Biotechnology Letters最新文献

{"title":"Optimization of esterification reactions of perillyl alcohol with different fatty acids catalyzed by Novozym® 435 lipase.","authors":"Gustavo Dos Santos Martins, Daniel Luiz Reis Simas, Patrick Gabry Soares, Maíra Barcellos Marini, Michelle Frazão Muzitano, Ivana Correa Ramos Leal","doi":"10.1007/s10529-025-03581-9","DOIUrl":"https://doi.org/10.1007/s10529-025-03581-9","url":null,"abstract":"<p><p>Perillyl alcohol is a monoterpene known for its potent antitumor activity. In this study, we focused on esterification reactions of perillyl alcohol catalyzed by the immobilized enzyme Novozym 435 from Pseudozyma antarctica. Initially, the reaction conditions using a molar ratio of 1:4 (2:5 mmol of monoterpene: 10 mmol of octanoic acid), 40 mg of Novozym 435, 30 °C, and 150 rpm, 10 mL of cyclohexane, resulted in conversions up to 90% by 24 h. To explore the impact of different fatty acid carbon chain lengths on conversion, we conducted reactions with fatty acids containing chain lengths with 3, 6, 8, 12, and 18 carbons, evaluating the impact of the molar ratio of monoterpene:fatty acid. The observed impacts on conversion led to the categorization of fatty acids into two groups: the first group (C₃ and C₆) showed a positive impact with an increased molar ratio, while the second group (C₈-C₁₈) exhibited the opposite behavior. Based on these findings, we performed an experimental design (CCRD-Central Composite Rotatable Design) using octanoic acid (representing fatty acids with chains equal to or greater than eight carbons) and propionic acid (representing acids with chains lower than six carbons). The optimized conditions in CCRD resulted in conversions of 95.22% ± 0.61% with octanoic acid (substrate: acid, 2.5 mmol: 3.87 mM, enzyme: 48 mg) and 90.38% ± 0.99% with propionic acid (substrate: acid, 2.5 mmol: 3.10 mM, enzyme: 66.6 mg), at 30 °C and 150 rpm for 24 h. Finally, we assessed the reusability of Novozym 435 (48 mg) in the esterification reaction of (S)-(-)-perillyl alcohol (2.5 mmol) with octanoic acid (3.87 mM) for 24 h at 30 °C and 150 rpm. Remarkably, even after 10 cycles of 24 h, no loss of enzyme activity was detected, suggesting the potential for industrial applications.</p>","PeriodicalId":8929,"journal":{"name":"Biotechnology Letters","volume":"47 2","pages":"39"},"PeriodicalIF":2.0,"publicationDate":"2025-04-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143794307","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}

{"title":"Anti-liver fibrotic effects of small extracellular vesicle microRNAs from human umbilical cord-derived mesenchymal stem cells and their differentiated hepatocyte-like cells.","authors":"Min-Seok Choi, Jae-Sang Hong, Do-Hoon Lee, Yu Jin Jang, Jong-Hoon Kim, Young Sik Lee","doi":"10.1007/s10529-025-03579-3","DOIUrl":"10.1007/s10529-025-03579-3","url":null,"abstract":"<p><strong>Objective: </strong>The aim of this study is to identify therapeutic cargos within mesenchymal stem cell (MSC)-derived small extracellular vesicles (sEVs) for the treatment of liver fibrosis, a condition that poses significant health risks.</p><p><strong>Results: </strong>sEVs from human umbilical cord-derived MSCs (UCMSCs) and their differentiated hepatocyte-like cells (hpUCMSCs) were found to alleviate liver fibrosis in mouse models, reduce fibrogenic gene expression in the liver, and inhibit hepatic stellate cell (HSC) activation, a central driver of liver fibrosis, in vitro. Deep sequencing identified differentially abundant microRNAs (miRNAs) (high-abundance: 57, low-abundance: 22) in both UCMSC- and hpUCMSC-derived sEVs, compared to HeLa cell-derived sEVs, which lack anti-liver fibrotic activity. Functional enrichment analysis of the high-abundance sEV miRNA targets revealed their involvement in transcriptional regulation, apoptosis, and cancer-related pathways, all of which are linked to liver fibrosis and hepatocellular carcinoma. Notably, many of the top 10 most abundant miRNAs reduced pro-fibrotic marker levels in activated HSCs in vitro.</p><p><strong>Conclusion: </strong>The therapeutic potential of the high-abundance miRNAs shared by UCMSC- and hpUCMSC-derived sEVs in treating liver fibrosis is highlighted.</p>","PeriodicalId":8929,"journal":{"name":"Biotechnology Letters","volume":"47 2","pages":"38"},"PeriodicalIF":2.0,"publicationDate":"2025-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143771298","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}

{"title":"Single-cell protein production from photosynthetic bacteria wastewater treatment.","authors":"Wei Zhao, Mingyue Zhao, Sijia Zheng, Guangming Zhang","doi":"10.1007/s10529-025-03582-8","DOIUrl":"https://doi.org/10.1007/s10529-025-03582-8","url":null,"abstract":"<p><p>The production of single-cell protein (SCP) from microorganisms holds significant importance due to its potential as an alternative protein source. Photosynthetic bacteria (PSB) wastewater treatment and resource recovery method stands out as an effective means to produce SCP, protein content is usually in the 40-60% range, thereby making it a highly valuable byproduct. This comprehensive review not only summarizes the current methods for the production and utilization of SCP but also traces the historical evolution of protein production from PSB wastewater treatment. It delves into the various factors that influence the yield of SCP, meticulously analyzing aspects such as the specific PSB strain employed, the type of wastewater processed, and the light-oxygen conditions under which the process occurs.While this technology has garnered increasing attention in recent years owing to its dual benefits of wastewater treatment and SCP production, the number of studies conducted in this field remains relatively scarce. Furthermore, the majority of these studies have primarily focused on the utilization of the Rhodopseudomonas genus for treating food wastewater treatment under light-anaerobic conditions. Despite these advancements, challenges to economic viability and limitations to industrial-scale production remain. At the conclusion of this review, we discuss the existing problems within the technology, such as the need for optimized conditions for different PSB strains and wastewater types, as well as the potential future prospects for its widespread adoption and commercialization.</p>","PeriodicalId":8929,"journal":{"name":"Biotechnology Letters","volume":"47 2","pages":"37"},"PeriodicalIF":2.0,"publicationDate":"2025-03-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143750819","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}

{"title":"The pedal-like loop of (R)-selective transaminases plays a critical role to the functionality of the enzyme.","authors":"Chao Xiang, Yu-Ke Ce, Ya-Ping Xue, Yu-Guo Zheng","doi":"10.1007/s10529-025-03577-5","DOIUrl":"https://doi.org/10.1007/s10529-025-03577-5","url":null,"abstract":"<p><p>In enzyme engineering, a lot of studies have focused on engineering the active site to broaden substrate specificity or enhance transaminase activity; however, relatively little is known about the mechanisms by which substrates are recognized and enter the binding pocket. Transaminases play a crucial role in the synthesis of chiral amines due to their exceptional stereoselectivity and catalytic efficiency. In this study, we explored how the pedal-like loop at the active site influences (R)-transaminase (ATA) activity and substrate recognition by modulating the substrate channel. The pedal-like loop at the active site was swapped with loops from other well-characterized transaminases, and the best-performing variant exhibited a 5.2-fold increase in activity toward (R)-phenylethylamine ((R)-PEA) and an 11.8-fold increase in activity toward isopropylamine (IPA). Additionally, some variants showed significant changes in substrate preference. Homology modeling and molecular docking analysis provided compelling evidence that the pedal-like loop is a critical determinant of both substrate recognition and catalytic activity in (R)-ATA.</p>","PeriodicalId":8929,"journal":{"name":"Biotechnology Letters","volume":"47 2","pages":"35"},"PeriodicalIF":2.0,"publicationDate":"2025-03-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143655962","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}

{"title":"Optimization of recombinant neurturin expression in Escherichia coli using response surface methodology.","authors":"Zahra Hajihassan, Aysan Yaseri, Mina Yazdi","doi":"10.1007/s10529-025-03575-7","DOIUrl":"https://doi.org/10.1007/s10529-025-03575-7","url":null,"abstract":"<p><p>Neurturin, a neurotrophic growth factor, has been identified as a potential treatment or reversal agent for neurodegenerative conditions. Although Escherichia coli is an appropriate host for recombinant protein expression, the production of proteins with disulfide bonds, such as neurturin, in this strain is frequently accompanied by the formation of inclusion bodies. In this study, the Rosetta-gami strain, which is well-suited for the accurate formation of disulfide bonds was employed for the soluble production of neurturin. Response surface methodology (RSM) was also used to investigate the effects of IPTG concentration, post-induction time and temperature on the soluble production of neurturin. The results showed that the highest yield of neurturin production occurred in the presence of 0.8 mM of IPTG after 5.5 h at 26 ºC. Fractional Factorial Design was used in the subsequent stage to screen the effects of culture medium components on the protein production. The best concentrations of yeast extract, tryptone and MgSO₄ to have a significant effect on total protein concentration were determined by RSM design to be 15 g/l for both tryptone and yeast extract and 2.2 g/l for MgSO₄. Finally, an experiment was carried out under optimized conditions to evaluate the yield of the process. The results demonstrated a notable enhancement in neurturin production following optimization, with an increase of 8.6-fold compared to the normal condition.</p>","PeriodicalId":8929,"journal":{"name":"Biotechnology Letters","volume":"47 2","pages":"36"},"PeriodicalIF":2.0,"publicationDate":"2025-03-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143655961","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}

{"title":"Biochemical characterization of a bilfunctional endoglucanase/glucomannanase derived from mountain soil.","authors":"Justice Kipkorir Rono, Qingyun Zhang, Yong He, Shaochen Wang, Yunbin Lyu, Zhi Min Yang, Zhiyang Feng","doi":"10.1007/s10529-025-03574-8","DOIUrl":"https://doi.org/10.1007/s10529-025-03574-8","url":null,"abstract":"<p><p>Metagenomics is increasingly recognized as a vital technique for exploring uncultured microorganisms, with one key application being the discovery of novel enzymes for industrial use. This study identified an endoglucanase gene from soil metagenome, termed ZFEG1801, which was expressed in E. coli BL21, purified, and characterized for its biochemical properties. The 72.8 kDa recombinant protein exhibited hydrolytic activity against sodium carboxymethyl cellulose (CMC) and konjac glucomannan (KG), with activities of 12.1 U/mg and 42.1 U/mg, respectively. The enzyme displayed optimal activity at pH 5 for CMC and pH 6 for KG, with broad pH stability ranging from 5 to 9. The optimal temperature was 40 °C, and it remained thermally stable between 20 and 40 °C, retaining over 60% of its activity. The enzyme activity remained stable in the presence of most metal ions; however, CMCase activity was inhibited by Cu²⁺, while glucomannanase activity was inhibited by Mn²⁺, Fe³⁺, and Ca²⁺. The catalytic efficiency towards both substrates was reduced by addition of SDS, DMSO, ethanol, isopropanol and acetonitrile. The V_max and K_m of the purified recombinant enzyme were 106.4 μmol/L/min and 4.9 mg/mL for CMC, and 833.3 μmol/L/min and 11.1 mg/mL for KG, respectively. The dual catalytic properties of ZFEG1801, broad pH stability and resistance to additives, demonstrate its potential for use in various biomass degradation processes.</p>","PeriodicalId":8929,"journal":{"name":"Biotechnology Letters","volume":"47 2","pages":"33"},"PeriodicalIF":2.0,"publicationDate":"2025-03-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143630173","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}

{"title":"Metabolic engineering of Escherichia coli for enhanced production of hyaluronic acid.","authors":"Syafira Rizqi Eskasalam, Selim Ashoor, Hyeon Jeong Seong, Yu-Sin Jang","doi":"10.1007/s10529-025-03578-4","DOIUrl":"https://doi.org/10.1007/s10529-025-03578-4","url":null,"abstract":"<p><strong>Objectives: </strong>To enhance hyaluronic acid (HA) production in Escherichia coli by utilizing hasA genes from Streptococcus pyogenes and Streptococcus parauberis, and employing metabolic engineering strategies.</p><p><strong>Results: </strong>The expression of the hasA (SpaHasA) gene from S. parauberis in E. coli K12 W3110 led to higher HA production compared to the other gene. Knockout of the zwf and pfkA genes in the engineered E. coli expressing SpaHasA gene, further increased HA production to 891 mg l^-1. Overexpression of the galU and ugd genes in the zwf and pfkA double mutant harboring the SpaHasA gene elevated HA output to 1017 mg l^-1. Using the same engineered E. coli strain, optimizing the MgSO₄ concentration in the culture medium enhanced production to 1187 mg l^-1, and in fed-batch fermentation, it achieved 2283 mg HA l^-1.</p><p><strong>Conclusions: </strong>The hasA genes from various Streptococcus groups, especially S. parauberis, significantly boost HA production in E. coli, demonstrating their potential for microbial fermentation applications.</p>","PeriodicalId":8929,"journal":{"name":"Biotechnology Letters","volume":"47 2","pages":"34"},"PeriodicalIF":2.0,"publicationDate":"2025-03-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143630105","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}

{"title":"Multiplicity of infection and culture medium on the SARS-CoV-2 virus like-particles production by baculovirus/insect system.","authors":"Luis Giovani de Oliveira Guardalini, Felipe Moura Dias, Samanta Omae Camalhonte, Jaci Leme, Thaissa Consoni Bernardino, Felipe Soares Sposito, Eduardo Dias, Renato Mancini Astray, Aldo Tonso, Soraia Attie Calil Jorge, Eutimio Gustavo Fernández Núñez","doi":"10.1007/s10529-025-03572-w","DOIUrl":"https://doi.org/10.1007/s10529-025-03572-w","url":null,"abstract":"<p><p>This work aimed to assess the SARS-CoV-2 structural proteins' expression and virus-like particles (VLP) production by Baculovirus/Insect cell platform using two levels of Multiplicity of Infection (MOI), and two culture media, one of them a serum-free medium and the other one chemically defined. Two SARS-CoV-2 VLP were obtained from Sf9 cells coinfection using in both cases, three monocistronic recombinant baculoviruses holding the genes of Nucleocapsid (N; MOI = 2 or 0.2), Membrane (M; MOI = 1 or 0.1), and Envelope (E; MOI = 1 or 0.1) viral proteins, and the fourth one was changed between a baculovirus bearing Spike protein (S; MOI = 3 or 0.3) or receptor-binding domain (RBD; MOI = 3 or 0.3) genes of SARS-CoV-2. Similar performance was verified for both culture media in SARS-CoV-2 VLP production bearing four structural virus proteins or RBD domain. The SARS-CoV-2 structural proteins' expression was comparable at different MOIs (tenfold) as well as SARS-CoV-2 VLP size (around 100 nm). The increase in specific death rates over the coinfection phase was confirmed in relatively high MOI assays. This finding was related to an exponential virus titer profile for high MOIs over the entire infection phase, meanwhile, a viral peak was observed at low MOIs, confirming a secondary infection. The SARS-CoV-2 VLP improved production carrying immunogenic S protein was confirmed concerning others holding RBD. However, the protein composition of produced VLP should be studied further to assess the VLP homogeneity when different culture media and MOIs are used.</p>","PeriodicalId":8929,"journal":{"name":"Biotechnology Letters","volume":"47 2","pages":"32"},"PeriodicalIF":2.0,"publicationDate":"2025-03-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143565999","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}

{"title":"Development of a fully automated latex-enhanced immunoturbidimetric method for quantitative serum Lp(a) measurement.","authors":"Yanyan Liu, Meijiao Li, Hao Zhang, Le Gao, Jitao Liu, Yue Hou, Jiancheng Xu","doi":"10.1007/s10529-025-03564-w","DOIUrl":"https://doi.org/10.1007/s10529-025-03564-w","url":null,"abstract":"<p><strong>Background: </strong>Lipoprotein (a) [Lp(a)] is a critical factor in cardiovascular health, composed of low-density lipoprotein-like particles bound to apolipoprotein (a). Elevated Lp(a) levels are associated with an increased risk of cardiovascular diseases (CVD), accelerating disease progression and raising CVD-related mortality. However, the lack of standardized measurement methods for Lp(a) contributes to diagnostic uncertainties in this area.</p><p><strong>Method: </strong>A quantitative measurement method for serum Lp(a) was developed using fully automated latex-enhanced particle immunoturbidimetry, marking a significant advancement in diagnostic capabilities. Key parameters, including repeatability, stability, linearity, detection limit, interference, and method comparison, were evaluated to ensure the assay's reliability and accuracy.</p><p><strong>Result: </strong>Lp(a) in samples was detected by carboxylated latex particles (95 nm in diameter) covalently coated with anti-Lp(a) antibodies. Lp(a) concentration was quantified by measuring the turbidity changes caused by agglutination at 600 nm. This method provides rapid, accurate, and fully automated measurements on the Hitachi 7100 automatic biochemical analyzer. With intra-batch precision CV% of 1.10% and inter-batch precision CV% of 1.79%, the method demonstrates reliable performance with Randox biochemical quality control samples. It has a detection limit of 7 mg/L and a high correlation coefficient (R² = 0.9946) within the 0-1500 mg/L range. Minimal interference from bilirubin, fat emulsion, hemoglobin, and ascorbic acid was observed. Additionally, it shows strong correlation (R² = 0.9972) with a commercially available latex-enhanced immunoturbidimetric Lp(a) assay reagent, confirming its comparability and clinical suitability.</p><p><strong>Conclusion: </strong>The quantitative serum Lp(a) determination method based on latex-enhanced immunoturbidimetry offers numerous advantages. It provides rapid, accurate, and automated results, making it ideal for routine clinical testing. The method effectively measures Lp(a) in serum samples by leveraging the interaction between Lp(a) and latex particles.</p>","PeriodicalId":8929,"journal":{"name":"Biotechnology Letters","volume":"47 2","pages":"31"},"PeriodicalIF":2.0,"publicationDate":"2025-03-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143565968","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}

{"title":"Improving the hydrophilic microenvironment surrounding the catalytic site of fructosyltransferase enhances its catalytic ability.","authors":"Fanzhi Wang, Suren Singh, Kugen Permaul","doi":"10.1007/s10529-025-03566-8","DOIUrl":"10.1007/s10529-025-03566-8","url":null,"abstract":"<p><p>The hydrophilic microenvironment surrounding an enzyme's active site can influence its catalytic activity. This study examines the effect of enhancing this environment in the Aspergillus niger fructosyltransferase, SucC. Bioinformatics analysis identified a cysteine residue (C66) near the catalytic triad (D64, D194, E271) as vital for maintaining the active site's structure and facilitating substrate transport. Simulated mutagenesis suggested that mutating cysteine to serine (C66S) could increase hydrophilicity without altering the structure significantly. This mutation was predicted to enhance substrate affinity, with binding energy changing from -3.65 to -4.14 kcal mol^-1. The C66S mutant, expressed in Pichia pastoris GS115, showed a 61.3% increase in specific activity, a 13.5% decrease in K_m (82.20/71.14 mM), and a 21.6% increase in k_cat (112.23/136.48 min^-1), resulting in a 40.1% increase in catalytic efficiency (1.37/1.92 min^-1 mM^-1). For fructooligosaccharides (FOS) production, C66S demonstrated enhanced transfructosylation, particularly in the initial stages of the reaction, achieving higher overall FOS yields. These findings highlight that modifying the active site hydrophilicity, without causing major structural changes, is a promising strategy for improving an enzyme's catalytic efficiency.</p>","PeriodicalId":8929,"journal":{"name":"Biotechnology Letters","volume":"47 2","pages":"30"},"PeriodicalIF":2.0,"publicationDate":"2025-02-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11865173/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143514671","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}