Natural Sciences And Engineering Research Council Of Canada Research Articles

Glucocorticoid Receptor Orchestrates Interactions Between Glucocorticoid ‐Responsive and ‐Unresponsive Enhancers to Modulate Gene Expression

RATIONALEGlucocorticoids (GCs) are stress hormones that act on the GC receptor (GR; NR3C1) to elicit effects, including repression of inflammatory gene expression. The mechanisms by which GR modulate gene expression are not completely understood. GR binds to thousands of genomic loci, but only regulates the expression of hundreds of genes. Genome‐wide chromatin interaction studies revealed that the proximal promotor of various GC‐regulated genes interacts with multiple distal enhancer elements that harbour GC‐dependent GR binding. The significance of such interactions and the contribution of each locus to the achieved gene expression are yet to be investigated. The current work provides a focused perspective on genomic events that lead to the induction of KLF9 by GCs, a gene expression feature that is highly conserved in multiple cell types.RESULTSGC‐mediated induction of KLF9 was confirmed in the airway epithelial cell lines (A549 and BEAS‐2B), as well as in primary culture of various airway structural cells. This induction was abolished in A549 cells either by pretreating cells with the GR antagonist, Org34517, or by knocking down GR with siRNA. Chromatin immunoprecipitation of GR following GC treatment in A549, BEAS‐2B, and primary human bronchial epithelial (HBE) cells showed GR binding to sites 5.9, 6.7, 25, and 65 kb upstream of the KLF9 gene. Global nascent transcript analysis of BEAS‐2B cells following GC treatment showed marked induction of enhancer RNA (eRNA) from each of these sites. This observation was validated using qPCR in A549 and primary HBE cells. GR binding regions were then cloned upstream to a luciferase reporter to test their enhancer activity due to GCs in isolation from their genomic context. There was a clear distinction between proximal regions (i.e. 5.9 and 6.7 kb from KLF9 promotor) and the distal regions (25 and 65 kb) in terms of basal activity and response to GCs. Proximal regions showed very strong basal reporter activity that was not affected by GC treatment. Distal regions, however, showed minimal basal activity but produced dose‐dependent increase in reporter activity by GCs, which was abolished by mutating the GC response elements (GRE) from these regions. Kinetics of long‐range genomic interactions identified a significant interaction between the distal and the proximal regions in a slightly delayed fashion (4h onward), this was temporally associated with enhanced binding of EP300 (histone acetylase associated with gene activation) to the proximal region.CONCLUSIONSOur data suggests that each GR binding site upstream to KLF9 responds differently to GCs, but all sites may collectively contribute to the basal transcriptional activity, induction, and maintenance of KLF9 expression. Co‐localization of other transcriptional regulators may explain the distinction between different sites. Our data are essential in establishing a model to explain GR genomic binding behaviours and how they correlate with gene expression outcomes, thus providing insights on how to modulate GC actions to achieve clinical benefits.Support or Funding InformationFunding: Canadian Institutes of Health Research (CIHR) and Natural Sciences and Engineering Research Council of Canada (NSERC)

Tafazzin contributes to IgE‐mediated mast cell degranulation and cytokine secretion

Prevalence of allergic pathologies, such as food allergies, asthma, and atopic dermatitis, have been on the rise over the past several decades with approximately 40% of children in developed nations suffering one of these chronic inflammatory diseases. These pathologies, driven largely by sentinel immune cells known as mast cells, occur in tissues that interface with the external environment. Induction of the allergic response is initiated through the activation of FceRI by receptor‐bound allergen‐specific IgE. Subsequently, mast cells rapidly modulate various metabolic pathways to meet the energy demands associated with the processes directing both the early and late phases of the allergic response. The growing field of immunometabolism suggests that immune cell function can be modulated through effective regulation of metabolic processes. The purpose of this study was to assess the role of tafazzin, a mitochondrial cardiolipin remodeler, in modulating FceRI‐mediated mast cell activation. Primary mast cell cultures were established using fetal livers generated through breeding of heterozygotes carrying the doxycycline‐inducible TAZ shRNA knockdown cassette. Hematopoietic progenitors from homozygote and wild‐type fetal liver, were differentiated into mature mast cells under the direction of IL‐3, PGE2, and stem cell factor. TAZ knockdown was initiated by 1 μg/ml doxycycline (dox) for 5 days, resulting in a 99.9% reduction in tafazzin protein content relative to baseline expression and a 99.7% reduction relative to wild‐type control. Flow cytometric analysis of mast cell receptor expression demonstrated that FcɛRI was unimpacted by dox treatment, allowing for interrogation of induced signaling‐mediated responses. Following tafazzin knockdown, anti‐TNP IgE‐sensitized liver‐derived mast cells (LMC) were assessed in b‐hexosaminidase degranulation assays identifying a 31.4% reduction in degranulation (n = 4, p < 0.05) between the TAZ shRNA+/+ cells and the wild‐type LMCs activated with TNP‐BSA (allergen) with SCF potentiation. ELISA (n = 6) were conducted to analyze the secretion of de novo synthesized inflammatory mediators. TAZ shRNA+/+ cells secreted lower levels of CCL1 (p < 0.01), CCL2 (p < 0.05), and TNF (p < 0.001) when compared to wild‐type cells treated with dox. Induced transcription was analyzed using qPCR (n = 4) to assess if differences detected in mediator secretion were due to altered transcription. Transcript levels following a knockdown in tafazzin protein levels were significantly reduced at the 60‐minute timepoint for CCL1 (p < 0.05); while other genes analyzed, such as CCL2 and TNF, were not significantly impacted. These results suggest that tafazzin contributes to FceRI‐mediated mast cell secretory mechanisms in both the early (degranulation) and late phases (secretion of de novo synthesized mediators) of the allergic response. Collectively, this work supports the notion that regulation of mast cell metabolism is a potentially viable approach for ameliorating the severity of mast cell‐mediated pathologies.Support or Funding InformationSupported by the Natural Sciences and Engineering Research Council of Canada (NSERC); Canada Foundation for Innovation (CFI); Government of Ontario; and, Brock University.

Unfolding the Mysteries of Oxygen Sensing ‐ A Comprehensive Analysis of the Hypoxic Response in Zebrafish Gills One Cell at a Time via Single Cell RNA Sequencing

All vertebrates “sense” changes in O2 through specialized cells, called chemoreceptors, which interact with the peripheral and central nervous systems to maintain O2 homeostasis. Aquatic vertebrates are especially prone to fluctuations in environmental O2 availability. Therefore, it is vital for them to readily detect changes in PO2 and make appropriate respiratory and cardiovascular adjustments. Teleost fish, such as zebrafish (Danio rerio), have O2 chemoreceptors, called neuroepithelial cells (NECs), present in the gill epithelium and undergo membrane depolarization and vesicular recycling when exposed to acute hypoxia. NECs have also been shown to increase in number and size following chronic hypoxia. Here we present a comprehensive analysis of the effects of hypoxia on gill cells including NECs through a transcriptomic study. Using the transgenic line ETvmat2:GFP, zebrafish were acclimated to normoxia (PO2 = 160 mmHg) or severe hypoxia (PO2 = 35 mmHg ) and maintained for 14 days. Isolated cells from the distal ends of gill filaments, areas rich in NECs, were analyzed using single cell RNA sequencing (scRNA‐seq) analysis. Four normoxia (control) and four hypoxia‐treated biological samples were individually labelled before scRNA‐seq library construction using the 10X Genomics chromium technology. A total of 45,000 gill cells were consequently sequenced on a NextSeq500 (Illumina) sequencer with a read depth of 17,000 per cell. Our preliminary results separated 21 cell clusters and identified one cluster to be NECs based on gene expression. Highly differentially expressed genes in NECs included tph1a encoding tryptophan hydroxylase 1, the rate limiting enzyme in 5‐HT biosynthesis, and sv2 encoding synaptic vesicle 2 protein. Other differentially expressed genes in NECs are involved in mitochondrial oxidative metabolism and ATP synthesis. Moreover, differential expression analysis showed a clear shift in the transcriptome of NECs following 14 days of chronic hypoxia. NECs in the hypoxia group showed a high expression of genes involved in stress‐regulated polyubiquitination in contrast to the control group. The transcriptomic change implies a change in dynamics of the NEC population. We are the first group to conduct a single cell transcriptomic study in zebrafish gill cells. Our study is expected to provide a complete atlas for the hypoxic response in single gill cells and serve as a platform for future work on peripheral oxygen sensing.Support or Funding InformationThis research project is funded by Natural Sciences and Engineering Research Council of Canada (NSERC).

Negative Modulation of α4β2* Nicotinic Acetylcholine Receptors During Postnatal Maturation of the Mouse Prefrontal Cortex

Acetylcholine (ACh) signalling within the medial prefrontal cortex (mPFC) is important for the normal development and function of prefrontal cognitive networks. Layer VI of the mPFC is highly populated with pyramidal neurons that express the α4β2* subtype of nicotinic acetylcholine receptor (nAChR), and the activation of these receptors by ACh is central to its function in this region. These receptors can be negatively modulated through several forms of inhibition. For example, nicotine potently desensitizes nAChRs to ACh activation, while the endogenous progesterone‐derived neurosteroid 3α‐hydroxy‐5α‐prenan‐20‐one (allopregnanolone; ALLO) inhibits nAChR activation by ACh through a presumed negative allosteric modulation. Both forms of inhibition mediated by nicotine and ALLO have been demonstrated for α4β2* nAChRs located on mPFC layer VI neurons in young postnatal mice. However, the relative strength for both forms of inhibition in mature mice, compared with the young postnatal age, is not clear. In addition, the mechanism of action for ALLO inhibition of α4β2* nAChRs is not well understood. Using whole‐cell electrophysiology, we measured the ability of both nicotine (300 nM) and ALLO (10 μM) to inhibit 1 mM ACh activation of α4β2* nAChRs located on mPFC layer VI neurons from male and female CD1‐strain mice at postnatal day (P)15‐20 (young postnatal age) and P80‐120 (adulthood). For nicotine, nAChR desensitization was greater at P15‐20 than at P80‐120, and this result was driven by an effect of postnatal age in male mice only. Conversely, ALLO inhibited nAChR function to a similar degree at both P15‐20 and P80‐120. Ongoing ACh binding experiments aim to determine the mechanism of action for ALLO inhibition of these receptors at both ages. Results from this study confirm that the endogenous neurosteroid ALLO inhibits mPFC α4β2* nAChRs in adulthood as it does in young postnatal life. Developmental changes to the magnitude of nicotine desensitization but not ALLO inhibition suggests that different factors regulate each type of negative modulation at α4β2* nAChRs, and that these factors are differentially altered during postnatal maturation of the mPFC.Support or Funding InformationNatural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant 2019‐04989 to CDCB

The Long‐Term Effects of Exercise Mitigate Deleterious Healthspan Changes and Cancer Incidence in Mice Following Diet‐Induced Obesity and Radiation Exposure

There are nearly 16 million cancer survivors living in the United States today, with two‐thirds of them having received radiation therapy as part of their treatment. Furthermore, there is a concurrent increase in obesity and physical inactivity among this population. Combined with the effects of ionizing radiation (IR), these host factors are increasing the risk of secondary, treatment‐induced cancers and may contribute to poor healthspan outcomes in cancer survivors. As such, a critical need exists to develop a better understanding of how these host factors interact with radiation exposure, such that we can provide cancer survivors with better lifestyle recommendations to enhance their overall quality of life and decrease their secondary cancer risk. Male CBA mice were randomly divided into a control diet group (CTRL) or a 45% high fat diet group (HFD) to induce obesity. At nine weeks of age, half of the mice in each dietary group began a treadmill exercise training intervention (EX), or remained sedentary (SED), and were then all exposed to ionizing radiation (3 Gy) at 13 weeks of age. Healthspan was measured in each group at 17 months of age by assessing: endurance performance, metabolic and locomotor activity, depression‐like behaviour, forelimb grip strength and plasma clinical chemistry. Endurance performance was significantly greater in CTRL/EX compared to both HFD groups 5 days pre‐IR and compared to both SED groups 5 months post‐IR (both p<0.05). Metabolic analysis revealed a significant increase in the respiratory exchange ratio in CTRL compared to HFD mice (p<0.05), but there were no significant differences in O2 consumption, CO2, or heat production (p>0.05). There was also a trend for an increase in locomotor activity in the CTRL/EX compared to CTRL/SED group during their light and dark cycle (p = 0.06 and p = 0.108, respectively). Further, there was a significant decrease in depression‐like behaviour among HFD compared to CTRL mice, and EX compared to SED mice (p<0.05), as well as a significant decrease in forelimb grip strength normalized to body weight among HFD compared to CTRL mice (p<0.05). Additionally, there was a significant increase in total cholesterol levels among HFD compared to CTRL mice (p<0.05). Interestingly, the CTRL/EX and HFD/EX groups had a 20% decrease in cancer incidence compared to the HFD/SED group, and EX mice had a 17.5% decrease in cancer incidence compared to SED mice, independent of diet (both p>0.05). Our current findings suggest that long‐term exercise and dietary interventions should be implemented in cancer survivors exposed to radiation therapy in order to improve healthspan and potentially protect against secondary cancer incidence.Support or Funding InformationThe American Institute of Cancer Research (AICR), The Natural Sciences and Engineering Research Council of Canada (NSERC), and The Canadian Institutes of Health Research (CIHR)

Inhibition of JNK Signaling During Mast Cell Differentiation Promotes Altered Cytokine Secretion and Degranulation Profiles

Mast cells are large granulated leukocytes active in both innate and acquired immunity. These cells differentiate from hematopoietic stem cells, found in the medullary cavity of long trabecular bones. Increased inflammatory activity and numbers of mast cells contribute to allergic disease, which is of increasing concern worldwide. C‐jun N‐terminal kinase (JNK) is a major signaling node in the map kinase pathway that has been shown to contribute to IL‐3 signaling, known to promote murine mast cell differentiation. The aim of this research is to determine the role of JNK signaling in mast cell differentiation. Primary bone marrow‐derived mast cell (BMMC) cultures were established from C57BL/6 mice and differentiated for 7 weeks in the presence or absence of a JNK inhibitor (JNK‐IN‐8). Following removal of the inhibitor for 5 days, phenotypic and functional characteristics of BMMCs were probed using flow cytometry, ELISA, and β‐hexominidase release assays. Flow cytometric analysis of surface expression of the mast cell marker c‐kit, and levels of IgE bound to FcɛRI, a measure of mast cell sensitization, were unaffected by impaired JNK signaling compared to cells grown in the absence of inhibitor over mast cell differentiation, suggesting a normal mast cell phenotype with the potential to be activated via the allergen‐bound IgE‐dependent pathway. Cells differentiated under JNK inhibition showed a significant impairment in the secretion of IL‐6 (p<0.05) and IL‐13 (p<0.05) and a non‐significant impairment in TNF and CCL1 secretion at 24 hrs post‐activation compared to cells grown in the absence of inhibitor. Degranulation was also significantly decreased (by 25%; p<0.05) in BMMCs grown under JNK inhibition. To probe an epigenetic mechanistic role for histone acetylation changes in JNK‐impaired differentiation, expression of HDAC and HAT enzymes were considered by qPCR and western blotting, but were not modulated by JNK. These results suggest that JNK plays an important role in the development of a fully functional mast cell in the context of IgE‐mediated allergic inflammation and may warrant consideration as a strategy to regulate mast cell development in associated pathologies.Support or Funding InformationSupported by the Natural Sciences and Engineering Research Council of Canada (NSERC); Canada Foundation for Innovation (CFI); Government of Ontario and Brock University.

THE EFFECTS OF PSYCHOTROPIC MEDICATIONS ON GAIT STABILITY OF OLDER ADULTS WITH DEMENTIA

THE EFFECTS OF PSYCHOTROPIC MEDICATIONS ON GAIT STABILITY OF OLDER ADULTS WITH DEMENTIA

Imaging porphyry copper alteration using aeromagnetic data at Highland Valley Copper, British Columbia, Canada

ABSTRACTWe evaluated imaging alteration associated with porphyry copper style mineralisation using aeromagnetic data in the Highland Valley Copper (HVC) district as part of the Natural Sciences and Engineering Research Council of Canada (NSERC) and Canada Mining Innovation Council (CMIC) Footprints project. The first step of the investigation involved construction of a 3D geological model based on surface and regional information, and petrophysical data of the host rocks. From the petrophysics, it was observed that hydrothermal alteration affected the magnetic susceptibility of some batholithic host rocks at HVC, and low susceptibility values were associated with the altered rocks. From this observation, an alteration index was devised, differencing, based on the magnetic susceptibility, between unaltered and altered batholith host rocks. Three-dimensional inversions were conducted using geological constraints obtained from surface and borehole geology as well as physical property data. Then the alteration index methodology was tested using 3D inversions and residual models within five overlapping blocks covering portions of the batholith. The indices inferred from inverted susceptibility were compared to surface mapped alteration. For some felsic facies of the batholith, the alteration index derived from inverted magnetic susceptibility corresponds to the mapped alteration and delineates the known porphyry Cu system district. However, it is not true for others where alteration was less magnetite-destructive and more focused in structures.

Anisotropic planar Hall effect in the type-II topological Weyl semimetal WTe2

The research reported in this publication was supported by King Abdullah University of Science & Technology (KAUST) with Grants No. CRF-2015-SENSORS-2708 and No. URF-2015-2549 and by the Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant No. RGPIN-04178, the Ontario Early Researcher Award, and the Canada First Research Excellence Fund.

Localization and tracking of fish sounds with a 4-element underwater passive acoustic array

Localization and tracking are parameter estimation problems. As such, estimates are evaluated by their statistical distribution, in particular, their bias (equal to the mean of estimated positions minus the true position) and variance (variability in the measured positions about their mean). A variety of methods are used to localize sounds recorded by acoustic arrays. Here, we quantitatively compare the performance of four algorithms: (1) plane-wave-front frequency-domain beamforming; (2) curved-wave-front frequency domain beamforming; (3) time-difference-of-arrivals (TDOAs) using waveform cross-correlation; and (4) TDOAs using spectrogram cross-correlation, for fish sounds generated with a controlled underwater source at various GPS-located positions around a 4-element array. The array consisted of a SoundTrap ST4300 (Ocean Instruments, Auckland, NZ) four-channel acoustic recorder, equipped with four HTI-96-MIN hydrophones (High Tech, Inc., Long Beach, MS). The hydrophones were arranged in a tetrahedral-shaped configuration with a 20-m inter-element spacing. The objective is to optimize the localization and tracking of individual soniferous fish to better understand their small-scale spawning movements and reproductive behavior. Research supported by the Office of Naval Research, the Scripps graduate department, and a Natural Sciences and Engineering Research Council of Canada (NSERC) Postgraduate Scholarship-Doctoral (PGS D-3).

The GFDL Global Ocean and Sea Ice Model OM4.0: Model Description and Simulation Features

AbstractWe document the configuration and emergent simulation features from the Geophysical Fluid Dynamics Laboratory (GFDL) OM4.0 ocean/sea ice model. OM4 serves as the ocean/sea ice component for the GFDL climate and Earth system models. It is also used for climate science research and is contributing to the Coupled Model Intercomparison Project version 6 Ocean Model Intercomparison Project. The ocean component of OM4 uses version 6 of the Modular Ocean Model and the sea ice component uses version 2 of the Sea Ice Simulator, which have identical horizontal grid layouts (Arakawa C‐grid). We follow the Coordinated Ocean‐sea ice Reference Experiments protocol to assess simulation quality across a broad suite of climate‐relevant features. We present results from two versions differing by horizontal grid spacing and physical parameterizations: OM4p5 has nominal 0.5° spacing and includes mesoscale eddy parameterizations and OM4p25 has nominal 0.25° spacing with no mesoscale eddy parameterization. Modular Ocean Model version 6 makes use of a vertical Lagrangian‐remap algorithm that enables general vertical coordinates. We show that use of a hybrid depth‐isopycnal coordinate reduces the middepth ocean warming drift commonly found in pure z* vertical coordinate ocean models. To test the need for the mesoscale eddy parameterization used in OM4p5, we examine the results from a simulation that removes the eddy parameterization. The water mass structure and model drift are physically degraded relative to OM4p5, thus supporting the key role for a mesoscale closure at this resolution.

Mechanical Characterization of Catalyst Coated Membranes Subjected to Isolated Chemical Degradation in PEM Fuel Cells

The lifetime of polymer electrolyte membrane (PEM) fuel cells is governed by the operational stability of the membrane electrode assembly (MEA). Under dynamic automotive operating conditions and duty cycles, the membrane is subjected to chemical and mechanical degradation, which cause hydrogen leaks and ultimate cell failure. Chemical degradation is generally accepted as the key factor in the membrane decay process where membrane stability is affected by the formation of reactive radicals [1] and their attack on the ionomer molecular structure in the membrane [2]. H2O2 formed at the electrodes during fuel cell operation may diffuse into the membrane and decompose into hydroxyl radical ( . OH) via the Fenton’s reaction mechanism in presence of Fe2+ [3], commonly present as traces in membranes due to fabrication and/or operation-induced contamination. Low relative humidity (RH), and high temperature, reactant gas pressures and cell voltages accelerate the chemical degradation [1,4]. Steady-state open circuit voltage (SOCV) conditions are widely used in accelerated stress tests (AST) to intensify chemical stressors. In SOCV conditions, an Fe-ion redox cycle is generated in the MEA to preserve a relatively high Fe2+ concentration in the membrane, which leads to the most severe chemical membrane degradation through the Fenton mechanism [5,6]. Previously, the effects of isolated chemical degradation were exhibited at relatively mild stress levels at 90% RH (lifetime: 497 hours) and 100% RH (lifetime: 643 hours) where end-of-life (EOL) CCMs fractured at low strains right after passing their yield point during ex situ tensile tests [9]. In this work, an in situ SOCV-based AST with high stress levels and moderate humidity conditions were applied to induce pure chemical membrane degradation and establish its induced gradual decay in mechanical properties. Fluoride loss, an indication of global chemical degradation, increased steadily for the entire duration of the AST upto 140 hours (EOL). SEM investigations revealed gradual thinning of the membrane; however, no cracks were observed in the membrane. Previously, membranes subjected to pure mechanical degradation [7] and combined chemical and mechanical degradation [8-9] depicted localized damage (cracks and holes) that were incorporated due to RH cycling. Such features were not evident in the present work under isolated chemical stress. Thereafter, ex situ tensile experiments were performed with periodically extracted, partially AST-degraded CCM samples under both room (25oC, 50% RH) and fuel cell conditions (70oC, 90% RH). A dynamic mechanical analyzer (TA Instruments Q800 DMA) equipped with an environmental chamber was used. Reductions in ultimate tensile strength and fracture strain were observed as a function of AST operation time. Hygrothermal expansion test results revealed an overall decay in hygral expansion at 70oC of 33%, whereas the decay in thermal expansion at 90% RH was 40%. This was comparable to the results of CCMs subjected to pure mechanical degradation [7], where the hygral expansion decay from BOL upto 20,000 RH cycles was 33-50%, and a 50%-decay occurred in thermal expansion. For CCMs subjected to combined chemical and mechanical degradation, the decay in hygral expansion was only 25-30%, whereas 80% decay in thermal expansion was observed [8]. In summary, the observed microstructure-property relationship revealed the crucial role of chemical degradation by means of membrane thinning. Acknowledgements This research was supported by Mitacs through the Accelerate program, Ballard Power Systems, Natural Sciences and Engineering Research Council of Canada (NSERC), Canada Foundation for Innovation, British Columbia Knowledge Development Fund, Western Economic Diversification Canada and Simon Fraser University. This research was undertaken, in part, thanks to funding from the Canada Research Chairs program.

Hydrogen Bubble Templating of Fractal Ni Catalysts for Water Oxidation in Alkaline Media

This research was conducted as part of the Engineered Nickel Catalysts for Electrochemical Clean Energy project administered from Queen’s University and supported by Grant No. RGPNM 477963-2015 under the Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Frontiers Program. One of us (V.C.) would also like to acknowledge the Natural Sciences and Engineering Research Council (NSERC) for an Undergraduate Research Award.

CrossTalk proposal: Exercise training intensity is more important than volume to promote increases in human skeletal muscle mitochondrial content.

CrossTalk proposal: Exercise training intensity is more important than volume to promote increases in human skeletal muscle mitochondrial content.

Rebuttal from Martin MacInnis, Lauren Skelly and Martin Gibala.

We commend Bishop et al. (2019) for their comprehensive summary but respectfully disagree with their conclusion that training volume is more important than exercise intensity for increasing mitochondrial content in human skeletal muscle. Their position, based on the compiled data, centres on: (1) a positive correlation between training volume and citrate synthase (CS) activity and mitochondrial volume density (MitoVD); and (2) no correlation between training intensity and CS activity or MitoVD. The analysis presented by Bishop et al. (2019) is problematic for isolating the importance of exercise intensity per se. Firstly, on average, ∼37% more training sessions were performed for the moderate-intensity continuous training (MICT) as compared to high-intensity interval training (HIIT) and sprint interval training (SIT) (Granata et al. 2018): the HIIT and SIT interventions involved ∼15 sessions (lasting ∼15 min) compared to 21 sessions (lasting ∼70 min) for MICT (Granata et al. 2018). As indicated by time-course data (e.g. Murias et al. 2011), mitochondrial content increases as a training intervention progresses, which complicates comparisons among the intervention types. Secondly, despite the interval protocols being performed at higher intensities, the training volume for MICT (n = 33) was ∼60% and ∼700% greater than for HIIT (n = 9) and SIT (n = 20), respectively (Granata et al. 2018). As Bishop et al. (2019) demonstrated that training volume was positively associated with changes in mitochondrial content, the similar increase in CS activity across training types, despite differences in training volume, is strong evidence that HIIT and SIT were more efficient than MICT for increasing mitochondrial content. Given the disparities across intervention types, the lack of correlation between exercise intensity and the change in mitochondrial content is unsurprising and is not evidence against the importance of exercise intensity. Finally, while we agree that studies comparing different training protocols are important (Daussin et al. 2008; Granata et al. 2016; MacInnis et al. 2017; Montero & Lundby, 2017; Shepherd et al. 2017), we wish to clarify that the training interventions employed by Gillen et al. (2016) elicited similar increases in CS activity despite SIT involving about one-fifth the total volume of MICT (i.e. not equal volumes), demonstrating that high intensities of exercise can ‘compensate’ for low training volumes. Thus, in addition to our initial arguments (MacInnis et al. 2019), we contend that the pooled analysis from Bishop et al. (2019) supports our position that training intensity is more important than training volume for increasing human skeletal muscle mitochondrial content. Readers are invited to give their views on this and the accompanying CrossTalk articles in this issue by submitting a brief (250 word) comment. Comments may be submitted up to 6 weeks after publication of the article, at which point the discussion will close and the CrossTalk authors will be invited to submit a ‘LastWord’. Please email your comment, including a title and a declaration of interest, to [email protected] Comments will be moderated and accepted comments will be published online only as ‘supporting information’ to the original debate articles once discussion has closed. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. The authors declare that they have no competing interests. M.J.M., L.E.S. and M.J.G. contributed equally to the writing and critical revision of this manuscript. All authors have read and approved the final version of this manuscript and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed. The research programmes of M.J.M. (ID: RGPIN-2018-06424) and M.J.G. (ID: RGPIN-2015-04632) are funded through Discovery Grants from the Natural Sciences and Engineering Research Council of Canada (NSERC). L.E.S. was funded by an NSERC Vanier Canada Graduate Scholarship.

Correction to: Incorporating citizen science, museum specimens, and field work into the assessment of extinction risk of the American Bumble bee (Bombus pensylvanicus De Geer 1773) in Canada

We thank all museum collections and contributors of bumble bee species occurrence data to the Bumble Bees of North America database managed by LLR (see http://www.leifrichardson.org/bbna.html ). We thank everyone who submitted and/or verified observations for the citizen science program Bumble Bee Watch, especially Rich Hatfield of the Xerces Society, and to the partners and funders of that program. We thank Environment and Climate Change Canada, the Ontario Ministry of Natural Resources and Forestry, The W. Garfield Weston Foundation, the Rogers Foundation, the Schad Foundation, and Wildlife Preservation Canada for funding and supporting the field research by VJM and SRC. We acknowledge the support of the Natural Sciences and Engineering Research Council of Canada (NSERC), reference numbers RGPIN-2017-05642 and CGSD-503997-2017. VJM also thanks the rare Charitable Research Preserve for a scholarship.

H Sorption Based Materials for the Hydrogenation of CO2 to Added Value Fuel Products

Energy transformation through technological systems has always been a critical issue regarding how our society works. With the environmental consideration taking over these last years, the establishment of efficient energy conversion technology allowing storage of renewably produced electricity in simple non-environmentally harmful chemicals is one of the great challenges of our times. The interest in CO2 in this context is simple; it represents a largely distributed low-energy chemical available for green energy storage. The electrochemical reduction of CO2 to small fuel molecule has been largely investigated, but the reduction process inefficiency and the cost of the highly specialized catalysts act as drawback to the implementation of this technology to industrial applications. In this work, we explore bimetallic catalyst that moves away from the simple optimization of reaction rate by adjustment of adsorption energies of species (i.e. volcano plot). We propose to combine a material active for H adsorption to a conventional CO2 electrocatalyst to explore the effect of potential enhanced availability and transfer of H during the carbon species conversion. The Cu/Pd system has been selected as a model material for the studies. Although many structures are possible to combine the metals, we chose to plate the Cu on the Pd substrate by UPD technique [1] to reduce the large hydrogen evolution occurring at Pd. The system has been studied by conventional electrochemistry, including a diversity of potentiostatic measurements and cyclic voltammetry (CV). The isotherm of H sorption has been assessed using Lasia methodology [2] to insure that the H sorption on Pd and the CO2 reduction on Cu occur in the same potential region. The UPD has been optimized and combined to dilution method to allow CO2 reduction at Cu-free solution after the deposition of a designed monolayer (ML) content of Cu on Pd. CVs has been conducted to access the general behavior of the Cu/Pd system. Activity of the underlying substrate for H has been observed to occur at the 1ML Cu on Pd. Measurements point toward the preservation of some H sorption activity by the Pd through the Cu ML although activity of bare Pd in contact with the solution through ML defect is not to exclude. We propose that the preservation of the Pd structure and electronic effects at the 1 ML Cu explains the possibility of enhanced H adsorption after the reduction by Cu at the interface. Differential electrochemical mass spectrometry (DEMS) measurements are conducted to study the effect of different content of Cu ML at the surface of the catalyst on the product distribution during the electrolysis. The specific adsorption of the anion from the electrolyte being known to affect the reaction pathway by partial and scattered surface coverage [3], different electrolytes were used to gain insight on pathways that are privileged by the different Cu/Pd surfaces. To further rationalize the links between surface properties and reaction products, X-ray photoelectron spectroscopy at near-ambient pressures (APXPS) measurements has been collected. This technique uses X-rays that generate photoelectrons that enter a differentially pumped XPS analyzer with electronoptics that facilitate the characterization of a solid material that is exposed to gases to enable the observation of their interaction during the spectrum collection. This method has previously been used to highlight different modes of CO2 adsorption on Cu surface [4]. Several ML content of Cu over Pd has been exposed to CO2 and CO under H-present and H-absent regimes. The affinity of the CO for Pd dominant surfaces has been observed. The coverage of Cu on the Pd substrate also influences the charge transfer between the adsorbed CO and the surface. We believe that the close containment of domains active for H sorption and CO2 reduction can be used as a tool to control selectivity over CO2 reduction and to favor the production of small added value molecules. Acknowledgement The financial support of the Fonds de recherche du Québec – Nature et technologies (FRQNT), of the Natural Sciences and Engineering Research Council of Canada (NSERC) and of Rio Tinto Alcan are gratefully acknowledged. This research used resources of the Advanced Light Source, which is a DOE Office of Science User Facility under contract no. DE-AC02-05CH11231. References A. Kumar and D. A. Buttry, The Journal of Physical Chemistry C, 2015, 119(29), 16927-16933H. Duncan and A. Lasia, J. Electroanal. Chem., 2008, 621(1), 62−68.P. Dubé, G. Brisard, J. Electroanal. Chem., 2008, 582(1-2), 230-240M. Favaro, H. Xiao, T. Cheng, W. A. Goddard, J. Yano et E. J. Crumlin, PNAS, 2017, 201701405

SUN-014 Identification of a New SUMOylation Process Regulating Nuclear Receptor Nor-1

Nuclear receptors are transcriptional factors controlling critical gene expression networks essential to the developmental and homeostatic regulation of several physiological processes. Transcriptional control of nuclear receptors is achieved by interaction with specific ligands and in response to cellular signalling pathways, which often results in post-translational modifications of receptors. Among such modifications, SUMOylation has emerged as a key process regulating nuclear receptor function. Our recent work has revealed an atypical SUMOylation process that requires phosphorylation, thereby defining a novel SUMO conjugating motif termed pSuM. Functional pSuM has been described for estrogen nuclear receptor ERβ and bile acid receptor FXR, and sequence analysis has indicated a conserved pSuM in the orphan nuclear receptor Nor-1. This study aims to characterize the function of Nor-1 pSuM-mediated SUMOylation. Nor-1 has various functions in the nervous system, including a role in neuronal survival and hippocampal development. In line with an implication of the SUMOylation process in neurodegenerative diseases, we speculate that pSuM SUMOylation might regulate Nor-1 activity and participate in the maintenance of neuronal integrity. The SUMOylation potential of Nor-1 and the mechanism regulating this process were investigated using immunoprecipitation and Western blot analysis. Our results indicate that Nor-1 was SUMOylated at the pSuM motif as well as at another consensus SUMOylation motif, thereby conjugating SUMO-1 and SUMO-2. We also found that the phosphorylation of the pSuM motif was essential for pSuM activation and SUMO conjugation. Furthermore, phosphorylation of adjacent residues to the pSuM appeared to potentiate the SUMOylation of Nor-1, suggesting a role for pSuM extension. This demonstrates the implication of specific kinase pathways required to promote pSuM activation. Using luciferase reporter assays and qPCR analysis, we also showed that SUMOylation regulates Nor-1 transcriptional activity and responsive gene expression in neuronal cells. Our findings provide new insights on the mechanistic regulation of Nor-1 function by phosphorylation and SUMOylation. We also identify a new atypical and highly regulated SUMOylation process as an additional targeting possibility for SUMO conjugation to coordinate transcriptional competence. This study contributes to expand the intricate dynamics of the SUMOylation process used by incoming signals to govern gene networks by nuclear receptors and possibly other transcription factors. Sources of Research Support: This work is supported by the Natural Sciences and Engineering Research Council of Canada (NSERC), Canadian Institute of Health Research (CIHR) award, and Fonds de la recherche en santé du Québec (FRQS) award.

In Vivo Knockdown of the IP3R1 Calcium Channel Disrupts ER and Actin Morphology, as well as TBC Bulb Maturation in the Apical Processes of Sertoli Cells in Rat Testes

Spermatogenesis is the process by which sperm cells are produced, but at one phase of this process, late stage spermatids are temporarily attached to the apex of Sertoli cells in the seminiferous epithelium. Late spermatids are anchored by large adhesion like junction complexes termed ‘ectoplasmic specializations’ (ESs) that are proposed to be disassembled by novel endocytic structures only found in mammalian Sertoli cells, called ‘tubulobulbar complexes’ (TBCs). Both ESs and TBCs are heavily actin related, with parallel actin arrays and dendritic actin networks respectively being hallmarks of their identities. The endoplasmic reticulum (ER) in the Sertoli cell apical processes surrounding the late spermatids forms an extensive and continuous network. Cisternae of this network form part of the structure of the ESs, participate in a membrane contact site with the bulbs of TBCs, and have a parallel alignment with the surrounding Sertoli cell plasma membrane. Previously, we've localized the IP3R calcium channel to the TBC bulb‐ER contacts as well as other calcium exchange machinery to various parts of the ER in the apical process. Calcium release and exchange have been implicated in processes related to both actin dynamics as well as in fusion and acidification of endocytic components such as endosomes. We've proposed that calcium exchange may be a crucial mechanism in regulating both the actin networks of ESs and TBCs, as well as in facilitating the maturation and fusion of TBC bulbs as they develop into putative endosomes. If calcium exchange from the ER is responsible for regulating the local actin networks and maturation of endocytic components in the apical process, then knocking down the primary calcium release channel of the ER should alter local actin and TBC bulb morphology. To test this, we injected the testes of Sprague Dawley rats with siRNAs against IP3R1 (ITPR1) and collected tissues at 1 day, 2 day, and 3 days post injection and processed the tissues for either western blot, immunofluorescence, or electron microscopy. ITPR1 siRNA treated tissues showed significant morphological alterations to the ER, actin, and TBC bulbs in the apical processes of late spermatids compared to controls. This data supports the hypothesis that ER calcium exchange is an integral component to actin dynamics and TBC bulb maturation in the apical processes of Sertoli cells.Support or Funding InformationNatural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant awarded to AWV; Alexander Graham Bell Scholarship (NSERC) awarded to AAThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cell scientist to watch – Pleasantine Mill

ABSTRACT Pleasantine Mill graduated in microbiology and immunology from McGill University, Montréal, Canada, and completed her PhD in medical and molecular genetics at the University of Toronto, Canada. There, under the supervision of Chi-chung Hui, she studied Hedgehog signalling pathways in skin development and tumorigenesis. For her postdoctoral research, Pleasantine moved to Ian Jackson's laboratory at the MRC Human Genetics Unit, Edinburgh, UK, with a Natural Sciences and Engineering Research Council of Canada (NSERC) fellowship, followed by a Caledonian Research fellowship. Initially focusing on neural crest development, she identified and studied several mouse mutants that displayed defects in ciliogenesis and cilia structure that went on to be implicated in human disease. Pleasantine established her own research program at the MRC in 2014. Her group investigates the genetic programme for cilia structure and function and the links to human ciliopathies. She is the recipient of the 2019 Women in Cell Biology Early Career Award Medal from the British Society for Cell Biology (BSCB).