Journal of biotechnology最新文献

{"title":"Monitoring small-scale bioreactor studies for media development using polarized total synchronous fluorescence spectroscopy (pTSFS) and synchronous light scattering (SyLS)","authors":"Bernard O. Boateng,&nbsp;Alan G. Ryder","doi":"10.1016/j.jbiotec.2024.10.002","DOIUrl":"10.1016/j.jbiotec.2024.10.002","url":null,"abstract":"<div><div>Biopharmaceutical process development often involves the use of small-scale bioreactors (SSBR) for optimizing media formulations and process conditions during scale up to commercial scale production. Two key process parameters (CPP) used in SSBR studies are protein titre and viable cell density (VCD). Here, we explore the efficacy of parallel polarized total synchronous fluorescence spectroscopy (TSFS_||) and Synchronous Light Scattering (SyLS_||) to qualitatively monitor these CPPs and quantitatively predict titre and VCD for a large-scale cell culture media optimization SSBR study. The study involved 71 different media formulations (50+ components each), and the bioprocess was run for 13 days or more. Samples were extracted at set times (Day 0, 3, 9, and 13) and clarified by centrifugation. TSFS_|| spectra showed significant emission changes along with increased light scatter over the course of the bioprocess. SyLS_|| measurements strongly correlated with particle size data obtained from Dynamic Light Scattering but did not correlate well with VCD probably because of the centrifugation-based sample preparation. Statistical and principal component analysis (PCA) of the pTSFS data showed that spectral variation was greater between media formulations than due to the evolving bioprocess. This prevented the development of accurate global prediction models for media performance (e.g., predicting product titre at day 9 from media spectra measured at day 0). However, classification methods were successfully used to select media subsets with better quantitative prediction accuracy based on spectral similarities. A practical binary (high/low performance) classification model based on Support Vector Machines was generated for media formulation screening. Combining emission and scatter measurements with multivariate data analysis provides a more holistic, multi-attribute bioprocess monitoring method that minimizes the need to use different offline analytical methods. This methodology can be used to monitor process trajectories and deviations, and ultimately be used to predict bioprocess CPPs when implemented on production scale processes where there is much less compositional variation in the media. We believe this SSBR-pTSFS/SyLS approach will provide a valuable resource to develop the design/parameter space for in-process monitoring at production scale from early-stage process/media development studies.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"395 ","pages":"Pages 205-215"},"PeriodicalIF":4.1,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142400370","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Harnessing nature's catalysts: Advances in enzymatic alkene cleavage","authors":"Lukas Schober ,&nbsp;Astrid Schiefer ,&nbsp;Margit Winkler ,&nbsp;Florian Rudroff","doi":"10.1016/j.jbiotec.2024.09.020","DOIUrl":"10.1016/j.jbiotec.2024.09.020","url":null,"abstract":"<div><div>Double bonds are prevalent in various substrates and renewable feedstocks, and their cleavage typically necessitates harsh reaction conditions involving high temperatures, organic solvents, and hazardous catalysts such as heavy metals or ozone. This review explores the sustainable enzymatic alternatives developed by nature for alkene cleavage. It provides a comprehensive overview of alkene-cleaving enzymes, detailing their mechanisms, substrate specificities, and applications. The enzymes discussed include those acting on aliphatic, cyclic, and activated aromatic systems. Emphasizing the significance of these biocatalysts in green chemistry and biocatalysis, this review highlights their potential to replace traditional chemical oxidants with safer, cost-effective, and environmentally friendly options. Future research directions include expanding enzyme substrate scopes, enhancing their operational stability and activity, and integrating them into scalable processes for broader application in the pharmaceutical, flavor, and fragrance industries.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"395 ","pages":"Pages 189-204"},"PeriodicalIF":4.1,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142371877","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Production of recombinant human type I collagen homotrimers in CHO cells and their physicochemical and functional properties","authors":"Chuan Wang ,&nbsp;Xiaolei Guo ,&nbsp;Mingtao Fan ,&nbsp;Long Yue ,&nbsp;Hang Wang ,&nbsp;Jiadao Wang ,&nbsp;Zhengqi Zha ,&nbsp;Hongping Yin","doi":"10.1016/j.jbiotec.2024.09.011","DOIUrl":"10.1016/j.jbiotec.2024.09.011","url":null,"abstract":"<div><div>Collagen is the most abundant protein in human and mammalian structures and is a component of the mammalian extracellular matrix (ECM). Recombinant collagen is a suitable alternative to native collagen extracted from animal tissue for various biomaterials. However, due to the limitations of the expression system, most recombinant collagens are collagen fragments and lack triple helix structures. In this study, Chinese hamster ovary (CHO) cells were used to express the full-length human type I collagen α1 chain (rhCol1α1). Moreover, Endo180 affinity chromatography and pepsin were used to purify pepsin-soluble rhCol1α1 (PSC1). The amino acid composition of PSC1 was closer to that of native human type I collagen, and PSC1 contained 9.1 % hydroxyproline. Analysis of the CD spectra and molecular weight distribution results revealed that PSC1 forms a stable triple helix structure that is resistant to pepsin hydrolysis and has some tolerance to MMP1, MMP2 and MMP8 hydrolysis. Atomic force microscopy (AFM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM) revealed that PSC1 can self-assemble into fibers at a concentration of 1 mg/ml; moreover, PSC1 can promote the proliferation and migration of NIH 3T3 cells. In conclusion, our data suggest that PSC1 is a highly similar type of recombinant collagen that may have applications in biomaterials and other medical fields.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"395 ","pages":"Pages 149-160"},"PeriodicalIF":4.1,"publicationDate":"2024-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142365347","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Intensified functional expression of recombinant Zymomonas mobilis zinc-dependent alcohol dehydrogenase I","authors":"Klaudia Žigová ,&nbsp;Zuzana Marčeková ,&nbsp;Tatiana Petrovičová ,&nbsp;Katarína Lorková ,&nbsp;František Čacho ,&nbsp;Vladimír Krasňan ,&nbsp;Martin Rebroš","doi":"10.1016/j.jbiotec.2024.09.012","DOIUrl":"10.1016/j.jbiotec.2024.09.012","url":null,"abstract":"<div><div>Alcohol dehydrogenase I from <em>Zymomonas mobilis</em> (zmADH1) is a zinc-dependent oxidoreductase that catalyses the oxidation of primary or secondary alcohols to the corresponding aldehydes or ketones using NAD⁺/NADH as a cofactor. Efforts to express zmADH1 in <em>Escherichia coli</em> in a soluble form have been laden with solubility difficulties. A soluble form of recombinant zmADH1 was achieved by the addition of 1 mM zinc into media. Zinc addition facilitates the proper folding of recombinant zmADH1 and significantly reduces the formation of inclusion bodies. The yield of recombinant zmADH1 represents approximately 30 mg/1 L Luria-Bertani media. Intensified production in fermenters showed a striking difference between the specific and total activities of zmADH1 produced at different zinc concentrations. The zmADH1 showed an affinity to medium-chain alcohols, especially 1-pentanol, which could be used in new greener routes for preparation of aldehydes and alcohols.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"395 ","pages":"Pages 141-148"},"PeriodicalIF":4.1,"publicationDate":"2024-09-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142347202","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Microbial production of levulinic acid from glucose by engineered Pseudomonas putida KT2440","authors":"Hyun Jin Kim ,&nbsp;Byung Chan Kim ,&nbsp;Hanna Park ,&nbsp;Geunsang Cho ,&nbsp;Taekyu Lee ,&nbsp;Hee Taek Kim ,&nbsp;Shashi Kant Bhatia ,&nbsp;Yung-Hun Yang","doi":"10.1016/j.jbiotec.2024.09.015","DOIUrl":"10.1016/j.jbiotec.2024.09.015","url":null,"abstract":"<div><div>Levulinic acid(LA) is produced through acid-catalyzed hydrolysis and dehydration of lignocellulosic biomass. It is a key platform chemical used as an intermediate in various industries including biofuels, cosmetics, pharmaceuticals, and polymers. Traditional LA production uses chemical conversion, which requires high temperatures and pressures, strong acids, and produces undesirable side reactions, repolymerization products, and waste problems Therefore, we designed an integrated process to produce LA from glucose through metabolic engineering of <em>Pseudomonas putida</em> KT2440. As a metabolic engineering strategy, codon optimized phospho-2-dehydro-3-deoxyheptonate aldolase (AroG), 3-dehydroshikimate dehydratase (AsbF), and acetoacetate decarboxylase (Adc) were introduced to express genes of the shikimate and β-ketoadipic acid pathways, and the 3-oxoadipate CoA-transferase (<em>pcaIJ)</em> gene was deleted to prevent loss of biosynthetic intermediates. To increase the accumulation of the produced LA, the <em>lva</em> operon encoding levulinyl-CoA synthetase (LvaE) was deleted resulting in the high LA-producing strain <em>P. putida</em> HP203. Culture conditions such as medium, temperature, glucose concentration, and nitrogen source were optimized, and under optimal conditions, <em>P. putida</em> HP203 strain biosynthesized 36.3 mM (4.2 g/L) LA from glucose in a fed-batch fermentation system. When lignocellulosic biomass hydrolysate was used as the substrate, this strain produced 7.31 mM of LA. This is the first report of microbial production of LA from glucose by <em>P. putida</em>. This study suggests the possibility of manipulating biosynthetic pathway to produce biological products from glucose for various applications.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"395 ","pages":"Pages 161-169"},"PeriodicalIF":4.1,"publicationDate":"2024-09-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142347213","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Photocurable biomaterials labeled with luminescent sensors dedicated to bioprinting","authors":"Paweł Jamróz ,&nbsp;Andrzej Świeży ,&nbsp;Małgorzata Noworyta ,&nbsp;Katarzyna Starzak ,&nbsp;Patrycja Środa ,&nbsp;Weronika Wielgus ,&nbsp;Patryk Szymaszek ,&nbsp;Małgorzata Tyszka-Czochara ,&nbsp;Joanna Ortyl","doi":"10.1016/j.jbiotec.2024.09.017","DOIUrl":"10.1016/j.jbiotec.2024.09.017","url":null,"abstract":"<div><div>In the present study, we focused on the development and characterization of formulations that function as biological inks. These inks were doped with coumarin derivatives to act as molecular luminescent sensors that allow the monitoring of the kinetics of in situ photopolymerization in 3D (DLP) printing and bioprinting using pneumatic extrusion techniques, making it possible to study the changes in the system in real time. The efficiency of the systems was tested on compositions containing monomers: poly(ethylene glycol) diacrylates and photoinitiators: 2,4,6-trimethylbenzoyldi-phenylphosphinate and lithium phenyl-2,4,6-trimethylbenzoylphosphinate. The selected formulations were spectroscopically characterized and examined for their photopolymerization kinetics and rheological properties. This is important because of the fact that spectroscopic characterization, examination of photopolymerization kinetics, and rheological properties provide valuable insights into the behaviour of photocurable resin dedicated for 3D printing processes. The next step involved printing tests on commercially available 3D printers. In turn, printing carried out as part of the work on commercially available 3D printers further verified the effectiveness of the formulations. Moreover the formulation components and the resulting 3D objects were tested for their antiproliferative effects on the selected Chinese hamster ovary cell line, CHO-K1.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"395 ","pages":"Pages 122-140"},"PeriodicalIF":4.1,"publicationDate":"2024-09-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142347215","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Millet as a promising C4 model crop for sustainable biofuel production","authors":"Pooja R. Aggarwal ,&nbsp;Mehanathan Muthamilarasan ,&nbsp;Pooja Choudhary","doi":"10.1016/j.jbiotec.2024.09.019","DOIUrl":"10.1016/j.jbiotec.2024.09.019","url":null,"abstract":"<div><div>The rapid depletion of conventional fuel resources and rising energy demand has accelerated the search for alternative energy sources. Further, the expanding need to use bioenergy crops for sustainable fuel production has enhanced the competition for agricultural land, raising the “food vs. fuel” competition. Considering this, producing bioenergy crops on marginal land has a great perspective for achieving sustainable bioenergy production and mitigating the negative impacts of climate change. C4 crops are dual-purpose crops with better efficiency to fix atmospheric CO₂ and convert solar energy into lignocellulosic biomass. Of these, millets have gained worldwide attention due to their climate resilience and nutraceutical properties. Due to close synteny with contemporary C4 bioenergy crops, millets are being considered a model crop for studying diverse agronomically important traits associated with biomass production. Millets can be cultivated on marginal land with minimum fertilizer inputs and maximum biomass production. In this regard, advanced molecular approaches, including marker-assisted breeding, multi-omics approaches, and gene-editing technologies, can be employed to genetically engineer these crops for enhanced biofuel production efficiency. The current study aims to provide an overview of millets as a sustainable bioenergy source and underlines the significance of millets as a C4 model to elucidate the genes and pathways involved in lignocellulosic biomass production using advanced molecular biology approaches.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"395 ","pages":"Pages 110-121"},"PeriodicalIF":4.1,"publicationDate":"2024-09-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142347214","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Enhanced activation of signaling pathway by recombinant human adiponectin from genome-edited chickens","authors":"Eunhui Yoo,&nbsp;Hee Jung Choi,&nbsp;Jae Yong Han","doi":"10.1016/j.jbiotec.2024.09.016","DOIUrl":"10.1016/j.jbiotec.2024.09.016","url":null,"abstract":"<div><div>Adiponectin (ADPN) exerts various cellular and metabolic functions by activating signaling pathways, including extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) pathways, the protein kinase B (Akt) pathway, and the p38 mitogen-activated protein kinase (MAPK) pathway. However, generating functional recombinant human adiponectin (hADPN) in bacterial or mammalian cells is challenging. Although ADPN agonist peptides have been developed, problems like stability, solubility, and affinity for receptors remain. Recently, a genome-edited chicken bioreactor system was established, ensuring efficient ADPN production with optimal post-transcriptional modifications. We assessed the ability of egg white (EW)-derived hADPN, commercial hADPN, various ADPN agonist peptides, and globular ADPN on activation of the ERK1/2, Akt, and p38 MAPK pathways. EW-derived hADPN, abundant in hexamers and high molecular weight multimers, significantly phosphorylated ERK1/2 in serum-starved HEK293 cells after 15 min of treatment. Comparative analysis revealed that EW-derived hADPN and commercial hADPN induced greater phosphorylation of ERK1/2, Akt, and p38 MAPK than ADPN agonist peptides and globular ADPN, with EW-derived hADPN showing the highest activation. In summary, the finding that EW-derived hADPN strongly activates the ERK1/2, Akt, p38 MAPK signaling pathways highlights that an ADPN production system based on genome-edited chickens is an advantageous alternative to existing methods.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"395 ","pages":"Pages 95-99"},"PeriodicalIF":4.1,"publicationDate":"2024-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142327763","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Expediting adenovirus titer assays via an algorithmic live-cell imaging technique","authors":"Claire Velikonja ,&nbsp;Landon Steenbakkers ,&nbsp;Joshua How ,&nbsp;Mackenzie Enns ,&nbsp;Brandon Corbett ,&nbsp;Chris McCready ,&nbsp;Jake Nease ,&nbsp;Prashant Mhaskar ,&nbsp;David Latulippe","doi":"10.1016/j.jbiotec.2024.09.018","DOIUrl":"10.1016/j.jbiotec.2024.09.018","url":null,"abstract":"<div><div>Interest in virus-based therapeutics for the treatment of genetic and oncolytic diseases has created a demand for high-yield, low-cost virus-manufacturing processes. However, traditional analytical methods of assessing infectious virus titer require multiple processing steps and manual counting, limiting sample throughput, and increasing human error. This bottleneck severely limits the development of new manufacturing unit operations to drive down costs. In this work, we utilize an Incucyte Live-Cell Analysis System to develop a high-throughput infectious titer assay for adenovirus expressing a GFP-transgene. Although previous studies have demonstrated live-cell imaging’s potential for use with other viruses, they provide little guidance regarding the selection of the viewing and analysis parameters. To fill this gap, we develop an algorithmic approach to identify the optimum viewing and analysis parameters and create a statistical workflow for quantifying infectious adenovirus in a sample dilution series in a standard 24-well microplate. The developed assay is comparable to Hexon staining, the gold-standard for adenovirus infectious titer, with a Pearson correlation coefficient of 0.9. Finally, the developed algorithmic approach and statistical workflow were applied to create an assay for adenovirus titer using a 96-well microplate, allowing five times more samples to be quantified compared to the standard 24-well plate. While this assay uses a GFP-insert that precludes its use in a clinical environment, the key learnings surrounding the careful use of viewing and analysis parameters, and the statistical workflow are widely applicable to implementing life-cell imaging for dilution-series-based assays. Moreover, this method directly enables the fast and accurate evaluation of virus samples in a preclinical environment.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"395 ","pages":"Pages 216-227"},"PeriodicalIF":4.1,"publicationDate":"2024-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142347200","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Valorization of levulinic acid by esterification with 1-octanol using a novel biocatalyst derived from Araujia sericifera","authors":"Jaime E. Bayona Solano ,&nbsp;Daniel A. Sánchez ,&nbsp;Gabriela M. Tonetto","doi":"10.1016/j.jbiotec.2024.09.014","DOIUrl":"10.1016/j.jbiotec.2024.09.014","url":null,"abstract":"<div><div>Levulinic acid, which can be obtained from biomass, has sparked great interest as a biologically-based chemical building block with wide versatility and potential. Its esterification with alcohols of different chain lengths is a promising valorization process for obtaining esters with various applications in the areas of biofuels/biolubricants, food and cosmetics, among others. In this work, the enzymatic esterification of levulinic acid and 1-octanol using a biocatalyst derived from <em>Araujia sericifera</em> latex was studied in systems with and without solvent. The influence of the molar ratio between alcohol and acid (ranging from 2:1–1:9), the biocatalyst loading (between 7.5 % and 17.5 % relative to the acid), the volume of <em>n</em>-heptane used as reaction solvent (from 0 to 4 ml), and the reaction time (6 hours) were investigated. The activity and stability of the biocatalyst in successive uses were also analyzed. A conversion of 49 % was achieved when the reaction was carried out in a solvent-free system, using an alcohol/acid molar ratio of 1:7 and after 5 h of reaction. On the other hand, the conversion was 65.1 % when the reaction was conducted in a system containing 1 ml of <em>n</em>-heptane as solvent, an alcohol/acid molar ratio of 1:8, and 5 h of reaction. In both cases, a temperature as low as 30 °C and an agitation speed of 300 RPM were used.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"395 ","pages":"Pages 100-109"},"PeriodicalIF":4.1,"publicationDate":"2024-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142347216","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}