卵巢细胞培养物中 Fc 融合蛋白滴度的影响

IF 3.2 4区生物学

Biotechnology and Bioprocess Engineering Pub Date : 2024-05-07 DOI: 10.1007/s12257-024-00105-6

Jungae Lim, Jin-Hyuk Lim, Ji-Hoon Lee, Su-Hwan Cheon, Guewha Lee, Z-Hun Kim, Dong-Il Kim

引用次数: 0

Efficient biodegradation of low-density polyethylene by Pseudomonas plecoglossicida SYp2123 was observed through FT-IR and FE-SEM analysis 通过 FT-IR 和 FE-SEM 分析观察褶皱假单胞菌 SYp2123 对低密度聚乙烯的高效生物降解作用

IF 3.2 4区生物学

Biotechnology and Bioprocess Engineering Pub Date : 2024-05-06 DOI: 10.1007/s12257-024-00108-3

Ye-Jin Kim, Yeon-Hwa Kim, Ye-Rim Shin, Su-Yeong Choi, Jeong-Ann Park, Hyun-Ouk Kim, Kwang Suk Lim, Suk-Jin Ha

{"title":"Efficient biodegradation of low-density polyethylene by Pseudomonas plecoglossicida SYp2123 was observed through FT-IR and FE-SEM analysis","authors":"Ye-Jin Kim, Yeon-Hwa Kim, Ye-Rim Shin, Su-Yeong Choi, Jeong-Ann Park, Hyun-Ouk Kim, Kwang Suk Lim, Suk-Jin Ha","doi":"10.1007/s12257-024-00108-3","DOIUrl":"https://doi.org/10.1007/s12257-024-00108-3","url":null,"abstract":"Plastics have been consistently produced for their practicality and convenience; however, unmanaged plastics often end up in the ocean and decompose into nano-plastics through photolytic decomposition and weathering, negatively affecting marine life. This can eventually affect humans via the food chain, highlighting the need for effective solutions. Microbial biodegradation has been proposed as a solution to minimize the impact of nano-plastics on the environment, and degradation byproducts can be used in microbial metabolic pathways. In this study, 57 bacterial strains were isolated and identified from a waste treatment facility. Bacterial strains with lipase activity were selected on Tween80 agar plates. Additionally, strains capable of growing on minimal salt agar plates supplemented with low-density polyethylene (LDPE) beads were selected. Incubation in a minimal salt medium with LDPE beads as the sole carbon source led to the selection of Pseudomonas plecoglossicida SYp2123, which is capable of degrading LDPE. This strain was subjected to high cell density culture, and Fourier-transform infrared spectroscopy revealed chemical changes on the surface of LDPE beads. Additionally, field-emission scanning electron microscopy confirmed substantial biodegradation of the surface. P. plecoglossicida SYp2123 was able to degrade LDPE beads. This discovery shows that P. plecoglossicida can potentially be used as an environmentally friendly approach for tackling issues associated with polyethylene waste. ","PeriodicalId":8936,"journal":{"name":"Biotechnology and Bioprocess Engineering","volume":"29 1","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-05-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140884948","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Inhibitory effects of bromoindoles on Escherichia coli O157:H7 biofilms 溴化吲哚对大肠杆菌 O157:H7 生物膜的抑制作用

IF 3.2 4区生物学

Biotechnology and Bioprocess Engineering Pub Date : 2024-04-18 DOI: 10.1007/s12257-024-00097-3

Hyejin Jeon, Bharath Reddy Boya, Gyuwon Kim, Jin-Hyung Lee, Jintae Lee

{"title":"Inhibitory effects of bromoindoles on Escherichia coli O157:H7 biofilms","authors":"Hyejin Jeon, Bharath Reddy Boya, Gyuwon Kim, Jin-Hyung Lee, Jintae Lee","doi":"10.1007/s12257-024-00097-3","DOIUrl":"https://doi.org/10.1007/s12257-024-00097-3","url":null,"abstract":"Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is responsible for outbreaks of hemorrhagic colitis worldwide, but no effective therapy exists for EHEC infections. EHEC readily forms antimicrobial-tolerant biofilms on various biotic and abiotic surfaces. Here, we investigated the antimicrobial and antibiofilm abilities of 16 halogenated (fluoro-, chloro-, bromo-, or iodo-) indoles and indole against a pathogenic EHEC strain. Antibiofilm activities followed the order chloroindoles > bromoindoles > indole > fluoroindoles. For example, the minimum inhibitory concentrations (MICs) of 4-bromoindole and 5-bromoindole were 100 and 200 μg/mL, respectively, and at 20 μg/mL, they both inhibited EHEC biofilm formation by more than 61% without affecting planktonic cell growth. However, at concentrations greater than their MICs, both showed bactericidal activity. Antibiofilm effects were confirmed by scanning electron microscopy. Both 4-bromoindole and 5-bromoindole reduced swimming and swarming motility and curli formation, which are important factors for EHEC biofilm formation. Furthermore, quantitative structure–activity relationship analysis demonstrated that halogenation of indole with chlorine, bromine, or iodine at positions C-4 or C-5 promotes antimicrobial activity but that substitution at C-7 is detrimental. The study shows that halogenated indoles, particularly bromoindoles, have potential use as antimicrobial and antibiofilm therapies against EHEC.","PeriodicalId":8936,"journal":{"name":"Biotechnology and Bioprocess Engineering","volume":"28 1","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-04-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140617124","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Assessment of Damnacanthus major Siebold & Zucc callus for antioxidative and moisturizing capacities using an artificial skin alternative 使用人工皮肤替代品评估大芒萁（Damnacanthus major Siebold & Zucc）胼胝的抗氧化和保湿能力

IF 3.2 4区生物学

Biotechnology and Bioprocess Engineering Pub Date : 2024-04-13 DOI: 10.1007/s12257-024-00103-8

Byung Man Lee, Hyun Ju Woo, Bum Jun Jang, Jae Ahn Shin, Young Min Ham, Eun Bi Jang, Sung Chun Kim, Jeong Mi Kim, Hwa Sung Shin

{"title":"Assessment of Damnacanthus major Siebold & Zucc callus for antioxidative and moisturizing capacities using an artificial skin alternative","authors":"Byung Man Lee, Hyun Ju Woo, Bum Jun Jang, Jae Ahn Shin, Young Min Ham, Eun Bi Jang, Sung Chun Kim, Jeong Mi Kim, Hwa Sung Shin","doi":"10.1007/s12257-024-00103-8","DOIUrl":"https://doi.org/10.1007/s12257-024-00103-8","url":null,"abstract":"Plants contain many useful substances; however, their availability and uniformity are constrained. Controlled cell culture is attracting attention as a method for identifying useful substances with better quality control than conventional plant extraction techniques. Artificial skin is widely used as an alternative to animal testing to evaluate the safety and efficacy of cosmetic materials. The Damnacanthus major Siebold & Zucc is an evergreen shrub of the madder family, and the anthraquinone series substances present in its roots have antioxidant and whitening effects, implying its potential to be used in cosmetics. In this study, the antioxidant, moisturizing, and phototoxic properties of the ethanol extract of its callus were assessed using an artificial skin alternative. It showed a distinct antioxidant effect over 600 μg/mL and a moisturizing effect over 125 μg/mL. Phospho-extracellular signal-regulated kinase 1/2 and cAMP response element-binding protein were found to be upregulated in the artificial skin due to the moisturizing mechanism, and phospho-NFκB repressing factor was observed to be upregulated in human epidermal keratinocytes (HaCaT cells) due to the antioxidant mechanism. Additionally, analysis of the artificial skin revealed no phototoxicity up to 1 mg/mL. The results of this study demonstrate that the ethanol extract of Damnacanthus major Siebold & Zucc can be used as a cosmetic material.","PeriodicalId":8936,"journal":{"name":"Biotechnology and Bioprocess Engineering","volume":"38 1","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-04-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140573821","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0