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Targeted mutagenesis in wheat microspores using CRISPR/Cas9
No full textCRISPR/Cas9 genome editing is a transformative technology that will facilitate the development of crops to meet future demands. However, application of gene editing is hindered by the long life cycle of many crop species and because desired genotypes generally require multiple generations to achieve. Single-celled microspores are haploid cells that can develop into double haploid plants and have been widely used as a breeding tool to generate homozygous plants within a generation. In this study, we combined the CRISPR/Cas9 system with microspore technology and developed an optimized haploid mutagenesis system to induce genetic modifications in the wheat genome. We investigated a number of factors that may affect the delivery of CRISPR/Cas9 reagents into microspores and found that electroporation of a minimum of 75,000 cells using 10\u201320\u2009\ub5g DNA and a pulsing voltage of 500\u2009V is optimal for microspore transfection using the Neon transfection system. Using multiple Cas9 and sgRNA constructs, we present evidence for the seamless introduction of targeted modifications in an exogenous DsRed gene and two endogenous wheat genes, including TaLox2 and TaUbiL1. This study demonstrates the value and feasibility of combining microspore technology and CRISPR/Cas9-based gene editing for trait discovery and improvement in plants.Peer reviewed: YesNRC publication: Ye
Testing the role of recollision in N2+ air lasing
No full textIt has been known for many years that during filamentation of femtosecond light pulses in air, gain is observed on the B to X transition in N+2. While the gain mechanism remains unclear, it has been proposed that recollision, a process that is fundamental to much of strong field science, is critical for establishing gain. We probe this hypothesis by directly comparing the influence of the ellipticity of the pump light on gain in air filaments. Then, we decouple filamentation from gain by measuring the gain in a thin gas jet that we also use for high harmonic generation. The latter allows us to compare the dependence of the gain on the ellipticity of the pump with the dependence of the high harmonic signal on the ellipticity of the fundamental. We find that gain and harmonic generation have very different behavior in both filaments and in the jet. In fact, in a jet we even measure gain with circular polarization. Thus, we establish that recollision does not play a significant role in creating the inversion.Peer reviewed: YesNRC publication: Ye
The benefit of kitchen exhaust fan use after cooking: an experimental assessment
No full textCooking is one of the main sources of indoor air pollutants, and may even exceed the contribution from outdoor sources. This pilot study examines the use of different flow-rate fans during cooking and tests whether continuing to run the fan after cooking significantly improves pollutant removal rates and integrated exposures. Tests were carried out in the Canadian Centre for Housing Technology's twin research houses, in Ottawa, Ontario. We completed the same cooking protocol 60 times on a gas stove, testing 6 different flow rates on three different over-the-range exhaust fans, while continuously measuring UFP, PM2.5, NO2, and NO. The fan was operated during cooking for all tests and then either turned off or left on after cooking for the duration of the three hour test. We estimated decay rates, source emission rates, and integrated exposures to measured pollutants following the cooking test. The results showed that while leaving the fan on after cooking generally increased decay rates, it had a relatively small effect on integrated exposures compared to the effects of fan flow rate and the specific fan used during cooking. For PM2.5, the effect of running an exhaust fan for 15\u202fmin after cooking was similar in magnitude to the impact of a 100 cfm increase in the flow rate used while cooking: both were associated with a decrease in 15-min integrated exposure of roughly 3\u202f\u202f\u3bcg\u202fm 123. This suggests that one can partially compensate for a low flow rate exhaust fan by continuing to run the fan after cooking.Peer reviewed: YesNRC publication: Ye
Combustion performance and unburned hydrocarbon emissions of a natural gas\u2013diesel dual fuel engine at a low load condition
No full textThe combustion of natural gas reduces fuel cost and generates less emissions of carbon dioxide and particulate matter (PM) than diesel and gasoline. Replacing diesel by natural gas in internal combustion engines is of great interest for transportation and stationary power generation. Dual fuel combustion is an efficient way to burn natural gas in internal combustion engines. In natural gas\u2013diesel dual fuel engines, unburned hydrocarbon emissions increase with increasing natural gas fraction. Many studies have been conducted to improve the performance of natural gas\u2013diesel dual fuel engines and reported the performance of combustion and emissions of regulated pollutants and total unburned hydrocarbon at various engine operating strategies. However, little has been reported on the emissions of different unburned hydrocarbon components. In this paper, an experimental investigation was conducted to investigate the combustion performance and emissions of various unburned hydrocarbon components, including methane, ethane, ethylene, acetylene, propylene, formaldehyde, acetaldehyde, and benzaldehyde, at a low engine load condition. The operating conditions, such as engine speed, load, intake temperature, and pressure, were well controlled during the experiment. The combustion and emissions performance of pure diesel and natural gas\u2013diesel dual fuel combustion were compared. The effect of diesel injection timing was analyzed. The results show that appropriately advancing diesel injection timing to form a homogeneous charge compression ignition (HCCI)-like combustion is beneficial to natural gas\u2013diesel dual fuel combustion at low load conditions. The emissions of different unburned hydrocarbon components changed in dual fuel combustion, with emissions of some unburned hydrocarbon components being primarily due to the combustion of natural gas, while those of others being more related to diesel combustion.Peer reviewed: YesNRC publication: Ye
Development of a small modular multi-stage axial compressor for ice crystal icing research
No full textThis paper describes a Small Modular Axial Compressor Rig (SMACR) being developed by the National Research Council of Canada (NRC) for use in research into ice crystal icing (ICI) of jet engine compressors. This compressor will allow a variety of effects that cannot be simulated with static rigs, such as those used in previous ICI research at NRC, to be investigated. These include particle fragmentation, centrifuging and melting by the heat of compression. The paper describes the design of a single stage that can be replicated to obtain a multi-stage compressor by simply changing the rotor and stator and other minor hardware items. The modularity is achieved by using an independent high speed electric motor for each stage and mounting the (overhung) rotor directly on the motor output shaft. Preliminary test results are presented for the modular drive assembly combined with the 1st stage rotor and stator for sea level pressure and lower pressures of ~69kPa and 47kPa obtained by testing in a purpose-built low pressure (LP) plenum. These results show that the 1st stage can deliver the target power of approximately 15kW at rotational speeds over 30000RPM, which should be sufficient for fragmentation studies. Simulation results for ice particle melting in the compressor with 2 and 3 stages and fully glaciated inlet conditions show that 2 stages will be able to produce a discharge melt ratio (MR) above the lower end of the \u201cplateau\u201d for ICI, at least at the lowest targeted operating pressure of 34.5kPa (5psia) where low melting will result from high evaporative cooling. An additional (3rd) stage will likely be required to produce an MR approaching the upper end of the plateau at these conditions.Peer reviewed: YesNRC publication: Ye
Characterization of an engine icing tunnel and comparison to liquid water content from an evaporation model
No full textThe National Research Council Canada (NRC) operates several engine icing facilities. This paper discusses recent improvements to an intermediate size engine icing tunnel and describes the characterisation of the liquid water content (LWC) provided by the spray mast. The measured values are compared to the LWC predicted by the NRC evaporation model, which is used to control the tunnel cloud conditions.Peer reviewed: YesNRC publication: Ye
Enter the matrix: factorization uncovers knowledge from omics
No full textOmics data contain signals from the molecular, physical, and kinetic inter- and intracellular interactions that control biological systems. Matrix factorization (MF) techniques can reveal low-dimensional structure from high-dimensional data that reflect these interactions. These techniques can uncover new biological knowledge from diverse high-throughput omics data in applications ranging from pathway discovery to timecourse analysis. We review exemplary applications of MF for systems-level analyses. We discuss appropriate applications of these methods, their limitations, and focus on the analysis of results to facilitate optimal biological interpretation. The inference of biologically relevant features with MF enables discovery from high-throughput data beyond the limits of current biological knowledge \u2013 answering questions from high-dimensional data that we have not yet thought to ask.Peer reviewed: YesNRC publication: Ye
Abstract A189: identification of antibody-drug conjugate targets using curated public data, in-house glycoproteomics, and a surrogate in vitro ADC assay
No full textAntibody-drug conjugates (ADCs) are a promising approach for cancer therapy, combining the specificity of an antibody with the potency of small-molecule toxins. To identify cellular targets for the development of new ADCs, we have set out to identify proteins that (1) are expressed on the cell surface; (2) have high specificity for tumors, with relatively low expression on normal tissues; and (3) can internalize into the tumor cell by a mechanism that enables the delivery and activation of sufficient amounts of toxin to kill cancer cells. Here at the NRC, we have built a pipeline to identify new ADC targets, incorporating public gene expression data mining and glycoproteomic profiling, followed by in vitro screening through a surrogate ADC assay. Public data enable the analysis of large numbers of human tumors and normal tissues, providing a population-based estimate of gene expression. Through curation of the Gene Expression Omnibus, we have built a microarray database that contains >26,000 tumor samples and >8,800 normal samples, all on the Affymetrix HGU133 Plus 2.0 platform. We have also collected RNA-seq data for >5,000 normal samples from the GTEx database, and 6,900 tumor samples from The Cancer Genome Atlas. These samples cover a broad range of tissues: blood, bone marrow, brain, breast, colon, heart, kidney, liver, lung, muscle, ovary, pancreas, prostate, skin, stomach, and uterus. To identify candidates for ADC development, we first classify tumors into subtypes through consensus clustering followed by a Monte Carlo implementation of our iterative ensemble classification methods. Next, we perform differential gene expression analysis between normal tissues and known or novel cancer subtypes. In one example, we have identified 50 breast cancer targets, 7 of which have already been developed as ADCs to the clinical trial stage by others, demonstrating the validity and promise of this approach (Fauteux et al., 2016). Glycoproteomics data are typically derived from small numbers of samples, making a population-based analysis less informative. Therefore, we have integrated glycoproteomic data into our target selection pipeline in two ways. First, glycoproteomics has been used to profile the cell surface of 11 tumor cell lines. Using an approach with high specificity for cell-surface glycoproteins, over 200 cell-surface proteins have been identified for each cell line. This data enables the selection of targets that are amenable to our in vitro functional assay for ADC activity, based on expression in our screen-adapted cell lines. Glycoproteomics has also been used to identify and prioritize targets upregulated during hypoxia or epithelial-mesenchymal transition, two important aspects of tumor biology. For example, cellular glycoproteins from four pancreatic cell lines were profiled under normoxic and hypoxic conditions, identifying >70 proteins upregulated under hypoxic conditions. These glycoproteomic datasets, in conjunction with the public data analysis, are being used to identify promising ADC targets. Based on these target selection methods, we are currently producing and screening thousands of NRC monoclonal and single-domain antibodies generated against a variety of cancer-associated cell surface targets and screening them for ADC activity, in vitro and in vivo.Peer reviewed: YesNRC publication: Ye