Natural Sciences And Engineering Research Council Of Canada Research Articles

Impact of Spinal Cord Injury and Chronically Induced Orthostatic Hypotension on Left Ventricular Contractility and Stiffness

IntroductionComplete spinal cord injury (SCI) above the T3 spinal level is associated impaired left ventricular (LV) systolic function. However, the effect on diastolic structure and function has yet to be examined. Furthermore, high thoracic SCI impairs centrally‐derived cardiovascular responses to postural change, leading to the development of orthostatic hypotension (OH) post‐SCI. While it has been demonstrated that these frequent bouts of OH cause impairments in cerebrovascular function, it is not known whether this hemodynamic instability also impairs cardiac function.MethodsThis study examined the effect of SCI on LV systolic and diastolic function in the presence or absence of daily bouts of acutely induced OH. 22 rats were divided into four groups: 1. Rats with a sham injury and no daily OH (SHAM, n=5), 2. Rats with a sham injury and daily induced OH (SHAM‐OH, n=6), 3. Rats with a complete T3 transection injury and daily induced OH (T3‐OH, n=5), 4. Rats with a complete T3 transection and no daily OH (T3‐SCI, n=6). 9 weeks after surgery, LV pressure‐volume (PV) catheterization was performed to assess LV basal PV indices, as well as cardiac contractility and stiffness. Furthermore, the coupling of the arterial system and the left ventricle was characterized to examine the influence of SCI and/or OH on the efficiency of the interaction between the LV and arterial system.ResultsCompared to both SHAM and SHAM‐OH, the T3‐SCI and T3‐OH groups experienced significant reductions in systolic blood pressure, LV stroke work and cardiac output (all p<0.023). The T3‐OH group demonstrated an increase in arterial elastance compared to both the SHAM‐OH and SHAM groups (p<0.014). T3‐SCI and T3‐OH groups exhibited reduced end‐systolic elastance compared to both SHAM and SHAM‐OH (p<0.04). No difference was found in LV stiffness between the four groups. Finally, the ratio of ventricular‐vascular coupling was found to be significantly increased in the T3‐OH group compared to all other groups (all P<0.019).ConclusionComplete high thoracic and cervical SCI causes a reduction in LV contractile function. Combined high thoracic SCI and daily OH impairs ventricular‐vascular coupling via changes in arterial compliance.Support or Funding InformationHeart and Stroke Foundation of Canada (HSFC), International Collaborations on Repair Discoveries (ICORD), Natural Sciences and Engineering Research Council of Canada (NSERC), Blusson Integrated Cures Partnership (BICP).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Repeated mild traumatic brain injury in the juvenile brain: Investigations on synaptic plasticity in the hippocampus

Traumatic Brain Injury (TBI) occurs when an impulsive force is transmitted to the head and affects the brain. Up to 75% of all brain injuries are classified as “mild” TBI (mTBI; also known as concussion). There is growing evidence that during a life time repeated mTBI (rmTBI) can produce cumulative structural damage and long‐term changes in behaviour. The juvenile brain is in a period of robust synaptic reorganization and myelination, making this a particularly vulnerable time to incur either mTBI or rmTBI. Although most children recover from mTBI incidents, a significant proportion experience learning and memory impairments after rmTBI. Memory formation is closely dependent on the capacity of the brain to regulate long‐lasting changes in neuronal communication (synaptic plasticity) with the hippocampus being an essential structure for this process. Therefore, we hypothesize that rmTBI causes learning and memory deficits through its effects on hippocampal synaptic plasticity.Using the Awake Closed‐Head Injury (ACHI) model we examined how male and female Long‐Evans rats (25–28 days of age) responded after 8 episodes of ACHI over a four day period in. Animals were assessed for changes in their state of consciousness and sensorimotor abilities immediately following each ACHI, allowing us to examine the acute effects of each injury. Synaptic plasticity was assessed using in vitro electrophysiology either one day or seven days post‐ACHI.Our results show that rmTBI impacts sensorimotor abilities and impairs hippocampal synaptic plasticity in both males and female subjects. These data suggest that learning and memory deficits from rmTBI may be due to impairments in hippocampal synaptic plasticity.Support or Funding InformationSupport or Funding Information Canadian Institutes of Health Research (CIHR) Natural Sciences and Engineering Research Council of Canada (NSERC) CP funded by Fundació Universitaria Agustí Pedro PonsThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Involvement of MURC/Cavin‐4 in store‐operated Ca2+ entry in neonatal cardiomyocytes

STIM1 is a regulatory protein of store‐operated calcium entry (SOCE) and its expressions of in the cardiomyocytes suggests that it may play a role in cardiomyocytes function. To better understand this role, we sought to identify unknown protein partners of STIM1. We fused the ERM domain of STIM1 (amino acids 219–548) to GST and used it for a GST‐pulldown assay combined with analytical liquid chromatography‐mass spectrometry. From the lysate of RIPA‐solubilized proteins from adult rat hearts, we identified the muscle‐related coiled‐coil protein (MURC), also known as Cavin‐4, as a potential candidate. Also, we show that is the HR1 domain of MURC interacts with the ERM domains of STIM1.To understand the role of this interaction, we overexpressed MURC in neonatal rat ventricular myocytes (NRVM) and determined is impact on SOCE. NRVM were loaded with the Ca2+ probe, FURA‐2AM, and incubated with nifedipine to prevent Ca2+ entry at the plasma membrane via L‐type calcium channels. To induce intracellular store depletion and SOCE activation, 10mM caffeine and 2μM thapsigargin were added to the perfusion buffer in the absence of extracellular Ca2+ and Ca2+ entry was determined when Ca2+ was re‐introduced in the extracellular medium. Using this protocol, we demonstrated that the Ca2+ entry in NRVM overexpressing MURC is 1.8‐fold higher that control bGal condition. This effect is reduced by SOCE inhibitors (SKF‐96365, 2‐APB, and YM‐58483). The Ca2+ entry measured in NRVM overexpressing ΔHR1‐MURC was similar to the Ca2+ entry in the bGal control. These results indicate that MURC overexpression relies on its HR1 domain to potentiate SOCE in these cells.The increase in SOCE in MURC overexpressing NRVM may be the result from an enhanced interaction between Orai1 and STIM1. To test this possibility, we assessed the extent of STIM1‐Orai1 complex formation with a co‐immunoprecipitation assay using HA‐Orai1 as the target protein. The STIM1‐Orai1 interaction was evaluated in resting conditions or upon SOCE activation. As expected, SOCE activation enhanced the interaction between STIM1 and Orai1 by 2.80‐fold compared to the control condition. MURC overexpression was also sufficient to increase the STIM1‐Orai1 complex formation in resting (1.78‐fold) and Ca2+ store‐depleted (2.44‐fold) conditions. These results show that MURC overexpression in NRVM favors the interaction of STIM1 with Orai1.Also, we show that R140W‐MURC mutant, a missense variant of the HR1 domain associated with human dilated cardiomyopathy, exacerbates the SOCE increase in NRVM. Our study provides novel evidence that MURC regulates SOCE by interacting with STIM1 in cardiomyocytes. In addition, we identified a first potential mechanism by which the R140W mutation may contribute to Ca2+ mishandling and the development of cardiomyopathies.Support or Funding InformationSupported by the Natural Sciences and Engineering Research Council of Canada (NSERC) [M.A.M, and G.B.], CRMUS graduate student research award [J.M.], FRQS doctoral training award [J.D.], CIHR graduate scholarship [H.G.], André‐Lussier Research Chair [J.‐L.P.], and FRQS and Heart and Stroke Foundation of Canada Investigator awards [M.A‐M.].This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Characterization of Ca2+‐Based Action Potentials in Horizontal Cells in the Goldfish (Carassius auratus) Retina

About 65 years ago, the first intracellular recordings ever to be made in the retina were made in horizontal cells (HCs) of goldfish (Carassius auratus). These recordings were some of the first examples of graded potentials (where membrane potential changes in proportion with a stimulus), and it was assumed that outer retinal neurons did not present action potentials (APs). HCs are interneurons which provide inhibitory feedback to photoreceptors, and in vision they are responsible for improving contrast, edge detection, and color opponency. Since these first recordings, Ca2+‐based APs and spontaneous increases in intracellular Ca2+ concentration ([Ca2+]i) have been found in fish HCs, although they are poorly understood and their role in vision (if any) is unknown. We characterized spontaneous, Ca2+‐based APs in isolated goldfish HCs by measuring changes in [Ca2+]i and membrane potential (Vm). Using Fura‐2 Ca2+ imaging, we found spontaneous Ca2+ activity characterized by transient elevation in [Ca2+]i of short (seconds) and long (minutes) duration, in 157/177 cells (89%). These APs were similar in amplitude to Ca2+ responses evoked by glutamate. APs were reversibly eliminated in Ca2+‐free solutions (n=18), and were abolished by the L‐type Ca2+ channel blocker, nifedipine (100 μM; n=5). APs were also dependent on intracellular Ca2+ stores: they were abolished by the ryanodine receptor antagonists, ryanodine (20 μM; in 7/8 cells) and dantrolene (50 μM; n=7). The ryanodine receptor agonist, caffeine (n=10), increased AP frequency 2.8‐fold (p<0.0002) and reduced median duration (29.3 s to 18.7 s), time to peak (12.3 s to 7.8 s), and area under the curve (by 59.6%). When caffeine was co‐applied with the store‐operated channel antagonist, 2‐aminoethyldiphenyl borate (2‐APB), frequency was unaffected, further confirming a role for stores. We tracked changes in Vm with whole‐cell current‐clamp recording, and with the voltage‐sensitive dye, FluoVolt. Phenotypes of APs in FluoVolt (n=6) and current‐clamp (48/57 cells; 84%) experiments displayed a sharp rise to peak, followed by a slow decline for the duration of the transient, and a steep return to baseline. In current‐clamp experiments, APs were dependent on L‐type Ca2+ channel activity. APs were blocked by Co2+ (5 mM; n=5) and nifedipine (100 μM; n=5), and were amplified and prolonged by the L‐type channel‐permeant ion, Ba2+ (15 mM; n=6). In addition, APs were abolished in 6/9 cells in Ca2+‐free solution. Collectively, our data suggest that activation of voltage‐dependent L‐type Ca2+ channels leads to Ca2+ influx, depolarization of Vm, and additional Ca2+ release from stores via ryanodine receptors. Understanding this phenomenon is a first step to elucidating a possible role for APs in vision or retinal physiology. This work also challenges the long‐held belief that outer retinal neurons do not present APs, and may lead to new paradigms about how visual information is encoded in the vertebrate retina.Support or Funding InformationWe acknowledge the support of the Natural Sciences and Engineering Research Council of Canada (NSERC), grant 342303.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Glucocorticoid‐Driven Transcriptomes in Human Airway Epithelial Cells: Commonalities, Differences and Functional Insight

RATIONALEGlucocorticoids act on the glucocorticoid receptor (GR; NR3C1) to resolve inflammation and, as inhaled corticosteroids (ICS), are the cornerstone of treatment for asthma. However, reduced efficacy in severe disease and during exacerbations indicates a need to improve ICS actions. Since ICS target the epithelium, glucocorticoid‐driven transcriptomes were compared between primary human bronchial epithelial (HBE) cells and common cell lines, pulmonary type II A549 and bronchial epithelial BEAS‐2B cells.METHODSGene expression profiling of RNA extracted from A549, BEAS‐2B and HBE cells following budesonide treatment was performed using Affymetrix PrimeView microarrays. Genes showing significant induction (fold ≥2, P ≤0.05) or repression (fold ≤0.5, P ≤0.05) compared to untreated cells, in any of the cell variants were used for further analyses.RESULTSIn BEAS‐2B cells, budesonide induced or, in a delayed fashion, repressed the expression of 63, 133, 240, and 257 or 15, 56, 236, and 344 mRNAs at 1, 2, 6, and 18 h, respectively. Within the early‐induced mRNAs were multiple transcriptional activators and repressors, thereby providing mechanisms for the subsequent modulation of gene expression. Using the above criteria, 17 (BCL6, BIRC3, CEBPD, ERRFI1, FBXL16, FKBP5, GADD45B, IRS2, KLF9, PDK4, PER1, RGCC, RGS2, SEC14L2, SLC16A12, TFCP2L1, TSC22D3) induced and 8 (ARL4C, FLRT2, IER3, IL11, PLAUR, SEMA3A, SLC4A7, SOX9) repressed mRNAs were common between A549, BEAS‐2B and HBE cells at 6 h. As absolute gene expression change showed greater commonality, lowering the cut‐off (≥1.25 or ≤0.8‐fold) within these groups produced 93 and 82 genes induced or repressed in common. Since large changes in few mRNAs and/or small changes in many mRNAs may drive function, gene ontology (GO)/pathway analyses were performed using both stringency criteria. Budesonide‐induced genes showed GO term enrichment for positive and negative regulation of transcription, signaling, proliferation, apoptosis, and movement, as well as FOXO and PI3K‐Akt signaling pathways. Repressed genes were enriched for inflammatory signaling pathways (TNF, NF‐κB) and GO terms for cytokine activity, chemotaxis and cell signaling. Reduced growth factor expression and effects on proliferation and apoptosis were highlighted.CONCLUSIONSWhile glucocorticoids repress mRNAs associated with inflammation, prior induction of transcriptional activators and repressors may explain longer‐term responses to these agents. Positive and negative effects on signaling, proliferation, migration and apoptosis were revealed. Since many such gene expression changes occurred in human airways post‐ICS inhalation, effects in cell lines and primary HBE cells in vitro may be relevant to ICS in vivo.Support or Funding InformationThis work was supported by: RN grants: Canadian Institutes of Health Research (CIHR), Natural Sciences and Engineering Research Council of Canada (NSERC) discovery grant, and AstraZeneca; MMM studentships: NSERC Postgraduate Scholarship – Doctoral, Queen Elizabeth II Doctoral scholarship, and The Lung Association – Alberta & NWT studentship award.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Sociocultural Exploration of Sex-Bias in NSERC-funded Human Cardiovascular Research at Ontario Universities

It is well established that sex-bias against the inclusion of women exists in human cardiovascular research, where women have been excluded from or under-represented in the research process, despite the high prevalence of cardiovascular disease among this population. To address the sex-bias against the inclusion of women in federally-funded research, including research funded by the Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant (DG) program, the Tri-Council Policy Statement: Ethical Conduct for Research Involving Humans was introduced in 2010. However, despite the introduction of this policy, it remains unknown whether a sex-bias persists in NSERC DG-funded basic human cardiovascular research at Ontario universities. The purpose of this research is twofold. (1) Using a quantitative analysis, this research will determine the presence or absence of a sex-bias against the inclusion of women in NSERC DG-funded basic human cardiovascular research at Ontario universities from policy implementation to the present. After analysis of all NSERC DG-funded publications (n=96), female exclusion or under-representation was evident in 63% of publications. (2) By conducting semi-structured interviews with Ontario university basic human cardiovascular researchers (n=5) and by using thematic analyses, this study will characterize the sex-bias against the inclusion of women, and by using a sociocultural lens, will explore how research, as a social institution, may act to construct, maintain and reinforce sex inequalities. This research will highlight practical changes that could occur to challenge this sex-bias and increase female inclusion in cardiovascular research, ultimately with the goal of improving female cardiovascular health.

Government programs for university–industry partnerships: Logics, design, and implications for academic science

Canada’s research policy over the past three decades has steered national science toward industrial innovation. Yet, the outcomes of this policy orientation are debatable. Macro-level indicators continue to show weak performance in firm innovation, and there is also evidence that this policy stance has had material effects on science at the meso and micro levels. If so much public investment has gone into harnessing university–industry collaborations for a substantial period of time, why has Canada not seen an uptick in innovative activity? To address this puzzle, we turned our gaze to the policy machinery that promotes academic–industrial collaboration. We carried out case studies of two long-standing federal programs supporting research and development activities between universities and industry. Both programs are administered by the Natural Sciences and Engineering Research Council of Canada (NSERC), Canada’s largest funder of natural sciences and engineering research. Drawing from multiple sources of data, we find that underneath the apparently successful implementation of these programs lies a misalignment between micro- and meso-level incentives for collaboration, and macro-level policy goals related to technological innovation and economic renewal. Our article makes two main contributions. First, it calls into question the escalating federal investments in university–industry partnership programs, as long as their ability to meet operational milestones remains the main source of evaluation. Second, it demonstrates the relevance and need for the literature to go beyond a focus on narrow technical questions at the micro level, and address broader questions about the purpose, goals, and beneficiaries of university–industry collaboration policy.

Regional Assessment of Current and Future States of GHGs Emissions: Impacts of Transportation Policy Scenarios

Estimating the future state of greenhouse gases (GHGs) and air quality associated with transportation policies and infrastructure investments is key to the development of meaningful transportation and planning decisions. This study describes the design and application of an integrated transportation emission model for the prediction of GHGS in CO2eq in the Greater Montreal Region as a result of transport policy scenarios and Land use scenarios. For this purpose, a travel demand model linked with models for traffic assignment and emissions, was used to simulate GHG emissions in a base year (2008) and a horizon year (2031) while incorporating population and demographic projections. Various stakeholders were consulted in the development of future scenarios affecting land-use and transportation through a web-based survey and workshop.In the 2031 business as usual scenario, an average decrease of 30% in GHG emissions was estimated compared to the 2008 base case. This decrease is primarily attributed to projected improvements in vehicle technology. The modelling system was used to evaluate the impact of a 20% market penetration in electric vehicles, revealing significant reductions in GHG emissions across the region. Work is currently underway to estimate the emissions of nitrogen oxides (NOx) and NO2 concentrations to assess air quality and individuals’ daily exposure by tracking activity locations and trajectories of the population and observe the level of reduction in daily exposures compared to the base case.This study is funded by a collaborative grant from the Canadian Institutes of Health Research (CIHR) and the Natural Sciences and Engineering Research Council of Canada (NSERC) and Ouranos.

VaCATE: A Platform for Automating Data Output from Compartmental Analysis by Tracer Efflux

Compartmental analysis by tracer efflux (CATE) is fundamental to examinations of membrane transport, allowing study of solute movement among subcellular compartments with high temporal, spatial, and chemical resolution. CATE can provide a wealth of information about fluxes and pool sizes in complex systems, but is a mathematically intensive procedure, and there is a need for software designed to fully, easily, and dynamically analyse results from CATE experiments. Here we present vaCATE (Visualized Automation of Compartmental Analysis by Tracer Efflux), a software package that meets these criteria. A robust suite of test cases using CATE datasets from experiments with intact rice (Oryza sativa L.) root systems reveals the high fidelity of vaCATE and the ease with which parameters can be extracted, using a three-compartment model and a curve-stripping procedure to distinguish them on the basis of variable exchange rates. vaCATE was developed using Python 2.7 and can be used in most situations where compartmental analysis is required. Funding Statement: This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Ontario Graduate Scholarship Fund (OGS).

On the development of a predictive functional trait approach for studying terrestrial arthropods.

The characterization of ecological communities with functional traits allows to consider simultaneously the ability of a species to survive and reproduce in an environment, its interactions with other species and its effects on the ecosystem. Functional traits have been studied mainly by plant ecologists, but are increasingly common in the study of other taxa including arthropods. Arthropods represent a group for which a functional trait approach could be highly profitable because of their high diversity, abundance, ubiquity and role in many important ecological processes. This review synthesizes two decades of functional trait research on terrestrial arthropods. We show that while the approach has gained popularity, particularly in the last decade, the absence of clearly postulated hypotheses is a recurrent problem limiting generalization. Furthermore, studied traits are often poorly related to studied functions. To address these problems, we propose a step-by-step protocol to postulate clear hypotheses prior to trait selection and emphasize the need for a common set of more generalizable traits in future studies. Extending the functional trait approach to arthropods opens the door to improving our understanding of interspecific interactions and potential links between response and effect traits. We present the concept of trait-matching with several examples of arthropod traits known to be effective predictors of consumer-resource interactions. The development of a successful functional trait approach for terrestrial arthropods will necessitate an understanding of relevant traits, standardized measurement protocols and open access databases to share this information. Such progress will provide ecologists with a new set of tools to answer broad questions in several fields including the study of community assembly, ecological networks and multitrophic functionality.

A Dynamic Impedance Study of the Initial Stages of Nickel Oxidation

The nature of the species that form on oxidizing Ni electrode in alkaline solution has been studied for a long time. Bode summarized the formation and interconversion of these hydroxide and oxyhydroxide phases in terms of time and potential [1], but their structure and kinetics remain an active area of study [2]. The initially formed oxide is believed to be a hydrated hydroxide, α-Ni(OH)2, which irreversibly interconverts to a less hydrated β-Ni(OH)2 at higher potentials. The formation and interconversion of these hydroxide phases is studied here by dynamic electrochemical impedance spectroscopy (dEIS) [3,4], which applies a multi-frequency waveform during a cyclic voltammetry sweep or during a chronoamperometry experiment. It enables the study of non-steady states of the surface that cannot be accessed by conventional potentiostatic EIS. It is particularly suited to study the irreversible α-Ni(OH)2 to β-Ni(OH)2 interconversion. An electrochemical polishing procedure was used to remove the oxide layer in order to have a clean and oxide-free surface before doing the experiment. This step consisted of galvanostatic holding in phosphoric acid solution. Impedance data were collected during voltammograms at different slow sweep rates, with selected hold periods before, during, or after the peaks, and were correlated to charges under the voltammograms. The impedance data were fitted to equivalent circuits. In most cases the faradaic impedance approximated a charge-transfer resistance, but the use of complex capacitance plots showed evidence for additional structure at low frequencies. Comparison of the the polarization resistance determined from the slope of the voltammograms with the estimated low-frequency intercept of the impedance showed a discrepancy in the peak region, which also suggests that a slow process exists that whose kinetics is not determinable by dEIS. The sweep-hold experiments followed the conversion of the α phase to the β phase, and found that the oxidation is not complete in the potential range of 0.2-0.3 V. Voltammograms of the admittance at selected frequencies were found to be sensitive to the surface condition, and showed that the surface condition did not return to the same value after cycling. 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. [1] H. Bode, K. Dehmelt and J. Witte. Electrochim. Acta., 11, 8 (1966). [2] M. Alsabet, M. Grden and G. Jerkiewicz, Electrocatalysis, 2, 4 (2011). [3] R.L. Sacci, F. Seland and D.A. Harrington, Electrochim. Acta., 131, 13 (2014). [4] R.L. Sacci and D.A. Harrington, ECS Transactions, 19, 31 (2009).

A Film Maturation Process for Improving the Cycle Life of Calendered Silicon Negative Electrodes

Increasing the energy density of Li-ion batteries (LiB) is a key issue. A promising approach is to replace graphite in LIB anodes with silicon. The main challenge is to deal with the large silicon volume expansion induced by its lithiation, which damages the mechanical integrity (electronic network) of the electrode, and produces an unstable solid electrolyte interphase (SEI). We have successfully improved the performance of silicon based anodes by working on various aspects. First, our ball-milled silicon offers the right nanostructure to limit Si particle cracking in addition to be produced using an industrially viable process [1]. Second, the use of a mixture of carboxymethylcellulose (CMC) and citric acid (CA) as binder system buffer favors the formation of strong bonds between the binder and the Si particles [2] and a protective layer on the Si nanoparticles that significantly decreases electrolyte reduction [3]. Lastly, the use of carbon nanoplatelets as conductive additive insures better ability of the electrode architecture to reversibly expand/contract upon cycling [4]. In this communication, we would like to present a postprocessing treatment (called maturation) that we have recently developed. Indeed, maturation very significantly improves the mechanical and electrochemical stabilities of silicon electrodes made with the CMC/CA binder [5]. This treatment consists of storing the electrode in a humid atmosphere for a few days before drying and cell assembly. This results in a beneficial in situ reactive modification of the interfaces within the electrode. Our investigations suggest that the binder tends to concentrate at the silicon interparticle contacts. As a result, the cohesion of the composite film is strengthened. Moreover, the corrosion of the copper current collector, inducing the formation of copper carboxylate bonds, improves the adhesion of the composite film. This results in an impressive improvement of the electrode cycle life. The calendering of Si-based electrodes is required to obtain a substantial gain in their volumetric capacity compared to conventional graphite electrode. However, the calendering of silicon/carbon nanoplatelets/CMC/CA electrodes induces a major decrease of their cycling stability. This can be attributed to the rupture of the particle-binder bridges during the calendering, lowering the mechanical strength of the electrode. Interestingly, we found that these cohesive bonds can be restored through the maturation treatment. From in-operando dilatometric experiments, it appears that the volumetric expansion is lower and more reversible than for a standard (not-calendered, not-matured) electrode. As a result, a remarkable improvement of the cycle life is observed [6]. Finally, we found that the maturation process is also efficient for silicon electrodes made with the polyacrylic acid (PAA) binder [7]. Acknowledgements The authors thank the Natural Sciences and Engineering Research Council of Canada (NSERC) (grant RGPIN-2016-04524) and Transition Énergétique Québec (TEQ) (grant Techno-0040-0001) for financial support of this work.

Detection of Electrooxidation Products in Microfluidic Devices Using Raman Spectroscopy

Microfluidics combined with Raman Spectroscopy has great promise for monitoring reaction processes with very little volume of liquid provided. This advanced method for in-situ and online detection allows for flexible manipulation of fluids, micro/nano-particles, and biological samples1. Lab-on-an-chip applications are often used for chemical analysis, but Raman microscopy has also been used to monitor reactions within microreactors2. In electrochemistry, the use of SERS to analyse surface species is common. However the combination of the controlled mass transport in microfluidics with detection of soluble intermediate and product species by Raman offers the possibility of mechanistic kinetic studies of electrocatalytic reactions. We here show the feasibility of this method for studying electrocatalysis of oxidation of alcohols. The top figure shows the cell, which consists of a channel where the laser focuses at or near a Pt wire/mesh working electrode. The channel, which is built with 1 mm height Teflon spacers topped with cover glass, is connected to downstream cylindrical reservoirs with a reference electrode and counter electrode. A Teflon tube with a diameter of 1 mm connected to a syringe pump flows the liquid through the channel at a pre-set flow rate to oxidize alcohol for downstream or on-wire measurements. The width of the channel is fixed at 3 mm. Concentrated KOH (5 M) is used as the electrolyte, and in the proof-of-concept studies here 5 M methanol is used to ensure a high S/N ratio in the Raman spectra. The cell went through several design iterations, with detection above/beside the WE, WE above reflective roughened Au, or capture downstream above a roughened Au/Ag SERS substrate. A time sequence of spectra taken with the laser focused on the Pt WE shows production of formate (see bottom figure). Formate was the main product, and its dependence on time, potential, flow rate and laser position were investigated. The areas of the C-O stretching peaks for methanol and C=O stretching peaks for formate were calibrated with standard concentration solutions, to enable quantitative operation. A peak characteristic of carbonate was present but was too small to quantitate. Under the assumption that carbonate was the only other carbon-containing product, the calculated electron count from the products agreed with that from the charge passed electrochemically, indicating the feasibility of the method for qunatitative detection under flow conditions. One conclusion is that at higher potentials, the reaction tends to produce more formate than carbonate. Work is ongoing to optimize the conditions to improve the detection limit and to extend these studies to the oxidation of other alcohols. 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.[1] A.F. Chrimes, K. Khoshmanesh, P.R. Stoddart, A. Mitchell, K. Kalantar-zadeh, Microfluidics and Raman microscopy: current applications and future challenges, Chem. Soc. Rev., 42, 5880 (2013). [2] P.D. Fletcher, S.J. Haswell, X. Zhang, Monitoring of chemical reactions within microreactors using an inverted Raman microscopic spectrometer, Electrophoresis, 24, 3239 (2003). Figure 1

High Temperature Electrooxidation of Glycerol on Nickel

This work involves the long term electrooxidation of glycerol in a custom autoclave setup. Experiments are usually performed over approximately 48 hours. The custom autoclave setup was adapted from work done by Thomas Holm [1,2]. Within the autoclave a typical three electrode cell is built, wherein all glass pieces have been replaced with alkaline stable counterparts. This is largely done with Teflon, however a Nafion membrane is used to separate the working and reference electrode volumes from the bulk volume at the counter electrode to avoid contamination at the reference, and to keep the oxidation volume small. A small oxidation volume leads to higher concentrations of products, allowing them to be more easily detected using HPLC.Analysis of the oxidized products is performed via HPLC using an Aminex HPX-87H column. Using these techniques we have been able to show that the temperature at which electrooxidation occurs significantly alters the selectivity of products, as well as causing some new products to form (See Figure 1). Calibration curves have been made up for each of these products using known standards, and so we are able to quantitatively determine how much of each product is formed. However, the carbonate peak is not quantitative, and so carbonate was separately analysed by gravimetric or titrimetric methods.With this information we have been attempting to carry out a carbon balance between products and reactants to ensure all products has been accounted for. Though some reasonable values have been attained for this, problems arise when an electron balance is attempted. Much more oxidation is carried out than is expected from the number of electrons used during the experiment, and so we can conclude that some chemical interactions are occurring in solution. It is likely that some products are reacting with the alkaline solution, perhaps through chemical disproproportionation reactions. The existance of such reactions can be confirmed for glyceraldehyde, which has been shown to exist on a short time scale using PM-IRRAS [3] even though no glyceraldehyde is detected by HPLC. We find that HPLC glyceraldehyde standards decompose into other compounds that we see here. Work is ongoing to establish the identity and distribution of products as a function of the reaction conditions. 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. 1. T. Holm, P.K. Dahlstrøm, S. Sunde, D. A. Harrington, F. Seland, ECS Trans, 75, 1055, (2016).2. T. Holm, P.K. Dahlstrøm, O.S. Burheim, S. Sunde, D.A. Harrington, and F. Seland, Electrochim. Acta., 222, 1792 (2016).3. M.S.E. Houache, E. Cossar, S. Ntais, E.A. Baranova, J. Power Sources, on the web, doi:10.1016/j.jpowsour.2017.08.089. Figure 1

Kinetic Analysis of PRMT1 Reveals Multifactorial Processivity and a Sequential Ordered Mechanism

BACKGROUNDProtein arginine N‐methyltransferases (PRMTs) are responsible for the transfer of one or two methyl groups from the methyl donor S‐adenosyl‐L‐methionine (SAM) to produce mono‐ or dimethylated arginine residues and the spent cofactor S‐adenosyl‐L‐homocysteine (SAH). Methylated arginines in eukaryotic cells are involved in several crucial biological processes, including transcription control, DNA damager repair, and RNA processing. However, contrasting findings for two major questions in the field have generated debate regarding PRMT mechanisms. The purpose of this research was to elucidate the PRMT1 mechanism and answer these two highly debated questions: do PRMTs dimethylate their substrates processively or distributively, and do PRMTs bind their substrates using a random or sequential method of substrate binding?METHODSTo explore these mechanisms, we assayed PRMT1 with well‐characterized peptide substrates. We used tandem mass spectrometry to quantitate the ratios of mono‐ and dimethylarginine species produced to assess PRMT1 processivity. We determined the mechanism of bisubstrate binding using radioactive SAM in enzyme assays to measure methyl transfer to a target peptide in the presence of various concentrations of product inhibitors.RESULTSWe demonstrate that PRMT1 processivity differs depending on the substrate sequence, which is congruent with previous reports. However, we show for the first time that as cofactor and/or enzyme concentration increases, the ratio of dimethylarginine to monomethylarginine substantially increases, suggesting the enzyme becomes more processive. Further, the steady‐state inhibition patterns favour a sequential ordered mechanism over a random mechanism, indicating that for catalysis to occur, SAM binding must precede peptide binding.CONCLUSIONSThe degree of PRMT1 processivity is a multifactorial effect that is heavily influenced by experimental design considerations, so previous conclusions about processivity may require reassessment. We also find that PRMT1 uses a sequential ordered substrate binding mechanism, in contrast with recent findings. Use of nonlinear regression and presentation of our data using three linear plots strengthens our conclusions. Considering the conserved active sites within the human PRMT family, we anticipate that our results regarding PRMT1 kinetic and processive mechanisms may extend to other human PRMTs.Support or Funding InformationThis work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) RGPIN‐2015–04450 Discovery Grant (A.F.), NSERC CGS‐M (J.I.B.), the Shaughnessy Hospital Volunteer Society Fellowship in Health Care (J.I.B.), and the Walter C. Koerner Fellowship (J.I.B.). Additional support provided by Utrecht University and the Netherlands Organization for Scientific Research is acknowledged (T.K., J.v.S., and N.I.M.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Carnosol increases skeletal muscle cell glucose uptake via AMPK‐dependent GLUT 4 glucose transporter translocation

Skeletal muscle is highly important in glucose homeostasis since it is quantitatively a major insulin‐target tissue. Insulin action in muscle cells activates the phosphatidylinositol‐3 kinase (PI3K)/Akt signaling pathway causing the translocation of intracellularly stored GLUT4 glucose transporters to the plasma membrane leading to increased glucose uptake. Impaired insulin action in muscle leads to insulin resistance and type 2 diabetes mellitus (T2DM).AMP‐activated kinase (AMPK) is a cellular energy sensor and its activation increases glucose uptake by skeletal muscle cells. Finding AMPK activators is viewed as an effective approach to combat insulin resistance and T2DM. Rosemary extract (RE) has been shown to increase muscle glucose uptake and AMPK activity but the components responsible for these effects have not been identified yet. In the current study, we investigated the effect of carnosol, a polyphenol found in high concentrations in RE. L6 rat muscle cells were used to measure uptake of [3H]‐2‐deoxy‐D‐glucose and the signaling molecules involved were investigated by immunoblotting. Carnosol stimulated glucose uptake in L6 myotubes in a dose‐ and time‐dependent manner. A response comparable to maximum insulin stimulation (196±9.2 % of control) was seen with 50μM of carnosol (2h) (182±7.8 % of control). Carnosol did not affect Akt phosphorylation while it significantly increased AMPK phosphorylation. Furthermore, the increase in glucose uptake in the presence of carnosol was significantly reduced by the AMPK inhibitor compound C (CC) while it was not affected by the PI3K inhibitor wortmannin. Carnosol increased plasma membrane GLUT4 glucose transporter levels in GLUT4myc overexpressing L6 cells and this response was abolished by the AMPK inhibitor CC. Our study is the first to show a significant increase in muscle glucose uptake by carnosol via a mechanism that involves AMPK.Support or Funding InformationSupported by a Natural Sciences and Engineering Research Council of Canada (NSERC) grant to ET.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Attenuation of FFA‐induced skeletal muscle insulin resistance by rosemary extract

Elevated blood free fatty acids (FFAs), as seen in obesity, impair insulin action in muscle leading to insulin resistance and Type 2 diabetes mellitus. Serine phosphorylation of the insulin receptor substrate (IRS) reduces insulin‐stimulated tyrosine phosphorylation leading to insulin resistance. A number of serine/threonine kinases including JNK, mTOR and p70 6SK have been implicated in serine phosphorylation of IRS‐1 and insulin resistance. Activation of the energy sensor AMP‐activated protein kinase (AMPK) increases glucose uptake and has become an important target to counteract insulin resistance. We reported recently that rosemary extract (RE) increased skeletal muscle cell glucose uptake and activated AMPK. However, its effect on FFA‐induced insulin resistant muscle cells has never been elucidated. In this study, we investigated the effect of RE in palmitate‐induced insulin resistant L6 muscle cells. Glucose uptake was measured using [3H]‐2‐deoxy‐D‐glucose and the signaling molecules involved were investigated by immunoblotting.Exposure of L6 myotubes to the FFA palmitate (P) (0.2 mM, 16 hours) resulted in significant reduction in insulin‐stimulated glucose uptake (I: 201±1.21% and P+I: 117±15.6% of control, P<0.001 respectively) indicating insulin resistance. Importantly, exposure to RE resulted in significant restoration of insulin‐stimulated glucose uptake (179±10.5% of control, P<0.001). Treatment with palmitate increased serine phosphorylation of IRS‐1 and significantly decreased the insulin‐stimulated phosphorylation of Akt and these effects were completely abolished in the presence of RE. Treatment with palmitate increased the phosphorylation/activation of JNK, mTOR and p70 6SK whereas RE completely abolished these effects. RE increased the phosphorylation of AMPK even in the presence of palmitate. Overall, our data indicate that treatment with RE attenuates the FFA‐induced insulin resistance. RE and RE polyphenols may have potent effects against insulin resistance and deserve further study.Support or Funding InformationSupported by a Natural Sciences and Engineering Research Council of Canada (NSERC) grant to ET.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

H1/H2 histamine receptor blockade lowers substrate‐dependent mitochondrial H2O2 emission in deep gastrocnemius muscle following a bout of prolonged exercise

Prolonged exercise increases oxidative metabolism in skeletal muscle mitochondria, which is facilitated, in part, by increased blood flow and delivery of nutrients and oxygen. Histamine mediates many acute exercise responses, including sustained vasodilation and associated delivery of substrates to muscle. We tested the hypothesis that H1 and H2 histamine receptor blockade would attenuate exercise‐induced changes in skeletal muscle mitochondrial function. Adult male Wistar rats (75–94 days old; 441.2 ± 39.5 g) were randomly assigned to one of three groups (n = 12/group): exercise, in the form of one hour of continuous treadmill running at 20 cm/sec (Group 1); mass‐specific oral gavage with H1 antagonist fexofenadine (7.11 mg/kg) and H2 antagonist ranitidine (3.95 mg/kg) one hour prior to the same exercise protocol (Group 2); control, which received neither antihistamines nor exercise (Group 3). An hour post exercise, mitochondrial function was assessed in saponin‐permeabilized myofibers from deep gastrocnemius (mixed fiber types) and soleus (oxidative) skeletal muscle. A sequential substrate‐inhibitor titration protocol was used to examine multiple mitochondrial metabolic pathways (i.e., malate + octanoyl‐carnitine, ADP, lactate + NAD+, pyruvate, glutamate, succinate, inhibitors of antioxidant defense glutathione peroxidase and thioredoxin). Substrate‐dependent oxygen consumption (JO2; high‐resolution respirometry) was not different between treatments; however, H1 and H2 histamine receptor blockade significantly decreased hydrogen peroxide emission (mH2O2; fluorometric monitoring of Amplex Red oxidation) in deep gastrocnemius fibres (P < .05). Given the widespread and routine use of antihistamines, further research into the effects of these antagonists on mitochondrial function, in the context of exercise, is warranted.Support or Funding InformationThe Natural Sciences and Engineering Research Council of Canada (NSERC), The Nova Scotia Health Research Foundation (NSHRF), and Canada Foundation for Innovation (CFI)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inhibition of allergen‐mediated mast cell activation by rosemary extract (Rosmarinus officinalis L.)

The prevalence of allergic inflammatory disorders is increasing at an alarming rate, with 40–50% of school‐aged children suffering today and more than 25% of people living in a developed nation presently diagnosed or expected to develop, an allergic disease in their lifetime. This trend places a large economic burden on healthcare systems with $20B spent on treating these disorders in the USA in 2007. Mast cells are immune sentinels and a driving force in both normal and pathological contexts of inflammation. Allergen detection by the immune system results in production of allergen‐specific immunoglobulin E (IgE) antibodies. These antibodies bind to their high‐affinity receptor FcɛRI, which is constitutively expressed on the surface of mast cells. Crosslinking of FcɛRI by allergen‐bound IgE antibodies leads to mast cell degranulation (e.g. histamine) resulting in an early phase response, and the release of newly synthesized pro‐inflammatory mediators, contributing to a late phase response. The mitogen‐activated protein kinase (MAPK) family and, nuclear factor‐κ‐light‐chain‐enhancer of activated B cells (NF‐κB) pathways have been established to be driving mechanisms behind mast cell‐induced inflammation. Rosemary extract (RE) is rich in polyphenols, including rosmarinic acid (RA), carnosic acid (CA), and carnosol (CO), which have been shown to inhibit the MAPK and NF‐κB pathways in other cellular contexts in vitro and in in vivo animal models. However, the effect of these polyphenols on mast cell activation and degranulation has not been explored. Therefore, the aim of this study was to evaluate the potential of RE in modulating mast cell activation and FcɛRI signaling via these pathways toward understanding the mechanism of action and functional outcomes. Mast cells were sensitized with anti‐TNP IgE and were stimulated with the cognate allergen (TNP‐BSA) under stem cell factor (SCF) potentiation, and treated with 0 – 25 μg/mL RE. Samples were then collected for β‐hexosaminidase assay, quantitative polymerase chain reaction (qPCR), enzyme‐linked immunosorbent assay (ELISA) and, western blotting analysis. The β‐hexosaminidase assay demonstrated that RE treatment inhibited mast cell degranulation dose‐dependently to a maximum (down to 15% of control) at 25 μg/mL (p<0.001) RE. qPCR analysis showed that RE treatment at 25 μg/mL resulted in decreased gene expression of IL6 at both 60 and 120 mins (p<0.05), IL13 at 120 mins (p<0.05), TNF at 120 mins (p<0.05), CCL1 at both 60 (p<0.01) and 120 mins (p<0.05), CCL3 at both 60 and 120 minutes (p<0.05), and Rcan1 at 120 mins (p<0.05). ELISA analysis further supported the qPCR data showing decreases in pro‐inflammatory IL‐6 at 12 hrs (p<0.05) and TNF at 24 hrs (p<0.05). Western blot analysis demonstrated that RE treatment at both 5 and 25 μg/mL inhibited phosphorylation of p38‐MAPK at 5 mins. This work enhances our current understanding of mast cell functional modulation, mast cell signaling, and mechanistic activities of RE with relevance to innate defences and allergy.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.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The First Characterization of a Novel Stem Cell Population and the Temporal Relationship with Satellite Cells in Human Skeletal Muscle

Skeletal muscle possesses the remarkable ability to repair itself in response to a damaging stimulus. Muscle repair or myogenesis is driven by the activation and proliferation of muscle-resident stem cells known as satellite cells (SC) and the subsequent fusion or donation of myonuclei into skeletal muscle fibers. Although SC have been identified as the main skeletal muscle progenitor, several other unique cell populations have been recognized as contributing to skeletal muscle plasticity. PW1+ interstitial cells or PICs have been recently defined as a muscle-resident stem cell population that is anatomically and functionally distinct from Pax7+ expressing SC. This population has previously only been identified in murine and porcine models. PICs are derived from a different cell lineage than that of SC and are located in the interstitium outside of the SC niche. Yet, PW1+ expression has also been colocalized with Pax7+ expressing SC indicating a possible interaction or transitive property of SC to exhibit PW1 expression. Therefore the objective of this investigation was to identify PICs in human skeletal muscle tissue and to examine the temporal relationship and quantify the proliferation of both PICs and SC in response to a single bout of muscle damaging eccentric exercise. Ten young adults (25 ± 3yr; mean ± SEM) participated in a muscle damaging protocol involving eccentric loading of the quadriceps muscle on an isokinetic dynamometer. A total of 300 contractions, separated into 30 sets of 10 repetitions, was performed at 180°/s over a range of 60° of movement with one-minute of rest between consecutive sets. Percutaneous needle biopsies from the vastus lateralis were taken pre-damage and 6h, 24h, 72h, and 96h post eccentric damage. In response to single bout of eccentric exercise, total Pax7+ cells/100 myofibers remained unchanged at 6h (8.9 ± 1.8 cells/100 myofibers), 24h (9.1 ± 1.1 cells/100 myofibers), but significantly increased at 72h (11.2 ± 1.4 cells/100 myofibers) and 96h (10.2 ± 1.2 cells/100 myofibers) as compared to Pre (7.6 ± 1.0 cells/100 myofibers). PW1+ interstitial cells/100 myofibers remained unchanged at all timepoints following damage. However, total PW1+ cells/100 myofibers increased at 72h (3.1 ± 1.2 cells/100 myofibers) and 96h (4.5 ± 1.7/100 myofibers), with no change at 6h (0.9 ± 0.3 cells/100 myofibers) and 24h (2.4 ± 0.9 cells/100 myofibers) as compared to Pre (0.6 ± 0.2 cells/100 myofibers). The expression of Pax7+/PW1+ in myofibers remained unchanged at 6h (0.2 ± 0.1 cells/100 myofibers) and 24h (0.2 ± 0.1 cells/100 myofibers) and 96h (0.9 ± 0.3 cells/100 myofibers), but significantly changed at 72h (1.0 ± 0.3 cells/100 myofibers). There was also a trend in the percentage of total SC expressing PW1 at 72h (10.4 ± 3.2 % SC expressing PW1), p=0.082) when compared to Pre (4.1± 2.1% SC expressing PW1). Here, for the first time in human skeletal muscle, we identify a population of PW1+ interstitial cells that appear to be exercise-responsive. We also show that PW1 colocalizes with a subset of muscle SC. The exact contribution of PW1 to skeletal muscle repair/adaptation in humans is not fully understood, however this data supports the notion that atypical myogenic progenitors may contribute to physiological cues in humans. Support or Funding Information The Natural Sciences and Engineering Research Council of Canada (NSERC) Representative image of a DAPI/Laminin/Pax7/PW1 stain of a muscle cross section. *; represents SC, arrow represents PIC This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.