Pathogenic variability of Ascochyta rabiei in Saskatchewan

Non-Peer ReviewedForty isolates of Ascochyta rabiei collected in Saskatchewan Canada and 18 obtained from other countries were assessed for pathogenicity on eight chickpea differential lines/cultivars under controlled conditions. Each assessment was repeated once. Based on the differential by isolate interactions, 15 distinct pathotypes were identified among the isolates from Saskatchewan. This demonstrates that many races of A. rabiei are present in Saskatchewan. Formation of new virulent races through gene recombination is possible. This will have large impact on Ascochyta blight development and its control. Plant breeders should anticipate a highly diverse A. rabiei population with a high potential for change

Osmotic pressure modulates single cell cycle dynamics inducing reversible growth arrest and reactivation of human metastatic cells

Biophysical cues such as osmotic pressure modulate proliferation and growth arrest of bacteria, yeast cells and seeds. In tissues, osmotic regulation takes place through blood and lymphatic capillaries and, at a single cell level, water and osmoregulation play a critical role. However, the effect of osmotic pressure on single cell cycle dynamics remains poorly understood. Here, we investigate the effect of osmotic pressure on single cell cycle dynamics, nuclear growth, proliferation, migration and protein expression, by quantitative time-lapse imaging of single cells genetically modified with fluorescent ubiquitination-based cell cycle indicator 2 (FUCCI2). Single cell data reveals that under hyperosmotic stress, distinct cell subpopulations emerge with impaired nuclear growth, delayed or growth arrested cell cycle and reduced migration. This state is reversible for mild hyperosmotic stress, where cells return to regular cell cycle dynamics, proliferation and migration. Thus, osmotic pressure can modulate the reversible growth arrest and reactivation of human metastatic cells

Variability of Optical Counterparts in the Chandra Galactic Bulge Survey

We present optical lightcurves of variable stars consistent with the positions of X-ray sources identified with the Chandra X-ray Observatory for the Chandra Galactic Bulge Survey. Using data from the Mosaic-II instrument on the Blanco 4m Telescope at CTIO, we gathered time-resolved photometric data on timescales from $\sim2$ hr to 8 days over the $\frac{3}{4}$ of the X-ray survey containing sources from the initial GBS catalog. Among the lightcurve morphologies we identify are flickering in interacting binaries, eclipsing sources, dwarf nova outbursts, ellipsoidal variations, long period variables, spotted stars, and flare stars. $87\%$ of X-ray sources have at least one potential optical counterpart. $24\%$ of these candidate counterparts are detectably variable; a much greater fraction than expected for randomly selected field stars, which suggests that most of these variables are real counterparts. We discuss individual sources of interest, provide variability information on candidate counterparts, and discuss the characteristics of the variable population.Comment: Accepted for publication in the Astrophysical Journal Supplement

Use of cultivar resistance and crop rotation with Bacillus subtilis for clubroot control in canola

Non-Peer ReviewedThis study was conducted to assess additional strategies potentially complimentary to cultivar resistance or biocontrol in control of clubroot. New granular Bacillus subtilis formulations and a seed dressing method were developed to facilitate biofungicide delivery in field trials. The granular formulations were applied in furrow during seeding at 50 kg/ha to a clubroot resistant (CR) and susceptible (CS) canola cultivar, respectively, in three field trials. The seed dressing applied approximately 1×105 to 5×106 cfu/seed doses of the biocontrol agent, and was evaluated on the CS cultivar seeded to different crop-rotation scenarios where the plots had a 1-year, 3-year, or 11-year break from last canola crop. Clubroot disease pressure was high at all trial sites with disease severity indexes (DSI) ranging from 69% to 98% on the CS cultivar. None of the granular formulations reduced clubroot substantially, whereas the CR cultivar showed a high effect, reducing DSI to below 15% and doubling the yield over that of CS cultivar. Plots of varying rotation showed a pattern of clubroot pathogen pressure, with those of 1-year break from canola being the highest. The DSI for all rotational scenarios was high, reaching 100% in short-rotation plots. Biofungicide seed dressing did not reduce DSI, but longer crop rotation often reduced gall size slightly, showed much milder above-ground damage, and increased the yield significantly relative to short rotation in two separate trials. Even a 3-year break from canola was highly beneficial, with the yield doubled as opposed to that with only 1-year break from canola

Abrogated cryptic activation of lentiviral transfer vectors

Despite significant improvements in lentivirus (LV) vector-based gene therapy there are still several safety risks using LV vectors including the potential formation of replication-competent LV particles. To address this shortcoming, we constructed a novel and safer gene transfer system using modified SIN-based LV gene transfer vectors. Central to our approach is a conditional deletion of the Ψ packaging signal after integration in the target genome. Here we demonstrate that after transduction of target cells, conventional SIN-based LV transfer vectors can still be mobilized. However mobilization is rendered undetectable if transductions are followed by a Cre/loxP-mediated excision of Ψ. Thus conditional elimination of the packaging signal may represent another advance in increasing the safety of LV vectors for gene therapeutic treatment of chronic diseases

Silencing and Un-silencing of Tetracycline-Controlled Genes in Neurons

To identify the underlying reason for the controversial performance of tetracycline (Tet)-controlled regulated gene expression in mammalian neurons, we investigated each of the three components that comprise the Tet inducible systems, namely tetracyclines as inducers, tetracycline-transactivator (tTA) and reverse tTA (rtTA), and tTA-responsive promoters (Ptets). We have discovered that stably integrated Ptet becomes functionally silenced in the majority of neurons when it is inactive during development. Ptet silencing can be avoided when it is either not integrated in the genome or stably-integrated with basal activity. Moreover, long-term, high transactivator levels in neurons can often overcome integration-induced Ptet gene silencing, possibly by inducing promoter accessibility

An Inducible and Reversible Mouse Genetic Rescue System

Inducible and reversible regulation of gene expression is a powerful approach for uncovering gene function. We have established a general method to efficiently produce reversible and inducible gene knockout and rescue in mice. In this system, which we named iKO, the target gene can be turned on and off at will by treating the mice with doxycycline. This method combines two genetically modified mouse lines: a) a KO line with a tetracycline-dependent transactivator replacing the endogenous target gene, and b) a line with a tetracycline-inducible cDNA of the target gene inserted into a tightly regulated (TIGRE) genomic locus, which provides for low basal expression and high inducibility. Such a locus occurs infrequently in the genome and we have developed a method to easily introduce genes into the TIGRE site of mouse embryonic stem (ES) cells by recombinase-mediated insertion. Both KO and TIGRE lines have been engineered for high-throughput, large-scale and cost-effective production of iKO mice. As a proof of concept, we have created iKO mice in the apolipoprotein E (ApoE) gene, which allows for sensitive and quantitative phenotypic analyses. The results demonstrated reversible switching of ApoE transcription, plasma cholesterol levels, and atherosclerosis progression and regression. The iKO system shows stringent regulation and is a versatile genetic system that can easily incorporate other techniques and adapt to a wide range of applications

Transplantation of canine olfactory ensheathing cells producing chondroitinase ABC promotes chondroitin sulphate proteoglycan digestion and axonal sprouting following spinal cord injury

Olfactory ensheathing cell (OEC) transplantation is a promising strategy for treating spinal cord injury (SCI), as has been demonstrated in experimental SCI models and naturally occurring SCI in dogs. However, the presence of chondroitin sulphate proteoglycans within the extracellular matrix of the glial scar can inhibit efficient axonal repair and limit the therapeutic potential of OECs. Here we have used lentiviral vectors to genetically modify canine OECs to continuously deliver mammalian chondroitinase ABC at the lesion site in order to degrade the inhibitory chondroitin sulphate proteoglycans in a rodent model of spinal cord injury. We demonstrate that these chondroitinase producing canine OECs survived at 4 weeks following transplantation into the spinal cord lesion and effectively digested chondroitin sulphate proteoglycans at the site of injury. There was evidence of sprouting within the corticospinal tract rostral to the lesion and an increase in the number of corticospinal axons caudal to the lesion, suggestive of axonal regeneration. Our results indicate that delivery of the chondroitinase enzyme can be achieved with the genetically modified OECs to increase axon growth following SCI. The combination of these two promising approaches is a potential strategy for promoting neural regeneration following SCI in veterinary practice and human patients

Cost of Mating and Insemination Capacity of a Genetically Modified Mosquito Aedes aegypti OX513A Compared to Its Wild Type Counterpart

The idea of implementing genetics-based insect control strategies modelled on the traditional SIT is becoming increasingly popular. In this paper we compare a genetically modified line of Aedes aegypti carrying a tetracycline repressible, lethal positive feedback system (OX513A) with its wild type counterpart with respect to their insemination capacities and the cost of courtship and mating. Genetically modified males inseminated just over half as many females as the wild type males during their lifetime. Providing days of rest from mating had no significant effect on the total number of females inseminated by males of either line, but it did increase their longevity. Producing sperm had a low cost in terms of energy investment; the cost of transferring this sperm to a receptive female was much higher. Continued mating attempts with refractory females suggest that males could not identify refractory females before investing substantial energy in courtship. Although over a lifetime OX513A males inseminated fewer females, the number of females inseminated over the first three days, was similar between males of the two lines, suggesting that the identified cost of RIDL may have little impact on the outcome of SIT-based control programmes with frequent releases of the genetically modified males

Cardiac-Specific Expression of the Tetracycline Transactivator Confers Increased Heart Function and Survival Following Ischemia Reperfusion Injury

Mice expressing the tetracycline transactivator (tTA) transcription factor driven by the rat α-myosin heavy chain promoter (α-MHC-tTA) are widely used to dissect the molecular mechanisms involved in cardiac development and disease. However, these α-MHC-tTA mice exhibit a gain-of-function phenotype consisting of robust protection against ischemia/reperfusion injury in both in vitro and in vivo models in the absence of associated cardiac hypertrophy or remodeling. Cardiac function, as assessed by echocardiography, did not differ between α-MHC-tTA and control animals, and there were no noticeable differences observed between the two groups in HW/TL ratio or LV end-diastolic and end-systolic dimensions. Protection against ischemia/reperfusion injury was assessed using isolated perfused hearts where α-MHC-tTA mice had robust protection against ischemia/reperfusion injury which was not blocked by pharmacological inhibition of PI3Ks with LY294002. Furthermore, α-MHC-tTA mice subjected to coronary artery ligation exhibited significantly reduced infarct size compared to control animals. Our findings reveal that α-MHC-tTA transgenic mice exhibit a gain-of-function phenotype consisting of robust protection against ischemia/reperfusion injury similar to cardiac pre- and post-conditioning effects. However, in contrast to classical pre- and post-conditioning, the α-MHC-tTA phenotype is not inhibited by the classic preconditioning inhibitor LY294002 suggesting involvement of a non-PI3K-AKT signaling pathway in this phenotype. Thus, further study of the α-MHC-tTA model may reveal novel molecular targets for therapeutic intervention during ischemic injury