Biotechnology and Bioengineering最新文献

{"title":"One-Pot Hetero-Di-C-Glycosylation of the Natural Polyphenol Phloretin by a Single C-Glycosyltransferase With Broad Sugar Substrate Specificity","authors":"Tuo Li,&nbsp;Annika J. E. Borg,&nbsp;Leo Krammer,&nbsp;Rolf Breinbauer,&nbsp;Bernd Nidetzky","doi":"10.1002/bit.28948","DOIUrl":"10.1002/bit.28948","url":null,"abstract":"<p>The structural motif of hetero-di-<i>C</i>-glycosyl compound is prominent in plant polyphenol natural products and involves two different glycosyl residues (e.g., β-<span>d</span>-glucosyl, β-<span>d</span>-xylosyl) attached to carbons of the same phenolic ring. Polyphenol hetero-di-<i>C</i>-glycosides attract attention as specialized ingredients of herbal medicines and their tailored synthesis by enzymatic <i>C</i>-glycosylation is promising to overcome limitations of low natural availability and to expand molecular diversity to new-to-nature glycoside structures. However, installing these di-<i>C</i>-glycoside structures with synthetic precision and efficiency is challenging. Here we have characterized the syntheses of <i>C</i>-β-galactosyl-<i>C</i>-β-glucosyl and <i>C</i>-β-glucosyl-<i>C</i>-β-xylosyl structures on the phloroglucinol ring of the natural polyphenol phloretin, using kumquat (<i>Fortunella crassifolia</i>) <i>C-</i>glycosyltransferase (<i>Fc</i>CGT). The <i>Fc</i>CGT uses uridine 5'-diphosphate (UDP)-galactose (5 mU/mg) and UDP-xylose (0.3 U/mg) at lower activity than UDP-glucose (3 U/mg). The 3'-<i>C</i>-β-glucoside (nothofagin) is ~10-fold less reactive than non-glycosylated phloretin with all UDP-sugars, suggesting the practical order of hetero-di-<i>C</i>-glycosylation as <i>C</i>-galactosylation or <i>C</i>-xylosylation of phloretin followed by <i>C</i>-glucosylation of the resulting mono-<i>C</i>-glycoside. Each <i>C</i>-glycosylation performed in the presence of twofold excess of UDP-sugar proceeds to completion and appears to be effectively irreversible, as evidenced by the absence of glycosyl residue exchange at extended reaction times. Synthesis of <i>C</i>-β-glucosyl-<i>C</i>-β-xylosyl phloretin is shown at 10 mM concentration in quantitative conversion using cascade reaction of <i>Fc</i>CGT and UDP-xylose synthase, allowing for in situ formation of UDP-xylose from the more expedient donor substrate UDP-glucuronic acid. The desired di-<i>C</i>-glycoside with Xyl or Gal was obtained as a single product of the synthesis and its structure was confirmed by NMR.</p>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 5","pages":"1296-1304"},"PeriodicalIF":3.5,"publicationDate":"2025-02-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/bit.28948","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143257834","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":"Enhancing Biomass Productivity by Forecast-Informed Pond Operations","authors":"Hongxiang Yan,&nbsp;Mark S. Wigmosta,&nbsp;Ning Sun,&nbsp;Song Gao,&nbsp;Michael H. Huesemann","doi":"10.1002/bit.28952","DOIUrl":"10.1002/bit.28952","url":null,"abstract":"<p>Microalgal cultivation for biofuels and proteins holds significant promise but faces challenges in achieving economically viable biomass productivity under variable environmental conditions. This study introduces a forecast-informed pond operation (FIPO) system that uses numerical weather prediction (NWP) ensemble forecasts and the biomass assessment tool (BAT) to optimize daily dilution rates for enhanced biomass production. In contrast to the current practice, where fixed dilution rates are based on operator experience, the FIPO system determines the optimal dilution rate based on future weather forecasts and biomass growth conditions. Our experiments validate the effectiveness of FIPO in both short- and long-term growth scenarios. In short-term experiments, FIPO increased biomass production by 21.3% compared to batch growth and 7.4% over fixed dilution (60% every 3 days) operations. The NWP forecast-informed operations achieved biomass production nearly identical to that using perfect weather forecasts, highlighting the accuracy of current NWP forecasts for guiding pond operations. In long-term experiments, FIPO resulted in biomass production increases of 13.3% and 17.8% compared to two fixed dilution rates (60% every 3 days and 20% daily). These findings underscore the viability of using NWP forecasts to optimize microalgal cultivation systems. By adjusting daily dilution rates in response to forecasted weather, operators can achieve higher biomass yields and mitigate risks associated with environmental variability. This study provides a foundation for future research and practical applications in commercial-scale microalgal production.</p>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 5","pages":"1245-1257"},"PeriodicalIF":3.5,"publicationDate":"2025-02-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/bit.28952","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143257833","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":"Systematic Development of a Detergent Toolbox as an Alternative to Triton X-100","authors":"Varsha Yadav,&nbsp;Mary Lunson,&nbsp;Hannah Shore,&nbsp;Jean Aucamp","doi":"10.1002/bit.28947","DOIUrl":"10.1002/bit.28947","url":null,"abstract":"<p>Detergents are routinely included in protein purification processes to inactivate enveloped viruses that may arise from adventitious or endogenous contamination. The detergent Triton X-100 (TX-100) has been widely used as part of the production process for therapeutic proteins. However, recent ecological studies indicate that TX-100 and its metabolites detrimentally impact aquatic organisms, thus alternative detergents for viral inactivation are required. The overall aim of this study was to identify one or more detergents that are a suitable replacement for TX-100 in the viral inactivation step. In stage one, 16 potential alternatives were identified and screened against TX-100 using multiple criteria such as solubility, feasibility of virus inactivation, critical micelle concentration, and storage conditions. The multi-criteria decision analysis (MCDA) methodology was used to identify four candidates for the second stage assessment. In stage two, a detailed evaluation was undertaken and two candidates C16-AO, and C11/15-sEO9, were found to be practical alternatives to TX-100 for use in protein therapeutic production processes for inactivating enveloped viruses. In addition, C13-EO8 demonstrated good viral inactivation capability and warrants further investigation in detergent clearance and impact on product quality.</p>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 5","pages":"1096-1104"},"PeriodicalIF":3.5,"publicationDate":"2025-02-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/bit.28947","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143192638","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":"Construction of a CRISPR-Cas9-Based Genetic Editing Tool for Serratia marcescens Using a Stationary Phase Promoter and Its Application in Putrescine Production","authors":"Linbo Gou,&nbsp;Di Liu,&nbsp;Tai-Ping Fan,&nbsp;Huaxiang Deng,&nbsp;Yujie Cai","doi":"10.1002/bit.28949","DOIUrl":"10.1002/bit.28949","url":null,"abstract":"<div>\u0000 \u0000 <p>Putrescine plays a significant role in green food production and agriculture by promoting plant growth and enhancing crop quality. Its application reduces the reliance on chemical fertilizers and pesticides, thereby supporting the advancement of sustainable agricultural practices. This study achieved efficient production of putrescine in <i>Serratia marcescens</i>. <i>S. marcescens</i> has been extensively used to synthesize antimicrobial substances and express proteins, but its application has been limited by the lack of efficient genome-editing tools. This study presents a CRISPR-Cas9-based tool for gene editing in <i>S. marcescens</i>. A dual-plasmid system was constructed, incorporating an editing template into the plasmid pEdit with target-specific sgRNA. A stationary phase promoter was used to express Cas9 from <i>Streptococcus pyogenes</i> protein, avoiding the need for additional inducers and ensuring efficient one-step gene knockout and integration. The tool demonstrated over 80% editing efficiency across various <i>S. marcescens</i> strains and enabled successful single-base mutations. Using this tool, we enhanced putrescine production in <i>S. marcescens</i> HBQA7, optimizing the expression of ornithine decarboxylase from <i>Clostridium aceticum</i> DSM1496 with the P2 promoter and identifying the optimal integration site. Putrescine production reached 8.46 g/L within 48 h. This study significantly advances <i>S. marcescens</i> gene editing and metabolic engineering.</p></div>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 5","pages":"1233-1244"},"PeriodicalIF":3.5,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143192639","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":"Demonstrating the Effectiveness of an Alternative to Triton X-100 for Detergent-Mediated Viral Inactivation in Biomanufacturing","authors":"Kakolie Banerjee,&nbsp;Alice Antonello,&nbsp;Sandra Johnson,&nbsp;Anja Licht,&nbsp;Almut Rapp,&nbsp;Corinne Miller","doi":"10.1002/bit.28940","DOIUrl":"10.1002/bit.28940","url":null,"abstract":"<div>\u0000 \u0000 <p>Detergent-mediated viral inactivation is an important process step for ensuring viral safety of parenteral biotherapeutics, including plasma proteins and monoclonal antibodies (mAb). The conventional Triton X-100 detergent has ecological toxicity concerns and REACH classification that mandate replacement in the biopharmaceutical industry. Criteria for a replacement detergent include viral inactivation efficacy, acceptable safety and biodegradation profile, process removal, and quality suitable for parenteral drug product manufacturing. A non-ionic, C11-15 secondary alcohol ethoxylate, Deviron 13-S9 detergent, has been demonstrated to meet the necessary requirements for detergent performance. Benchmarking studies with Triton X-100 detergent demonstrate comparable performance with a panel of enveloped viruses in multiple matrices, including human IgG, clarified cell culture harvest, and fractionated plasma. Deviron 13-S9 detergent demonstrated viral inactivation efficiency comparable to or better than Triton X-100 detergent, achieving &gt; 5 log reduction values. Critical micelle concentration was determined across different temperatures and media. Deviron 13-S9 detergent was demonstrated to be readily biodegradable according to OECD 301B guidelines. The absence of detergent binding to typical chromatography resins used in downstream purification was confirmed. The process removal of Deviron 13-S9 detergent from a protein-containing matrix was demonstrated using a protein A resin. These findings support Deviron 13-S9 detergent as a viable alternative to Triton X-100 detergent, ensuring robust viral inactivation, environmental compatibility, and alignment with regulatory requirements.</p></div>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 5","pages":"1087-1095"},"PeriodicalIF":3.5,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143124766","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":"Chromatography in Downstream Processing of Recombinant Adeno-Associated Viruses: A Review of Current and Future Practises","authors":"Julius Klemens Lorek,&nbsp;Madelène Isaksson,&nbsp;Bernt Nilsson","doi":"10.1002/bit.28932","DOIUrl":"10.1002/bit.28932","url":null,"abstract":"<p>Recombinant adeno-associated virus (rAAV) has emerged as an attractive gene delivery vector platform to treat both rare and pervasive diseases. With more and more rAAV-based therapies entering late-stage clinical trials and commercialization, there is an increasing pressure on the rAAV manufacturing process to accelerate drug development, account for larger trials, and commercially provide high doses. Still, many of the pre-clinical and clinical manufacturing processes are tied to outdated technologies, which results in substantial production expenses. Those processes face challenges including low productivity and difficult scalability, which limits its ability to provide for required dosages which in turn influences the accessibility of the drug. And as upstream efforts are expected to increase productivities, the downstream part needs to adapt with more scalable and efficient technologies. In this review, both traditional and novel rAAV downstream technologies are presented and discussed. Traditional rAAV downstream processes are based on density gradient ultracentrifugation and have been shown to effectively purify rAAVs with high yields and purities. However, those processes lack scalability and efficiency, which is why novel rAAV downstream processes based on column-chromatography have emerged as an attractive alternative and show potential for integration in continuous processes, following the principle of next-generation manufacturing.</p>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 5","pages":"1067-1086"},"PeriodicalIF":3.5,"publicationDate":"2025-02-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/bit.28932","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143124893","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":"Effects of Air Temperature on Transient Expression of Influenza Hemagglutinin in Nicotiana benthamiana: Analysis of Transgene Transcription and Plant Stress Responses","authors":"Patthasarun Pruksarojanakul,&nbsp;Kazune Atsumi,&nbsp;Youngjun Oh,&nbsp;Nobuyuki Matoba,&nbsp;Ryo Matsuda","doi":"10.1002/bit.28942","DOIUrl":"10.1002/bit.28942","url":null,"abstract":"<p>Postinfiltration air temperature is known to affect the accumulation of recombinant protein in <i>Agrobacterium</i>-mediated transient expression in <i>Nicotiana benthamiana</i> plants, including the number of days needed to reach maximum content and the rate of reduction thereafter. This study aimed to clarify whether the transcript levels of the transgenes and those of plant stress response markers (i.e., hypersensitive response and endoplasmic reticulum [ER] stress) could be primary determinants of the accumulation of recombinant influenza hemagglutinin (HA) at 21 or 26°C. We found no correlation between the transgene expression levels (<i>HA, RdRP</i>, and <i>MP</i>) and the number of days needed to reach the maximum HA protein content at both temperatures. Regardless of the accumulation compartment, HA protein content peaked earlier at 26°C than at 21°C. The rapid reduction of HA content after reaching the maximum, observed only in the ER at 26°C, correlated with severe necrosis and high transcript levels of two representative ER stress markers, <i>bZIP60</i> and <i>BiP</i>. This correlation suggests that high postinfiltration air temperature affects HA accumulation primarily through ER stress, a key factor in the rapid reduction of HA content after the peak.</p>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 5","pages":"1142-1152"},"PeriodicalIF":3.5,"publicationDate":"2025-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/bit.28942","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143063719","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":"Machine Learning-Powered Optimization of a CHO Cell Cultivation Process","authors":"Jannik Richter,&nbsp;Qimin Wang,&nbsp;Ferdinand Lange,&nbsp;Phil Thiel,&nbsp;Nina Yilmaz,&nbsp;Dörte Solle,&nbsp;Xiaoying Zhuang,&nbsp;Sascha Beutel","doi":"10.1002/bit.28943","DOIUrl":"10.1002/bit.28943","url":null,"abstract":"<p>Chinese Hamster Ovary (CHO) cells are the most widely used cell lines to produce recombinant therapeutic proteins such as monoclonal antibodies (mAbs). However, the optimization of the CHO cell culture process is very complex and influenced by various factors. This study investigates the use of machine learning (ML) algorithms to optimize an established industrial CHO cell cultivation process. A ML algorithm in the form of an artificial neural network (ANN) was used and trained on datasets from historical and newly generated CHO cell cultivation runs. The algorithm was then used to find better cultivation conditions and improve cell productivity. The selected artificial intelligence (AI) tool was able to suggest optimized cultivation settings and new condition combinations, which promised both increased cell growth and increased mAb titers. After performing the validation experiments, it was shown that the ML algorithm was able to successfully optimize the cultivation process and significantly improve the antibody production. The best results showed an increase in final mAb titer up to 48%, demonstrating that the use of ML algorithms is a promising approach to optimize the productivity of bioprocesses like CHO cell cultivation processes clearly.</p>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 5","pages":"1153-1164"},"PeriodicalIF":3.5,"publicationDate":"2025-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/bit.28943","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143063720","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":"Modeling the Performance of an Anaerobic Moving Bed Biofilm Reactor","authors":"Yuhang Cai,&nbsp;Joshua P. Boltz,&nbsp;Bruce E. Rittmann","doi":"10.1002/bit.28938","DOIUrl":"10.1002/bit.28938","url":null,"abstract":"<div>\u0000 \u0000 <p>Sub-models representing transformation processes by microorganisms and hydrolases, a one-dimensional (1-D) biofilm, and a bioreactor were integrated to simulate organic-matter fermentation and methane (CH₄) production in an anaerobic moving bed biofilm reactor (AnMBBR). The integrated models correctly represented all experimental observations and identified mechanisms underlying how and why AnMBBR performance changed when the volumetric loading rate (VLR) of total chemical oxygen demand (TCOD) increased from 3.9 to 19.5 kg COD_T/m³-d. The fractional removal of TCOD and CH₄ production decreased as the VLR of TCOD increased, in part, due to an increasing biofilm thickness that filled the protected channels in the interior of the plastic carriers and led to a decrease in biofilm surface area and an increase in the mass-transfer boundary layer. Also, the ~25-day duration for each VLR of TCOD was too brief to allow the biofilm to establish a new quasi-steady state with respect to biofilm thickness. The mechanistic understanding of how biofilm characteristics and process performance respond to increased VLR of TCOD can be applied in engineering practice to improve AnMBBR process design and operation.</p></div>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 5","pages":"1130-1141"},"PeriodicalIF":3.5,"publicationDate":"2025-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143063721","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":"pH Threshold Impacts Chalcopyrite Bioleaching Dynamics for the Extreme Thermoacidophile Sulfurisphaera ohwakuensis","authors":"Daniel J. Willard,&nbsp;Mohammad J. H. Manesh,&nbsp;Kaitlyn M. John,&nbsp;Robert M. Kelly","doi":"10.1002/bit.28945","DOIUrl":"10.1002/bit.28945","url":null,"abstract":"<p>The extremely thermoacidophilic archaeon <i>Sulfurisphaera ohwakuensis</i> served as the basis for probing how initial pH (pH_initial) affects copper mobilization from chalcopyrite. Screening of small-scale cultures (75 mL) at 75°C revealed that ~pH 3.0 was a maximal threshold for bioleaching onset. Subsequently, chalcopyrite at 10 g/L in 750 mL culture media, containing small amounts of ferric ion, adjusted to pH 2.5 with sulfuric acid and incubated for 24 h at 75°C before inoculation, brought the pH to approximately 3.0 through abiotic chemical reactions. However, the resulting subtle differences in pH_initial (3.0 ± 0.15) in bioleaching cultures, while not affecting microbial growth, were critical to bioleaching onset and progress. Initial iron levels were less important than pH_initial in starting the bioleaching process. X-Ray Diffraction (XRD) surface analysis informed bioleaching trajectories over 21 days and reinforced the impact of pH_initial. The subtle differences in pH_initial markedly affected <i>S. ohwakuensis</i> onset and outcomes, as it presumably would for other bioleaching thermoacidophilic archaea. Furthermore, the findings here highlight the challenges faced in replicating bioleaching experiments across, and even within, laboratories as well as in achieving consistent results in bioleaching processes.</p>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 5","pages":"1165-1173"},"PeriodicalIF":3.5,"publicationDate":"2025-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/bit.28945","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143063723","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}