Biotechnology Letters最新文献

{"title":"Multiplicity of infection and culture medium on the SARS-CoV-2 virus like-particles production by baculovirus/insect system.","authors":"Luis Giovani de Oliveira Guardalini, Felipe Moura Dias, Samanta Omae Camalhonte, Jaci Leme, Thaissa Consoni Bernardino, Felipe Soares Sposito, Eduardo Dias, Renato Mancini Astray, Aldo Tonso, Soraia Attie Calil Jorge, Eutimio Gustavo Fernández Núñez","doi":"10.1007/s10529-025-03572-w","DOIUrl":"10.1007/s10529-025-03572-w","url":null,"abstract":"<p><p>This work aimed to assess the SARS-CoV-2 structural proteins' expression and virus-like particles (VLP) production by Baculovirus/Insect cell platform using two levels of Multiplicity of Infection (MOI), and two culture media, one of them a serum-free medium and the other one chemically defined. Two SARS-CoV-2 VLP were obtained from Sf9 cells coinfection using in both cases, three monocistronic recombinant baculoviruses holding the genes of Nucleocapsid (N; MOI = 2 or 0.2), Membrane (M; MOI = 1 or 0.1), and Envelope (E; MOI = 1 or 0.1) viral proteins, and the fourth one was changed between a baculovirus bearing Spike protein (S; MOI = 3 or 0.3) or receptor-binding domain (RBD; MOI = 3 or 0.3) genes of SARS-CoV-2. Similar performance was verified for both culture media in SARS-CoV-2 VLP production bearing four structural virus proteins or RBD domain. The SARS-CoV-2 structural proteins' expression was comparable at different MOIs (tenfold) as well as SARS-CoV-2 VLP size (around 100 nm). The increase in specific death rates over the coinfection phase was confirmed in relatively high MOI assays. This finding was related to an exponential virus titer profile for high MOIs over the entire infection phase, meanwhile, a viral peak was observed at low MOIs, confirming a secondary infection. The SARS-CoV-2 VLP improved production carrying immunogenic S protein was confirmed concerning others holding RBD. However, the protein composition of produced VLP should be studied further to assess the VLP homogeneity when different culture media and MOIs are used.</p>","PeriodicalId":8929,"journal":{"name":"Biotechnology Letters","volume":"47 2","pages":"32"},"PeriodicalIF":2.0,"publicationDate":"2025-03-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143565999","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development of a fully automated latex-enhanced immunoturbidimetric method for quantitative serum Lp(a) measurement.","authors":"Yanyan Liu, Meijiao Li, Hao Zhang, Le Gao, Jitao Liu, Yue Hou, Jiancheng Xu","doi":"10.1007/s10529-025-03564-w","DOIUrl":"10.1007/s10529-025-03564-w","url":null,"abstract":"<p><strong>Background: </strong>Lipoprotein (a) [Lp(a)] is a critical factor in cardiovascular health, composed of low-density lipoprotein-like particles bound to apolipoprotein (a). Elevated Lp(a) levels are associated with an increased risk of cardiovascular diseases (CVD), accelerating disease progression and raising CVD-related mortality. However, the lack of standardized measurement methods for Lp(a) contributes to diagnostic uncertainties in this area.</p><p><strong>Method: </strong>A quantitative measurement method for serum Lp(a) was developed using fully automated latex-enhanced particle immunoturbidimetry, marking a significant advancement in diagnostic capabilities. Key parameters, including repeatability, stability, linearity, detection limit, interference, and method comparison, were evaluated to ensure the assay's reliability and accuracy.</p><p><strong>Result: </strong>Lp(a) in samples was detected by carboxylated latex particles (95 nm in diameter) covalently coated with anti-Lp(a) antibodies. Lp(a) concentration was quantified by measuring the turbidity changes caused by agglutination at 600 nm. This method provides rapid, accurate, and fully automated measurements on the Hitachi 7100 automatic biochemical analyzer. With intra-batch precision CV% of 1.10% and inter-batch precision CV% of 1.79%, the method demonstrates reliable performance with Randox biochemical quality control samples. It has a detection limit of 7 mg/L and a high correlation coefficient (R² = 0.9946) within the 0-1500 mg/L range. Minimal interference from bilirubin, fat emulsion, hemoglobin, and ascorbic acid was observed. Additionally, it shows strong correlation (R² = 0.9972) with a commercially available latex-enhanced immunoturbidimetric Lp(a) assay reagent, confirming its comparability and clinical suitability.</p><p><strong>Conclusion: </strong>The quantitative serum Lp(a) determination method based on latex-enhanced immunoturbidimetry offers numerous advantages. It provides rapid, accurate, and automated results, making it ideal for routine clinical testing. The method effectively measures Lp(a) in serum samples by leveraging the interaction between Lp(a) and latex particles.</p>","PeriodicalId":8929,"journal":{"name":"Biotechnology Letters","volume":"47 2","pages":"31"},"PeriodicalIF":2.0,"publicationDate":"2025-03-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143565968","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Improving the hydrophilic microenvironment surrounding the catalytic site of fructosyltransferase enhances its catalytic ability.","authors":"Fanzhi Wang, Suren Singh, Kugen Permaul","doi":"10.1007/s10529-025-03566-8","DOIUrl":"10.1007/s10529-025-03566-8","url":null,"abstract":"<p><p>The hydrophilic microenvironment surrounding an enzyme's active site can influence its catalytic activity. This study examines the effect of enhancing this environment in the Aspergillus niger fructosyltransferase, SucC. Bioinformatics analysis identified a cysteine residue (C66) near the catalytic triad (D64, D194, E271) as vital for maintaining the active site's structure and facilitating substrate transport. Simulated mutagenesis suggested that mutating cysteine to serine (C66S) could increase hydrophilicity without altering the structure significantly. This mutation was predicted to enhance substrate affinity, with binding energy changing from -3.65 to -4.14 kcal mol^-1. The C66S mutant, expressed in Pichia pastoris GS115, showed a 61.3% increase in specific activity, a 13.5% decrease in K_m (82.20/71.14 mM), and a 21.6% increase in k_cat (112.23/136.48 min^-1), resulting in a 40.1% increase in catalytic efficiency (1.37/1.92 min^-1 mM^-1). For fructooligosaccharides (FOS) production, C66S demonstrated enhanced transfructosylation, particularly in the initial stages of the reaction, achieving higher overall FOS yields. These findings highlight that modifying the active site hydrophilicity, without causing major structural changes, is a promising strategy for improving an enzyme's catalytic efficiency.</p>","PeriodicalId":8929,"journal":{"name":"Biotechnology Letters","volume":"47 2","pages":"30"},"PeriodicalIF":2.0,"publicationDate":"2025-02-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11865173/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143514671","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Enhancement hispolon production from Phellinus linteus via epigenetic-modified culture to inhibit human breast cancer cells.","authors":"Phongsakorn Chueaphromsri, Phongsakorn Kunhorm, Nipha Chaicharoenaudomrung, Parinya Noisa","doi":"10.1007/s10529-025-03561-z","DOIUrl":"10.1007/s10529-025-03561-z","url":null,"abstract":"<p><p>Phellinus linteus (PL) is a medicinal fungus known for producing hispolon, a bioactive compound with antioxidant, anti-inflammatory, and anticancer properties. However, the natural scarcity of PL and the unsuccessful cultivation of its fruiting bodies have led to the exploration of alternative methods for enhancing its bioactive compound production. In this study, static fermentation was employed, and Valproic acid (VPA), a histone deacetylase inhibitor (HDACi), was added to the culture medium to induce epigenetic modifications and enhance hispolon production. After 30 days of fermentation, the hispolon concentration was analyzed using high-performance liquid chromatography (HPLC), mycelial dry weight was measured, and the expression of hispolon synthesis-related enzymes was quantified using quantitative PCR (qPCR). Additionally, the anticancer potential of the fermented media was assessed in human breast adenocarcinoma HTB-26 cells using assays for cytotoxicity, reactive oxygen species (ROS) formation, apoptosis, antioxidant activity, and autophagy markers. The results revealed that the addition of 400 µM VPA increased hispolon production by 120% and mycelial dry weight by 41%, likely due to enhanced transcriptional accessibility. Furthermore, the PL fermentation media significantly inhibited HTB-26 cell growth through the induction of ROS formation, autophagy, and apoptosis. These findings suggest that VPA-enhanced static fermentation of PL offers a promising strategy for optimizing hispolon production and developing effective anticancer therapeutics.</p>","PeriodicalId":8929,"journal":{"name":"Biotechnology Letters","volume":"47 2","pages":"29"},"PeriodicalIF":2.0,"publicationDate":"2025-02-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143514655","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Genome-wide screening of mitogen-activated protein kinase (MAPK) gene family and expression profile under heavy metal stress in Solanum lycopersicum.","authors":"Hasan Can, Ilhan Dogan, Fatih Tabanli, Mehmet Emin Uras, Asli Hocaoglu-Ozyigit, Ibrahim Ilker Ozyigit","doi":"10.1007/s10529-025-03567-7","DOIUrl":"10.1007/s10529-025-03567-7","url":null,"abstract":"<p><p>MAPKs are one of the essential signal transduction complexes which are responsible for the perception of abiotic stress and for the producing of related transcripts for responding to abiotic stress. For systematical analyzes of the mitogen-activated protein (MAP) kinase gene families and their expression profiles in Solanum lycopersicum L. exposed to diverse heavy metal stresses, 17 SlMAPK genes were studied in comparison with their 159 orthologs from various plant species. The result of phylogenetic analysis revealed that SlMAPKs were divided into four different subgroups (A, B, C, and D) based on their nucleic acid and protein sequence alignments. SlMAPKs including A, B and C group had lower molecular weights and more hydrophobic structures than D group SlMAPKs, while possible extra phosphorylation sites predicted in D-group SLMAPKs. 24 cis regulating elements such as Box 4, TATA-box, ABRE and CAAT-box were detected in their upstream parts of DNA sequences. Also, it was determined that SlMAPKs show interactions with important proteins such as Guanine nucleotide-binding protein beta subunit, heterotrimeric G-protein, protein phosphatase 2C and HY5. The results from our gene expression analyzes, significant increases were found in the expressions of the selected SLMAPK gene with applications of a range of increasing heavy metal concentrations (e.g., by the application of the 400 mM Ni + Pb exposure, a 16-fold increase in the expression of SlMAPK gene was noted). Overall, SlMAPK genes and proteins known were re-evaluated, and the SlMAPKs interactions with some other important proteins were observed. Also, some predictions about the extra phosphorylation sites of SlMAPKs having effects on their functions were done. In addition, the expression levels of SlMAPK genes were monitored under different levels of heavy metal stress exposures.</p>","PeriodicalId":8929,"journal":{"name":"Biotechnology Letters","volume":"47 2","pages":"27"},"PeriodicalIF":2.0,"publicationDate":"2025-02-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143447746","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"O₂-microbubble of iron-porphyrin conjugated polyaspartamide for molecular ultrasound contrast effect.","authors":"Yoon Na Cho, Jun Woo Lim, Seung Joo Oh, Sa Ra Han, Sungwoo Cho, Jimin Jeong, Byoung Hee Han, Jae Hyun Jeong","doi":"10.1007/s10529-025-03571-x","DOIUrl":"10.1007/s10529-025-03571-x","url":null,"abstract":"<p><strong>Objective: </strong>This study aimed to prepare oxygen-microbubbles incorporating ferrous porphyrin to emulate the oxygen-capturing ability of hemoglobin porphyrin in red blood cells.</p><p><strong>Results: </strong>We synthesized poly(2-hydroxyethyl aspartamide) (PHEA) grafted with ferrous porphyrins (Iron-P-PHEA) and created microbubbles using 1,2-dipalmitoyl-sn-glycero-3-phosphocholine. These microbubbles trapped oxygen and retained it over a 2 h period. The O₂-microbubbles demonstrated an enhanced photoacoustic effect as an ultrasound contrast agent, as confirmed by Doppler ultrasound testing.</p><p><strong>Conclusions: </strong>The innovative strategy for O₂-microbubble preparation enhances the efficiency of targeted delivery in molecular optical and ultrasound imaging.</p>","PeriodicalId":8929,"journal":{"name":"Biotechnology Letters","volume":"47 2","pages":"28"},"PeriodicalIF":2.0,"publicationDate":"2025-02-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143447748","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Evaluation of secretory signal peptides for heterologous protein secretion in Cyanobacterium aponinum PCC10605.","authors":"Rajesh Nandru, Bhaskar Bhadra, Nilanjan Roy, Anshul Nigam, Penna Suprasanna","doi":"10.1007/s10529-025-03569-5","DOIUrl":"10.1007/s10529-025-03569-5","url":null,"abstract":"<p><p>Biomanufacturing of recombinant proteins in the microalgae has become an important field of research owing to sustainability, scalability, safety, and metabolic diversity of the microalgal system. Recovery of the recombinant protein from the host system needs to be devised and established, since the conventional downstream process for recombinant protein extraction is associated with high costs and resources. In a previous study, we have reported two putative signal peptides of C. aponinum using in silico approach. Herein, we evaluated the two secretory signal peptides for heterologous protein secretion in C. aponinum PCC10605. The green fluorescent protein was used as secretory protein and as a reporter. Signal peptides, thermitase and porin, fused with GFP were transformed in to C. aponinum for studying the expression and secretion. Following the antibiotic screening and GFP fluorescence analysis, transformants secreting GFP in the supernatant were validated by using western blotting. The results showed that fluorescence, as measured by FACS analysis and TECAN reader, varied among the two signal peptides and, higher fluorescence was recorded in the 'thermitase SP secreted GFP' supernatant. The thermitase signal peptide may offer as a new gateway for recombinant protein production and secretion in C. aponinum.</p>","PeriodicalId":8929,"journal":{"name":"Biotechnology Letters","volume":"47 2","pages":"25"},"PeriodicalIF":2.0,"publicationDate":"2025-02-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143439767","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"In-silico evaluation of potential plant-based tyrosinase inhibitors for cosmetic and pharmaceutical applications.","authors":"Jyoti Srivastava, Sukhendra Singh, Rupika Sinha","doi":"10.1007/s10529-025-03570-y","DOIUrl":"10.1007/s10529-025-03570-y","url":null,"abstract":"<p><p>Tyrosinase is involved in a critical step of melanin synthesis; therefore, tyrosinase inhibitors are gaining more importance in the medicinal and cosmetic industry for the treatment of different pigmentary disorders. In the last decades, mushroom tyrosinase was used as a standard enzyme for the identification and advancement of most tyrosinase inhibitors. Due to differences in structure and substrate specificity between mushroom and human tyrosinase, there is a need for a more specific study with human tyrosinase. Additionally, the tyrosinase inhibitors which are currently in use have various side effects, therefore, safer inhibitors from natural sources are required. Different tyrosinase inhibitors from natural sources (aloesin, norartocarpetin, hesperetin, morin and taxifolin) were evaluated for an effective eco-friendly whitening agent using different bioinformatics tools. To check the efficacy and safety of the selected compounds ADME analysis was performed which showed that all the selected compounds fulfilled most of the parameters of general drug discovery. Docking of selected ligands was performed against the predicted structure of human tyrosinase; and the binding affinity (in kcal/mol) of kojic acid, aloesin, norartocarpetin, hesperetin, morin and taxifolin were obtained to be - 5.6, - 7.2, - 7.6, - 7.5, - 7.3 and - 7.2 respectively. Among all the selected ligands, norartocarpetin had the lowest binding affinity, i.e., - 7.6 kcal/mol, which showed that norartocarpetin could be used as a potent tyrosinase inhibitor. This bioactive compound is widely distributed in Moraceae plants and therefore, poses as a natural solution to various melanin-based dermatological issues and it can have a potential application in pharmaceuticals and cosmetic industries for the treatment of pigmentary disorders.</p>","PeriodicalId":8929,"journal":{"name":"Biotechnology Letters","volume":"47 2","pages":"26"},"PeriodicalIF":2.0,"publicationDate":"2025-02-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143439768","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mycoremediation of Sulfamethoxazole and metabolic pathway by Aspergillus tubingensis strain.","authors":"Raveena Ann Alex, Jayanthi Abraham","doi":"10.1007/s10529-025-03568-6","DOIUrl":"10.1007/s10529-025-03568-6","url":null,"abstract":"<p><p>Over the last few decades sulfonamides are being prescribed on a large scale for treating human beings and livestock. Contaminants of sulfonamide antibiotics are present in various environments and these residues can enter the food web, leading to health threat. The purpose of this study was to assess sulfamethoxazole degradation using a novel strain of Aspergillus sp. and demonstrates the degradation pathway of sulfamethoxazole. To the best of our knowledge, this marks the first detailed biodegradation pathway for Aspergillus sp. AJC4 proposed. The biodegradation pattern of sulfamethoxazole was assessed using High Performance Liquid Chromatography (UPLC) and validated through Gas Chromatography Mass Spectroscopy (GC-MS), Liquid Chromatography Mass Spectrometry (LC-MS) and Fourier Transform Infrared Spectroscopy (FTIR). The fungal isolate was able to degrade 99.42% of sulfamethoxazole at a concentration of 150 mg/l within 7 d. Three metabolic compounds were identified throughout the Sulfamethoxazole biodegradation process. The degradation pathway was shown to follow first order kinetics model according to the kinetics energy.</p>","PeriodicalId":8929,"journal":{"name":"Biotechnology Letters","volume":"47 1","pages":"23"},"PeriodicalIF":2.0,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143188040","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Bacillus velezensis CE 100 controls anthracnose disease in walnut trees (Juglans regia L.) by inhibiting Colletotrichum gloeosporioides and eliciting induced systemic resistance.","authors":"Vantha Choub, Sang-Jae Won, Jae-Hyun Moon, Su-In Choi, Henry B Ajuna, Young Sang Ahn","doi":"10.1007/s10529-025-03560-0","DOIUrl":"10.1007/s10529-025-03560-0","url":null,"abstract":"<p><p>Biological control of plant diseases is recognized as an effective and environmental friendly alternative to chemical fungicides. We demonstrated the dual biocontrol strategy of Bacillus velezensis CE 100 through the hydrolytic activity of chitinase and β-1,3-glucanase and the elicitation of induced systemic resistance (ISR) against Colletotrichum gloeosporioides that causes anthracnose disease in walnut trees. B. velezensis CE 100 produced a maximum of 62.1 units mL^-1 (132.9 units mL^-1) chitinase and 5.2 units mL^-1 (9.4 units mL^-1) β-1,3-glucanase enzymes in the broth culture (crude enzyme fraction), and inhibited spore germination and mycelial growth of C. gloeosporioides by 81.6% and 22.6%, respectively, at 100 µl mL^-1 of crude enzyme fraction. The inoculation of B. velezensis CE 100 induced the production of pathogenesis-related (PR) chitinase in walnuts by 2.1-fold, and to a lesser extent PR β-1,3-glucanase, and reduced anthracnose disease severity by 3.0-fold compared to the control group. The bacterium produced a maximum of 11.4 µg mL^-1 indole-3-acetic acid (IAA) and improved the chlorophyll content, shoot length, and root collar diameter of walnut trees compared to the fungicide treatment and control groups. B. velezensis CE 100 demonstrated the prospect of controlling walnut anthracnose by direct antagonism and ISR against C. gloeosporioides, while simultaneously enhancing walnut growth through IAA production.</p>","PeriodicalId":8929,"journal":{"name":"Biotechnology Letters","volume":"47 1","pages":"20"},"PeriodicalIF":2.0,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143188030","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}