bioRxiv - Molecular Biology最新文献

{"title":"Cryo-EM uncovers a sequential mechanism for RNA polymerase I pausing and stalling at abasic DNA lesions","authors":"Alicia Santos-Aledo, Adrian Plaza-Pegueroles, Marta Sanz-Murillo, Federico M. Ruiz, Jun Xu, David Gil-Carton, Dong Wang, Carlos Fernandez-Tornero","doi":"10.1101/2024.09.09.611646","DOIUrl":"https://doi.org/10.1101/2024.09.09.611646","url":null,"abstract":"RNA polymerase I (Pol I) transcribes ribosomal DNA (rDNA) to produce the rRNA precursor, which accounts for up to 60% of the total transcriptional activity in growing cells. Pol I monitors rDNA integrity and influences cell survival, but little is known about how this enzyme processes abasic DNA lesions. Here, we report electron cryo-microscopy (cryo-EM) structures of Pol I at different stages of stalling at abasic sites, supported by in vitro transcription studies. Our results show that templating abasic sites slow nucleotide, which occurs by addition by base sandwiching between the RNA 3-prime end and the Pol I bridge helix. However, the presence of a templating abasic site induces opening of the Pol I cleft for either enzyme dissociation from DNA or for access of A12-Ct into the active site to stimulate RNA cleavage. Nucleotide addition opposite the lesion induces an early translocation intermediate that is different from previously-described RNA polymerase paused states, as DNA bases in the hybrid tilt to form hydrogen bonds with the newly-added RNA base. While in this state nucleotide addition is strongly disfavoured, intrinsic Pol I RNA cleavage activity acts as a failsafe route to minimize lesion bypass. Our results uncover a two-step mechanism leading to persistent Pol I stalling after nucleotide addition opposite Ap sites, which is distinct from arrest by CPD lesions and from Pol II blockage at Ap sites.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"98 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142212807","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The landscape of Arabidopsis tRNA aminoacylation","authors":"Luis F Ceriotti, Jessica M Warren, M. Virginia Sanchez-Puerta, Daniel B Sloan","doi":"10.1101/2024.09.09.612099","DOIUrl":"https://doi.org/10.1101/2024.09.09.612099","url":null,"abstract":"The function of tRNAs depends on enzymes that cleave primary transcript ends, add a 3' CCA tail, introduce post-transcriptional base modifications, and charge (aminoacylate) mature tRNAs with the correct amino acid. Maintaining an available pool of the resulting aminoacylated tRNAs is essential for protein synthesis. High-throughput sequencing techniques have recently been developed to provide a comprehensive view of aminoacylation state in a tRNA-specific fashion. However, these methods have never been applied to plants. Here, we treated Arabidopsis thaliana RNA samples with periodate and then performed tRNA-seq to distinguish between aminoacylated and uncharged tRNAs. This approach successfully captured every tRNA isodecoder family and detected expression of additional tRNA-like transcripts. We found that estimated aminoacylation rates and CCA tail integrity were significantly higher on average for organellar (mitochondrial and plastid) tRNAs than for nuclear/cytosolic tRNAs. Reanalysis of previously published human cell line data showed a similar pattern. Base modifications result in nucleotide misincorporations and truncations during reverse transcription, which we quantified and used to test for relationships with aminoacylation levels. We also determined that the Arabidopsis tRNA-like sequences (t-elements) that are cleaved from the ends of some mitochondrial mRNAs have post-transcriptionally modified bases and CCA-tail addition. However, these t-elements are not aminoacylated, indicating that they are only recognized by a subset of tRNA-interacting enzymes and do not play a role in translation. Overall, this work provides a characterization of the baseline landscape of plant tRNA aminoacylation rates and demonstrates an approach for investigating environmental and genetic perturbations to plant translation machinery.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"178 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142212810","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Leveraging CRISPR-Cas13d in an inducible knockdown system to interrogate Drosophila germ granule mRNAs","authors":"Zoya A. Gauhar, Cameron Myhrvold, Elizabeth R. Gavis","doi":"10.1101/2024.09.09.611872","DOIUrl":"https://doi.org/10.1101/2024.09.09.611872","url":null,"abstract":"Ribonucleoprotein (RNP) germ granules are hallmarks of germ cells across the animal kingdom and are thought to be hubs for post-transcriptional regulation that promote formation of the germ cell precursors. While numerous RNAs are associated with germ granules in Drosophila, the functions of many in germline development are poorly understood. Current methods for RNA knockdown, such as RNAi, do not allow local depletion of transcripts such as those found in the germ granules. We leveraged CRISPR-Cas13 to create a subcellular RNA knockdown system and tested it on two mRNAs, nanos (nos) and sarah (sra), whose abundance in germ granules differs. Because Cas13 has both cis and trans cleavage activities, we evaluated the effect of target abundance on off-target RNA depletion. We show on and off-target RNA depletion is coupled when targeting the more abundant nos germ granule transcripts. Off-target RNA knockdown is less potent when the system is used for less abundant sra transcripts. When sra is knocked down in germ granules, we observe defective primordial germ cell migration, and an increase in the calcium indicator GCaMP at the posterior, consistent with sra encoding a negative regulator of calcium signaling. In sum, we report an in vivo Cas13-based system for subcellular knockdown, evaluate its feasibility, and uncover a novel function for sra germ granule transcripts in promoting germline development.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"21 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142212808","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"SIRT6 loss causes intervertebral disc degeneration in mice by promoting senescence and SASP status","authors":"Pranay Ramteke, Bahiyah Watson, Mallory Toci, Victoria Tran, Shira Johnston, Maria Tsingas, Ruteja Barve, Ramkrishna Mitra, Richard Loeser, John Collins, Makarand Risbud","doi":"10.1101/2024.09.09.612072","DOIUrl":"https://doi.org/10.1101/2024.09.09.612072","url":null,"abstract":"Intervertebral disc degeneration is a major risk factor contributing to chronic low back and neck pain. While the etiological factors for disc degeneration vary, age is still one of the most important risk factors. Recent studies have shown the promising role of SIRT6 in mammalian aging and skeletal tissue health, however its role in the intervertebral disc health remains unexplored. We investigated the contribution of SIRT6 to disc health by studying the age-dependent spinal phenotype of mice with conditional deletion of Sirt6 in the disc (AcanCreERT2; Sirt6fl/fl). Histological studies showed a degenerative phenotype in knockout mice compared to Sirt6fl/fl control mice at 12 months which became pronounced at 24 months. RNA-Seq analysis of NP and AF tissues, quantitative histone analysis, and in vitro multiomics employing RNA-seq with ATAC-seq revealed that SIRT6-loss resulted in changes in acetylation and methylation status of specific Histone 3 lysine residues, thereby affecting DNA accessibility and transcriptomic landscape. A decrease in autophagy and an increase in DNA damage were also noted in Sirt6-deficient cells. Further mechanistic insights revealed that loss of SIRT6 increased senescence and SASP burden in the disc characterized by increased p21, yH2AX, IL-6, and TGF-b; abundance. Taken together our study highlights the contribution of SIRT6 in modulating DNA damage, autophagy and cell senescence, and its importance in maintaining disc health during aging thereby underscoring it as a potential therapeutic target to treat intervertebral disc degeneration.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"3 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142212809","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"High-quality peptide evidence for annotating non-canonical open reading frames as human proteins","authors":"Eric W Deutsch, Leron W Kok, Jonathan M Mudge, Jorge Ruiz-Orera, Ivo Fierro-Monti, Zhi Sun, Jennifer G Abelin, M Mar Alba, Julie L Aspden, Ariel A Bazzini, Elspeth Bruford, Marie A Brunet, Lorenzo Calviello, Steven A Carr, Anne-Ruxandra Carvunis, Sonia Chothani, Jim Clauwaert, Kellie Dean, Pouya Faridi, Adam Frankish, Norbert Hubner, Nicholas Ingolia, Michele Magrane, Maria Jesus Martin, Thomas F Martinez, Gerben Menschaert, Uwe Ohler, Sandra Orchard, Owen Rackham, Xavier Roucou, Sarah A Slavoff, Eivind Valen, Aaron C Wacholder, Jonathan S. Weissman, Wei Wu, Zhi Xie, Jyoti Choudhary, Michal Bassani-Sternberg, Juan Antonio Vizcaino, Nicola Ternette, Robert L. Moritz, John Prensner, Sebastiaan van Heesch","doi":"10.1101/2024.09.09.612016","DOIUrl":"https://doi.org/10.1101/2024.09.09.612016","url":null,"abstract":"A major scientific drive is to characterize the protein-coding genome as it provides the primary basis for the study of human health. But the fundamental question remains: what has been missed in prior genomic analyses? Over the past decade, the translation of non-canonical open reading frames (ncORFs) has been observed across human cell types and disease states, with major implications for proteomics, genomics, and clinical science. However, the impact of ncORFs has been limited by the absence of a large-scale understanding of their contribution to the human proteome. Here, we report the collaborative efforts of stakeholders in proteomics, immunopeptidomics, Ribo-seq ORF discovery, and gene annotation, to produce a consensus landscape of protein-level evidence for ncORFs. We show that at least 25% of a set of 7,264 ncORFs give rise to translated gene products, yielding over 3,000 peptides in a pan-proteome analysis encompassing 3.8 billion mass spectra from 95,520 experiments. With these data, we developed an annotation framework for ncORFs and created public tools for researchers through GENCODE and PeptideAtlas. This work will provide a platform to advance ncORF-derived proteins in biomedical discovery and, beyond humans, diverse animals and plants where ncORFs are similarly observed.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"39 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142212811","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Topology-Driven Discovery of Transmembrane Protein S-Palmitoylation","authors":"Michael T Forrester, Jacob R. Egol, Sinan Ozbay, Rohit Singh, Purushothama Rao Tata","doi":"10.1101/2024.09.08.611865","DOIUrl":"https://doi.org/10.1101/2024.09.08.611865","url":null,"abstract":"Protein <em>S</em>-palmitoylation is a reversible lipophilic posttranslational modification regulating a diverse number of signaling pathways. Within transmembrane proteins (TMPs), <em>S</em>-palmitoylation is implicated in conditions from inflammatory disorders to respiratory viral infections. Many small-scale experiments have observed <em>S</em>-palmitoylation at juxtamembrane Cys residues. However, most large-scale <em>S</em>-palmitoyl discovery efforts rely on trypsin-based proteomics within which hydrophobic juxtamembrane regions are likely underrepresented. Machine learning, by virtue of its freedom from experimental constraints, is particularly well suited to address this discovery gap surrounding TMP <em>S</em>-palmitoylation. Utilizing a UniProt-derived feature set, a gradient boosted machine learning tool (TopoPalmTree) was constructed and applied to a holdout dataset of viral <em>S</em>-palmitoylated proteins. Upon application to the mouse TMP proteome, 1591 putative <em>S</em>-palmitoyl sites (i.e. not listed in SwissPalm or UniProt) were identified. Two lung-expressed <em>S</em>-palmitoyl candidates (synaptobrevin Vamp5 and water channel Aquaporin-5) were experimentally assessed. Finally, TopoPalmTree was used for rational design of an <em>S</em>-palmitoyl site on KDEL-Receptor 2. This readily interpretable model aligns the innumerable small-scale experiments observing juxtamembrane <em>S</em>-palmitoylation into a proteomic tool for TMP <em>S</em>-palmitoyl discovery and design, thus facilitating future investigations of this important modification.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"7 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142212815","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Investigation of risk factors associated with Ancylostoma spp. infection and the benzimidazole F167Y resistance marker polymorphism in dogs from the United States","authors":"Pablo David Jimenez Castro, Christian Leutenegger, Christian Savard, Cecilia Lozoya, Holly Brown, Jennifer Willcox, Haresh Rochani, Heather Martinez","doi":"10.1101/2024.09.08.611871","DOIUrl":"https://doi.org/10.1101/2024.09.08.611871","url":null,"abstract":"Ancylostoma caninum is the most significant intestinal nematode parasite of dogs. We acquired fecal surveillance data using molecular diagnostics in a large population of dogs in the United States (US). A diagnostic test using real-time PCR (qPCR) for Ancylostoma spp. and allele-specific qPCR detecting the SNP F167Y was used in 885,424 canine fecal samples collected between March 2022 and December 2023. Overall, Ancylostoma spp. had a prevalence of 1.76% (15,537/885,424), with the highest observed in the South 3.73% (10,747/287,576), and the lowest in the West 0.45% (632/140,282). Within the subset of Ancylostoma spp.-detected dogs used for further analysis, the F167Y SNP had an overall prevalence of 14.2% with the highest in the West and the lowest in the Midwest (10.76%). The greyhound breed exhibited a higher prevalence of Ancylostoma spp. infections (17.03%) and a higher prevalence of the F167Y polymorphism (33.6%) compared to non-greyhound breeds (13.7% and 2.08%), respectively, but were not associated with the highest breed risk for the F167Y polymorphism. Sex did not influence hookworm infection nor F167Y polymorphism prevalence. Intact dogs had a prevalence of hookworm infection and F167Y polymorphism of 2.51% and 14.6%, respectively. Puppies showed increased prevalence of hookworms (3.70%) and the F167Y SNP (17.1%). Greyhounds, bluetick coonhounds, and boerboels had the highest relative risks (RR) for hookworm infection, while Cavalier King Charles spaniels, Havanese, and shiba inus had the lowest. The top and bottom three with the highest and lowest RR for the F167Y SNP were the old English sheepdog, American foxhound, and toy poodle Toy, and shih tzu, Maltese, and Australian cattle dogs, respectively. This study highlights the value of an accessible diagnostic qPCR test with fast turnaround times in unraveling the molecular epidemiology of hookworms and benzimidazole resistance, as well as explore potentially important risk factors associated with infection in medicalized dogs.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"3 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142212812","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Protein degradation by small tag artificial bacterial E3 ligase","authors":"Soren Warming, Zhenyi Liu, Ming-Chi Tsai, Soumitra Ghosh, Jessica Lawrence, Sarah Chu, Baris Bingol","doi":"10.1101/2024.09.07.611797","DOIUrl":"https://doi.org/10.1101/2024.09.07.611797","url":null,"abstract":"Targeting of proteins for degradation in a reversible manner is a powerful approach to decipher gene function and mimic drug effects, with great potential for drug target discovery and validation. A generalized approach is to tag a protein of interest and then use this tag to recruit an endogenously or exogenously expressed E3 ligase for its polyubiquitination and subsequent degradation via 26S proteasome. However, the often bulky size of the tag and the great variability of substrate-dependent degradation efficiency of mammalian E3 ligases pose great challenges in practice. Here we show that small tags (10-15 amino acids) can be used to efficiently tag endogenous proteins for degradation when coupled with an exogenously expressed artificial bacterial E3 ligase (ABEL) consisting of a tag-interacting moiety and the catalytic domain of the bacterial E3 ligase IpaH9.8. We name this versatile and efficient platform degradation by small tag ABEL (DESTABEL). Furthermore, we show that an ABEL containing a nanobody against human α-synuclein mediates efficient degradation in primary neurons as well as in the adult mouse brain. Taken together, our data show that tag-dependent and independent ABELs are powerful yet flexible tools for studies of protein function and drug target validation.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"273 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142212813","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Optimizing Digital PCR Assays for Multiplexed Microbial Source Tracking in Surface Waters","authors":"Shimul Ghosh, Aaron W Bivins","doi":"10.1101/2024.09.07.611803","DOIUrl":"https://doi.org/10.1101/2024.09.07.611803","url":null,"abstract":"Digital PCR (dPCR) shows great promise for precise, sensitive, and inhibition-resilient measurement of nucleic acids in surface water, providing an advantage for applications such as microbial source tracking (MST). Herein, we describe our empirical optimization of two triplex format dPCR reactions (Triplex 1 - Cow M2, Rum2Bac, Cow M3; Triplex 2 - HF183/BacR287, Pig2Bac, Entero1a) on the QIAcuity One system for MST. Each triplex produced gene copy measurements similar to single-plex format assays for a standard reference material (SRM 2917) at a fixed concentration and along a concentration gradient. For achieved water samples previously tested by single-plex assays, each triplex also produced MST marker measurements comparable to the single-plex results. The triplexes described here can be directly adopted for MST on the QIAcuity, or the optimization protocol we demonstrate can be used to develop additional multiplex assays on the QIAcuity system.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"6 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142212814","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Modulating NLRP3 splicing with antisense oligonucleotides to control pathological inflammation","authors":"Roni Klein, Janset Onyuru, Estela M. Viera, Christopher D. Putnam, Hal M. Hoffman, Michelle L. Hastings","doi":"10.1101/2024.09.06.611206","DOIUrl":"https://doi.org/10.1101/2024.09.06.611206","url":null,"abstract":"Inflammation has an essential role in healing. However, over-active inflammation disrupts normal cellular functions and can be life-threatening when not resolved. The NLRP3 inflammasome, a component of the innate immune system, is an intracellular multiprotein complex that senses stress-associated signals, and, for this reason is a promising therapeutic target for treating unresolved, pathogenic inflammation. Alternative splicing of NLRP3 RNA has been suggested as a regulatory mechanism for inflammasome activation, as some spliced isoforms encode NLRP3 proteins with compromised function. Here, we take advantage of this natural regulatory mechanism and devise a way to control pathogenic inflammation using splice-switching antisense oligonucleotides (ASOs). To identify and induce NLRP3 spliced isoforms lacking inflammatory activity, we tested a series of ASOs, each targeting a different exon, to determine the most effective strategy for down-regulating NLRP3. We identify several ASOs that modulate NLRP3 splicing, reduce NLRP3 protein, and decrease inflammasome signaling in vitro. The most effective ASO suppresses systemic inflammation in vivo in mouse models of acute inflammation and cryopyrin-associated periodic syndrome (CAPS). Our results demonstrate a systematic approach to protein engineering using splice-switching ASOs to generate isoforms with altered activity, and identify an ASO that can treat pathological inflammation in mice by reducing functional NLRP3.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"52 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142212665","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}