Biotechnology Journal最新文献

{"title":"Efficient production of 22(R)-hydroxycholesterol via combination optimization of Saccharomyces cerevisiae","authors":"Yaru Pang,&nbsp;Xu Cheng,&nbsp;Yali Ban,&nbsp;Yue Li,&nbsp;Bo Lv,&nbsp;Chun Li","doi":"10.1002/biot.202400286","DOIUrl":"10.1002/biot.202400286","url":null,"abstract":"<p>22(R)-hydroxycholesterol (22(R)-HCHO) is a crucial precursor of steroids biosynthesis with various biological functions. However, the production of 22(R)-HCHO is expensive and unsustainable due to chemical synthesis and extraction from plants or animals. This study aimed to construct a microbial cell factory to efficiently produce 22(R)-HCHO through systems metabolic engineering. First, we tested 7-dehydrocholesterol reductase (Dhcr7s) and cholesterol C22-hydroxylases from different sources in <i>Saccharomyces cerevisiae</i>, and the titer of 22(R)-HCHO reached 128.30 mg L⁻¹ in the engineered strain expressing Dhcr7 from <i>Columba livia</i> (ClDhcr7) and cholesterol 22-hydroxylase from <i>Veratrum californicum</i> (VcCyp90b27). Subsequently, the 22(R)-HCHO titer was significantly increased to 427.78 mg L⁻¹ by optimizing the critical genes involved in 22(R)-HCHO biosynthesis. Furthermore, hybrid diploids were constructed to balance cell growth and 22(R)-HCHO production and to improve stress tolerance. Finally, the engineered strain produced 2.03 g L⁻¹ of 22(R)-HCHO in a 5-L fermenter, representing the highest 22(R)-HCHO titer reported to date in engineered microbial cell factories. The results of this study provide a foundation for further applications of 22(R)-HCHO in various industrially valuable steroids.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 7","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141625464","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Synthetic biology for Monascus: From strain breeding to industrial production","authors":"Junping Zhou,&nbsp;Qilu Pan,&nbsp;Yinan Xue,&nbsp;Yaping Dong,&nbsp;Yihong Chen,&nbsp;Lianggang Huang,&nbsp;Bo Zhang,&nbsp;Zhi-Qiang Liu,&nbsp;Yuguo Zheng","doi":"10.1002/biot.202400180","DOIUrl":"10.1002/biot.202400180","url":null,"abstract":"<p>Traditional Chinese food therapies often motivate the development of modern medicines, and learning from them will bring bright prospects. <i>Monascus</i>, a conventional Chinese fungus with centuries of use in the food industry, produces various metabolites, including natural pigments, lipid-lowering substances, and other bioactive ingredients. Recent <i>Monascus</i> studies focused on the metabolite biosynthesis mechanisms, strain modifications, and fermentation process optimizations, significantly advancing <i>Monascus</i> development on a lab scale. However, the advanced manufacture for <i>Monascus</i> is lacking, restricting its scale production. Here, the synthetic biology techniques and their challenges for engineering filamentous fungi were summarized, especially for <i>Monascus</i>. With further in-depth discussions of automatic solid-state fermentation manufacturing and prospects for combining synthetic biology and process intensification, the industrial scale production of <i>Monascus</i> will succeed with the help of <i>Monascus</i> improvement and intelligent fermentation control, promoting <i>Monascus</i> applications in food, cosmetic, agriculture, medicine, and environmental protection industries.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 7","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141625466","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"UV-based dynamic control improves the robustness of multicolumn countercurrent solvent gradient purification of oligonucleotides","authors":"Ismaele Fioretti,&nbsp;Thomas Müller-Späth,&nbsp;Lars Aumann,&nbsp;Mattia Sponchioni","doi":"10.1002/biot.202400170","DOIUrl":"10.1002/biot.202400170","url":null,"abstract":"<p>Therapeutic oligonucleotides (ONs) have great potential to treat many diseases due to their ability to regulate gene expression. However, the inefficiency of standard purification techniques to separate the target sequence from molecularly similar variants is hindering development of large scale ON manufacturing at a reasonable cost. Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) is a valuable process able to bypass the purity-yield tradeoff typical of single-column operations, and hence to make the ON production more sustainable from both an economic and environmental point of view. However, operating close to the optimum of MCSGP can be challenging, resulting in unstable process performance and in a drift in product quality, especially when running a continuous process for extended periods where process parameters such as temperature are prone to variation. In this work, we demonstrate how greater process robustness is introduced in the design and execution of MCSGP for the purification of a 20mer single-stranded DNA sequence through the implementation of UV-based dynamic control. With this novel approach, the cyclic steady state was reached already in the third cycle and disturbances coming from fluctuations in the feed quality, loading amount and temperature were effectively compensated allowing a stable operation close to the optimum. In response to the perturbations, the controlled process kept the standard deviation on product recovery below 3.4%, while for the non-controlled process it increased up to 27.5%.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 7","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202400170","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141625468","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Efficient generation of recombinant anti-HER2 scFv with high yield and purity using a simple method","authors":"Hanool Yun,&nbsp;Sun-Hee Kim,&nbsp;Seung-Hee Kim,&nbsp;Hae-Min Park,&nbsp;Hee-Jin Jeong","doi":"10.1002/biot.202300745","DOIUrl":"10.1002/biot.202300745","url":null,"abstract":"<p>We developed a method to produce a soluble form of a single-chain fragment variable (scFv) targeting human epithelial growth factor receptor 2 (HER2) in <i>Escherichia coli</i>. By optimizing the orientations of the variable heavy (VH) and variable light (VL) domains and the His-tag, we identified the HL-His type antibody with the highest HER2-binding activity. Purification of HL-His yielded 40.7 mg from a 1 L culture, achieving &gt;99% purity. The limit of detection was determined to be 2.9 ng, demonstrating high production yield, purity, and sensitivity. Moreover, we successfully labeled HER2⁺ cell lines with fluorescent dye-conjugated scFv, resulting in a significantly higher observed signal-to-background ratio, compared to that of HER2⁻ cell lines. This highlights the potential of these fluorescent scFvs as valuable probes for HER2⁺ breast cancer diagnostics. Notably, the process for the complete scFv production was streamlined and required only 4–5 days. Additionally, the product maintained its activity after freeze storage, allowing for large-scale production and a wide range of practical applications.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 7","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202300745","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141625463","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Novel calibration design improves knowledge transfer across products for the characterization of pharmaceutical bioprocesses","authors":"Laura M. Helleckes,&nbsp;Claus Wirnsperger,&nbsp;Jakub Polak,&nbsp;Gonzalo Guillén-Gosálbez,&nbsp;Alessandro Butté,&nbsp;Moritz von Stosch","doi":"10.1002/biot.202400080","DOIUrl":"10.1002/biot.202400080","url":null,"abstract":"<p>Modern machine learning has the potential to fundamentally change the way bioprocesses are developed. In particular, horizontal knowledge transfer methods, which seek to exploit data from historical processes to facilitate process development for a new product, provide an opportunity to rethink current workflows. In this work, we first assess the potential of two knowledge transfer approaches, meta learning and one-hot encoding, in combination with Gaussian process (GP) models. We compare their performance with GPs trained only on data of the new process, that is, local models. Using simulated mammalian cell culture data, we observe that both knowledge transfer approaches exhibit test set errors that are approximately halved compared to those of the local models when two, four, or eight experiments of the new product are used for training. Subsequently, we address the question whether experiments for a new product could be designed more effectively by exploiting existing knowledge. In particular, we suggest to specifically design a few runs for the novel product to calibrate knowledge transfer models, a task that we coin <i>calibration design</i>. We propose a customized objective function to identify a set of calibration design runs, which exploits differences in the process evolution of historical products. In two simulated case studies, we observed that training with calibration designs yields similar test set errors compared to common design of experiments approaches. However, the former requires approximately four times fewer experiments. Overall, the results suggest that process development could be significantly streamlined when systematically carrying knowledge from one product to the next.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 7","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-07-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141597931","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Engineering a 3-dimensional tissue construct with adipose-derived stem cells for healing bone defect: An ex vivo study with femur head","authors":"Aditi Mahajan,&nbsp;Siddhartha Sharma,&nbsp;Sanjay Kumar Bhadada,&nbsp;Aditya Aggarwal,&nbsp;Shalmoli Bhattacharyya","doi":"10.1002/biot.202300751","DOIUrl":"10.1002/biot.202300751","url":null,"abstract":"<div>\u0000 \u0000 <p>The compatibility of bone graft substitutes (BGS) with mesenchymal stem cells (MSCs) is an important parameter to consider for their use in repairing bone defects as it eventually affects the clinical outcome. In the present study, a few commercially available BGS – β-tricalcium phosphate (β-TCP), calcium sulfate, gelatin sponge, and different forms of hydroxyapatite (HAP) were screened for their interactions with MSCs from adipose tissue (ADSCs). It was demonstrated that HAP block favorably supported ADSC viability, morphology, migration, and differentiation compared to other scaffolds. The results strongly suggest the importance of preclinical evaluation of bone scaffolds for their cellular compatibility. Furthermore, the bone regenerative potential of HAP block with ADSCs was evaluated in an ex vivo bone defect model developed using patient derived trabecular bone explants. The explants were cultured for 45 days in vitro and bone formation was assessed by expression of osteogenic genes, ALP secretion, and high resolution computed tomography. Our findings confirmed active bone repair process in ex vivo settings. Addition of ADSCs significantly accelerated the repair process and improved bone microarchitecture. This ex vivo bone defect model can emerge as a viable alternative to animal experimentation and also as a potent tool to evaluate patient specific bone therapeutics under controlled conditions.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 7","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-07-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141578405","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Human platelet lysate supports SH-SY5Y neuroblastoma cell proliferation and differentiation into a dopaminergic-like neuronal phenotype under xenogeneic-free culture conditions","authors":"Miguel Ribeiro,&nbsp;Jonas Campos,&nbsp;Tiffany S. Pinho,&nbsp;Belém Sampaio-Marques,&nbsp;Sandra Barata-Antunes,&nbsp;Jorge Ribeiro Cibrão,&nbsp;Ricardo Araújo,&nbsp;Sara Duarte-Silva,&nbsp;Elsa Moreira,&nbsp;Rui A. Sousa,&nbsp;Pedro M. Costa,&nbsp;António J. Salgado","doi":"10.1002/biot.202400068","DOIUrl":"10.1002/biot.202400068","url":null,"abstract":"<p>SH-SY5Y is a human neuroblastoma cell line that can be differentiated into several neuronal phenotypes, depending on culture conditions. For this reason, this cell line has been widely used as an in vitro model of neurodegenerative conditions, such as Parkinson's disease (PD). However, most studies published to date used fetal bovine serum (FBS) as culture medium supplement for SH-SY5Y cell differentiation. We report on the testing of human platelet lysate (hPL) as a culture medium supplement to support SH-SY5Y cell culture. Both standard hPL and a fibrinogen-depleted hPL (FD-hPL) formulation, which does not require the addition of anticoagulants to culture media, promoted an increase in SH-SY5Y cell proliferation in comparison to FBS, without compromising metabolic activity. SH-SY5Y cells cultured in hPL or FD-hPL also displayed a higher number of neurite extensions and stained positive for MAP2 and synaptophysin, in the absence of differentiation stimuli; reducing hPL or FD-hPL concentration to 1% v/v did not affect cell proliferation or metabolic activity. Furthermore, following treatment with retinoic acid (RA) and further stimulation with brain-derived neurotrophic factor (BDNF) and nerve growth factor beta (NGF-β), the percentage of SH-SY5Y cells stained positive for dopaminergic neuronal differentiation markers (tyrosine hydroxylase [TH] and Dopamine Transporter [DAT]) was higher in hPL or FD-hPL than in FBS, and gene expression of dopaminergic markers TH, DAT, and DR2 was also detected.</p><p>Overall, the data herein presented supports the use of hPL to differentiate SH-SY5Y cells into a neuronal phenotype with dopaminergic features, and the adoption of FD-hPL as a fully xenogeneic free alternative to FBS to support the use of SH-SY5Y cells as a neurodegeneration model.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 7","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-07-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141578353","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Toehold region triggered CRISPR/Cas12a trans-cleavage for detection of uracil-DNA glycosylase activity","authors":"Chenyu Cui,&nbsp;Guihuan Guo,&nbsp;Ting-Hsuan Chen","doi":"10.1002/biot.202400097","DOIUrl":"10.1002/biot.202400097","url":null,"abstract":"<p>DNA glycosylases are a group of enzymes that play a crucial role in the DNA repair process by recognizing and removing damaged or incorrect bases from DNA molecules, which maintains the integrity of the genetic information. The abnormal expression of uracil-DNA glycosylase (UDG), one of significant DNA glycosylases in the base-excision repair pathway, is linked to numerous diseases. Here, we proposed a simple UDG activity detection method based on toehold region triggered CRISPR/Cas12a <i>trans</i>-cleavage. The toehold region on hairpin DNA probe (HP) produced by UDG could induce the <i>trans</i>-cleavage of ssDNA with fluorophore and quencher, generating an obvious fluorescence signal. This protospacer adjacent motif (PAM)-free approach achieves remarkable sensitivity and specificity in detecting UDG, with a detection limit as low as 0.000368 U mL⁻¹. Moreover, this method is able to screen inhibitors and measure UDG in complex biological samples. These advantages render it highly promising for applications in clinical diagnosis and drug discovery.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 7","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-07-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202400097","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141578355","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Fast-track adaptive laboratory evolution of Cupriavidus necator H16 with divalent metal cations","authors":"Sepwin Nosten Sitompul,&nbsp;Laura Andrea Diaz Garcia,&nbsp;Joseph Price,&nbsp;Kang Lan Tee,&nbsp;Tuck Seng Wong","doi":"10.1002/biot.202300577","DOIUrl":"10.1002/biot.202300577","url":null,"abstract":"<p>Microbial strain improvement through adaptive laboratory evolution (ALE) has been a key strategy in biotechnology for enhancing desired phenotypic traits. In this Biotech Method paper, we present an accelerated ALE (aALE) workflow and its successful implementation in evolving <i>Cupriavidus necator</i> H16 for enhanced tolerance toward elevated glycerol concentrations. The method involves the deliberate induction of genetic diversity through controlled exposure to divalent metal cations, enabling the rapid identification of improved variants. Through this approach, we observed the emergence of robust variants capable of growing in high glycerol concentration environments, demonstrating the efficacy of our aALE workflow. When cultivated in 10% v/v glycerol, the adapted variant Mn-C2-B11, selected through aALE, achieved a final OD₆₀₀ value of 56.0 and a dry cell weight of 15.2 g L⁻¹, compared to the wild type (WT) strain's final OD₆₀₀ of 39.1 and dry cell weight of 8.4 g L⁻¹. At an even higher glycerol concentration of 15% v/v, Mn-C2-B11 reached a final OD₆₀₀ of 48.9 and a dry cell weight of 12.7 g L⁻¹, in contrast to the WT strain's final OD₆₀₀ of 9.0 and dry cell weight of 3.1 g L⁻¹. Higher glycerol consumption by Mn-C2-B11 was also confirmed by high-performance liquid chromatography (HPLC) analysis. This adapted variant consumed 34.5 times more glycerol compared to the WT strain at 10% v/v glycerol. Our method offers several advantages over other reported ALE approaches, including its independence from genetically modified strains, specialized genetic tools, and potentially carcinogenic DNA-modifying agents. By utilizing divalent metal cations as mutagens, we offer a safer, more efficient, and cost-effective alternative for expansion of genetic diversity. With its ability to foster rapid microbial evolution, aALE serves as a valuable addition to the ALE toolbox, holding significant promise for the advancement of microbial strain engineering and bioprocess optimization.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 7","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-07-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202300577","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141578352","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Biomass specific perfusion rate as a control lever for the continuous manufacturing of biosimilar monoclonal antibodies from CHO cell cultures","authors":"Jiayu Leong,&nbsp;Wen Qin Tang,&nbsp;Jake Chng,&nbsp;Wei Xuan Ler,&nbsp;Norhaizat Abdul Manan,&nbsp;Lyn Chiin Sim,&nbsp;Zi Ying Zheng,&nbsp;Wei Zhang,&nbsp;Ian Walsh,&nbsp;Gerben Zijlstra,&nbsp;Maarten Pennings,&nbsp;Say Kong Ng","doi":"10.1002/biot.202400092","DOIUrl":"10.1002/biot.202400092","url":null,"abstract":"<p>Continuous manufacturing enables high volumetric productivities of biologics such as monoclonal antibodies. However, it is challenging to maintain both high viable cell densities and productivities at the same time for long culture durations. One of the key controls in a perfusion process is the perfusion rate which determines the nutrient availability and potentially controls the cell metabolism. Cell Specific Perfusion Rate (CSPR) is a feed rate proportional to the viable cell density while Biomass Specific Perfusion Rate (BSPR) is a feed rate proportional to the biomass (cell volume multiply by cell density). In this study, perfusion cultures were run at three BSPRs in the production phase. Low BSPR favored a growth arresting state that led to gradual increase in cell volume, which in turn led to an increase in net perfusion rate proportional to the increase in cell volume. Consequently, at low BSPR, while the cell viability and cell density decreased, high specific productivity of 55 pg per cell per day was achieved. In contrast, the specific productivity was lower in bioreactors operating at a high BSPR. The ability to modulate the cell metabolism by using BSPR was confirmed when the specific productivity increased after lowering the BSPR in one of the bioreactors that was initially operating at a high BSPR. This study demonstrated that BSPR significantly influenced cell growth, metabolism, and productivity in cultures with variable cell volumes.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 7","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-07-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202400092","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141578403","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}