NAR Cancer最新文献

{"title":"PDXNet portal: patient-derived Xenograft model, data, workflow and tool discovery.","authors":"Soner Koc, Michael W Lloyd, Jeffrey W Grover, Nan Xiao, Sara Seepo, Sai Lakshmi Subramanian, Manisha Ray, Christian Frech, John DiGiovanna, Phillip Webster, Steven Neuhauser, Anuj Srivastava, Xing Yi Woo, Brian J Sanderson, Brian White, Paul Lott, Lacey E Dobrolecki, Heidi Dowst, Yvonne A Evrard, Tiffany A Wallace, Jeffrey A Moscow, James H Doroshow, Nicholas Mitsiades, Salma Kaochar, Chong-Xian Pan, Moon S Chen, Luis Carvajal-Carmona, Alana L Welm, Bryan E Welm, Michael T Lewis, Ramaswamy Govindan, Li Ding, Shunqiang Li, Meenhard Herlyn, Michael A Davies, Jack Roth, Funda Meric-Bernstam, Peter N Robinson, Carol J Bult, Brandi Davis-Dusenbery, Dennis A Dean, Jeffrey H Chuang","doi":"10.1093/narcan/zcac014","DOIUrl":"10.1093/narcan/zcac014","url":null,"abstract":"<p><p>We created the PDX Network (PDXNet) portal (https://portal.pdxnetwork.org/) to centralize access to the National Cancer Institute-funded PDXNet consortium resources, to facilitate collaboration among researchers and to make these data easily available for research. The portal includes sections for resources, analysis results, metrics for PDXNet activities, data processing protocols and training materials for processing PDX data. Currently, the portal contains PDXNet model information and data resources from 334 new models across 33 cancer types. Tissue samples of these models were deposited in the NCI's Patient-Derived Model Repository (PDMR) for public access. These models have 2134 associated sequencing files from 873 samples across 308 patients, which are hosted on the Cancer Genomics Cloud powered by Seven Bridges and the NCI Cancer Data Service for long-term storage and access with dbGaP permissions. The portal includes results from freely available, robust, validated and standardized analysis workflows on PDXNet sequencing files and PDMR data (3857 samples from 629 patients across 85 disease types). The PDXNet portal is continuously updated with new data and is of significant utility to the cancer research community as it provides a centralized location for PDXNet resources, which support multi-agent treatment studies, determination of sensitivity and resistance mechanisms, and preclinical trials.</p>","PeriodicalId":18879,"journal":{"name":"NAR Cancer","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2022-04-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9026194/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9777076","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":"Synergistic action of WDR5 and HDM2 inhibitors in SMARCB1-deficient cancer cells.","authors":"Andrea C Florian, Chase M Woodley, Jing Wang, Brian C Grieb, Macey J Slota, Kiana Guerrazzi, Chih-Yuan Hsu, Brittany K Matlock, David K Flaherty, Shelly L Lorey, Stephen W Fesik, Gregory C Howard, Qi Liu, April M Weissmiller, William P Tansey","doi":"10.1093/narcan/zcac007","DOIUrl":"10.1093/narcan/zcac007","url":null,"abstract":"<p><p>Rhabdoid tumors (RT) are rare and deadly pediatric cancers driven by loss of <i>SMARCB1</i>, which encodes the SNF5 component of the SWI/SNF chromatin remodeler. Loss of <i>SMARCB1</i> is associated with a complex set of phenotypic changes including vulnerability to inhibitors of protein synthesis and of the p53 ubiquitin-ligase HDM2. Recently, we discovered small molecule inhibitors of the 'WIN' site of WDR5, which in MLL-rearranged leukemia cells decrease the expression of a set of genes linked to protein synthesis, inducing a translational choke and causing p53-dependent inhibition of proliferation. Here, we characterize how WIN site inhibitors act in RT cells. As in leukemia cells, WIN site inhibition in RT cells causes the comprehensive displacement of WDR5 from chromatin, resulting in a decrease in protein synthesis gene expression. Unlike leukemia cells, however, the growth response of RT cells to WIN site blockade is independent of p53. Exploiting this observation, we demonstrate that WIN site inhibitor synergizes with an HDM2 antagonist to induce p53 and block RT cell proliferation <i>in vitro</i>. These data reveal a p53-independent action of WIN site inhibitors and forecast that future strategies to treat RT could be based on dual WDR5/HDM2 inhibition.</p>","PeriodicalId":18879,"journal":{"name":"NAR Cancer","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2022-03-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/76/58/zcac007.PMC8892060.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10246020","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":"Editorial: NAR Cancer and epigenetics and cancer.","authors":"Danzhou Yang,&nbsp;Jonathan Dickerhoff,&nbsp;William S Dynan","doi":"10.1093/narcan/zcac003","DOIUrl":"https://doi.org/10.1093/narcan/zcac003","url":null,"abstract":"Over the past 20 years, breakthrough discoveries in epigenetics have transformed our knowledge of chromatin structure alterations and regulation mechanisms in response to physiological or pathological signals. Without changing the gene sequence, epigenetic changes affect the way our genes work, leading to heritable phenotypes. Cancer epigenetics is one of the most active areas of cancer research and represents the single largest category of article submissions to NAR Cancer . NAR Cancer is pleased to publish a thematic collection of articles, ‘Cancer Epigenetics’ that encapsulates exciting discoveries in this area. Ten Surveys and Summaries provide insights into the ways in which cancer progression, diagnosis and therapy are interwoven with epigenetics. These review articles, together with relevant recent standard articles, are now made available as a special collection on the journal’s website. This is the journal’s first-ever thematic collection. We plan for these collections to become regular features in the future. Chemical DNA and histones is the and other enzymes","PeriodicalId":18879,"journal":{"name":"NAR Cancer","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2022-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8894078/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10588475","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":"Transcriptional regulators of human oncoviruses: structural and functional implications for anticancer therapy.","authors":"Ivona Nečasová,&nbsp;Martin Stojaspal,&nbsp;Edita Motyčáková,&nbsp;Tomáš Brom,&nbsp;Tomáš Janovič,&nbsp;Ctirad Hofr","doi":"10.1093/narcan/zcac005","DOIUrl":"https://doi.org/10.1093/narcan/zcac005","url":null,"abstract":"<p><p>Transcription is often the first biosynthetic event of viral infection. Viruses produce preferentially viral transcriptional regulators (vTRs) essential for expressing viral genes and regulating essential host cell proteins to enable viral genome replication. As vTRs are unique viral proteins that promote the transcription of viral nucleic acid, vTRs interact with host proteins to suppress detection and immune reactions to viral infection. Thus, vTRs are promising therapeutic targets that are sequentially and structurally distinct from host cell proteins. Here, we review vTRs of three human oncoviruses: HBx of hepatitis B virus, HBZ of human T-lymphotropic virus type 1, and Rta of Epstein-Barr virus. We present three cunningly exciting and dangerous transcription strategies that make viral infections so efficient. We use available structural and functional knowledge to critically examine the potential of vTRs as new antiviral-anticancer therapy targets. For each oncovirus, we describe (i) the strategy of viral genome transcription; (ii) vTRs' structure and binding partners essential for transcription regulation; and (iii) advantages and challenges of vTR targeting in antiviral therapies. We discuss the implications of vTR regulation for oncogenesis and perspectives on developing novel antiviral and anticancer strategies.</p>","PeriodicalId":18879,"journal":{"name":"NAR Cancer","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2022-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8892037/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9693246","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":"The contribution of uncharted RNA sequences to tumor identity in lung adenocarcinoma.","authors":"Yunfeng Wang,&nbsp;Haoliang Xue,&nbsp;Marine Aglave,&nbsp;Antoine Lainé,&nbsp;Mélina Gallopin,&nbsp;Daniel Gautheret","doi":"10.1093/narcan/zcac001","DOIUrl":"https://doi.org/10.1093/narcan/zcac001","url":null,"abstract":"<p><p>The identity of cancer cells is defined by the interplay between genetic, epigenetic transcriptional and post-transcriptional variation. A lot of this variation is present in RNA-seq data and can be captured at once using reference-free, k-mer analysis. An important issue with k-mer analysis, however, is the difficulty of distinguishing signal from noise. Here, we use two independent lung adenocarcinoma datasets to identify all reproducible events at the k-mer level, in a tumor versus normal setting. We find reproducible events in many different locations (introns, intergenic, repeats) and forms (spliced, polyadenylated, chimeric etc.). We systematically analyze events that are ignored in conventional transcriptomics and assess their value as biomarkers and for tumor classification, survival prediction, neoantigen prediction and correlation with the immune microenvironment. We find that unannotated lincRNAs, novel splice variants, endogenous HERV, Line1 and Alu repeats and bacterial RNAs each contribute to different, important aspects of tumor identity. We argue that differential RNA-seq analysis of tumor/normal sample collections would benefit from this type k-mer analysis to cast a wider net on important cancer-related events. The code is available at https://github.com/Transipedia/dekupl-lung-cancer-inter-cohort.</p>","PeriodicalId":18879,"journal":{"name":"NAR Cancer","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2022-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/13/fe/zcac001.PMC8807116.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10318377","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":"PP2A and cancer epigenetics: a therapeutic opportunity waiting to happen.","authors":"Samantha L Tinsley,&nbsp;Brittany L Allen-Petersen","doi":"10.1093/narcan/zcac002","DOIUrl":"https://doi.org/10.1093/narcan/zcac002","url":null,"abstract":"<p><p>The epigenetic state of chromatin is altered by regulators which influence gene expression in response to environmental stimuli. While several post-translational modifications contribute to chromatin accessibility and transcriptional programs, our understanding of the role that specific phosphorylation sites play is limited. In cancer, kinases and phosphatases are commonly deregulated resulting in increased oncogenic signaling and loss of epigenetic regulation. Aberrant epigenetic states are known to promote cellular plasticity and the development of therapeutic resistance in many cancer types, highlighting the importance of these mechanisms to cancer cell phenotypes. Protein Phosphatase 2A (PP2A) is a heterotrimeric holoenzyme that targets a diverse array of cellular proteins. The composition of the PP2A complex influences its cellular targets and activity. For this reason, PP2A can be tumor suppressive or oncogenic depending on cellular context. Understanding the nuances of PP2A regulation and its effect on epigenetic alterations can lead to new therapeutic avenues that afford more specificity and contribute to the growth of personalized medicine in the oncology field. In this review, we summarize the known PP2A-regulated substrates and potential phosphorylation sites that contribute to cancer cell epigenetics and possible strategies to therapeutically leverage this phosphatase to suppress tumor growth.</p>","PeriodicalId":18879,"journal":{"name":"NAR Cancer","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2022-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8807117/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39888787","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":"Comprehensive profiling of mRNA splicing indicates that GC content signals altered cassette exon inclusion in Ewing sarcoma.","authors":"Garrett T Graham,&nbsp;Saravana P Selvanathan,&nbsp;Stefan K Zöllner,&nbsp;Emily Stahl,&nbsp;Adam Shlien,&nbsp;Natasha J Caplen,&nbsp;Aykut Üren,&nbsp;Jeffrey A Toretsky","doi":"10.1093/narcan/zcab052","DOIUrl":"https://doi.org/10.1093/narcan/zcab052","url":null,"abstract":"<p><p>Ewing sarcoma (EwS) is a small round blue cell tumor and is the second most frequent pediatric bone cancer. 85% of EwS tumors express the fusion oncoprotein EWS-FLI1, the product of a t(11;22) reciprocal translocation. Prior work has indicated that transcription regulation alone does not fully describe the oncogenic capacity of EWS-FLI1, nor does it provide an effective means to stratify patient tumors. Research using EwS cell lines and patient samples has suggested that EWS-FLI1 also disrupts mRNA biogenesis. In this work we both describe the underlying characteristics of mRNA that are aberrantly spliced in EwS tumor samples as well as catalogue mRNA splicing events across other pediatric tumor types. Here, we also use short- and long-read sequencing to identify <i>cis</i>-factors that contribute to splicing profiles we observe in Ewing sarcoma. Our analysis suggests that GC content upstream of cassette exons is a defining factor of mRNA splicing in EwS. We also describe specific splicing events that discriminate EwS tumor samples from the assumed cell of origin, human mesenchymal stem cells derived from bone marrow (hMSC-BM). Finally, we identify specific splicing factors PCBP2, RBMX, and SRSF9 by motif enrichment and confirm findings from tumor samples in EwS cell lines.</p>","PeriodicalId":18879,"journal":{"name":"NAR Cancer","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2022-01-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/da/5e/zcab052.PMC8759570.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39835831","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":"A comprehensive framework for analysis of microRNA sequencing data in metastatic colorectal cancer.","authors":"Eirik Høye,&nbsp;Bastian Fromm,&nbsp;Paul H M Böttger,&nbsp;Diana Domanska,&nbsp;Annette Torgunrud,&nbsp;Christin Lund-Andersen,&nbsp;Torveig Weum Abrahamsen,&nbsp;Åsmund Avdem Fretland,&nbsp;Vegar J Dagenborg,&nbsp;Susanne Lorenz,&nbsp;Bjørn Edwin,&nbsp;Eivind Hovig,&nbsp;Kjersti Flatmark","doi":"10.1093/narcan/zcab051","DOIUrl":"https://doi.org/10.1093/narcan/zcab051","url":null,"abstract":"<p><p>Although microRNAs (miRNAs) contribute to all hallmarks of cancer, miRNA dysregulation in metastasis remains poorly understood. The aim of this work was to reliably identify miRNAs associated with metastatic progression of colorectal cancer (CRC) using novel and previously published next-generation sequencing (NGS) datasets generated from 268 samples of primary (pCRC) and metastatic CRC (mCRC; liver, lung and peritoneal metastases) and tumor adjacent tissues. Differential expression analysis was performed using a meticulous bioinformatics pipeline, including only bona fide miRNAs, and utilizing miRNA-tailored quality control and processing. Five miRNAs were identified as up-regulated at multiple metastatic sites Mir-210_3p, Mir-191_5p, Mir-8-P1b_3p [mir-141-3p], Mir-1307_5p and Mir-155_5p. Several have previously been implicated in metastasis through involvement in epithelial-to-mesenchymal transition and hypoxia, while other identified miRNAs represent novel findings. The use of a publicly available pipeline facilitates reproducibility and allows new datasets to be added as they become available. The set of miRNAs identified here provides a reliable starting-point for further research into the role of miRNAs in metastatic progression.</p>","PeriodicalId":18879,"journal":{"name":"NAR Cancer","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2022-01-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8759566/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39835830","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":"High-grade ovarian cancer associated H/ACA snoRNAs promote cancer cell proliferation and survival.","authors":"Laurence Faucher-Giguère,&nbsp;Audrey Roy,&nbsp;Gabrielle Deschamps-Francoeur,&nbsp;Sonia Couture,&nbsp;Ryan M Nottingham,&nbsp;Alan M Lambowitz,&nbsp;Michelle S Scott,&nbsp;Sherif Abou Elela","doi":"10.1093/narcan/zcab050","DOIUrl":"https://doi.org/10.1093/narcan/zcab050","url":null,"abstract":"<p><p>Small nucleolar RNAs (snoRNAs) are an omnipresent class of non-coding RNAs involved in the modification and processing of ribosomal RNA (rRNA). As snoRNAs are required for ribosome production, the increase of which is a hallmark of cancer development, their expression would be expected to increase in proliferating cancer cells. However, assessing the nature and extent of snoRNAs' contribution to cancer biology has been largely limited by difficulties in detecting highly structured RNA. In this study, we used a dedicated midsize non-coding RNA (mncRNA) sensitive sequencing technique to accurately survey the snoRNA abundance in independently verified high-grade serous ovarian carcinoma (HGSC) and serous borderline tumour (SBT) tissues. The results identified SNORA81, SNORA19 and SNORA56 as an H/ACA snoRNA signature capable of discriminating between independent sets of HGSC, SBT and normal tissues. The expression of the signature SNORA81 correlates with the level of ribosomal RNA (rRNA) modification and its knockdown inhibits 28S rRNA pseudouridylation and accumulation leading to reduced cell proliferation and migration. Together our data indicate that specific subsets of H/ACA snoRNAs may promote tumour aggressiveness by inducing rRNA modification and synthesis.</p>","PeriodicalId":18879,"journal":{"name":"NAR Cancer","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2022-01-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8759569/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39835829","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":"Characterization of the consensus mucosal microbiome of colorectal cancer.","authors":"Lan Zhao, Susan M Grimes, Stephanie U Greer, Matthew Kubit, HoJoon Lee, Lincoln D Nadauld, Hanlee P Ji","doi":"10.1093/narcan/zcab049","DOIUrl":"10.1093/narcan/zcab049","url":null,"abstract":"<p><p>Dysbioisis is an imbalance of an organ's microbiome and plays a role in colorectal cancer pathogenesis. Characterizing the bacteria in the microenvironment of a cancer through genome sequencing has advantages compared to culture-based profiling. However, there are notable technical and analytical challenges in characterizing universal features of tumor microbiomes. Colorectal tumors demonstrate microbiome variation among different studies and across individual patients. To address these issues, we conducted a computational study to determine a consensus microbiome for colorectal cancer, analyzing 924 tumors from eight independent RNA-Seq data sets. A standardized meta-transcriptomic analysis pipeline was established with quality control metrics. Microbiome profiles across different cohorts were compared and recurrently altered microbial shifts specific to colorectal cancer were determined. We identified cancer-specific set of 114 microbial species associated with tumors that were found among all investigated studies. Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria were among the four most abundant phyla for the colorectal cancer microbiome. Member species of Clostridia were depleted and <i>Fusobacterium nucleatum</i> was one of the most enriched bacterial species in tumors. Associations between the consensus species and specific immune cell types were noted. Our results are available as a web data resource for other researchers to explore (https://crc-microbiome.stanford.edu).</p>","PeriodicalId":18879,"journal":{"name":"NAR Cancer","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2021-12-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/32/70/zcab049.PMC8693571.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39790372","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}