Silence最新文献

{"title":"Limited evidence for evolutionarily conserved targeting of long non-coding RNAs by microRNAs.","authors":"Babak Alaei-Mahabadi,&nbsp;Erik Larsson","doi":"10.1186/1758-907X-4-4","DOIUrl":"10.1186/1758-907X-4-4","url":null,"abstract":"<p><strong>Background: </strong>Long non-coding RNAs (lncRNAs) are emerging as important regulators of cell physiology, but it is yet unknown to what extent lncRNAs have evolved to be targeted by microRNAs. Comparative genomics has previously revealed widespread evolutionarily conserved microRNA targeting of protein-coding mRNAs, and here we applied a similar approach to lncRNAs.</p><p><strong>Findings: </strong>We used a map of putative microRNA target sites in lncRNAs where site conservation was evaluated based on 46 vertebrate species. We compared observed target site frequencies to those obtained with a random model, at variable prediction stringencies. While conserved sites were not present above random expectation in intergenic lncRNAs overall, we observed a marginal over-representation of highly conserved 8-mer sites in a small subset of cytoplasmic lncRNAs (12 sites in 8 lncRNAs at 56% false discovery rate, P = 0.10).</p><p><strong>Conclusions: </strong>Evolutionary conservation in lncRNAs is generally low but patch-wise high, and these patches could, in principle, harbor conserved target sites. However, while our analysis efficiently detected conserved targeting of mRNAs, it provided only limited and marginally significant support for conserved microRNA-lncRNA interactions. We conclude that conserved microRNA-lncRNA interactions could not be reliably detected with our methodology.</p>","PeriodicalId":88703,"journal":{"name":"Silence","volume":"4 1","pages":"4"},"PeriodicalIF":0.0,"publicationDate":"2013-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/1758-907X-4-4","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"31671840","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Analysis of hairpin RNA transgene-induced gene silencing in Fusarium oxysporum.","authors":"Ulrike Schumann, Neil A Smith, Kemal Kazan, Michael Ayliffe, Ming-Bo Wang","doi":"10.1186/1758-907X-4-3","DOIUrl":"10.1186/1758-907X-4-3","url":null,"abstract":"<p><strong>Background: </strong>Hairpin RNA (hpRNA) transgenes can be effective at inducing RNA silencing and have been exploited as a powerful tool for gene function analysis in many organisms. However, in fungi, expression of hairpin RNA transcripts can induce post-transcriptional gene silencing, but in some species can also lead to transcriptional gene silencing, suggesting a more complex interplay of the two pathways at least in some fungi. Because many fungal species are important pathogens, RNA silencing is a powerful technique to understand gene function, particularly when gene knockouts are difficult to obtain. We investigated whether the plant pathogenic fungus Fusarium oxysporum possesses a functional gene silencing machinery and whether hairpin RNA transcripts can be employed to effectively induce gene silencing.</p><p><strong>Results: </strong>Here we show that, in the phytopathogenic fungus F. oxysporum, hpRNA transgenes targeting either a β-glucuronidase (Gus) reporter transgene (hpGus) or the endogenous gene Frp1 (hpFrp) did not induce significant silencing of the target genes. Expression analysis suggested that the hpRNA transgenes are prone to transcriptional inactivation, resulting in low levels of hpRNA and siRNA production. However, the hpGus RNA can be efficiently transcribed by promoters acquired either by recombination with a pre-existing, actively transcribed Gus transgene or by fortuitous integration near an endogenous gene promoter allowing siRNA production. These siRNAs effectively induced silencing of a target Gus transgene, which in turn appeared to also induce secondary siRNA production. Furthermore, our results suggested that hpRNA transcripts without poly(A) tails are efficiently processed into siRNAs to induce gene silencing. A convergent promoter transgene, designed to express poly(A)-minus sense and antisense Gus RNAs, without an inverted-repeat DNA structure, induced consistent Gus silencing in F. oxysporum.</p><p><strong>Conclusions: </strong>These results indicate that F. oxysporum possesses functional RNA silencing machineries for siRNA production and target mRNA cleavage, but hpRNA transgenes may induce transcriptional self-silencing due to its inverted-repeat structure. Our results suggest that F. oxysporum possesses a similar gene silencing pathway to other fungi like fission yeast, and indicate a need for developing more effective RNA silencing technology for gene function studies in this fungal pathogen.</p>","PeriodicalId":88703,"journal":{"name":"Silence","volume":"4 1","pages":"3"},"PeriodicalIF":0.0,"publicationDate":"2013-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3733888/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"31550370","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"cWords - systematic microRNA regulatory motif discovery from mRNA expression data.","authors":"Simon H Rasmussen,&nbsp;Anders Jacobsen,&nbsp;Anders Krogh","doi":"10.1186/1758-907X-4-2","DOIUrl":"https://doi.org/10.1186/1758-907X-4-2","url":null,"abstract":"<p><strong>Background: </strong>Post-transcriptional regulation of gene expression by small RNAs and RNA binding proteins is of fundamental importance in development of complex organisms, and dysregulation of regulatory RNAs can influence onset, progression and potentially be target for treatment of many diseases. Post-transcriptional regulation by small RNAs is mediated through partial complementary binding to messenger RNAs leaving nucleotide signatures or motifs throughout the entire transcriptome. Computational methods for discovery and analysis of sequence motifs in high-throughput mRNA expression profiling experiments are becoming increasingly important tools for the identification of post-transcriptional regulatory motifs and the inference of the regulators and their targets.</p><p><strong>Results: </strong>cWords is a method designed for regulatory motif discovery in differential case-control mRNA expression datasets. We have improved the algorithms and statistical methods of cWords, resulting in at least a factor 100 speed gain over the previous implementation. On a benchmark dataset of 19 microRNA (miRNA) perturbation experiments cWords showed equal or better performance than two comparable methods, miReduce and Sylamer. We have developed rigorous motif clustering and visualization that accompany the cWords analysis for more intuitive and effective data interpretation. To demonstrate the versatility of cWords we show that it can also be used for identification of potential siRNA off-target binding. Moreover, cWords analysis of an experiment profiling mRNAs bound by Argonaute ribonucleoprotein particles discovered endogenous miRNA binding motifs.</p><p><strong>Conclusions: </strong>cWords is an unbiased, flexible and easy-to-use tool designed for regulatory motif discovery in differential case-control mRNA expression datasets. cWords is based on rigorous statistical methods that demonstrate comparable or better performance than other existing methods. Rich visualization of results promotes intuitive and efficient interpretation of data. cWords is available as a stand-alone Open Source program at Github https://github.com/simras/cWords and as a web-service at: http://servers.binf.ku.dk/cwords/.</p>","PeriodicalId":88703,"journal":{"name":"Silence","volume":"4 1","pages":"2"},"PeriodicalIF":0.0,"publicationDate":"2013-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/1758-907X-4-2","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"31534802","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Generation of a luciferase-based reporter for CHH and CG DNA methylation in Arabidopsis thaliana.","authors":"Thanh Theresa Dinh,&nbsp;Michael O'Leary,&nbsp;So Youn Won,&nbsp;Shengben Li,&nbsp;Lorena Arroyo,&nbsp;Xigang Liu,&nbsp;Andrew Defries,&nbsp;Binglian Zheng,&nbsp;Sean R Cutler,&nbsp;Xuemei Chen","doi":"10.1186/1758-907X-4-1","DOIUrl":"https://doi.org/10.1186/1758-907X-4-1","url":null,"abstract":"<p><strong>Background: </strong>DNA methylation ensures genome integrity and regulates gene expression in diverse eukaryotes. In Arabidopsis, methylation occurs in three sequence contexts: CG, CHG and CHH. The initial establishment of DNA methylation at all three sequence contexts occurs through a process known as RNA-directed DNA methylation (RdDM), in which small RNAs bound by Argonaute4 (AGO4) guide DNA methylation at homologous loci through the de novo methyltransferase DRM2. Once established, DNA methylation at each of the three sequence contexts is maintained through different mechanisms. Although some players involved in RdDM and maintenance methylation have been identified, the underlying molecular mechanisms are not fully understood. To aid the comprehensive identification of players in DNA methylation, we generated a transgenic reporter system that permits genetic and chemical genetic screens in Arabidopsis.</p><p><strong>Results: </strong>A dual 35S promoter (d35S) driven luciferase (LUC) reporter was introduced into Arabidopsis and LUCL, a line with a low basal level of luciferase activity, was obtained. LUCL was found to be a multi-copy, single-insertion transgene that contains methylated cytosines in CG, CHG and CHH contexts, with the highest methylation in the CG context. Methylation was present throughout the promoter and LUC coding region. Treatment with an inhibitor of cytosine methylation de-repressed luciferase activity. A mutation in MET1, which encodes the CG maintenance methyltransferase, drastically reduced CG methylation and de-repressed LUC expression. Mutations in AGO4 and DRM2 also de-repressed LUC expression, albeit to a smaller extent than loss of MET1. Using LUCL as a reporter line, we performed a chemical screen for compounds that de-repress LUC expression, and identified a chemical, methotrexate, known to be involved in biogenesis of the methyl donor.</p><p><strong>Conclusion: </strong>We developed a luciferase-based reporter system, LUCL, which reports both RdDM and CG maintenance methylation in Arabidopsis. The low basal level of LUCL expression provides an easy readout in genetic and chemical genetic screens that will dissect the mechanisms of RdDM and methylation maintenance.</p>","PeriodicalId":88703,"journal":{"name":"Silence","volume":" ","pages":"1"},"PeriodicalIF":0.0,"publicationDate":"2013-04-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/1758-907X-4-1","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40250782","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Strand-specific libraries for high throughput RNA sequencing (RNA-Seq) prepared without poly(A) selection.","authors":"Zhao Zhang,&nbsp;William E Theurkauf,&nbsp;Zhiping Weng,&nbsp;Phillip D Zamore","doi":"10.1186/1758-907X-3-9","DOIUrl":"https://doi.org/10.1186/1758-907X-3-9","url":null,"abstract":"<p><strong>Unlabelled: </strong></p><p><strong>Background: </strong>High throughput DNA sequencing technology has enabled quantification of all the RNAs in a cell or tissue, a method widely known as RNA sequencing (RNA-Seq). However, non-coding RNAs such as rRNA are highly abundant and can consume >70% of sequencing reads. A common approach is to extract only polyadenylated mRNA; however, such approaches are blind to RNAs with short or no poly(A) tails, leading to an incomplete view of the transcriptome. Another challenge of preparing RNA-Seq libraries is to preserve the strand information of the RNAs.</p><p><strong>Design: </strong>Here, we describe a procedure for preparing RNA-Seq libraries from 1 to 4 μg total RNA without poly(A) selection. Our method combines the deoxyuridine triphosphate (dUTP)/uracil-DNA glycosylase (UDG) strategy to achieve strand specificity with AMPure XP magnetic beads to perform size selection. Together, these steps eliminate gel purification, allowing a library to be made in less than two days. We barcode each library during the final PCR amplification step, allowing several samples to be sequenced in a single lane without sacrificing read length. Libraries prepared using this protocol are compatible with Illumina GAII, GAIIx and HiSeq 2000 platforms.</p><p><strong>Discussion: </strong>The RNA-Seq protocol described here yields strand-specific transcriptome libraries without poly(A) selection, which provide approximately 90% mappable sequences. Typically, more than 85% of mapped reads correspond to protein-coding genes and only 6% derive from non-coding RNAs. The protocol has been used to measure RNA transcript identity and abundance in tissues from flies, mice, rats, chickens, and frogs, demonstrating its general applicability.</p>","PeriodicalId":88703,"journal":{"name":"Silence","volume":" ","pages":"9"},"PeriodicalIF":0.0,"publicationDate":"2012-12-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/1758-907X-3-9","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40198388","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"MicroRNAs from the same precursor have different targeting properties.","authors":"Antonio Marco, Jamie I Macpherson, Matthew Ronshaugen, Sam Griffiths-Jones","doi":"10.1186/1758-907X-3-8","DOIUrl":"10.1186/1758-907X-3-8","url":null,"abstract":"<p><strong>Unlabelled: </strong></p><p><strong>Background: </strong>The processing of a microRNA results in an intermediate duplex of two potential mature products that derive from the two arms (5' and 3') of the precursor hairpin. It is often suggested that one of the sequences is degraded and the other is incorporated into the RNA-induced silencing complex. However, both precursor arms may give rise to functional levels of mature microRNA and the dominant product may change from species to species, from tissue to tissue, or between developmental stages. Therefore, both arms of the precursor have the potential to produce functional mature microRNAs.</p><p><strong>Results: </strong>We have investigated the relationship between predicted mRNA targets of mature sequences derived from the 5' and 3' arms of the same pre-microRNAs. Using six state-of-the-art target prediction algorithms, we find that 5'/3' microRNA pairs target different sites in 3' untranslated regions of mRNAs. We also find that these pairs do not generally target overlapping sets of genes, or functionally related genes.</p><p><strong>Conclusions: </strong>We show that alternative mature products produced from the same precursor microRNAs have different targeting properties and therefore different biological functions. These data strongly suggest that developmental or evolutionary changes in arm choice will have significant functional consequences.</p>","PeriodicalId":88703,"journal":{"name":"Silence","volume":"3 1","pages":"8"},"PeriodicalIF":0.0,"publicationDate":"2012-09-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3503882/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"30936993","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"RNA: methods and protocols - a new series.","authors":"Phillip D Zamore","doi":"10.1186/1758-907X-3-7","DOIUrl":"https://doi.org/10.1186/1758-907X-3-7","url":null,"abstract":"<p><p> This month, Silence launches a new series on methods and protocols to study silencing pathways and analyze nucleic acids and proteins.</p>","PeriodicalId":88703,"journal":{"name":"Silence","volume":"3 1","pages":"7"},"PeriodicalIF":0.0,"publicationDate":"2012-06-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/1758-907X-3-7","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"30673679","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development of a luciferase-based reporter of transcriptional gene silencing that enables bidirectional mutant screening in Arabidopsis thaliana.","authors":"So Youn Won,&nbsp;Shengben Li,&nbsp;Binglian Zheng,&nbsp;Yuanyuan Zhao,&nbsp;Dongming Li,&nbsp;Xin Zhao,&nbsp;Huilan Yi,&nbsp;Lei Gao,&nbsp;Thanh Theresa Dinh,&nbsp;Xuemei Chen","doi":"10.1186/1758-907X-3-6","DOIUrl":"https://doi.org/10.1186/1758-907X-3-6","url":null,"abstract":"<p><strong>Unlabelled: </strong></p><p><strong>Background: </strong>Cytosine methylation is an important chromatin modification that maintains genome integrity and regulates gene expression through transcriptional gene silencing. Major players in de novo methylation guided by siRNAs (known as RNA-directed DNA methylation, or RdDM), maintenance methylation, and active demethylation have been identified in Arabidopsis. However, active demethylation only occurs at a subset of RdDM loci, raising the question of how the homeostasis of DNA methylation is achieved at most RdDM loci. To identify factors that regulate the levels of cytosine methylation, we aimed to establish a transgenic reporter system that allows for forward genetic screens in Arabidopsis.</p><p><strong>Results: </strong>We introduced a dual 35 S promoter (d35S) driven luciferase reporter, LUCH, into Arabidopsis and isolated a line with a moderate level of luciferase activity. LUCH produced transgene-specific 24 nucleotide siRNAs and its d35S contained methylated cytosine in CG, CHG and CHH contexts. Treatment of the transgenic line with an inhibitor of cytosine methylation de-repressed luciferase activity. Mutations in several components of the RdDM pathway but not the maintenance methylation genes resulted in reduced d35S methylation, especially CHH methylation, and de-repression of luciferase activity. A mutation in MOM1, which is known to cooperate with RdDM to silence transposons, reduced d35S DNA methylation and de-repressed LUCH expression. A mutation in ROS1, a cytosine demethylation enzyme, increased d35S methylation and reduced LUCH expression.</p><p><strong>Conclusion: </strong>We developed a luciferase-based reporter, LUCH, which reports both DNA methylation directed by small RNAs and active demethylation by ROS1 in Arabidopsis. The moderate basal level of LUCH expression allows for bi-directional genetic screens that dissect the mechanisms of DNA methylation as well as demethylation.</p>","PeriodicalId":88703,"journal":{"name":"Silence","volume":"3 1","pages":"6"},"PeriodicalIF":0.0,"publicationDate":"2012-06-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/1758-907X-3-6","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"30674423","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Application of RNA silencing to plant disease resistance.","authors":"Cheng-Guo Duan,&nbsp;Chun-Han Wang,&nbsp;Hui-Shan Guo","doi":"10.1186/1758-907X-3-5","DOIUrl":"https://doi.org/10.1186/1758-907X-3-5","url":null,"abstract":"<p><p> To reduce the losses caused by plant pathogens, plant biologists have adopted numerous methods to engineer resistant plants. Among them, RNA silencing-based resistance has been a powerful tool that has been used to engineer resistant crops during the last two decades. Based on this mechanism, diverse approaches were developed. In this review, we focus on the application of RNA silencing to produce plants that are resistant to plant viruses such as RNA and DNA viruses, viroids, insects, and the recent expansion to fungal pathogens.</p>","PeriodicalId":88703,"journal":{"name":"Silence","volume":"3 1","pages":"5"},"PeriodicalIF":0.0,"publicationDate":"2012-05-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/1758-907X-3-5","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"30656499","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Reducing ligation bias of small RNAs in libraries for next generation sequencing.","authors":"Karim Sorefan, Helio Pais, Adam E Hall, Ana Kozomara, Sam Griffiths-Jones, Vincent Moulton, Tamas Dalmay","doi":"10.1186/1758-907X-3-4","DOIUrl":"10.1186/1758-907X-3-4","url":null,"abstract":"<p><strong>Background: </strong>The use of nucleic acid-modifying enzymes has driven the rapid advancement in molecular biology. Understanding their function is important for modifying or improving their activity. However, functional analysis usually relies upon low-throughput experiments. Here we present a method for functional analysis of nucleic acid-modifying enzymes using next generation sequencing.</p><p><strong>Findings: </strong>We demonstrate that sequencing data of libraries generated by RNA ligases can reveal novel secondary structure preferences of these enzymes, which are used in small RNA cloning and library preparation for NGS. Using this knowledge we demonstrate that the cloning bias in small RNA libraries is RNA ligase-dependent. We developed a high definition (HD) protocol that reduces the RNA ligase-dependent cloning bias. The HD protocol doubled read coverage, is quantitative and found previously unidentified microRNAs. In addition, we show that microRNAs in miRBase are those preferred by the adapters of the main sequencing platform.</p><p><strong>Conclusions: </strong>Sequencing bias of small RNAs partially influenced which microRNAs have been studied in depth; therefore most previous small RNA profiling experiments should be re-evaluated. New microRNAs are likely to be found, which were selected against by existing adapters. Preference of currently used adapters towards known microRNAs suggests that the annotation of all existing small RNAs, including miRNAs, siRNAs and piRNAs, has been biased.</p>","PeriodicalId":88703,"journal":{"name":"Silence","volume":"3 1","pages":"4"},"PeriodicalIF":0.0,"publicationDate":"2012-05-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3489589/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"30654215","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}