ACS Synthetic Biology最新文献

{"title":"<i>rm</i>Combi-OGAB for the Directed Evolution of a Biosynthetic Gene Cluster toward Productivity Improvement.","authors":"Naoki Miyamoto, Kentaro Hayashi, Naohisa Ogata, Naoyuki Yamada, Kenji Tsuge","doi":"10.1021/acssynbio.4c00734","DOIUrl":"10.1021/acssynbio.4c00734","url":null,"abstract":"<p><p><u><b>Combi</b></u>natorial <u><b>O</b></u>rdered <u><b>G</b></u>ene <u><b>A</b></u>ssembly in <u><b><i>B</i></b></u><i>acillus subtilis</i> (Combi-OGAB) enables construction of combinatorial libraries of various genetic elements, such as promoters in a biosynthetic gene cluster (BGC), and screening of highly productive combinations from the library. The combinations are limited by the library design, and the selectable productivity is defined within the combination. To refine the selected BGC using conventional Combi-OGAB with expanded diversity, we devised a directed evolutionary method called as <u><b>r</b></u>andom <u><b>m</b></u>utagenesis with <u><b>Combi-OGAB</b></u> (<i>rm</i>Combi-OGAB), which includes random mutagenesis by error-prone PCR and Combi-OGAB. In the present study, Gramicidin S (GS)-producing plasmids were used to examine the utility of <i>rm</i>Combi-OGAB. GS plasmids, originally generated using conventional Combi-OGAB, were successfully evolved using <i>rm</i>Combi-OGAB. <i>B. subtilis</i> carrying the evolved plasmid with unpredictable mutations showed a 1.5-fold improvement in the GS productivity. We thus expect that <i>rm</i>Combi-OGAB can be applied to various BGCs for useful products, such as antibiotics, to improve their productivity.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":"629-633"},"PeriodicalIF":3.7,"publicationDate":"2025-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11852201/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143254160","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 Non-standard Nucleic Acids for Highly Sensitive Icosaplex (20-Plex) Detection of Microbial Threats for Environmental Surveillance.","authors":"Hinako Kawabe, Luran Manfio, Sebastian Magana Pena, Nicolette A Zhou, Kevin M Bradley, Cen Chen, Chris McLendon, Steven A Benner, Karen Levy, Zunyi Yang, Jorge A Marchand, Erica R Fuhrmeister","doi":"10.1021/acssynbio.4c00619","DOIUrl":"10.1021/acssynbio.4c00619","url":null,"abstract":"<p><p>Environmental surveillance and clinical diagnostics heavily rely on the polymerase chain reaction (PCR) for target detection. A growing list of microbial threats warrants new PCR-based detection methods that are highly sensitive, specific, and multiplexable. Here, we introduce a PCR-based icosaplex (20-plex) assay for detecting 18 enteropathogen and two antimicrobial resistance genes. This multiplexed PCR assay leverages the self-avoiding molecular recognition system (SAMRS) to avoid primer dimer formation, the artificially expanded genetic information system (AEGIS) for amplification specificity, and next-generation sequencing for amplicon identification. Using parallelized multitarget TaqMan Array Cards (TAC) to benchmark performance of the 20-plex assay on wastewater, soil, and human stool samples, we found 90% agreement on positive calls and 89% agreement on negative calls. Additionally, we show how long-read and short-read sequencing information from the 20-plex can be used to further classify allelic variants of genes and distinguish subspecies. The strategy presented offers sensitive, affordable, and robust multiplex detection that can be used to support efforts in wastewater-based epidemiology, environmental monitoring, and human/animal diagnostics.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":"470-484"},"PeriodicalIF":3.7,"publicationDate":"2025-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11854376/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143078035","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":"Characterization of Rationally Designed CRISPR/Cas9-Based DNA Methyltransferases with Distinct Methyltransferase and Gene Silencing Activities in Human Cell Lines and Primary Human T Cells.","authors":"Rosa Selenia Guerra-Resendez, Samantha LeGoff Lydon, Alex J Ma, Guy C Bedford, Daniel R Reed, Sunghwan Kim, Erik R Terán, Tomoki Nishiguchi, Mario Escobar, Andrew R DiNardo, Isaac B Hilton","doi":"10.1021/acssynbio.4c00569","DOIUrl":"10.1021/acssynbio.4c00569","url":null,"abstract":"<p><p>Nuclease-deactivated Cas (dCas) proteins can be used to recruit epigenetic effectors, and this class of epigenetic editing technologies has revolutionized the ability to synthetically control the mammalian epigenome and transcriptome. DNA methylation is one of the most important and well-characterized epigenetic modifications in mammals, and while many different forms of dCas-based DNA methyltransferases (dCas-DNMTs) have been developed for programmable DNA methylation, these tools are frequently poorly tolerated and/or lowly expressed in mammalian cell types. Further, the use of dCas-DNMTs has largely been restricted to cell lines, which limits mechanistic insights in karyotypically normal contexts and hampers translational utility in the longer term. Here, we extend previous insights into the rational design of the catalytic core of the mammalian DNMT3A methyltransferase and test three dCas9-DNMT3A/3L variants across different human cell lines and in primary donor-derived human T cells. We find that mutations within the catalytic core of DNMT3A stabilize the expression of dCas9-DNMT3A/3L fusion proteins in Jurkat T cells without sacrificing DNA methylation or gene-silencing performance. We also show that these rationally engineered mutations in DNMT3A alter DNA methylation profiles at loci targeted with dCas9-DNMT3A/3L in cell lines and donor-derived human T cells. Finally, we leverage the transcriptionally repressive effects of dCas9-DNMT3A/3L variants to functionally link the expression of a key immunomodulatory transcription factor to cytokine secretion in donor-derived T cells. Overall, our work expands the synthetic biology toolkit for epigenetic editing and provides a roadmap for the use of engineered dCas-based DNMTs in primary mammalian cell types.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":"384-397"},"PeriodicalIF":3.7,"publicationDate":"2025-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11854388/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143078034","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":"Microbial Production of Ectoine: A Review.","authors":"Ke Wang, Boya Cui, Yi Wang, Wei Luo","doi":"10.1021/acssynbio.4c00490","DOIUrl":"10.1021/acssynbio.4c00490","url":null,"abstract":"<p><p>Ectoine is an important natural secondary metabolite widely used in biomedical fields, novel cosmetics development, and the food industry. Due to the increasing market demand for ectoine, more cost-effective production methods are being explored. With the rapid development of synthetic biology and metabolic engineering technologies, the production of ectoine using traditional halophilic bacteria is gradually being replaced by higher-yielding and environmentally friendly nonhalophilic engineered strains. By introducing the ectoine synthesis pathway into model strains and optimizing the fermentation process through various metabolic regulations, high-level production of ectoine can be achieved. This review focuses on strategies for the microbial production of ectoine, including screening of wild strains, mutation breeding, and metabolic engineering of model strains, to elucidate the current research status and provide insights for the industrial production of ectoine.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":"332-342"},"PeriodicalIF":3.7,"publicationDate":"2025-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142996174","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":"Living Dual Heat- and pH-Responsive Textiles.","authors":"Dalia Jane Saldanha, Simon James Alexander Rowat, Henry Stephenson, Noémie-Manuelle Dorval Courchesne","doi":"10.1021/acssynbio.4c00808","DOIUrl":"10.1021/acssynbio.4c00808","url":null,"abstract":"<p><p>Smart textiles that integrate multiple environmental sensing capabilities are an emerging frontier in wearable technology. In this study, we developed dual pH- and temperature-responsive textiles by combining engineered bacterial systems with bacterially derived proteins. For temperature sensing, we characterized the properties of a heat sensitive promoter, P_hs, in <i>Escherichia coli</i> (<i>E. coli</i>) using enhanced green fluorescent protein as a reporter. Our findings demonstrate that the P_hs promoter drives elevated gene expression at temperatures between 37 and 43 °C, maintaining sustained activity for several hours. Moreover, we found that short heat shocks can significantly boost expression levels of the P_hs promoter. We successfully integrated <i>E. coli</i> expressing P_hs-EGFP cells onto textiles and confirmed their ability to retain heat-responsive behavior after integration. To achieve pH responsiveness, we utilized curli fibers, genetically engineered to incorporate a pH-sensitive fluorescent protein, pHuji. pH-sensing curli fibers are bacterial proteins that have a proven track record of creating stable bioresponsive textile coatings. By embedding P_hs-EGFP-expressing bacteria within curli fiber coatings, we created a dual-responsive textile capable of differentiating between acidic and alkaline environments while simultaneously responding to thermal stimuli. These multifunctional textiles exhibited dual environmental response and sensing capabilities. This work establishes a proof-of-concept for creating smart living textiles with modular functionalities, paving the way toward advanced bioresponsive materials.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":"564-574"},"PeriodicalIF":3.7,"publicationDate":"2025-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142996170","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":"Simultaneous <i>In Vivo</i> Assembly and Targeted Genome Integration of Gene Clusters in <i>Trichoderma reesei</i>.","authors":"Yu Fang, Xiangfeng Meng, Lin Liu, Zhongye Li, Kaili Jia, Weifeng Liu","doi":"10.1021/acssynbio.4c00810","DOIUrl":"10.1021/acssynbio.4c00810","url":null,"abstract":"<p><p>The saprophytic filamentous fungus <i>Trichoderma reesei</i> represents one of the most prolific cellulase producers and also has the potential to be developed into a tractable fungal host for biosynthesizing secondary metabolite products. To expedite the genetic engineering of filamentous fungi, efficient DNA assembly processes that can facilitate the transfer of large-sized DNA to fungal hosts, including <i>T. reesei</i>, are still in demand. Here, we developed a method for the simultaneous <i>in vivo</i> assembly and targeted genome integration of multiple DNA fragments (SATIMD) in <i>T. reesei</i>. While efficient orderly DNA end fusions were achieved by homologous recombination (HR) with various lengths of sequence overlaps (100-500 bp), the assembled DNA was also precisely integrated into a specific locus when combined with CRISPR/Cas9-mediated genome cutting. Specifically, we have used this method to achieve the assembly and functional expression of <i>T. reesei</i> key transcriptional activator Xyr1 for cellulase genes. Moreover, fusions and targeted integration of up to 10 different DNA fragments comprising the 32.7 kb sorbicillinoids biosynthetic gene cluster via a single-step transformation was demonstrated. We envision that SATIMD is a powerful tool not only useful for direct large heterologous gene cluster assembly in <i>T. reesei</i> but also can facilitate large-scale fungal strain genetic engineering.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":"575-584"},"PeriodicalIF":3.7,"publicationDate":"2025-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143363267","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":"Engineering Exopolysaccharide Biosynthesis of <i>Shewanella oneidensis</i> to Promote Electroactive Biofilm Formation for Liquor Wastewater Treatment.","authors":"Zixuan You, Huan Yu, Baocai Zhang, Qijing Liu, Bo Xiong, Chao Li, Chunxiao Qiao, Longhai Dai, Jianxun Li, Wenwei Li, Guosheng Xin, Zhanying Liu, Feng Li, Hao Song","doi":"10.1021/acssynbio.4c00417","DOIUrl":"10.1021/acssynbio.4c00417","url":null,"abstract":"<p><p>Microbial electrochemical systems (MESs), as a green and sustainable technology, can decompose organics in wastewater to recover bioelectricity. Electroactive biofilms, a microbial community structure encased in a self-produced matrix, play a decisive role in determining the efficiency of MESs. However, as an essential component of the biofilm matrix, the role of exopolysaccharides in electroactive biofilm formation and their influence on extracellular electron transfer (EET) have been rarely studied. Herein, to explore the effects of exopolysaccharides on biofilm formation and EET rate, we first inhibited the key genes responsible for exopolysaccharide biosynthesis (namely, <i>so_3171</i>, <i>so_3172</i>, <i>so_3177</i>, and <i>so_3178</i>) by using antisense RNA in <i>Shewanella oneidensis</i> MR-1. Then, to explore the underlying mechanisms why inhibition of exopolysaccharide synthesis could enhance biofilm formation and promote the EET rate, we characterized cell physiology and electrophysiology. The results showed inhibition of exopolysaccharide biosynthesis not only altered cell surface hydrophobicity and promoted intercellular adhesion and aggregation, but also increased biosynthesis of <i>c</i>-type cytochromes and decreased interfacial resistance, thus promoting electroactive biofilm formation and improving the EET rate of <i>S. oneidensis</i>. Lastly, to evaluate and intensify the capability of exopolysaccharide-reduced strains in harvesting electrical energy from actual liquor wastewater, engineered strain Δ3171-as3177 was further constructed to treat an actual thin stillage. The results showed that the output power density reached 380.98 mW m^-2, 11.1-fold higher than that of WT strain, which exhibited excellent capability of harvesting electricity from actual liquor wastewater. This study sheds light on the underlying mechanism of how inhibition of exopolysaccharides impacts electroactive biofilm formation and EET rate, which suggested that regulating exopolysaccharide biosynthesis is a promising avenue for increasing the EET rate.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":"373-383"},"PeriodicalIF":3.7,"publicationDate":"2025-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142646143","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":"Novel Isothermal Amplification Integrated with CRISPR/Cas13a and Its Applications for Ultrasensitive Detection of SARS-CoV-2.","authors":"Jaemin Kim, Yo Rim Kim, Sang Mo Lee, Jinhwan Lee, Seoyoung Lee, Dongeun Yong, Hyun Gyu Park","doi":"10.1021/acssynbio.4c00605","DOIUrl":"10.1021/acssynbio.4c00605","url":null,"abstract":"<p><p>We herein developed an ultrasensitive and rapid strategy to identify genomic nucleic acids by integrating a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 13a (Cas13a) into our recently developed isothermal technique, nicking and extension chain reaction system-based amplification (NESBA) reaction. In this technique, named CESBA, the NESBA reaction isothermally produces a large amount of RNA amplicons from the initial target genomic RNA (gRNA). The RNA amplicons bind to the crispr RNA (crRNA) and activate the collateral cleavage activity of Cas13a, which would then cleave the reporter probe nearby, consequently producing the final signals. Based on this design principle, we successfully detected SARS-CoV-2 gRNA as a model target very sensitively down to even a single copy (0.05 copies/μL) in both fluorescence- and lateral flow assay (LFA)-based modes with excellent specificity against other human coronaviruses (H-CoVs). We further validated the clinical applicability of CESBA by testing the 20 clinical samples with 100% clinical sensitivity and specificity. This work represents a potent and innovative strategy for the identification of genomic nucleic acids in molecular diagnostics, delivering exceptional levels of sensitivity.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":"463-469"},"PeriodicalIF":3.7,"publicationDate":"2025-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142996176","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":"DRIMS: A Synthetic Biology Platform that Enables Deletion, Replacement, Insertion, Mutagenesis, and Synthesis of DNA.","authors":"Leidy D Caraballo G, Inci Cevher Zeytin, Purva Rathi, Che-Hsing Li, Ai-Ni Tsao, Yaery J Salvador L, Manish Ranjan, Brendan Magee Traynor, Andras A Heczey","doi":"10.1021/acssynbio.4c00649","DOIUrl":"10.1021/acssynbio.4c00649","url":null,"abstract":"<p><p>DNA modification and synthesis are fundamental to genetic engineering, and systems that enable time- and cost-effective execution of these processes are crucial. Iteration of genetic construct variants takes significant time, cost and effort to develop new therapeutic strategies to treat diseases including cancer. Thus, decreasing cost and enhancing simplicity while accelerating the speed of advancement is critical. We have developed a PCR-based platform that allows for <b>d</b>eletion, <b>r</b>eplacement, <b>i</b>nsertion, <b>m</b>utagenesis, and <b>s</b>ynthesis of DNA (DRIMS). These modifications rely on the recA-independent recombination pathway and are carried out in a single amplification step followed by DpnI digestion and transformation into competent cells. DNA synthesis is accomplished through sequential PCR amplification reactions without the need for a DNA template. Here, we provide proof-of-concept for the DRIMS platform by performing four deletions within DNA fragments of various sizes, sixty-four replacements of DNA binding sequences that incorporate repeat sequences, four replacements of chimeric antigen receptor components, fifty-one insertions of artificial microRNAs that form complex tertiary structures, five varieties of point mutations, and synthesis of eight DNA sequences including two with high GC content. Compared to other advanced cloning methods including Gibson and \"in vivo assembly\", we demonstrate the significant advantages of the DRIMS platform. In summary, DRIMS allows for efficient modification and synthesis of DNA in a simple, rapid and cost-effective manner to accelerate the synthetic biology field and development of therapeutics.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":"485-496"},"PeriodicalIF":3.7,"publicationDate":"2025-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143121669","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":"<i>De Novo</i> Synthesis of Reticuline and Taxifolin Using Re-engineered Homologous Recombination in <i>Yarrowia lipolytica</i>.","authors":"Changtai Zhang, Mengsu Liu, Xinglong Wang, Junyi Cheng, Jinbo Xiang, Mingyu Yue, Yang Ning, Zhengxuan Shao, Chalak Najat Abdullah, Jingwen Zhou","doi":"10.1021/acssynbio.4c00853","DOIUrl":"10.1021/acssynbio.4c00853","url":null,"abstract":"<p><p><i>Yarrowia lipolytica</i> has been widely engineered as a eukaryotic cell factory to produce various important compounds. However, the difficulty of gene editing and the lack of efficient neutral sites make rewiring of <i>Y. lipolytica</i> metabolism challenging. Herein, a Cas9 system was established to redesign the <i>Y. lipolytica</i> homologous recombination system, which caused a more than 56-fold increase in the HR efficiency. The fusion expression of the hBrex27 sequence in the C-terminus of Cas9 recruited more Rad51 protein, and the engineered Cas9 decreased NHEJ, achieving 85% single-gene positive efficiency and 25% multigene editing efficiency. With this system, neutral sites on different chromosomes were characterized, and a deep learning model was developed for gRNA activity prediction, thus providing the corresponding integration efficiency and expression intensity. Subsequently, the tool and platform strains were validated by applying them for the <i>de novo</i> synthesis of (<i>S</i>)-reticuline and (2<i>S</i>)-taxifolin. The developed platform strains and tools helped transform <i>Y. lipolytica</i> into an easy-to-operate model cell factory, similar to <i>Saccharomyces cerevisiae</i>.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":"585-597"},"PeriodicalIF":3.7,"publicationDate":"2025-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143121666","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}