Genetics最新文献

{"title":"A single mutation G454A in the P450 CYP9K1 drives pyrethroid resistance in the major malaria vector Anopheles funestus reducing bed net efficacy.","authors":"Carlos S Djoko Tagne, Mersimine F M Kouamo, Magellan Tchouakui, Abdullahi Muhammad, Leon J L Mugenzi, Nelly M T Tatchou-Nebangwa, Riccado F Thiomela, Mahamat Gadji, Murielle J Wondji, Jack Hearn, Mbouobda H Desire, Sulaiman S Ibrahim, Charles S Wondji","doi":"10.1093/genetics/iyae181","DOIUrl":"https://doi.org/10.1093/genetics/iyae181","url":null,"abstract":"<p><p>Metabolic mechanisms conferring pyrethroid resistance in malaria vectors are jeopardizing the effectiveness of insecticide-based interventions, and identification of their markers is a key requirement for robust resistance management. Here, using a field-lab-field approach, we demonstrated that a single mutation G454A in the P450 CYP9K1 is driving pyrethroid resistance in the major malaria vector Anopheles funestus in East and Central Africa. Drastic reduction in CYP9K1 diversity was observed in Ugandan samples collected in 2014, with selection of a predominant haplotype (G454A mutation at 90%), which was completely absent in the other African regions. However, six years later (2020) the Ugandan 454A-CYP9K1 haplotype was found predominant in Cameroon (84.6%), but absent in Malawi (Southern Africa) and Ghana (West Africa). Comparative in vitro heterologous expression and metabolism assays revealed that the mutant 454A-CYP9K1 (R) allele significantly metabolises more type II pyrethroid (deltamethrin) compared with the wild G454-CYP9K1 (S) allele. Transgenic Drosophila melanogaster flies expressing 454A-CYP9K1 (R) allele exhibited significantly higher type I and II pyrethroids resistance compared to flies expressing the wild G454-CYP9K1 (S) allele. Furthermore, laboratory testing and field experimental hut trials in Cameroon demonstrated that mosquitoes harbouring the resistant 454A-CYP9K1 allele significantly survived to pyrethroids exposure (Odds ratio = 567, p < 0.0001). This study highlights the rapid spread of pyrethroid resistant CYP9K1 allele, under directional selection in East and Central Africa, contributing to reduced bed net efficacy. The newly designed DNA-based assay here will add to the toolbox of resistance monitoring and improving its management strategies.</p>","PeriodicalId":48925,"journal":{"name":"Genetics","volume":null,"pages":null},"PeriodicalIF":3.3,"publicationDate":"2024-11-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142607160","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"An explanation for the sister repulsion phenomenon in Patterson's f-statistics.","authors":"Gözde Atağ, Shamam Waldman, Shai Carmi, Mehmet Somel","doi":"10.1093/genetics/iyae144","DOIUrl":"10.1093/genetics/iyae144","url":null,"abstract":"<p><p>Patterson's f-statistics are among the most heavily utilized tools for analyzing genome-wide allele frequency data for demographic inference. Beyond studying admixture, f3- and f4-statistics are also used for clustering populations to identify groups with similar histories. However, previous studies have noted an unexpected behavior of f-statistics: multiple populations from a certain region systematically show higher genetic affinity to a more distant population than to their neighbors, a pattern that is mismatched with alternative measures of genetic similarity. We call this counter-intuitive pattern \"sister repulsion\". We first present a novel instance of sister repulsion, where genomes from Bronze Age East Anatolian sites show higher affinity toward Bronze Age Greece rather than each other. This is observed both using f3- and f4-statistics, contrasts with archaeological/historical expectation, and also contradicts genetic affinity patterns captured using principal components analysis or multidimensional scaling on genetic distances. We then propose a simple demographic model to explain this pattern, where sister populations receive gene flow from a genetically distant source. We calculate f3- and f4-statistics using simulated genetic data with varying population genetic parameters, confirming that low-level gene flow from an external source into populations from 1 region can create sister repulsion in f-statistics. Unidirectional gene flow between the studied regions (without an external source) can likewise create repulsion. Meanwhile, similar to our empirical observations, multidimensional scaling analyses of genetic distances still cluster sister populations together. Overall, our results highlight the impact of low-level admixture events when inferring demographic history using f-statistics.</p>","PeriodicalId":48925,"journal":{"name":"Genetics","volume":null,"pages":null},"PeriodicalIF":3.3,"publicationDate":"2024-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11538414/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142299277","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 genetic architecture of polygenic local adaptation and its role in shaping barriers to gene flow.","authors":"Arthur Zwaenepoel, Himani Sachdeva, Christelle Fraïsse","doi":"10.1093/genetics/iyae140","DOIUrl":"10.1093/genetics/iyae140","url":null,"abstract":"<p><p>We consider how the genetic architecture underlying locally adaptive traits determines the strength of a barrier to gene flow in a mainland-island model. Assuming a general life cycle, we derive an expression for the effective migration rate when local adaptation is due to genetic variation at many loci under directional selection on the island, allowing for arbitrary fitness and dominance effects across loci. We show how the effective migration rate can be combined with classical single-locus diffusion theory to accurately predict multilocus differentiation between the mainland and island at migration-selection-drift equilibrium and determine the migration rate beyond which local adaptation collapses, while accounting for genetic drift and weak linkage. Using our efficient numerical tools, we then present a detailed study of the effects of dominance on barriers to gene flow, showing that when total selection is sufficiently strong, more recessive local adaptation generates stronger barriers to gene flow. We then study how heterogeneous genetic architectures of local adaptation affect barriers to gene flow, characterizing adaptive differentiation at migration-selection balance for different distributions of fitness effects. We find that a more heterogeneous genetic architecture generally yields a stronger genome-wide barrier to gene flow and that the detailed genetic architecture underlying locally adaptive traits can have an important effect on observable differentiation when divergence is not too large. Lastly, we study the limits of our approach as loci become more tightly linked, showing that our predictions remain accurate over a large biologically relevant domain.</p>","PeriodicalId":48925,"journal":{"name":"Genetics","volume":null,"pages":null},"PeriodicalIF":3.3,"publicationDate":"2024-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11538419/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142019206","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 Raf/LIN-45 C-terminal distal tail segment negatively regulates signaling in Caenorhabditis elegans.","authors":"Robert A Townley, Kennedy S Stacy, Fatemeh Cheraghi, Claire C de la Cova","doi":"10.1093/genetics/iyae152","DOIUrl":"10.1093/genetics/iyae152","url":null,"abstract":"<p><p>Raf protein kinases act as Ras-GTP sensing components of the ERK signal transduction pathway in animal cells, influencing cell proliferation, differentiation, and survival. In humans, somatic and germline mutations in the genes BRAF and RAF1 are associated with malignancies and developmental disorders. Recent studies shed light on the structure of activated Raf, a heterotetramer consisting of Raf and 14-3-3 dimers, and raised the possibility that a Raf C-terminal distal tail segment (DTS) regulates activation. We investigated the role of the DTS using the Caenorhabditis elegans Raf ortholog lin-45. Truncations removing the DTS strongly enhanced lin-45(S312A), a weak gain-of-function allele equivalent to RAF1 mutations found in patients with Noonan Syndrome. We genetically defined three elements of the LIN-45 DTS, which we termed the active site binding sequence (ASBS), the KTP motif, and the aromatic cluster. In the context of lin-45(S312A), the mutation of each of these elements enhanced activity. We used AlphaFold to predict DTS protein interactions for LIN-45, fly Raf, and human BRAF within the activated heterotetramer complex. We propose the following distinct functions for the LIN-45 DTS elements: (1) the ASBS binds the kinase active site as an inhibitor; (2) phosphorylation of the KTP motif modulates the DTS-kinase domain interaction; and (3) the aromatic cluster anchors the DTS in an inhibitory conformation. Human RASopathy-associated variants in BRAF affect residues of the DTS, consistent with these predictions. This work establishes that the Raf/LIN-45 DTS negatively regulates signaling in C. elegans and provides a model for its function in other Raf proteins.</p>","PeriodicalId":48925,"journal":{"name":"Genetics","volume":null,"pages":null},"PeriodicalIF":3.3,"publicationDate":"2024-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11538406/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142299279","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":"SKN-1 activation during infection of Caenorhabditis elegans requires CDC-48 and endoplasmic reticulum proteostasis.","authors":"Carolaing Gabaldón, Ozgur Karakuzu, Danielle A Garsin","doi":"10.1093/genetics/iyae131","DOIUrl":"10.1093/genetics/iyae131","url":null,"abstract":"<p><p>During challenge of Caenorhabditis elegans with human bacterial pathogens such as Pseudomonas aeruginosa and Enterococcus faecalis, the elicited host response can be damaging if not properly controlled. The activation of Nrf (nuclear factor erythroid-related factor)/CNC (Cap-n-collar) transcriptional regulators modulates the response by upregulating genes that neutralize damaging molecules and promote repair processes. Activation of the C. elegans Nrf ortholog, SKN-1, is tightly controlled by a myriad of regulatory mechanisms, but a central feature is an activating phosphorylation accomplished by the p38 mitogen-activated kinase (MAPK) cascade. In this work, loss of CDC-48, an AAA+ ATPase, was observed to severely compromise SKN-1 activation on pathogen and we sought to understand the mechanism. CDC-48 is part of the endoplasmic reticulum (ER)-associated degradation (ERAD) complex where it functions as a remodeling chaperone enabling the translocation of proteins from the ER to the cytoplasm for degradation by the proteosome. Interestingly, one of the proteins retrotranslocated by ERAD, a process necessary for its activation, is SKN-1A, the ER isoform of SKN-1. However, we discovered that SKN-1A is not activated by pathogen exposure in marked contrast to the cytoplasmic-associated isoform SKN-1C. Rather, loss of CDC-48 blocks the antioxidant response normally orchestrated by SKN-1C by strongly inducing the unfolded protein response (UPRER). The data are consistent with the model of these 2 pathways being mutually inhibitory and support the emerging paradigm in the field of coordinated cooperation between different stress responses.</p>","PeriodicalId":48925,"journal":{"name":"Genetics","volume":null,"pages":null},"PeriodicalIF":3.3,"publicationDate":"2024-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11538416/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142019204","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":"Deep mutational scanning of CYP2C19 in human cells reveals a substrate specificity-abundance tradeoff.","authors":"Gabriel E Boyle, Katherine A Sitko, Jared G Galloway, Hugh K Haddox, Aisha Haley Bianchi, Ajeya Dixon, Melinda K Wheelock, Allyssa J Vandi, Ziyu R Wang, Raine E S Thomson, Riddhiman K Garge, Allan E Rettie, Alan F Rubin, Renee C Geck, Elizabeth M J Gillam, William S DeWitt, Frederick A Matsen, Douglas M Fowler","doi":"10.1093/genetics/iyae156","DOIUrl":"10.1093/genetics/iyae156","url":null,"abstract":"<p><p>The cytochrome P450s enzyme family metabolizes ∼80% of small molecule drugs. Variants in cytochrome P450s can substantially alter drug metabolism, leading to improper dosing and severe adverse drug reactions. Due to low sequence conservation, predicting variant effects across cytochrome P450s is challenging. Even closely related cytochrome P450s like CYP2C9 and CYP2C19, which share 92% amino acid sequence identity, display distinct phenotypic properties. Using variant abundance by massively parallel sequencing, we measured the steady-state protein abundance of 7,660 single amino acid variants in CYP2C19 expressed in cultured human cells. Our findings confirmed critical positions and structural features essential for cytochrome P450 function, and revealed how variants at conserved positions influence abundance. We jointly analyzed 4,670 variants whose abundance was measured in both CYP2C19 and CYP2C9, finding that the homologs have different variant abundances in substrate recognition sites within the hydrophobic core. We also measured the abundance of all single and some multiple wild type amino acid exchanges between CYP2C19 and CYP2C9. While most exchanges had no effect, substitutions in substrate recognition site 4 reduced abundance in CYP2C19. Double and triple mutants showed distinct interactions, highlighting a region that points to differing thermodynamic properties between the 2 homologs. These positions are known contributors to substrate specificity, suggesting an evolutionary tradeoff between stability and enzymatic function. Finally, we analyzed 368 previously unannotated human variants, finding that 43% had decreased abundance. By comparing variant effects between these homologs, we uncovered regions underlying their functional differences, advancing our understanding of this versatile family of enzymes.</p>","PeriodicalId":48925,"journal":{"name":"Genetics","volume":null,"pages":null},"PeriodicalIF":3.3,"publicationDate":"2024-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11538415/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142330515","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":"Evidence for a hydrogen sulfide-sensing E3 ligase in yeast.","authors":"Zane Johnson, Yun Wang, Benjamin M Sutter, Benjamin P Tu","doi":"10.1093/genetics/iyae154","DOIUrl":"10.1093/genetics/iyae154","url":null,"abstract":"<p><p>In yeast, control of sulfur amino acid metabolism relies upon Met4, a transcription factor that activates the expression of a network of enzymes responsible for the biosynthesis of cysteine and methionine. In times of sulfur abundance, the activity of Met4 is repressed via ubiquitination by the SCFMet30 E3 ubiquitin ligase, but the mechanism by which the F-box protein Met30 senses sulfur status to tune its E3 ligase activity remains unresolved. Herein, we show that Met30 responds to flux through the trans-sulfuration pathway to regulate the MET gene transcriptional program. In particular, Met30 is responsive to the biological gas hydrogen sulfide, which is sufficient to induce ubiquitination of Met4 in vivo. Additionally, we identify important cysteine residues in Met30's WD-40 repeat region that sense the availability of sulfur in the cell. Our findings reveal how SCFMet30 dynamically senses the flow of sulfur metabolites through the trans-sulfuration pathway to regulate the synthesis of these special amino acids.</p>","PeriodicalId":48925,"journal":{"name":"Genetics","volume":null,"pages":null},"PeriodicalIF":3.3,"publicationDate":"2024-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11538405/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142394481","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":"Population size rescaling significantly biases outcomes of forward-in-time population genetic simulations.","authors":"Amjad Dabi, Daniel R Schrider","doi":"10.1093/genetics/iyae180","DOIUrl":"10.1093/genetics/iyae180","url":null,"abstract":"<p><p>Simulations are an essential tool in all areas of population genetic research, used in tasks such as the validation of theoretical analysis and the study of complex evolutionary models. Forward-in-time simulations are especially flexible, allowing for various types of natural selection, complex genetic architectures, and non-Wright-Fisher dynamics. However, their intense computational requirements can be prohibitive to simulating large populations and genomes. A popular method to alleviate this burden is to scale down the population size by some scaling factor while scaling up the mutation rate, selection coefficients, and recombination rate by the same factor. However, this rescaling approach may in some cases bias simulation results. To investigate the manner and degree to which rescaling impacts simulation outcomes, we carried out simulations with different demographic histories and distributions of fitness effects using several values of the rescaling factor, Ǫ, and compared the deviation of key outcomes (fixation times, allele frequencies, linkage disequilibrium, and the fraction of mutations that fix during the simulation) between the scaled and unscaled simulations. Our results indicate that scaling introduces substantial biases to each of these measured outcomes, even at small values of Ʈ. Moreover, the nature of these effects depends on the evolutionary model and scaling factor being examined. While increasing the scaling factor tends to increase the observed biases, this relationship is not always straightforward, thus it may be difficult to know the impact of scaling on simulation outcomes a priori. However, it appears that for most models, only a small number of replicates was needed to accurately quantify the bias produced by rescaling for a given Ʈ. In summary, while rescaling forward-in-time simulations may be necessary in many cases, researchers should be aware of the rescaling procedure's impact on simulation outcomes and consider investigating its magnitude in smaller scale simulations of the desired model(s) before selecting an appropriate value of Ʈ.</p>","PeriodicalId":48925,"journal":{"name":"Genetics","volume":null,"pages":null},"PeriodicalIF":3.3,"publicationDate":"2024-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142584680","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Defects in the central metabolism prevent thymineless death in Escherichia coli, while still allowing significant protein synthesis.","authors":"Sharik R Khan, Andrei Kuzminov","doi":"10.1093/genetics/iyae142","DOIUrl":"10.1093/genetics/iyae142","url":null,"abstract":"<p><p>Starvation of Escherichia coli thyA auxotrophs for the required thymine or thymidine leads to the cessation of DNA synthesis and, unexpectedly, to thymineless death (TLD). Previously, TLD-alleviating defects were identified by the candidate gene approach, for their contribution to replication initiation, fork repair, or SOS induction. However, no TLD-blocking mutations were ever found, suggesting a multifactorial nature of TLD. Since (until recently) no unbiased isolation of TLD suppressors was reported, we used enrichment after insertional mutagenesis to systematically isolate TLD suppressors. Our approach was validated by isolation of known TLD-alleviating mutants in recombinational repair. At the same time, and unexpectedly for the current TLD models, most of the isolated suppressors affected general metabolism, while the strongest suppressors impacted the central metabolism. Several temperature-sensitive (Ts) mutants in important/essential functions, like nadA, ribB, or coaA, almost completely suppressed TLD at 42°C. Since blocking protein synthesis completely by chloramphenicol prevents TLD, while reducing protein synthesis to 10% alleviates TLD only slightly, we measured the level of protein synthesis in these mutants at 42°C and found it to be 20-70% of the WT, not enough reduction to explain TLD prevention. We conclude that the isolated central metabolism mutants prevent TLD by affecting specific TLD-promoting functions.</p>","PeriodicalId":48925,"journal":{"name":"Genetics","volume":null,"pages":null},"PeriodicalIF":3.3,"publicationDate":"2024-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11538421/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142113822","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":"Linkage equilibrium between rare mutations.","authors":"Anastasia S Lyulina, Zhiru Liu, Benjamin H Good","doi":"10.1093/genetics/iyae145","DOIUrl":"10.1093/genetics/iyae145","url":null,"abstract":"<p><p>Recombination breaks down genetic linkage by reshuffling existing variants onto new genetic backgrounds. These dynamics are traditionally quantified by examining the correlations between alleles, and how they decay as a function of the recombination rate. However, the magnitudes of these correlations are strongly influenced by other evolutionary forces like natural selection and genetic drift, making it difficult to tease out the effects of recombination. Here, we introduce a theoretical framework for analyzing an alternative family of statistics that measure the homoplasy produced by recombination. We derive analytical expressions that predict how these statistics depend on the rates of recombination and recurrent mutation, the strength of negative selection and genetic drift, and the present-day frequencies of the mutant alleles. We find that the degree of homoplasy can strongly depend on this frequency scale, which reflects the underlying timescales over which these mutations occurred. We show how these scaling properties can be used to isolate the effects of recombination and discuss their implications for the rates of horizontal gene transfer in bacteria.</p>","PeriodicalId":48925,"journal":{"name":"Genetics","volume":null,"pages":null},"PeriodicalIF":3.3,"publicationDate":"2024-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11538400/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142113823","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}