ACS Synthetic Biology最新文献

{"title":"Liposome-Encapsulated <i>Escherichia coli</i> Lysates to Reconstitute Intracellular Macromolecular Crowding Effects.","authors":"Milara S Kalacheva, Nuno R da Silva, Arnold J Boersma","doi":"10.1021/acssynbio.4c00824","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00824","url":null,"abstract":"<p><p>Intracellular macromolecular crowding impacts biomacromolecule behavior, including oligomerization, phase separation, and diffusion. However, understanding crowding effects in cells is challenging as cells respond and adapt to perturbations. Therefore, replicating in-cell crowding in liposomes would provide a good alternative to studying the consequences of macromolecular crowding. Here, we achieve physiological macromolecular crowding levels using <i>Escherichia coli</i> lysates in liposomes, as verified with a macromolecular crowding sensor. We shrink liposomes with a gradient-wise osmotic upshift to reach the high macromolecular crowding effects. We see that lysate induces higher macromolecular crowding than BSA at the same mg/mL, showing the need to use lysates to replicate in-cell behavior. We study the consequences of small cosolutes on macromolecular crowding and see that sugars and ATP modulate the lysate macromolecular crowding, implying they would also affect macromolecular crowding in cells. These artificial cells display the same crowding as <i>E. coli</i> at 220-300 mg/mL lysate and the same crowding as HEK293T at 50-100 mg/mL lysate. Hence, these artificial cells are a platform for obtaining information on physiologically relevant macromolecular crowding effects in a controlled environment.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143466556","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":"MultiCRISPR-EGA: Optimizing Guide RNA Array Design for Multiplexed CRISPR Using the Elitist Genetic Algorithm.","authors":"Yangyu Zhang, Guanlin Chen, Ce Liang, Bin Yang, Xin Lei, Tao Chen, Huaiguang Jiang, Wei Xiong","doi":"10.1021/acssynbio.4c00860","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00860","url":null,"abstract":"<p><p>Multiplexed CRISPR design, which allows for the concurrent and efficient editing of multiple genomic sites, is a powerful tool for complex genetic modifications. However, designing effective multiplexed guide RNA (gRNA) arrays remains challenging due to the exponential increase in potential gRNA array candidates and the significant impact of different target site selections on efficiency and specificity. Recognizing that more stable gRNAs, characterized by lower minimum free energy (MFE), have prolonged activity and thus higher efficacy, we developed MultiCRISPR-EGA, a graphical user interface (GUI)-based tool that employs the Elitist Genetic Algorithm (EGA) to design optimized single-promoter-driven multiplexed gRNA arrays. Computational experiments on <i>Escherichia coli</i> gene targets demonstrate that the EGA can rapidly optimize multiplexed gRNA arrays, outperforming other intelligent optimization algorithms in CRISPR interference (CRISPRi) applications, while the GUI provides real-time design progress control and compatibility with various CRISPR-Cas systems. This tool aims to advance the multiplexed gRNA array design process, enabling more efficient and cost-effective genome editing for synthetic biology.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143456261","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":"Metabolic Engineering of <i>Corynebacterium glutamicum</i> for Producing Different Types of Triterpenoids.","authors":"Jingzhi Li, Xinxin Wang, Xahnaz Xokat, Ya Wan, Xiaopeng Gao, Ying Wang, Chun Li","doi":"10.1021/acssynbio.4c00737","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00737","url":null,"abstract":"<p><p>Triterpenoids widely exist in nature with diverse structures and possess various functional properties and biological effects. However, research on triterpenoids biosynthesis in <i>Corynebacterium glutamicum</i> is still limited to squalene, which restricts the development of <i>C. glutamicum</i> to produce high-value triterpenoids. In this study, <i>C. glutamicum</i> was developed as an efficient and flexible platform for the biosynthesis of different types of triterpenoids. Squalene was synthesized and the titer was improved to 400.1 mg/L in flask combining strategies of metabolic engineering and fermentation optimization. Particularly, intracellular squalene accounted for more than 97%, addressing the problem of leaking squalene in <i>C. glutamicum</i>, which may restrict the subsequent synthesis of other triterpenoids derived from squalene. Furthermore, 201.9 mg/L (3S)-2,3-oxidosqualene (SQO) and 264.9 mg/L (3S,22S)-2,3,22,23-dioxidosqualene (SDO) were successfully synthesized in strains harboring heterogeneous squalene epoxidase from <i>Arabidopsis thaliana</i> with different expression strengths. Therefore, a platform for de novo triterpenoids synthesis based on SQO or SDO was constructed in <i>C. glutamicum</i>. For instance, biosynthesis of α-amyrin and α-onocerin was achieved for the first time by introducing oxidosqualene cyclases in SQO- and SDO-producing <i>C. glutamicum</i> strains, respectively. After optimization, the titer of α-amyrin and α-onocerin was improved to 65.3 and 136.85 mg/L, respectively. Furthermore, ursolic acid, derived from α-amyrin, was synthesized after expressing cytochrome P450 enzyme and its compatible cytochrome P450 reductases with a titer of 486 μg/L. For the first time, reactions of epoxidation, cyclization, and oxidation from squalene were achieved in <i>C. glutamicum</i>, leading to the production of different types of triterpenoids. Our study provides a new platform for the production of triterpenoids, which will be helpful for the large-scale production of triterpenoids employing <i>C. glutamicum</i> as a chassis strain.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-02-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143447433","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":"LAMP Assay Coupled with a <i>Pyrococcus furiosus</i> Argonaute System for the Rapid Detection of Porcine Epidemic Diarrhea Virus.","authors":"Zhaorong Yu, Ying Shao, Yu Zhang, Fanyu Cheng, Peng Fang, Jian Tu, Xiangjun Song, Kezong Qi, Zhenyu Wang","doi":"10.1021/acssynbio.4c00446","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00446","url":null,"abstract":"<p><p>Porcine epidemic diarrhea virus (PEDV) infection can lead to serious acute intestinal infectious disease, bringing huge economic losses to the pig industry. In addition to triggering an extremely high mortality rate for lactating piglets, there is currently a lack of effective treatments and vaccines. Therefore, rapid, accurate, sensitive, and specific detection of PEDV is critical for timely control. In this study, a nucleic acid detection method combining reverse transcription loop-mediated isothermal amplification (RT-LAMP) and <i>Pyrococcus furiosus</i> Argonaute (<i>Pf</i>Ago) was established for the detection of PEDV and performed after optimizing the system (mainly for the design and screening of the LAMP primers and <i>Pf</i>Ago gDNA). The optimized system had a detection limit as low as 2.4 copies/μL. To reach more timely on-site detection of PEDV and overcome the reliance on bulky and complex equipment, a lateral flow strip was introduced into the system, which could detect the target as low as 24 copies/μL. This RT-LAMP-<i>Pf</i>Ago system took about 35 min to react, and the results could be observed and clarified with the naked eyes. Moreover, the method was highly specific and had no cross-reactivity with other swine pathogens. The detection results for the clinical samples were consistent with those obtained by the gold standard method, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), proving its applicability. In conclusion, the established RT-LAMP-<i>Pf</i>Ago system can provide a new solution for the development of a portable, visual PEDV testing platform.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-02-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143439317","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":"Metabolite-Responsive Control of Transcription by Phase Separation-Based Synthetic Organelles.","authors":"Carolina Jerez-Longres, Wilfried Weber","doi":"10.1021/acssynbio.4c00633","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00633","url":null,"abstract":"<p><p>Living natural materials have remarkable sensing abilities that translate external cues into functional changes of the material. The reconstruction of such sensing materials in bottom-up synthetic biology provides the opportunity to develop synthetic materials with life-like sensing and adaptation ability. Key to such functions are material modules that translate specific input signals into a biomolecular response. Here, we engineer a synthetic organelle based on liquid-liquid phase separation that translates a metabolic signal into the regulation of gene transcription. To this aim, we engineer the pyruvate-dependent repressor PdhR to undergo liquid-liquid phase separation <i>in vitro</i> by fusion to intrinsically disordered regions. We demonstrate that the resulting coacervates bind DNA harboring PdhR-responsive operator sites in a pyruvate dose-dependent and reversible manner. We observed that the activity of transcription units on the DNA was strongly attenuated following recruitment to the coacervates. However, the addition of pyruvate resulted in a reversible and dose-dependent reconstitution of transcriptional activity. The coacervate-based synthetic organelles linking metabolic cues to transcriptional signals represent a materials approach to confer stimulus responsiveness to minimal bottom-up synthetic biological systems and open opportunities in materials for sensor applications.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-02-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143424618","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":"Optogenetic Modification of Glycerol Production in Wine Yeast.","authors":"Diego Ruiz, Claudia Inzunza, Javiera Barría, Camila Baeza, Antonio Molina, Francisco A Cubillos, Francisco Salinas","doi":"10.1021/acssynbio.4c00654","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00654","url":null,"abstract":"<p><p>The wine strains of <i>Saccharomyces cerevisiae</i> transform glucose into ethanol and other byproducts such as glycerol and acetate. The balance of these metabolites is important during the fermentation process, which impacts the organoleptic properties of wines. Ethanol and glycerol productions are mainly controlled by the <i>ADH1</i> and <i>GPD1</i> genes, which encode for the alcohol dehydrogenase and glycerol-3-phosphate-dehydrogenase enzymes, respectively. Genetic modification of these genes can thus be used to alter the levels of the corresponding metabolites and to reroute fermentation. In this work, we used an optogenetic system named FUN-LOV (FUNgal-Light Oxygen Voltage) to regulate the expression of <i>ADH1</i> and <i>GPD1</i> in a wine yeast strain using light. Initially, we confirmed the light-controlled expression of <i>GPD1</i> and <i>ADH1</i> in the engineered strains via RT-qPCR and a translational reporter, respectively. To characterize the generated yeast strains, we performed growth curve assays and laboratory-scale fermentations, observing phenotypic differences between illumination conditions that confirm the optogenetic control of the target genes. We also monitored glucose consumption and ethanol and glycerol productions during a fermentation time course, observing that the optogenetic control of <i>GPD1</i> increased glycerol production under constant illumination without affecting ethanol production. Interestingly, the optogenetic control of <i>ADH1</i> showed an inverted phenotype, where glycerol production increased under constant darkness conditions. Altogether, our results highlight the feasibility of using optogenetic tools to control yeast fermentation in a wine yeast strain, which allows changing the balance of metabolic products of interest in a light-dependent manner.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-02-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143416710","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":"minChemBio: Expanding Chemical Synthesis with Chemo-Enzymatic Pathways Using Minimal Transitions.","authors":"Mohit Anand, Vikas Upadhyay, Costas D Maranas","doi":"10.1021/acssynbio.4c00692","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00692","url":null,"abstract":"<p><p>Chemo-enzymatic pathway design aims to combine the strengths of enzymatic with chemical synthesis to traverse biomolecular design space more efficiently. While chemical reactions often struggle with regioselectivity and stereoselectivity, enzymatic conversions often encounter limitations of low enzyme activity or availability. Optimally integrating both approaches provides an opportunity to identify efficient pathways beyond the capabilities of either modality. Recently, studies have shown the advantage of leveraging enzymatic steps into industrial-scale chemical processes, such as for the blood sugar regulator Sitagliptin (Merck) and the HIV protease inhibitor Darunavir (Prozomix). Designing optimal chemo-enzymatic pathways is a complex task. It requires navigating a high-dimensional search space of potential reactions that combine individual chemical and biochemical steps while at the same time minimizing transitions between chemical catalysis and bioreactions. Here, we introduce an algorithmic approach, minChemBio, that relies on solving a mixed-integer linear programming (MILP) problem by optimally searching through known chemical and enzymatic steps extracted from the United States Patent Office (USPTO) and MetaNetX databases, respectively. minChemBio allows for the minimization of transitions between chemical and biological reactions in the pathway, thus reducing the need for costly separation and purification steps required. minChemBio was benchmarked on three case studies involving the synthesis of 2-5-furandicarboxylic acid, terephthalate, and 3-hydroxybutyrate. Identified designs included both established literature pathways as well as unexplored ones which were compared against pathways identified by existing retrosynthetic tools. minChemBio fills a current gap in the space of pathway retrosynthesis tools by controlling and minimizing the transitions between chemical catalysis and biocatalytic steps. It is accessible to users through open-source code (https://github.com/maranasgroup/chemo-enz).</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-02-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143412321","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":"A High-Throughput Cell-Free Enzyme Screening System Using Redox-Responsive Hydrogel Beads as Artificial Compartments.","authors":"Taisei Koga, Yui Okawa, Tomoyuki Ito, Kensei Orita, Kosuke Minamihata, Mitsuo Umetsu, Noriho Kamiya","doi":"10.1021/acssynbio.4c00783","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00783","url":null,"abstract":"<p><p>We have developed a rapid, simple, and high-throughput screening system for recombinant enzymes using disulfide-bonded hydrogel beads (HBs) produced via a microfluidic method. These redox-responsive HBs were compatible with the biosynthesis of enzyme mutants via cell-free protein synthesis, fluorescent staining through an enzymatic reaction, and genetic information recovery after fluorescence-activated droplet sorting (FADS). The expression of microbial transglutaminase zymogen (MTGz) using cell-free protein synthesis and the cross-linking-reactivity-based staining of HBs with a fluorescent product were validated. Next-generation sequencing (NGS) analysis of the genes recovered from highly fluorescent HBs identified novel mutation sites (N25 and N27) in the propeptide domain. The introduction of these mutations allowed for the design of an engineered active MTGz, demonstrating the potential of HBs as artificial compartments for the FADS-based selection of enzymes that catalyze peptide and protein cross-linking reactions.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143412320","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":"De Novo Production of 1,6-Hexanediol and 1,6-Hexamethylenediamine from Glucose by Metabolic Engineered Escherichia coli","authors":"Nan Qin,&nbsp;Fanghuan Zhu,&nbsp;Youmeng Liu,&nbsp;Dehua Liu and Zhen Chen*,&nbsp;","doi":"10.1021/acssynbio.4c0088110.1021/acssynbio.4c00881","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00881https://doi.org/10.1021/acssynbio.4c00881","url":null,"abstract":"<p >1,6-Hexamethylenediamine (HMD) and 1,6-hexanediol (HDO) are pivotal C6 platform chemicals with extensive applications as key monomers in the synthesis of nylons, polyurethanes, and polyesters. The biological production of HMD and HDO from cheap and renewable bioresources represents an environmentally benign strategy for the sustainable chemical industry. Herein, we report the development of a novel biocatalytic route for the direct conversion of <span>d</span>-glucose to HMD and HDO in <i>Escherichia coli</i>. This was achieved through the integration of an adipic acid synthesis module with conversion modules tailored for HMD and HDO production. The study entailed a comprehensive optimization of pathway enzymes, protein expression, and precursor supply. Furthermore, a co-culture fermentation strategy was employed to enhance the efficiency of labor division, resulting in a two-strain cocultivation process that yielded 16.62 mg/L of HMD and 214.93 mg/L of HDO using glucose as the sole carbon source. This study establishes a foundational framework for the advancement of sustainable biological production processes for HMD and HDO from renewable resources.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"14 2","pages":"598–608 598–608"},"PeriodicalIF":3.7,"publicationDate":"2025-02-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143452715","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":"High Ectoine Production from Lignocellulosic Hydrolysate by Escherichia coli through Metabolic and Fermentation Engineering","authors":"Yifan Feng,&nbsp;Wenlong Xiao,&nbsp;Xinyi Li,&nbsp;Weiyu Cao,&nbsp;Yujia Jiang,&nbsp;Wenming Zhang,&nbsp;Wankui Jiang*,&nbsp;Fengxue Xin* and Min Jiang,&nbsp;","doi":"10.1021/acssynbio.4c0089910.1021/acssynbio.4c00899","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00899https://doi.org/10.1021/acssynbio.4c00899","url":null,"abstract":"<p >Ectoine, a major compatible solute in halophilic micro-organisms, shows great potential in cosmetics and pharmaceuticals areas owing to its water-binding properties and capability to prevent oxidative damage. In this study, the <i>ectABC</i> gene cluster responsible for the ectoine synthesis originated from halophilic bacterium <i>Halomonas venusta</i> was first assembled into <i>Escherichia coli</i>. Subsequently, the <i>crr</i> gene in PTS was knocked out to further drive the metabolic flux from phosphoenolpyruvate to oxaloacetate, resulting in 1.27 g/L of ectoine. Then, the rate-limiting enzyme LysC in the ectoine synthesis pathway was identified and modified. The recombinant <i>E. coli</i> with the further overexpression of feedback-insensitive mutant <i>EclysC</i>* increased the ectoine titer to 2.51 g/L with a yield of 0.37 g/g in shake flasks. After the medium optimization including the carbon and nitrogen source, sodium chloride, and magnesium sulfate concentration, the ectoine titer was improved to 4.55 g/L. 115.15 g/L of ectoine with a yield of 0.23 g/g was obtained in the 5.0 L bioreactor through the optimization of substrate feeding and IPTG supplementation in the fed-batch fermentation. To achieve the cost-effective production of ectoine, lignocellulosic hydrolysate from wheat straw was adopted. 134.08 g/L of ectoine with a yield of 0.33 g/g sugar and a productivity of 3.7 g/L/h was finally produced, representing a relatively high level of ectoine production from renewable resources compared to other studies. This study provides valuable insights into a cost-effective and efficient method for industrial-scale ectoine production.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":"14 2","pages":"609–620 609–620"},"PeriodicalIF":3.7,"publicationDate":"2025-02-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143452590","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}