Project description:Interventions: To measure the registration error in the target point indicated by laparoscopic forceps with infra-red tracking system.
Primary outcome(s): target registration error(TRE)
Study Design: Single arm Non-randomized
Project description:The continued development of novel genome editors calls for a universal method to analyze their off-target effects. Here we describe a versatile method, called Tracking-seq, for in situ identification of off-target effects that is broadly applicable to common genome-editing tools, including Cas9, base editors and prime editors. Through tracking replication protein A (RPA)-bound single-stranded DNA followed by strand-specific library construction, Tracking-seq requires a low cell input and is suitable for in vitro, ex vivo and in vivo genome editing, providing a sensitive and practical genome-wide approach for off-target detection in various scenarios. We show, using the same guide RNA, that Tracking-seq detects heterogeneity in off-target effects between different editor modalities and between different cell types, underscoring the necessity of direct measurement in the original system.
Project description:Daghestan, with its exceptional combination of linguistic, geographic, and cultural diversity, presents an excellent natural laboratory for tracking the influence of demographic processes on patterns of genetic variation. This study was designed to investigate the co-evolution of genes and languages, comparing and contrasting patterns of linguistic, genetic and geographic variation among Daghestani populations.
Project description:Understand the mechanisms of evolution in large-scale bio-production by tracking population dynamics leading to production decline in mevalonic acid-producing Escherichia coli. Industrial bioproduction entails growth of the production host to large bioreactors (e.g. 1-300 m3). This may put the organism at risk for generating non-producing subpopulations of genetic heterogeneity, which is not phenotypically detected at lab-scale (e.g. 2 L). To study these dynamics, we experimentally simulated these growth durations by passing mevalonic acid-producing E. coli to maintain the populations in exponential growth for 45 generations.
Project description:The model prokaryote Escherichia coli can exist as a either a commensal or a pathogen in the gut of diverse mammalian hosts. These associations, coupled with its ease of cultivation and genetic variability, have made E. coli a popular indicator organism for tracking the origin of fecal water contamination. Source tracking accuracy is predicated on the assumption that E. coli isolates recovered from contaminated water present a genetic signature characteristic of the host from which they originated. In this study, we compared the accuracy with which E. coli isolated from humans, bear, cattle and deer could be identified by standard fingerprinting methods used for library-based microbial source tracking (repetitive element PCR and pulsed-field gel electrophoresis) in relation to microarray-based analysis of genome content. Our results show that patterns of gene presence or absence were more useful for distinguishing E. coli isolates from different sources than traditional fingerprinting methods, particularly in the case of human strains. Host-associated differences in genome composition included the presence or absence of mobile IS1 elements as well as genes encoding the ferric dicitrate iron transporter (fec), E. coli common pilus (ECP), type 1 fimbriae and the CRISPR associated cas proteins. Many of these differences occurred in regions of the E. coli chromosome previously shown to be “hot spots” for the integration of horizontally-acquired DNA. PCR primers designed to amplify the IS1 and fec loci confirmed array results and demonstrated the ease with which gene presence/absence data can be converted into a diagnostic assay. The data presented here suggest that, despite the high level of genetic diversity observed among isolates by PFGE, human-derived strains may constitute a distinct ecotype distinguished by multiple potential library-independent source tracking markers.
Project description:The model prokaryote Escherichia coli can exist as a either a commensal or a pathogen in the gut of diverse mammalian hosts. These associations, coupled with its ease of cultivation and genetic variability, have made E. coli a popular indicator organism for tracking the origin of fecal water contamination. Source tracking accuracy is predicated on the assumption that E. coli isolates recovered from contaminated water present a genetic signature characteristic of the host from which they originated. In this study, we compared the accuracy with which E. coli isolated from humans, bear, cattle and deer could be identified by standard fingerprinting methods used for library-based microbial source tracking (repetitive element PCR and pulsed-field gel electrophoresis) in relation to microarray-based analysis of genome content. Our results show that patterns of gene presence or absence were more useful for distinguishing E. coli isolates from different sources than traditional fingerprinting methods, particularly in the case of human strains. Host-associated differences in genome composition included the presence or absence of mobile IS1 elements as well as genes encoding the ferric dicitrate iron transporter (fec), E. coli common pilus (ECP), type 1 fimbriae and the CRISPR associated cas proteins. Many of these differences occurred in regions of the E. coli chromosome previously shown to be M-bM-^@M-^\hot spotsM-bM-^@M-^] for the integration of horizontally-acquired DNA. PCR primers designed to amplify the IS1 and fec loci confirmed array results and demonstrated the ease with which gene presence/absence data can be converted into a diagnostic assay. The data presented here suggest that, despite the high level of genetic diversity observed among isolates by PFGE, human-derived strains may constitute a distinct ecotype distinguished by multiple potential library-independent source tracking markers. Twelve isolates of E. coli ( 3 from bear, 3 from cattle, 3 from deer and 3 from humans) were isolated from feces and/or raw sewage. Genome content for each strain was assessed in duplicate using comparative genome hybridization with E. coli K12 MG1655 as the reference for a total of 24 arrays.
Project description:This SuperSeries is composed of the following subset Series: GSE22171: Pacific salmon gill samples: fate tracking in river, sampled in ocean GSE22177: Pacific salmon gill samples: fate tracking in river GSE22347: Pacific salmon gill samples: fate tracking at spawning grounds Refer to individual Series
Project description:Genetic drivers of cancer can be dysregulated through epigenetic modifications of DNA. While the critical role of DNA 5-methylcytosine (5mC) in the regulation of transcription is recognized, the functions of other non-canonical DNA modifications remain obsure. Here, we report the identification of novel N(6)-methyladenine (N6-mA) DNA modifications in human tissues and implicate this epigenetic regulation in human disease, the highly malignant brain cancer, glioblastoma. Glioblastoma upregulates N6-mA levels, which co-localize with heterochromatic histone modifications, namely H3K9me3. N6-mA levels are dynamically regulated by the DNA demethylase, ALKBH1, to transcriptionally silence oncogenic pathways through decreasing chromatin accessibility. Targeting the N6-mA regulator, ALKBH1, in patient-derived human glioblastoma models inhibited tumor cell proliferation and extended survival of tumor-bearing mice, supporting this novel DNA modification as a potential new molecular therapeutic target for glioblastoma. Collectively, our results uncover a novel epigenetic node in cancer through the DNA modification, N6-mA.
Project description:Genetic drivers of cancer can be dysregulated through epigenetic modifications of DNA. While the critical role of DNA 5-methylcytosine (5mC) in the regulation of transcription is recognized, the functions of other non-canonical DNA modifications remain obscure. Here, we report the identification of novel N(6)-methyladenine (N6-mA) DNA modifications in human tissues and implicate this epigenetic regulation in human disease, the highly malignant brain cancer, glioblastoma. Glioblastoma upregulates N6-mA levels, which co-localize with heterochromatic histone modifications, namely H3K9me3. N6-mA levels are dynamically regulated by the DNA demethylase, ALKBH1, to transcriptionally silence oncogenic pathways through decreasing chromatin accessibility. Targeting the N6-mA regulator, ALKBH1, in patient-derived human glioblastoma models inhibited tumor cell proliferation and extended survival of tumor-bearing mice, supporting this novel DNA modification as a potential new molecular therapeutic target for glioblastoma. Collectively, our results uncover a novel epigenetic node in cancer through the DNA modification, N6-mA.
Project description:microRNAs (miRNAs) associating with Argonaute proteins (AGOs) regulate gene expression in mammals. miRNA 3' ends are subject to frequent sequence modifications, which have been proposed to affect miRNA stability. However, the underlying mechanism is not well understood. Here, by genetic and biochemical studies as well as deep sequencing analyses, we find that AGO mutations disrupting miRNA 3' binding are sufficient to trigger extensive miRNA 3’ modifications in HEK293T cells and in cancer patients. Comparing these modifications in TUT4, TUT7 and DIS3L2 knockout cells, we find that TUT7 is more robust than TUT4 in oligo-uridylating mature miRNAs, which in turn leads to their degradation by the DIS3L2 exonuclease. Our findings indicate a decay machinery removing AGO-associated miRNAs with an exposed 3' end. A set of endogenous miRNAs including miR-7 are targeted by this machinery presumably due to target-directed miRNA degradation.