Identification and characterization of Dlc1 isoforms in the mouse and study of the biological function of a single gene trapped isoform

<p>Abstract</p> <p>Background</p> <p>The Dlc1 (deleted in liver cancer 1) tumour suppressor gene codes for a RhoGTPase activating protein that is found inactivated in many tumour types. Several transcriptional isoforms have been described but the functional significance and tissue distribution of each form is presently poorly understood. Also, differences in the number of isoforms and splice variants reported still exist between different mammalian species. In order to better understand the number and function of the different variants of the Dlc1 gene in the mouse, we have carried out a detailed analysis. Extensive 3' RACE experiments were carried out in order to identify all possible Dlc1 isoforms and splice variants in the mouse. In addition, we have generated a gene trapped mouse that targets one of these isoforms in order to study its biological function. The effect of this gene trap insertion on the splicing of other isoforms has also been studied.</p> <p>Results</p> <p>In addition to the known 6.1 and 6.2 Kb transcripts of Dlc1, our study revealed the existence of a novel 7.6 Kb transcriptional isoform in the mouse, which corresponds to the human 7.4 Kb (KIAA1723) cDNA transcript. A gene trapped embryonic cell line, with an insertion between Exon 1 and 2 of the 6.1 Kb transcriptional isoform, was used to generate a transgenic mouse. This line showed a significant reduction in the expression of the trapped isoform. However, reduced expression of the other isoforms was not seen. Mice heterozygous for the gene trapped allele were phenotypically normal, but homozygous mutant embryos did not survive beyond 10.5 days post coitum. Dlc1^gt/gtembryos showed defects in the brain, heart, and placental blood vessels. Cultured serum-free mouse embryo cells from Dlc1 deficient embryos had elevated RhoA activity and displayed alterations in the organization of actin filaments and focal adhesions. The Dlc1 deficient cells also exhibited increased wound closure in an <it>in vitro </it>scratch assay.</p> <p>Conclusions</p> <p>The mouse has three major transcriptional isoforms of the Dlc1 gene that are differentially expressed in various tissues. A mouse with exon 1 of the 6.1 Kb transcript gt resulted in hypomorphic expression of Dlc1 protein and an embryonic lethal phenotype in the homozygous condition, which indicates that this isoform plays a major role in mouse development. The Dlc1 deficient cells showed altered cytoskeleton structure, increased RhoA activity and cellular migration.</p

The International Gene Trap Consortium Website: a portal to all publicly available gene trap cell lines in mouse

Gene trapping is a method of generating murine embryonic stem (ES) cell lines containing insertional mutations in known and novel genes. A number of international groups have used this approach to create sizeable public cell line repositories available to the scientific community for the generation of mutant mouse strains. The major gene trapping groups worldwide have recently joined together to centralize access to all publicly available gene trap lines by developing a user-oriented Website for the International Gene Trap Consortium (IGTC). This collaboration provides an impressive public informatics resource comprising ∼45 000 well-characterized ES cell lines which currently represent ∼40% of known mouse genes, all freely available for the creation of knockout mice on a non-collaborative basis. To standardize annotation and provide high confidence data for gene trap lines, a rigorous identification and annotation pipeline has been developed combining genomic localization and transcript alignment of gene trap sequence tags to identify trapped loci. This information is stored in a new bioinformatics database accessible through the IGTC Website interface. The IGTC Website () allows users to browse and search the database for trapped genes, BLAST sequences against gene trap sequence tags, and view trapped genes within biological pathways. In addition, IGTC data have been integrated into major genome browsers and bioinformatics sites to provide users with outside portals for viewing this data. The development of the IGTC Website marks a major advance by providing the research community with the data and tools necessary to effectively use public gene trap resources for the large-scale characterization of mammalian gene function

Increased 5-HT3-mediated signalling in pelvic afferent neurons from mice deficient in P2X2 and/or P2X3 receptor subunits

Extracellular ATP and 5-hydroxytryptamine (5-HT) are both involved in visceral sensory pathways by interacting with P2X and 5-HT3 receptors, respectively. We have investigated the changes in P2X and 5-HT3-mediated signalling in pelvic afferent neurons in mice deficient in P2X2 and/or P2X3 subunits by whole-cell recording of L6–S2 dorsal root ganglion (DRG) neurons and by multi-unit recording of pelvic afferents of the colorectum. In wildtype DRG neurons, ATP evoked transient, sustained or mixed (biphasic) inward currents. Transient currents were absent in P2X3−/− neurons, whereas sustained currents were absent in P2X2−/− DRG neurons. Neither transient nor sustained currents were observed following application of ATP or α,β-methylene ATP (α,β-meATP) in P2X2/P2X3Dbl−/− DRG neurons. 5-HT was found to induce a fast inward current in 63% of DRG neurons from wildtype mice, which was blocked by tropisetron, a 5-HT3 receptor antagonist. The percentage of DRG neurons responding to 5-HT was significantly increased in P2X 2−/−, P2X3−/− and P2X2/P2X3Dbl−/− mice, and the amplitude of 5-HT response was significantly increased in P2X2/P2X3Dbl−/− mice. The pelvic afferent response to colorectal distension was attenuated in P2X2/P2X3Dbl−/− mice, but the response to serosal application of 5-HT was enhanced. Furthermore, tropisetron resulted in a greater reduction in pelvic afferent responses to colorectal distension in the P2X2/P2X3Dbl−/− preparations. These data suggest that P2X receptors containing the P2X2 and/or P2X3 subunits mediate purinergic activation of colorectal afferents and that 5-HT signalling in pelvic afferent neurons is up-regulated in mice lacking P2X2 or P2X3 receptor genes. This effect is more pronounced when both subunits are absent

Thrombectomy Outcomes With General vs Nongeneral Anesthesia: A Pooled Patient-Level Analysis From the EXTEND-IA Trials and SELECT Study

BACKGROUND AND OBJECTIVES: The effect of anesthesia choice on endovascular thrombectomy (EVT) outcomes is unclear. Collateral status on perfusion imaging may help identify the optimal anesthesia choice. METHODS: In a pooled patient-level analysis of EXTEND-IA, EXTEND-IA TNK, EXTEND-IA TNK part II, and SELECT, EVT functional outcomes (modified Rankin Scale score distribution) were compared between general anesthesia (GA) vs non-GA in a propensity-matched sample. Furthermore, we evaluated the association of collateral flow on perfusion imaging, assessed by hypoperfusion intensity ratio (HIR) - Tmax \u3e 10 seconds/Tmax \u3e 6 seconds (good collaterals - HIR \u3c 0.4, poor collaterals - HIR ≥ 0.4) on the association between anesthesia type and EVT outcomes. RESULTS: Of 725 treated with EVT, 299 (41%) received GA and 426 (59%) non-GA. The baseline characteristics differed in presentation National Institutes of Health Stroke Scale score (median [interquartile range] GA: 18 [13-22], non-GA: 16 [11-20], DISCUSSION: GA was associated with worse functional outcomes after EVT, particularly in patients with poor collaterals in a propensity score-matched analysis from a pooled patient-level cohort from 3 randomized trials and 1 prospective cohort study. The confounding by indication may persist despite the doubly robust nature of the analysis. These findings have implications for randomized trials of GA vs non-GA and may be of utility for clinicians when making anesthesia type choice. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that use of GA is associated with worse functional outcome in patients undergoing EVT. TRIAL REGISTRATION INFORMATION: EXTEND-IA: ClinicalTrials.gov (NCT01492725); EXTEND-IA TNK: ClinicalTrials.gov (NCT02388061); EXTEND-IA TNK part II: ClinicalTrials.gov (NCT03340493); and SELECT: ClinicalTrials.gov (NCT02446587)

Thrombectomy Outcomes With General vs Nongeneral Anesthesia: A Pooled Patient-Level Analysis From the EXTEND-IA Trials and SELECT Study

Background and Objectives The effect of anesthesia choice on endovascular thrombectomy (EVT) outcomes is unclear. Collateral status on perfusion imaging may help identify the optimal anesthesia choice. Methods In a pooled patient-level analysis of EXTEND-IA, EXTEND-IA TNK, EXTEND-IA TNK part II, and SELECT, EVT functional outcomes (modified Rankin Scale score distribution) were compared between general anesthesia (GA) vs non-GA in a propensity-matched sample. Furthermore, we evaluated the association of collateral flow on perfusion imaging, assessed by hypoperfusion intensity ratio (HIR) – Tmax \u3e 10 seconds/Tmax \u3e 6 seconds (good collaterals – HIR \u3c 0.4, poor collaterals – HIR ≥ 0.4) on the association between anesthesia type and EVT outcomes. Results Of 725 treated with EVT, 299 (41%) received GA and 426 (59%) non-GA. The baseline characteristics differed in presentation National Institutes of Health Stroke Scale score (median [interquartile range] GA: 18 [13–22], non-GA: 16 [11–20], p \u3c 0.001) and ischemic core volume (GA: 15.0 mL [3.2–38.0] vs non-GA: 9.0 mL [0.0–31.0], p \u3c 0.001). In addition, GA was associated with longer last known well to arterial access (203 minutes [157–267] vs 186 minutes [138–252], p = 0.002), but similar procedural time (35.5 minutes [23–59] vs 34 minutes [22–54], p = 0.51). Of 182 matched pairs using propensity scores, baseline characteristics were similar. In the propensity score–matched pairs, GA was independently associated with worse functional outcomes (adjusted common odds ratio [adj. cOR]: 0.64, 95% CI: 0.44–0.93, p = 0.021) and higher neurologic worsening (GA: 14.9% vs non-GA: 8.9%, aOR: 2.10, 95% CI: 1.02–4.33, p = 0.045). Patients with poor collaterals had worse functional outcomes with GA (adj. cOR: 0.47, 95% CI: 0.29–0.76, p = 0.002), whereas no difference was observed in those with good collaterals (adj. cOR: 0.93, 95% CI: 0.50–1.74, p = 0.82), pinteraction: 0.07. No difference was observed in infarct growth overall and in patients with good collaterals, whereas patients with poor collaterals demonstrated larger infarct growth with GA with a significant interaction between collaterals and anesthesia type on infarct growth rate (pinteraction: 0.020). Discussion GA was associated with worse functional outcomes after EVT, particularly in patients with poor collaterals in a propensity score–matched analysis from a pooled patient-level cohort from 3 randomized trials and 1 prospective cohort study. The confounding by indication may persist despite the doubly robust nature of the analysis. These findings have implications for randomized trials of GA vs non-GA and may be of utility for clinicians when making anesthesia type choice. Classification of Evidence This study provides Class III evidence that use of GA is associated with worse functional outcome in patients undergoing EVT. Trial Registration Information EXTEND-IA: ClinicalTrials.gov (NCT01492725); EXTEND-IA TNK: ClinicalTrials.gov (NCT02388061); EXTEND-IA TNK part II: ClinicalTrials.gov (NCT03340493); and SELECT: ClinicalTrials.gov (NCT02446587)

The origins and genomic diversity of American Civil War Era smallpox vaccine strains

Abstract: Vaccination has transformed public health, most notably including the eradication of smallpox. Despite its profound historical importance, little is known of the origins and diversity of the viruses used in smallpox vaccination. Prior to the twentieth century, the method, source and origin of smallpox vaccinations remained unstandardised and opaque. We reconstruct and analyse viral vaccine genomes associated with smallpox vaccination from historical artefacts. Significantly, we recover viral molecules through non-destructive sampling of historical materials lacking signs of biological residues. We use the authenticated ancient genomes to reveal the evolutionary relationships of smallpox vaccination viruses within the poxviruses as a whole

The mammalian gene function resource: the International Knockout Mouse Consortium.

In 2007, the International Knockout Mouse Consortium (IKMC) made the ambitious promise to generate mutations in virtually every protein-coding gene of the mouse genome in a concerted worldwide action. Now, 5 years later, the IKMC members have developed high-throughput gene trapping and, in particular, gene-targeting pipelines and generated more than 17,400 mutant murine embryonic stem (ES) cell clones and more than 1,700 mutant mouse strains, most of them conditional. A common IKMC web portal (www.knockoutmouse.org) has been established, allowing easy access to this unparalleled biological resource. The IKMC materials considerably enhance functional gene annotation of the mammalian genome and will have a major impact on future biomedical research

Mapping genetic variations to three- dimensional protein structures to enhance variant interpretation: a proposed framework

The translation of personal genomics to precision medicine depends on the accurate interpretation of the multitude of genetic variants observed for each individual. However, even when genetic variants are predicted to modify a protein, their functional implications may be unclear. Many diseases are caused by genetic variants affecting important protein features, such as enzyme active sites or interaction interfaces. The scientific community has catalogued millions of genetic variants in genomic databases and thousands of protein structures in the Protein Data Bank. Mapping mutations onto three-dimensional (3D) structures enables atomic-level analyses of protein positions that may be important for the stability or formation of interactions; these may explain the effect of mutations and in some cases even open a path for targeted drug development. To accelerate progress in the integration of these data types, we held a two-day Gene Variation to 3D (GVto3D) workshop to report on the latest advances and to discuss unmet needs. The overarching goal of the workshop was to address the question: what can be done together as a community to advance the integration of genetic variants and 3D protein structures that could not be done by a single investigator or laboratory? Here we describe the workshop outcomes, review the state of the field, and propose the development of a framework with which to promote progress in this arena. The framework will include a set of standard formats, common ontologies, a common application programming interface to enable interoperation of the resources, and a Tool Registry to make it easy to find and apply the tools to specific analysis problems. Interoperability will enable integration of diverse data sources and tools and collaborative development of variant effect prediction methods

The mammalian gene function resource: The International Knockout Mouse Consortium

In 2007, the International Knockout Mouse Consortium (IKMC) made the ambitious promise to generate mutations in virtually every protein-coding gene of the mouse genome in a concerted worldwide action. Now, 5 years later, the IKMC members have developed highthroughput gene trapping and, in particular, gene-targeting pipelines and generated more than 17,400 mutant murine embryonic stem (ES) cell clones and more than 1,700 mutant mouse strains, most of them conditional. A common IKMC web portal (www.knockoutmouse.org) has been established, allowing easy access to this unparalleled biological resource. The IKMC materials considerably enhance functional gene annotation of the mammalian genome and will have a major impact on future biomedical research