G3: Genes|Genomes|Genetics最新文献

{"title":"Characterization and trans-generation dynamics of mitogene pool in the silver carp (Hypophthalmichthys molitrix).","authors":"Jinlin Li, Hengshu Wu, Yingna Zhou, Manhong Liu, Yongheng Zhou, Jianing Chu, Elizabeth Kamili, Wenhui Wang, Jincheng Yang, Lijun Lin, Qi Zhang, Shuhui Yang, Yanchun Xu","doi":"10.1093/g3journal/jkae101","DOIUrl":"10.1093/g3journal/jkae101","url":null,"abstract":"<p><p>Multicopied mitogenome are prone to mutation during replication often resulting in heteroplasmy. The derived variants in a cell, organ, or an individual animal constitute a mitogene pool. The individual mitogene pool is initiated by a small fraction of the egg mitogene pool. However, the characteristics and relationship between them has not yet been investigated. This study quantitatively analyzed the heteroplasmy landscape, genetic loads, and selection strength of the mitogene pool of egg and hatchling in the silver carp (Hypophthalmichthys molitrix) using high-throughput resequencing. The results showed heteroplasmic sites distribute across the whole mitogenome in both eggs and hatchlings. The dominant substitution was Transversion in eggs and Transition in hatching accounting for 95.23%±2.07% and 85.38%±6.94% of total HP sites, respectively. The total genetic loads were 0.293±0.044 in eggs and 0.228±0.022 in hatchlings (P=0.048). The dN/dS ratio was 58.03±38.98 for eggs and 9.44±3.93 for hatchlings (P=0.037). These results suggest that the mitogenomes were under strong positive selection in eggs with tolerance to variants with deleterious effects, while the selection was positive but much weaker in hatchlings showing marked quality control. Based on these findings, we proposed a trans-generation dynamics model to explain differential development mode of the two mitogene pool between oocyte maturation and ontogenesis of offspring. This study sheds light on significance of mitogene pool for persistence of populations and subsequent integration in ecological studies and conservation practices.</p>","PeriodicalId":12468,"journal":{"name":"G3: Genes|Genomes|Genetics","volume":null,"pages":null},"PeriodicalIF":2.1,"publicationDate":"2024-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11491513/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141450236","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Modeling the evolution of Schizosaccharomyces pombe populations with multiple killer meiotic drivers.","authors":"José Fabricio López Hernández, Boris Y Rubinstein, Robert L Unckless, Sarah E Zanders","doi":"10.1093/g3journal/jkae142","DOIUrl":"10.1093/g3journal/jkae142","url":null,"abstract":"<p><p>Meiotic drivers are selfish genetic loci that can be transmitted to more than half of the viable gametes produced by a heterozygote. This biased transmission gives meiotic drivers an evolutionary advantage that can allow them to spread over generations until all members of a population carry the driver. This evolutionary power can also be exploited to modify natural populations using synthetic drivers known as \"gene drives.\" Recently, it has become clear that natural drivers can spread within genomes to birth multicopy gene families. To understand intragenomic spread of drivers, we model the evolution of 2 or more distinct meiotic drivers in a population. We employ the wtf killer meiotic drivers from Schizosaccharomyces pombe, which are multicopy in all sequenced isolates, as models. We find that a duplicate wtf driver identical to the parent gene can spread in a population unless, or until, the original driver is fixed. When the duplicate driver diverges to be distinct from the parent gene, we find that both drivers spread to fixation under most conditions, but both drivers can be lost under some conditions. Finally, we show that stronger drivers make weaker drivers go extinct in most, but not all, polymorphic populations with absolutely linked drivers. These results reveal the strong potential for natural meiotic drive loci to duplicate and diverge within genomes. Our findings also highlight duplication potential as a factor to consider in the design of synthetic gene drives.</p>","PeriodicalId":12468,"journal":{"name":"G3: Genes|Genomes|Genetics","volume":null,"pages":null},"PeriodicalIF":2.1,"publicationDate":"2024-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11491527/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141467397","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Pathogen infection alters the gene expression landscape of transposable elements in Drosophila melanogaster.","authors":"Sabrina L Mostoufi, Nadia D Singh","doi":"10.1093/g3journal/jkae171","DOIUrl":"10.1093/g3journal/jkae171","url":null,"abstract":"<p><p>Transposable elements make up substantial proportions of eukaryotic genomes and many are thought to be remnants of ancient viral infections. Current research has begun to highlight the role transposable elements can play in the immune system response to infections. However, most of our knowledge about transposable element expression during infection is limited by the specific host and pathogen factors from each study, making it difficult to compare studies and develop broader patterns regarding the role of transposable elements during infection. Here, we use the tools and resources available in the model, Drosophila melanogaster, to analyze multiple gene expression datasets of flies subject to bacterial, fungal, and viral infections. We analyzed differences in pathogen species, host genotype, host tissue, and sex to understand how these factors impact transposable element expression during infection. Our results highlight both shared and unique transposable element expression patterns between pathogens and suggest a larger effect of pathogen factors over host factors for influencing transposable element expression.</p>","PeriodicalId":12468,"journal":{"name":"G3: Genes|Genomes|Genetics","volume":null,"pages":null},"PeriodicalIF":2.1,"publicationDate":"2024-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11373657/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141916534","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A genetic screen of transcription factors in the Drosophila melanogaster abdomen identifies novel pigmentation genes.","authors":"Sarah J Petrosky, Thomas M Williams, Mark Rebeiz","doi":"10.1093/g3journal/jkae097","DOIUrl":"10.1093/g3journal/jkae097","url":null,"abstract":"<p><p>Gene regulatory networks specify the gene expression patterns needed for traits to develop. Differences in these networks can result in phenotypic differences between organisms. Although loss-of-function genetic screens can identify genes necessary for trait formation, gain-of-function screens can overcome genetic redundancy and identify loci whose expression is sufficient to alter trait formation. Here, we leveraged transgenic lines from the Transgenic RNAi Project at Harvard Medical School to perform both gain- and loss-of-function CRISPR/Cas9 screens for abdominal pigmentation phenotypes. We identified measurable effects on pigmentation patterns in the Drosophila melanogaster abdomen for 21 of 55 transcription factors in gain-of-function experiments and 7 of 16 tested by loss-of-function experiments. These included well-characterized pigmentation genes, such as bab1 and dsx, and transcription factors that had no known role in pigmentation, such as slp2. Finally, this screen was partially conducted by undergraduate students in a Genetics Laboratory course during the spring semesters of 2021 and 2022. We found this screen to be a successful model for student engagement in research in an undergraduate laboratory course that can be readily adapted to evaluate the effect of hundreds of genes on many different Drosophila traits, with minimal resources.</p>","PeriodicalId":12468,"journal":{"name":"G3: Genes|Genomes|Genetics","volume":null,"pages":null},"PeriodicalIF":2.1,"publicationDate":"2024-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11373662/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141183981","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A chromosome-level reference genome assembly and a full-length transcriptome assembly of the giant freshwater prawn (Macrobrachium rosenbergii).","authors":"Wirulda Pootakham, Kanchana Sittikankaew, Chutima Sonthirod, Chaiwat Naktang, Tanaporn Uengwetwanit, Wasitthee Kongkachana, Kongphop Ampolsak, Nitsara Karoonuthaisiri","doi":"10.1093/g3journal/jkae146","DOIUrl":"10.1093/g3journal/jkae146","url":null,"abstract":"<p><p>The giant freshwater prawn (Macrobrachium rosenbergii) is a key species in the aquaculture industry in several Asian, African, and South American countries. Despite a considerable growth in its production worldwide, the genetic complexities of M. rosenbergii various morphotypes pose challenges in cultivation. This study reports the first chromosome-scale reference genome and a high-quality full-length transcriptome assembly for M. rosenbergii. We employed the PacBio High Fidelity (HiFi) sequencing to obtain an initial draft assembly and further scaffolded it with the chromatin contact mapping (Hi-C) technique to achieve a final assembly of 3.73-Gb with an N50 scaffold length of 33.6 Mb. Repetitive elements constituted nearly 60% of the genome assembly, with simple sequence repeats and retrotransposons being the most abundant. The availability of both the chromosome-scale assembly and the full-length transcriptome assembly enabled us to thoroughly probe alternative splicing events in M. rosenbergii. Among the 2,041 events investigated, exon skipping represented the most prevalent class, followed by intron retention. Interestingly, specific isoforms were observed across multiple tissues. Additionally, within a single tissue type, transcripts could undergo alternative splicing, yielding multiple isoforms. We believe that the availability of a chromosome-level reference genome for M. rosenbergii, along with its full-length transcriptome, will be instrumental in advancing our understanding of the giant freshwater prawn biology and enhancing its molecular breeding programs, paving the way for the development of M. rosenbergii with valuable traits in commercial aquaculture.</p>","PeriodicalId":12468,"journal":{"name":"G3: Genes|Genomes|Genetics","volume":null,"pages":null},"PeriodicalIF":2.1,"publicationDate":"2024-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11373640/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141554541","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Experimental evolution of Saccharomyces cerevisiae for caffeine tolerance alters multidrug resistance and target of rapamycin signaling pathways.","authors":"Renee C Geck, Naomi G Moresi, Leah M Anderson, Rebecca Brewer, Timothy R Renz, Matthew Bryce Taylor, Maitreya J Dunham","doi":"10.1093/g3journal/jkae148","DOIUrl":"10.1093/g3journal/jkae148","url":null,"abstract":"<p><p>Caffeine is a natural compound that inhibits the major cellular signaling regulator target of rapamycin (TOR), leading to widespread effects including growth inhibition. Saccharomyces cerevisiae yeast can adapt to tolerate high concentrations of caffeine in coffee and cacao fermentations and in experimental systems. While many factors affecting caffeine tolerance and TOR signaling have been identified, further characterization of their interactions and regulation remain to be studied. We used experimental evolution of S. cerevisiae to study the genetic contributions to caffeine tolerance in yeast, through a collaboration between high school students evolving yeast populations coupled with further research exploration in university labs. We identified multiple evolved yeast populations with mutations in PDR1 and PDR5, which contribute to multidrug resistance, and showed that gain-of-function mutations in multidrug resistance family transcription factors Pdr1, Pdr3, and Yrr1 differentially contribute to caffeine tolerance. We also identified loss-of-function mutations in TOR effectors Sit4, Sky1, and Tip41 and showed that these mutations contribute to caffeine tolerance. These findings support the importance of both the multidrug resistance family and TOR signaling in caffeine tolerance and can inform future exploration of networks affected by caffeine and other TOR inhibitors in model systems and industrial applications.</p>","PeriodicalId":12468,"journal":{"name":"G3: Genes|Genomes|Genetics","volume":null,"pages":null},"PeriodicalIF":2.1,"publicationDate":"2024-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11373655/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141579432","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A genetic screen in Drosophila uncovers a role for senseless-2 in surface glia in the peripheral nervous system to regulate CNS morphology.","authors":"Haluk Lacin, Yuqing Zhu, Jose T DiPaola, Beth A Wilson, Yi Zhu, James B Skeath","doi":"10.1093/g3journal/jkae152","DOIUrl":"10.1093/g3journal/jkae152","url":null,"abstract":"<p><p>Despite increasing in mass approximately 100-fold during larval life, the Drosophila CNS maintains its characteristic form. Dynamic interactions between the overlying basement membrane and underlying surface glia are known to regulate CNS structure in Drosophila, but the genes and pathways that establish and maintain CNS morphology during development remain poorly characterized. To identify genes that regulate CNS shape in Drosophila, we conducted an EMS-based, forward genetic screen of the second chromosome, uncovered 50 mutations that disrupt CNS structure, and mapped these alleles to 17 genes. Analysis of whole genome sequencing data wedded to genetic studies uncovered the affected gene for all but 1 mutation. Identified genes include well-characterized regulators of tissue shape, like LanB1, viking, and Collagen type IV alpha1, and previously characterized genes, such as Toll-2 and Rme-8, with no known role in regulating CNS structure. We also uncovered that papilin and C1GalTA likely act in the same pathway to regulate CNS structure and found that the fly homolog of a glucuronosyltransferase, B4GAT1/LARGE1, that regulates Dystroglycan function in mammals is required to maintain CNS shape in Drosophila. Finally, we show that the senseless-2 transcription factor is expressed and functions specifically in surface glia found on peripheral nerves but not in the CNS to govern CNS structure, identifying a gene that functionally subdivides a glial subtype along the peripheral-central axis. Future work on these genes should clarify the genetic mechanisms that ensure the homeostasis of CNS form during development.</p>","PeriodicalId":12468,"journal":{"name":"G3: Genes|Genomes|Genetics","volume":null,"pages":null},"PeriodicalIF":2.1,"publicationDate":"2024-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11373656/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141599086","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The Psathyrostachys juncea DWARF27 gene encodes an all-trans-/9-cis-beta-carotene isomerase in the control of plant branches in Arabidopsis thaliana by strigolactones.","authors":"Xiaomin Ren, Qian Ai, Zhen Li, Qiao Zhao, Lan Yun","doi":"10.1093/g3journal/jkae147","DOIUrl":"10.1093/g3journal/jkae147","url":null,"abstract":"<p><p>Strigolactones (SLs), carotenoid-derived plant hormones, govern the growth and development of both monocotyledonous and dicotyledonous plants. DWARF27 (D27), a plastid-targeted protein located at the initiation site of the core pathway in SL synthesis, plays a crucial role in regulating plant tillering (branching). In rice (Oryza sativa) and wheat (Triticum aestivum), OsD27 and TaD27-B proteins modulate the number of plant tillers by participating in SL biosynthesis. Similarly, AtD27 in Arabidopsis thaliana is required for SL production and has a significant impact on phenotypic changes related to branching. At the same time, TaD27 in wheat has been confirmed as a functional orthologue of AtD27 in Arabidopsis, and both Psathyrostachys juncea and wheat belong to the Triticeae, so we speculate that PjD27 gene may also have the same function as AtD27 in Arabidopsis. In this study, we initially screened the PjD27 gene significantly associated with tillering regulation through transcriptome data analysis and subsequently validated its expression levels using qRT-PCR analysis. Furthermore, we conducted phylogenetic analysis using amino acid sequences from 41 species, including P. juncea, to identify closely related species of P. juncea. Here, we analyze the conservation of D27 protein among P. juncea, rice, wheat, and Arabidopsis and provide preliminary evidence suggesting that PjD27 protein is an orthologue of D27 protein in Arabidopsis. Through reverse genetics, we demonstrate the crucial role of PjD27 in regulating plant branching, establishing it as a functional orthologue of D27 in Arabidopsis. Furthermore, following transient expression in tobacco (Nicotiana tabacum), we demonstrate that the subcellular location of the PjD27 protein is consistent with the cellular location of TaD27-B in wheat. Quantitative analysis of SLs shows that PjD27 is a key gene regulating tillering (branching) by participating in SL biosynthesis. By elucidating the function of the PjD27 gene, our findings provide valuable genetic resources for new germplasm creation and improving grain yield in P. juncea.</p>","PeriodicalId":12468,"journal":{"name":"G3: Genes|Genomes|Genetics","volume":null,"pages":null},"PeriodicalIF":2.1,"publicationDate":"2024-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11373637/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141619801","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Impact of tRNA-induced proline-to-serine mistranslation on the transcriptome of Drosophila melanogaster.","authors":"Joshua R Isaacson, Matthew D Berg, William Yeung, Judit Villén, Christopher J Brandl, Amanda J Moehring","doi":"10.1093/g3journal/jkae151","DOIUrl":"10.1093/g3journal/jkae151","url":null,"abstract":"<p><p>Mistranslation is the misincorporation of an amino acid into a polypeptide. Mistranslation has diverse effects on multicellular eukaryotes and is implicated in several human diseases. In Drosophila melanogaster, a serine transfer RNA (tRNA) that misincorporates serine at proline codons (P→S) affects male and female flies differently. The mechanisms behind this discrepancy are currently unknown. Here, we compare the transcriptional response of male and female flies to P→S mistranslation to identify genes and cellular processes that underlie sex-specific differences. Both males and females downregulate genes associated with various metabolic processes in response to P→S mistranslation. Males downregulate genes associated with extracellular matrix organization and response to negative stimuli such as wounding, whereas females downregulate aerobic respiration and ATP synthesis genes. Both sexes upregulate genes associated with gametogenesis, but females also upregulate cell cycle and DNA repair genes. These observed differences in the transcriptional response of male and female flies to P→S mistranslation have important implications for the sex-specific impact of mistranslation on disease and tRNA therapeutics.</p>","PeriodicalId":12468,"journal":{"name":"G3: Genes|Genomes|Genetics","volume":null,"pages":null},"PeriodicalIF":2.1,"publicationDate":"2024-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11373654/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141579433","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A random mutagenesis screen enriched for missense mutations in bacterial effector proteins.","authors":"Malene L Urbanus, Thomas M Zheng, Anna N Khusnutdinova, Doreen Banh, Harley O'Connor Mount, Alind Gupta, Peter J Stogios, Alexei Savchenko, Ralph R Isberg, Alexander F Yakunin, Alexander W Ensminger","doi":"10.1093/g3journal/jkae158","DOIUrl":"10.1093/g3journal/jkae158","url":null,"abstract":"<p><p>To remodel their hosts and escape immune defenses, many pathogens rely on large arsenals of proteins (effectors) that are delivered to the host cell using dedicated translocation machinery. Effectors hold significant insight into the biology of both the pathogens that encode them and the host pathways that they manipulate. One of the most powerful systems biology tools for studying effectors is the model organism, Saccharomyces cerevisiae. For many pathogens, the heterologous expression of effectors in yeast is growth inhibitory at a frequency much higher than housekeeping genes, an observation ascribed to targeting conserved eukaryotic proteins. Abrogation of yeast growth inhibition has been used to identify bacterial suppressors of effector activity, host targets, and functional residues and domains within effector proteins. We present here a yeast-based method for enriching for informative, in-frame, missense mutations in a pool of random effector mutants. We benchmark this approach against three effectors from Legionella pneumophila, an intracellular bacterial pathogen that injects a staggering >330 effectors into the host cell. For each protein, we show how in silico protein modeling (AlphaFold2) and missense-directed mutagenesis can be combined to reveal important structural features within effectors. We identify known active site residues within the metalloprotease RavK, the putative active site in SdbB, and previously unidentified functional motifs within the C-terminal domain of SdbA. We show that this domain has structural similarity with glycosyltransferases and exhibits in vitro activity consistent with this predicted function.</p>","PeriodicalId":12468,"journal":{"name":"G3: Genes|Genomes|Genetics","volume":null,"pages":null},"PeriodicalIF":2.1,"publicationDate":"2024-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11373652/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141727047","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}