Accelerating Gene Discovery by Phenotyping Whole-Genome Sequenced Multi-mutation Strains and Using the Sequence Kernel Association Test (SKAT).

Forward genetic screens represent powerful, unbiased approaches to uncover novel components in any biological process. Such screens suffer from a major bottleneck, however, namely the cloning of corresponding genes causing the phenotypic variation. Reverse genetic screens have been employed as a way to circumvent this issue, but can often be limited in scope. Here we demonstrate an innovative approach to gene discovery. Using C. elegans as a model system, we used a whole-genome sequenced multi-mutation library, from the Million Mutation Project, together with the Sequence Kernel Association Test (SKAT), to rapidly screen for and identify genes associated with a phenotype of interest, namely defects in dye-filling of ciliated sensory neurons. Such anomalies in dye-filling are often associated with the disruption of cilia, organelles which in humans are implicated in sensory physiology (including vision, smell and hearing), development and disease. Beyond identifying several well characterised dye-filling genes, our approach uncovered three genes not previously linked to ciliated sensory neuron development or function. From these putative novel dye-filling genes, we confirmed the involvement of BGNT-1.1 in ciliated sensory neuron function and morphogenesis. BGNT-1.1 functions at the trans-Golgi network of sheath cells (glia) to influence dye-filling and cilium length, in a cell non-autonomous manner. Notably, BGNT-1.1 is the orthologue of human B3GNT1/B4GAT1, a glycosyltransferase associated with Walker-Warburg syndrome (WWS). WWS is a multigenic disorder characterised by muscular dystrophy as well as brain and eye anomalies. Together, our work unveils an effective and innovative approach to gene discovery, and provides the first evidence that B3GNT1-associated Walker-Warburg syndrome may be considered a ciliopathy.

Anticoagulation stewardship: Improving adherence to clinical guidelines and reducing overuse of venous thromboembolism prophylaxis in hospitalized medical patients.

Adherence to guideline recommendations for venous thromboembolism prophylaxis (VTE) in hospitalized medical patients is suboptimal despite national policies and institutional interventions. The aim of this quality improvement project was to improve adherence to guidelines and decrease the overuse of VTE prophylaxis in order to reduce the institutional cost for heparins. A multidisciplinary anticoagulation stewardship program (ACSP) using the audit and feedback strategy was implemented on the medicine inpatient units at a teaching hospital in Canada. The primary outcome measure was a comparison, pre and post introduction of the ACSP, of the costs per 6-month period for prophylactic dose enoxaparin and unfractionated heparin on the medicine units. The balancing measures were the 90-day VTE rate and major bleeding rate during the hospitalization. Six months after the implementation of the ACSP, the cost was decreased by >50 % without any observed negative impact on patient safety. This study demonstrates the potential for anticoagulation stewardship programs to optimize the use of VTE prophylaxis and reduce the associated costs and risks.

Use of polyvalent equine anti-viper serum to treat delayed coagulopathy due to suspected Sistrurus miliarius streckeri envenomation in two children.

BACKGROUND: Western Pygmy Rattlesnake (WPR) envenomation reportedly causes refractory and persistent coagulopathy when treated with CroFab® (Crotalidae Polyvalent Immune Fab). We report two cases where polyvalent equine anti-viper serum (AntivipmynTRI®) was used to treat recurrent coagulopathy in children. CASE DETAILS: The first patient was a 16-month-old male who was bitten by a confirmed WPR. The patient received a total of 18 vials of CroFab®. His labs normalized, swelling gradually improved, and the child was discharged to home. On day 5, the child returned to the emergency department with a great deal of inguinal tenderness. Labs were obtained and the child's INR was >13.1, while the fibrinogen was <60 mg/dL and the d-dimer was 11.72 mg/L. A decision was made to administer Antivipmyn TRI®, and the child received a total of 10 vials. Lab values significantly improved: INR 1.2, fibrinogen 93 mg/dL, and d-dimer 4.21 mg/L. The second patient was a 20-month-old male who presented following snake envenomation. The child was administered a total of 22 vials of CroFab® over approximately 70 h following envenomation. Physical exam continued to improve, however, lab results showed an increasing INR 1.98, decreasing platelet count 124 × 103 per µL, fibrinogen <60 mg/dL, and d-dimer >20 ug/mL. A total of 15 vials of Antivipmyn TRI® were administered to this patient. Following this administration, labs and clinical exam both significantly improved. Labs revealed INR 1.16, fibrinogen 110 mg/dL, d-dimer 3.2 mg/L and platelet count 215 × 103/µL. DISCUSSION: CroFab® is still the first-line treatment for children bitten by a WPR, but in some cases patients develop a recurrent coagulopathy. The rapid response demonstrated by Antivipmyn TRI® leads us to conclude that this is a potential therapy for this clinical situation.

Using C. elegans Forward and Reverse Genetics to Identify New Compounds with Anthelmintic Activity.

BACKGROUND: The lack of new anthelmintic agents is of growing concern because it affects human health and our food supply, as both livestock and plants are affected. Two principal factors contribute to this problem. First, nematode resistance to anthelmintic drugs is increasing worldwide and second, many effective nematicides pose environmental hazards. In this paper we address this problem by deploying a high throughput screening platform for anthelmintic drug discovery using the nematode Caenorhabditis elegans as a surrogate for infectious nematodes. This method offers the possibility of identifying new anthelmintics in a cost-effective and timely manner. METHODS/PRINCIPAL FINDINGS: Using our high throughput screening platform we have identified 14 new potential anthelmintics by screening more than 26,000 compounds from the Chembridge and Maybridge chemical libraries. Using phylogenetic profiling we identified a subset of the 14 compounds as potential anthelmintics based on the relative sensitivity of C. elegans when compared to yeast and mammalian cells in culture. We showed that a subset of these compounds might employ mechanisms distinct from currently used anthelmintics by testing diverse drug resistant strains of C. elegans. One of these newly identified compounds targets mitochondrial complex II, and we used structural analysis of the target to suggest how differential binding of this compound may account for its different effects in nematodes versus mammalian cells. CONCLUSIONS/SIGNIFICANCE: The challenge of anthelmintic drug discovery is exacerbated by several factors; including, 1) the biochemical similarity between host and parasite genomes, 2) the geographic location of parasitic nematodes and 3) the rapid development of resistance. Accordingly, an approach that can screen large compound collections rapidly is required. C. elegans as a surrogate parasite offers the ability to screen compounds rapidly and, equally importantly, with specificity, thus reducing the potential toxicity of these compounds to the host and the environment. We believe this approach will help to replenish the pipeline of potential nematicides.