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Poster Abstracts

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Poster Number 1

A pipeline for reproducible behavioural analysis in the Open Field Test

Andre Telfer, Jackson McCormack, Frances Sherratt, Ahmad Alftieh, John Lewis, Oliver van Kaick, Alfonso Abizaid; Carleton University, Carleton University, Carleton University, Carleton University, University of Ottawa, Carleton University, Carleton University

In Neuroscience, behavioural studies in animals are a key component to understanding how the brain functions to generate complex cognitive, motivational, and emotional processes. Behavioural assays are a cornerstone of this behavioural research, yet, despite much standardisation, continue to produce highly variable data leading to reproducibility challenges. One aspect that influences this variability is the selection of parameters used to define behaviours. For example, if a behaviour measurement is how much time is spent in a zone, then the size of the zone is a parameter which can affect the results of the experiment. We explore the importance of these behavioural parameters in the Open Field Test (OFT) which is a common behavioural assay used as a test for the anxiolytic effects of drugs, motivation, locomotor activity, fear responses and exploration. Measuring the behaviours from dozens of OFT videos once often takes weeks, so we created an automated pipeline using deep learning and rule-based analysis that could analyse experiments many times for a range of behavioural parameter values in seconds. We were then able to demonstrate that the statistical significance between treatment groups was sensitive to the behavioural parameters which many researchers assume to be robust. The information produced by our pipeline can be used by researchers to reduce reproducibility issues tied to behavioural analysis. Furthermore, the pipeline is a free method for researchers to automate weeks of manual work or replace widely used and expensive commercial solutions still prone to this parameter-induced variability.

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Poster Number 2

Green Days

Malick Sylla, Erika Uzoegwu, Frank Li, Veronica Yung

Introduction: Our winning intervention for this year’s OneHealth Challenge is The Green Days Excursion Program. The Green Days program will partner with schools located in low-income neighborhoods with limited access to quality greenspace, and offer regular excursions to quality greenspace in their cities. Our program’s mission is to help bridge the gap between low-income youth and quality greenspace, whilst fostering a longer term relationship between the kids and the outdoors. Methodology: Several studies in recent years have highlighted inequalities in greenness within Canadian cities. These studies have found that a positive correlation often exists between socioeconomic status and vegetation cover - meaning that as family income lowers, so do the surrounding green areas. As such, we have narrowed our focus onto low-income communities, so as to try and help bridge the accessibility barrier. Results: We believe that Green Days has the potential to positively serve the communities in which it operates. By bridging the accessibility gap, Green Days will work to ensure that regardless of their socio-economic situation, children will be able to reap the variety of health benefits that come with spending quality time outdoors, and develop a lasting relationship with nature. Conclusion: Beyond the immediate benefits of the program, we believe Green Days can create lasting impact. Immersion in greenspace as a child has been shown to foster a strong concern for nature and environmental health as adults. Thus, the Green Days program hopes to encourage and empower future nature enthusiasts, environmental advocates, conservationists, and land defenders.

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Poster Number 3

Flowers for hope

Sofia Parra, Laurie Saphira Bijoux, Amirita Bhattacharyya, Margaret Hanna, Xiaoduo Lin

Flowers for hope is a project initially designed for children in terminal care. Setting in child hospital, the project consists of plant wall, gardening kits, and a series of activities related to the flowers. All the materials including seeds in this project we chose are feasible and sustainable. By attending the activities, we hope that they may have new attitude towards life by growing their own flowers, playing and reading surrounding by greens.

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Poster Number 4

Identification of Novel Nutrient-Gene interactions in Neural Tube Defects

Marina White1, Jayden Arif-Pardy1, Kristin L Connor1 1Carleton University, Ottawa, Canada

Neural tube defects (NTDs) remain among the most common congenital anomalies. Contributing risk factors include genetics and nutrient deficiencies, however, a comprehensive assessment of nutrient-gene interactions in NTDs is lacking. We hypothesised that multiple nutrient-gene interactions would be evident in NTD-associated gene signatures. Amniocyte gene expression data from fetuses with NTDs (cases; n=3) and fetuses with no congenital anomalies (controls; n=5) were obtained from GEO (GSE4182). Differentially expressed genes (DEG) were identified (eBayes; FDR q value<0.05, absolute fold change>2) and screened for having nutrient-cofactors (Scott-Boyer, Sci Rep 2016). Transcription factors (TFs) with nutrient cofactors that regulated DEG, and nutrient-sensitive miRNAs that had a previous link to NTDs, were identified and used to construct DEG regulatory networks (iRegulon v1.3, miRWalk2.0). Of the 880 DEG in cases (vs. controls), 10% (n=87) had at least one nutrient cofactor. DEG regulatory network analysis revealed that 39% and 52% of DEGs in cases were regulated by 22 nutrient-sensitive miRNAs and 10 nutrient-dependent TFs, respectively. Zinc- and B vitamin- dependent genes and gene regulatory networks (Zinc: 10 TFs targeting 50.6% of DEGs; B vitamins: 4 TFs targeting 37.7% of DEGs, 9 miRNAs targeting 17.6% of DEGs) were dysregulated in cases. Two nutrient-dependent TFs predicted to target DEGs in cases (Tumor Protein 63 and Churchill Domain Containing 1) have not been previously linked to NTDs. We identified multiple novel, nutrient-sensitive gene regulatory networks associated with spina bifida, which may relate to NTD pathogenesis, and indicate new targets to explore for NTD prevention or to optimise fetal development.

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Poster Number 5

Developing novel oncolytic virus therapies for pancreatic cancers

Sydney Vallati (1,2), Mathieu J.F. Crupi (2,3), Zaid Taha (2,3), Jack Whelan (2,3), Emily Lee (2,4), Betty Huang (2,4), John C. Bell (2,3) 1: Carleton University, 2: Ottawa Hospital Research Institute, 3: University of Ottawa, 4: University of British Columbia

RET is a cell-surface receptor tyrosine kinase involved in the regulation of proliferation, migration, and survival of neural crest derived tissues in normal development. However, aberrant activation of this protein can contribute to tumourigenesis, regional invasion and metastasis of many human cancers. Although RET overexpression is known to contribute to aggressive pancreatic cancers, the RET-specific immune-related mechanisms are still not well understood. Here, we identify immunosuppressive targets that the RET proto-oncogene can regulate, and strategically design novel therapeutic payloads to be encoded in oncolytic viruses to target RET-positive pancreatic cancers. We hypothesize that RET may promote the expression of different proteins that can allow tumours to evade detection and elimination by immune cells. The relationship between RET and immunosuppressive proteins was characterized by RNAseq and immunoblotting, using several RET-expressing cancer cell lines and their equivalent RET knockouts that we developed using CRISPR technology. Next, we determined the susceptibility of pancreatic cancer cell lines and their RET knockouts to different oncolytic viruses, including vaccinia and vesicular stomatitis virus. Advances in understanding the immune-related roles of RET in pancreatic cancer will be used to develop novel RET-targeting transgenes, including microRNA technology or Bispecific T cell Engagers (BiTEs), that we can encode into oncolytic viruses.

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Poster Number 6

Exploring resegmentation in the African clawed frog, Xenopus laevis with in-situ hybridization of the Tbx18 and Uncx genes

Sara Hassan, Dana E. Korneisel, & Hillary C. Maddin

Segmentation in the vertebrate embryo is identified in early development first through the formation of somites along the anteroposterior axis of an embryo and later by the development of vertebrae and muscle segments. The sclerotome cells in each somite that will form vertebrae are widely considered to shift relative to myotome, precursors of muscle, to enable spine movement. To account for that shift, resegmentation proposes that somitic sclerotome cells first polarize rostro-caudally, then recombine when the caudal half of one sclerotome combines with the rostral half of the next sclerotome. Under this model, each vertebra is derived from two somites. Previous studies have demonstrated that resegmentation occurs in some vertebrate model organisms through grafting surgeries. While resegmentation is well-supported in model organisms such as axolotl and chick, studies have yet to demonstrate resegmentation within Anura. As the somites of anuran embryos are very small, and sclerotome cells make up a much smaller portion of a somite in amphibians than they do in amniotes, we aim to use genetic landmarks rather than surgeries to assess whether resegmentation occurs in the African clawed frog, Xenopus laevis. The genes Uncx and Tbx18, both homeobox genes, have been shown to contribute to somite polarization in mice. In this study, we employ in situ hybridization of Uncx and Tbx18 in X. laevis embryos to examine whether this model amphibian in developmental biology exhibits evidence of sclerotome polarization within the somite, and thus whether the resegmentation model is a good candidate to explain vertebral segmentation in these unique vertebrates.

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Poster Number 7

Community Flyers

Lexie Armstrong, Nura Salsal, Tasha Burhunduli and Sydney Pedari

Introduction: Individuals with physical disabilities living in low-income housing are experiencing unique health disparities associated with the COVID-19 pandemic and are consequently experiencing barriers preventing them from having diverse social networks, social support, and greenspace exposure all of which are directly associated with individual health. A major contributor to these disparities is the current lack of funding regarding the maintenance and creation of infrastructure and urban greenspaces that suit the needs of individuals with physical disabilities. In addition, there is currently a lack of green spaces in the Ottawa downtown area that promote low-income community engagement, promote biodiversity, enrich the community, and actively promote mental and physical wellness all while accommodating the physical, social and emotional needs of physically disabled individuals living in low-income housing. Objective: Our project, the Community Flyers Program, aims to lessen the negative health impacts that low-income housed individuals with physical disabilities are experiencing during the pandemic by creating an integrative community program that increases biological diversity, mental wellness, social networks, greenspace exposure and physical activity. Results: The Community Flyers Program is expected to gather individuals living in low-income housing throughout the area surrounding Strathcona Park together, specifically residents who identify as being physically disabled. The program is expected to create social networks and support systems and increase social connectivity which has been shown to better mental health and increase individual’s sense of purpose [1]. The program also allows individuals to engage with birds and exposure to bird sounds has been associated with reduced stress and discomfort [2]. In addition, the program will be carried out in Strathcona park (a greenspace) and increase exposure to greenspaces is linked to reduced risks of dying from several common causes of death among urban Canadians [3]. Lastly, the program will promote biodiversity via the introduction of new birds to the ecosystem and in doing so participants will learn about bird ecology and improved cognitive functions such as memory retention [4]. Conclusion: Based on our research, the Community Flyers Program is expected to create community support, integration with greenspace, encourage sustainability, and biodiversity. The implementation of this annual program is predicted to produce systematic change within low-income communities within Strathcona Park that can be transferred to other greenspaces throughout Ottawa.

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Poster Number 8

Is mRNA Translation Compromised in SIFD?

Angelo Slade*, Martin Holcik* *Carleton University, Health Science Department

tRNA-nucleotidyltransferase 1 (TRNT1) is an essential enzyme responsible for the addition of 3’ CCA ends to all tRNA, a crucial process of tRNA maturation and amino acid attachment. Hypomorphic mutations within TRNT1 result in a rare and severe mitochondrial disease known as SIFD (sideroblastic anemia, B-cell immunodeficiency, periodic fevers, and developmental delay). The purpose of my research is to understand how hypomorphic mutations inTRNT1 cause SIFD. Preliminary patient data suggests a reduction in pools for certain tRNAs, I hypothesize that this reduction impairs translation of specific proteins, either through ribosomal stalling or translational infidelity, potentially impacting protein function. To investigate the molecular mechanism(s) of SIFD, the yeast model of SIFD that utilizes temperature sensitive allele of CCA1 (yeast homolog of TRNT1) is being used. Comparing healthy yeast to CCA1 deficient yeast, I observed reduction in tRNA abundances as in patient cells. Using yeast specific tRNA data, a bioinformatic screen of the yeast genome was conducted to identify genes which require large amounts of the reduced tRNAs to be translated. The abundance of these potentially impacted genes and their proteins will be investigated by western blotting and polysome profiling. To assess impacts on protein function, a B-galactosidase assay was conducted, showing that when compared to healthy yeast the CCA1 deficient yeast had a significant (P<0.001) reduction in the function of the B-gal protein. In addition, CCA1 deficient yeast showed a severely impacted ability to grow on galactose as an alternate carbon source, showing a potential impact on the ability to adapt to cellular stresses. By using the yeast model of SIFD we can better inform future experiments within patient cells, and potentially work towards the development of treatment options.

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Poster Number 9

Contribution of Bayes Structural Time Series models in Energy Prediction

Eralda Gjika , School of Mathematics and Statistics, Carleton University

Foresight is one of the challenges that has attracted the attention of researchers and professionals in various disciplines of science. Time series from different fields such as: finance, demography, energy, medicine, etc. have been in the attention of researchers who have focused on the suitability of different models based on the nature of time series and have evaluated the performance of these models. Time series methods have undergone significant developments since statistical models were proposed. In recent years, software developments and multidimensional time series computing capabilities have occupied an important place in scientific studies. Machine learning methods have already managed to offer high performance in long-term and short-term forecasts. This study will present the contribution of BSTS (Bayesian Structural Time Series) methods applied to energy time series data and other climacteric variables used as regressors in energy production.

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Poster Number 10

Uncovering the Basic Helix-Loop-Helix Transcription Factors Involved in Suberin Deposition.

Kassandra Fugard, Kyle Tapp, and Owen Rowland. Department of Biology and Institute of Biochemistry, Carleton University, Ottawa ON, Canada.

The projected impacts of climate change may dramatically affect the growth and development of plant species across the world. The formation of specialized cell wall-associated barriers is important for protecting plants by controlling water and solute movement through the root. Enhancement of these hydrophobic barriers will be important in generating more resilient crops. We are exploring the molecular mechanisms behind the deposition of suberin, a polyaliphatic and polyphenolic heteropolymer, that is deposited, for example, in the root endodermis and periderm. A connection between suberin and lateral root emergence has recently been revealed, but the molecular interconnectedness and physiological implications and mostly unexplored. A complex network of transcription factors (TFs) has been found to regulate suberin deposition in the model plant species Arabidopsis thaliana. The broad expression patterns of several suberin-associated TFs suggest multiple functions within suberin controlled processes. It is well established that MYB TFs typically interact with basic-helic-loop-helix (bHLH) TFs in transcriptional activating regulatory complexes. Our research here focusses on the role of bHLH TFs (bHLH111 and bHLH112) from Arabidopsis thaliana that we hypothesize regulate suberin synthesis under abiotic stress conditions. This study aims to provide insight into the regulatory functions of bHLH111 and bHLH112 by examining promoter activities under normal development and upon stress conditions. Loss-of-function mutants and transgenic overexpression lines are being used to determine whether bHLH111 and bHLH112 influence the deposition and/or composition of suberin. Finally, the potential role of bHLH111 and bHLH112 as regulators of abiotic signalling and lateral root development is being investigated.

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Poster Number 11

Impact of gestational hyperglycaemia on the development of the rat fetal hypothalamic melanocortin system

Ms. Kiara Ayoub1 , Dr. Marina Martins12 , Mr. Zachary Silver1 , Ms. Andrea Smith1 , Ms. Lindsay Hyland1 , Ms. Anna Carolina Kiss2 , Dr. Barbara Woodside3 , Dr. Alfonso Abizaid1 1Neuroscience, Carleton University, Ottawa, Canada, Department of Physiology, 2Institute of Biosciences of the University of São Paulo, São Paulo, Brazil, 3Psychology Department, Center for Studies in Behavioral Neurobiology, Concordia University, Montreal, Canada

Gestational Diabetes (GD) is associated with adverse metabolic outcomes in offspring, such as increased vulnerability to develop obesity and type 2 diabetes. Prior research has attributed obesity and other metabolic disorders by a dysregulation of the melanocortin system. Indeed, it is possible that GD may influence the development of this system and confer this vulnerability. We employed an experimental model of GD to examine how a maternal hyperglycemic state impacts the development of this system in the arcuate nucleus (ARC) in the developing fetus. To do this, we induced a mild diabetic state by injecting intraperitoneally pregnant Wistar rats with a low dose of streptozotocin (STZ; a pancreatic beta cell toxin), inducing a mild hyperglycemic and insulin deficient state. The injection of vehicle or 35 mg/kg of STZ was given (N=8/group) one week after impregnation was confirmed. GD was confirmed with a glucose tolerance test on day 15 and following this confirmation, on day 19, pregnant females were deeply anesthetized, and their fetuses were extracted via c-section. Fetuses were rapidly decapitated, and their heads were immediately submerged in 4% paraformaldehyde for fixation. Sections from these brains containing the ARC were processed for immunohistochemistry detecting the pro-opiomelanocortin (POMC) peptides. Results showed that pups harvested from STZ-treated rats had lower number of ARC POMC stained cells, suggesting that maternal hyperglycemia may be influencing the development of the melanocortin system, conferring vulnerability to metabolic disorders.

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Poster Number 12

Prevalence and Phenotypic Characterization of Patients with Bicuspid Aortic Valve

Casey Higginson *, Nadav Willner **, Laura Petruescu**, Luc Beauchesne **, Thais Coutinho **, Munir Boodhwani **, Kwan L Chan**, Ian Burwash **, David Messika-Zeitoun **. * Carleton University ** University of Ottawa Heart Institute

BACKGROUND: Bicuspid Aortic Valve (BAV) is a congenital condition with strong genetic determinants. It is well recognized that patients with BAV present with large annuluses; however, the prevalence is unclear, and the associated characteristics are unknown. OBJECTIVE: The purpose of this study was to (1) assess the prevalence of large annulus in bicuspid aortic valve (BAV) patients and (2) provide a phenotypic characterization of patients with BAV and large annulus. METHODS: Using the Echocardiography database of the University of Ottawa Heart Institute, we identified patients with BAV between 2015 and 2019. To compare the prevalence of enlarged annulus, patients with BAV were matched for age, sex, and body surface area (BSA) 1:1 with patients with tricuspid aortic valve identified from the same database. A large annulus was defined as a measured annulus above the 97.5% confidence interval of the predicted annulus size based on BSA calculated using the Leye formula (predicted annulus=5.7*BSA+12.1). RESULTS: We identified 598 adult patients with BAV. Prevalence of large annulus was 4 times higher in patients with BAV than TAV (22.6% vs. 5.2% respectively, p<0.0001). After matching for age, gender, and BSA, BAV patients presented larger annulus size than TAV patients (23.7 mm vs. 21.6 mm respectively, p<0.0001) and the prevalence of large annulus was more than 3 times higher (22.1% vs.8.0% respectively, OR 3.3 [2.2-4.9], p<0.001). Compared to BAV patients with normal annulus size, BAV patients with large annulus were younger (45±18 vs. 50±17 years, p=0.01), male sex (87% vs. 13%, p<0.0001), presented with larger aortic root diameter (38.8±5.5 vs. 34.9±5.4 mm, p<0.0001), larger ascending aorta diameter (38.1±6.6 mm vs. 36.6±6.4 mm, p = 0.01), more frequently regurgitant dysfunction (severe aortic regurgitation) (66.7% vs 33.3%, p < 0.0001), more frequently moderate aortic regurgitation (53.3% vs 46.7%, p < 0.0001), and less frequently stenotic dysfunction (moderate aortic stenosis) (23.5% vs 76.5%, p = 0.0448) (severe aortic stenosis) (6.7% vs 93.3%, p = 0.0448). There was no significant difference in BSA (1.95±0.23 vs 1.95±0.26 m2, p = 0.77). CONCLUSION: We confirm that BAV patients presented more often with large annulus size than patients with TAV but interestingly the vast majority of BAV patients had normal annular dimension. Most importantly patients with BAV and large annulus were more frequently male and presented with larger aorta dimension despite similar BSA and more frequently with regurgitant than stenotic dysfunction. Patients with BAV represent a heterogenous group and whether those with large annulus constitute a distinct genotypical subset remain to be evaluated.

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Poster Number 13

Integrating Coherent Anti-Stokes (CARS) Microscopy and Supervised Machine Learning to Classify Morphological Features of Atherosclerotic Plaques.

Natasha Kunchur, Leila Mostaço-Guidolin

Introduction. Atherosclerosis is a chronic inflammatory disease resulting in the accumulation of plaque within artery walls; rupturing the arteries and blocking blood flow. Through the combination of coherent anti-stokes Raman scattering (CARS) microscopy and machine learning, the stages of atherosclerotic plaque progression can be classified in a label-free manner. An automated classification pipeline employing image preprocessing, plaque segmentation, feature extraction and supervised machine learning algorithms were developed to classify CARS images of atherosclerotic plaques of Watanabe Heritable Hyperlipidemic rabbits aged zero between twenty-seven months. Methodology. The automated pipeline developed classifies plaque lesions based on their major clinical morphological features. A total of 24 features, inclusive of textural, shape and statistical features, were extracted from each CARS image of plaque; allowing the pipeline to classify the stage of plaque progression as Early Fatty Streak Development, Early Fibroatheroma or Advancing Atheroma. Results. A greater accuracy in classifying the stages of atherosclerosis development when using a multi-class support vector machine (MCSVM) was observed by employing features cable of capturing the most variation between plaque. Through the combination of independent foam cell segmentation and placing emphasis on extracted textural features, 80% classification accuracy of plaque progression was achieved by MCSVMs. Conclusion. Minute changes in the histological morphology of plaque can often be overlooked by the human eye. Through the combination of CARS microscopy and computational methods, a powerful classification tool was developed to identify the progression of atherosclerotic plaque in an automated manner.

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Poster Number 14

Role of ARK5 kinase in regulation of hnRNPA1 protein during cellular stress

Krishna Bhattarai, Martin Holcik, Department of Health Sciences, Carleton University, Ottawa, ON K1S 5B6

The hnRNP A1 is a nucleocytoplasmic shuttling RNA-binding protein that plays an important role in nucleic acid metabolism and gene expression regulation. The function of hnRNP A1 is, in part, determined by its specific location within the cell. Although some work has been done to elucidate the signalling pathways that regulate the cellular localization of hnRNP A1, the precise mechanism(s), including physiological and pathophysiological conditions that alter hnRNP A1 localization, are not known. We have previously conducted an unbiased RNAi-based kinome-wide screen to identify kinases that regulate hnRNP A1 localization during hypertonic stress. One of the hits from this screen is an AMPK-related protein kinase 5 (ARK5). Here we validate ARK5 as the kinase responsible for hnRNP A1 subcellular localization in response to hypertonic stress. We find that ARK5 directly interacts with and phosphorylates hnRNP A1 on the serines within the F-peptide region. We further show that the M9 motif of hnRNP A1 is essential for the ARK5-hnRNP A1 interaction and phosphorylation. Finally, the silencing of ARK5 increases the expression of an anti-apoptotic protein Bcl-xL and consequently delays caspase activation during hypertonic stress.

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Poster Number 15

The bun giveth, the hot-dog taketh away: Determinants of oxidation state specificity in the ALT family of hot-dog fold thioesterases from plants

Rebecca S. Kalinger, Owen Rowland, Department of Biology and Institute of Biochemistry, Carleton University

ACYL-LIPID THIOESTERASES (ALTs) are an unusual class of acyl-acyl carrier protein thioesterase enzymes from plants that terminate the fatty acid biosynthesis cycle prematurely. They belong to the “hot-dog fold” protein structure superfamily, named for a catalytic domain structure consisting of a central α-helical “hot-dog” enclosed in a β-sheet “bun." Unlike other plant thioesterases, ALTs can act on substrates of varied oxidation states, producing medium-chain (6-14 carbon) fatty acids, 3-hydroxy fatty acids, and methylketone precursors. While the products of ALTs have industrial value as ingredients in everyday products like plastics, fuels, and surfactants, little understanding of what dictates the widespread substrate preferences of ALTs currently limits their biotechnological use. Through targeted mutagenesis guided by computational modelling, we identified three amino acid motifs within the ALT catalytic domain that are central to oxidation state specificity: one on the central “hot-dog” α-helix, and two nearby motifs on the β-sheet surrounding it. By mutating these regions, we were able to manipulate the natural oxidation state preferences of ALT enzymes from diverse plant species. We successfully granted an ALT that normally cannot generate 3-hydroxy fatty acids or methylketone precursors the ability to do so, and restricted the product output of ALTs with broad oxidation state specificity to fully reduced fatty acids only. The findings of this study represent a significant advancement towards being able to engineer mutant ALTs with product profiles suited to specific biotechnological purposes.

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Poster Number 16

Deconstructing Trauma Experiences in University Students: Understanding the Associations of Trauma Type and Timing on Mental Health Symptomatologies

Ethier-Gagnon M.1,2; Daneshmend A.1,2; Jarkas D.1,2; McQuaid R.1,2; Department of Neuroscience, Carleton University1, The Royal’s Institute of Mental Health Research2

Introduction: Early life stress is a significant risk factor for the development of psychopathology in adulthood. Recent evidence suggests that both type and timing of trauma exposures before the age of 18 influence mental health outcomes. Yet, how these variations may differentially confer vulnerability to later psychopathological symptom dimensions remain unclear. This study aimed to delineate specific types and sensitive periods of trauma exposure that map onto distinct depressive and anxious symptom dimensions. Methodology: The association between trauma type and timing, and symptoms of depression and anxiety was examined among first- and second-year university students (N = 238, Mage = 19.0, 19.7% males, 80.3% females). Participants completed online questionnaires, including the Maltreatment and Abuse Chronology of Exposure scale, Beck Depression Inventory, Beck Anxiety Inventory, and Brief Resilience Scale. Results: Non-verbal emotional abuse was predictive of overall and atypical depressive symptoms, while parental verbal abuse was associated with overall depressive and somatic anxious symptoms. Emotional neglect predicted symptoms of overall and psychological anxiety. While, sexual abuse was predictive of all outcomes, except for atypical depression. Trauma exposure between the ages of 13-18 was most predictive of all mental health symptom dimensions assessed aside from sexual abuse. Conclusions: The present study suggests that both type and timing of trauma exposures in early life influence diverse depressive and anxious symptom profiles. Exposure to certain trauma types during critical periods of development should be considered in the development of early mental health interventions, particularly among adolescents.

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Poster Number 17

Bacillus Calmette-Guerin (BCG) induced trained immunity: One size does not fit all

Authors Mary-Elizabeth Sheridan1, Gerard Agbayani2, Duale Ahmed1, Zoya Versey1, Bassel Akache2, Edana Cassol1 Affiliations: 1. Department of Health Sciences, Carleton University 2. Human Health Therapeutics, National Research Council of Canada.

Immune memory has long been associated with the adaptive arm of the immune system, but recent work suggests innate cells are also capable of memory responses, termed trained immunity. This trained immunity is associated with metabolic and epigenetic reprogramming of bone marrow progenitors, which increases inflammatory responses to secondary stimuli in the tissue microenvironment. Among the most potent inducers of trained immunity is Bacillus Calmette-Guerin (BCG), the tuberculosis vaccine, which protects against viral respiratory infections. Emerging evidence suggests mitochondrial reprogramming plays a central role in BCG associated trained immunity, but the mechanisms remain unknown. Mice were intravenously treated with either BCG or PBS, sacrificed 27 days post treatment and the bone marrow was harvested from the tibia and femurs. Bone marrow cells were differentiated into bone marrow derived macrophages and stimulated with two viral ligands (TLR3 agonist Poly (I:C) and TLR7 agonist R837). These ligands differentially modulate mitochondrial function and inflammatory responses. Unlike bacterial ligands (LPS), BCG treatment was associated with decreased inflammatory cytokine production following stimulation with TLR3 and TLR7 agonists. Consistent with these findings, it also reduced mitochondrial reactive oxygen species production (ROS) and increased mitochondrial oxidative phosphorylation (OXPHOS) suggesting BCG may limit inflammatory responses in viral infections by reprogramming the mitochondria to support energy metabolism. They also indicate that the trained immune phenotype induced by BCG is not a “one size fits all” approach, and that it’s modulation of immune responsiveness is dependant on shifting the balance of the mitochondria between supporting OXPHOS vs. ROS production.

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Koziel Ly
Poster Number 18

Characterization of amygdala neurons activated by sleep deprivation

Nikita Koziel Ly (1), Chia-Luen Leu (2), Rhiannan Williams (2), and Melissa Chee (1); (1) Department of Neuroscience, Carleton University; (2) Institute of Neurogenomics, Helmholtz Zentrum Munich

INTRODUCTION: Chronic sleep deprivation (SD) is associated with increased risk of mental health disorders, metabolic syndrome, and cardiovascular disease. Little is known about the brain regions related to sleep, thus to overcome SD-related deficits, we must delineate the brain processes compromised by sleep loss. The objective of this study was to identify mouse brain structures activated by a 4-hour SD period. METHODS and RESULTS: Using the neuronal activity marker c-fos, we observed increased amygdala activation in 4-hour SD mice relative to well-rested control mice. We manually counted c-fos-labeled cells and found a robust increase in c-fos expression in specific amygdalar nuclei, including the basolateral (BLA) and central amygdala (CEA). We then generated brain atlas maps to visualize the distribution of this differential c-fos expression. To determine if these neurochemical changes corresponded with functional outcomes, we recorded the electrical activity of cells in the BLA and CEA. Interestingly, both BLA and CEA cells were more excitable after SD. Moreover, we found that SD cells had higher resistances and reduced current flow; these changes in current-voltage relationships implicate the dampening or closure of ion channels that stabilize cells at rest. CONCLUSION: In summary, we observed robust cell activation and increased cell excitability in several amygdalar nuclei following SD. The amygdala is an important brain structure for processing emotional events, thus our findings suggest that it may underlie worsened mood following sleep loss. Furthermore, as the amygdala is implicated in assessing threats, preserving amygdala function may help sustain decision-making, alertness, and health.

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Poster Number 19

Exploring interactions between macrophages and bacterial biofilms: Implications for cellular metabolism and wound healing.

Zoya Versey (1), Duale Ahmed (1), Carole Beaulieu (1), Mary-Elizabeth Sheridan (1), Katrina G. DeZeeuw (2), Jonah E. Marek (2), Joerg Overhage (1), Edana Cassol (1,3). (1) Department of Health Sciences, Carleton University, Ottawa, ON, Canada (2) Department of Complex Continuing Care, Saint Vincent Hospital, Ottawa, ON, Canada (3) Centre for Infection, Immunity and Inflammation, University of Ottawa, Ottawa, ON, Canada

Chronic wounds are difficult and expensive to treat, increase the risk of infection, and reduce quality of life. There is an urgent need to understand the pathophysiology of chronic wounds to develop new therapeutics to restore healing. One potential target is the macrophage. In chronic wounds, these cells are chronically activated in a proinflammatory state and are unable to promote tissue repair. However, the mechanisms underlying these processes remain poorly understood. We hypothesize that bioactive molecules secreted by the bacterial biofilms that colonize the wound bed drive this activation by reprogramming mitochondrial function, resulting in sustained production of inflammatory mediators and tissue damage. To investigate this, we exposed mouse bone-marrow derived macrophages to conditioned media from planktonic culture (PCM) or biofilm (BCM) of Staphylococcus aureus and/or Pseudomonas aeruginosa for 6h, 18h, and 48h. These strains are commonly found in chronic wounds. Changes in macrophage mitochondrial function and inflammatory cytokine production were assessed using flow cytometry, Seahorse XFp Analysis, and ELISA. We found that P. aeruginosa induced a strong inflammatory response associated with sustained mitochondrial reprogramming to support reactive oxygen species (ROS) production, which eventually resulted in cell death. Alternatively, S. aureus, induced a low-grade inflammatory response, associated with a transient reprogramming of the mitochondria and no cell death. Taken together, our results suggest that the activation status of macrophages may be highly dependent on the numbers and bacterial species colonizing the wound tissue. Understanding these processes may lead to new immunotherapies that reprogram macrophage function towards repair to restore healing.

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Poster Number 20

Elucidating the lipidomic dynamics of lentiviral production and infection

Joshua A Roberts (1,5), Elena Godbout (3), Christopher Boddy (2), Jean-Simon Diallo (3,4), Jeffrey C Smith (1,5), (1) Department of Chemistry, Carleton University, Ottawa, Canada (2) Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, Canada (3) Ottawa Hospital Research Institute, University of Ottawa, Ottawa, Canada (4) Virica Biotech Inc, Ottawa, Canada (5) Carleton Mass Spectrometry Centre, Carleton University, Ottawa, Canada

Lentiviral vector (LV) therapies are emerging as sophisticated and effective options for treating immunodeficiencies and cancer. Clinical-grade LV production for therapeutic use is expensive, requiring specialized packaging cells and bioreactor technology to produce harvestable quantities of virus. The lipid dynamics of producing LV in this setting are poorly understood. Reports from the closely related human immunodeficiency virus (HIV) have shown that altering its lipid composition lowers transducibility, suggesting that a better understanding of LV lipid dynamics may improve the quality, and potentially yield, of clinical-grade LV. To study this, HEK 293T packaging cells were transfected with LV plasmids followed by 48 hours of LV production. Cells were harvested and LV was purified from media by ultracentrifugation, lipids were extracted using a modified Bligh-Dyer protocol and analyzed by mass spectrometry (MS). Lipid were identified from tandem MS data using Agilent Lipid Annotator and were then quantified using MZmine 2; R and Microsoft Excel were used for statistical analysis. Production of harvestable quantities of LV resulted in a nearly comprehensive class-wide depletion of all HEK 293T lipids measured except for triacylglycerides, which increased in abundance. LV could also be successfully purified at quantities sufficient for LCMS-analysis, identifying 44 lipids present in LV. Future studies will focus on determining if specific lipids are depleted from HEK 293T cells in a time-dependent manner and if supplementing growth media with specific lipids allows them to be incorporated into LVs, with the aim of improving their rate of production or transducibility.

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Poster Number 21

Gut check: is there a role for the maternal gut microbiome in the development of fetal spina bifida?

Sheyholislami H1, Van Mieghem T2, Joris BR3, Gloor G3, Connor KL1 1Carleton University, Ottawa, Canada 2Sinai Health System, Toronto, Canada 3University of Western Ontario, London, Canada

Fetal neural tube defects (NTDs) are on the rise in Canada, driven by the most common NTD, spina bifida. Low folate and vitamin B12 levels during pregnancy contribute to the pathophysiology of spina bifida. However, periconceptional folic acid supplementation and mandatory fortification do not completely prevent the condition, suggesting other mechanisms. The maternal gut microbiome contributes to host micronutrient status through the synthesis, metabolism and transport of metabolites and micronutrients, and produces DNA nucleotides that are important for embryogenesis; despite this, it has not been previously considered in NTD pathogenesis. A case-control study was conducted to determine whether and how the maternal gut microbiome of pregnant people carrying a fetus with spina bifida (cases n=15) differed compared to the microbiome from uncomplicated pregnancies (controls n =18). We hypothesized that cases would have a dysfunctional gut metagenome, demonstrated by altered expression of genes/pathways that may be involved in neural tube closure. Bacterial DNA was extracted and sequenced (Illumina MiSeq) from maternal rectal swabs collected at ~25 weeks gestation. Metagenomic bins were taxonomically classified (PhyloPhlan v 3.0.58), annotated (KEGG v 4.3) and analyzed (ALDEx2). Twenty-two differentially abundant taxa were identified between cases and controls. Differences in pyridoxal-p and cysteine biosynthesis pathways, and associations between enriched taxa and estimated dietary intake of vitamin B12 and D, were observed. Thus, the gut microbiome of mothers carrying a fetus with spina bifida differs from healthy pregnancies, suggesting it may be a target for the prevention or treatment of NTDs.

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Poster Number 22

Enhancing the MS-based sensitivity of phosphopeptides by Trimethylation Enhancement using Diazomethane (TrEnDi)

Samiksha Vij*; Karl V. Wasslen*; Jeffrey M. Manthorpe*; Jeffrey C. Smith* *Department of Chemistry, Carleton University, Ottawa, ON

Mass spectrometry (MS) is widely regarded as the most powerful tool to study phosphopeptides. However, several challenges are commonly encountered in this field including difficulties detecting phosphopeptides with low stoichiometry, relatively high hydrophilicity and low ionization efficiency compared to their unmodified counterparts. Here we present the application of Trimethylation Enhancement using Diazomethane (TrEnDi) to improve the analytical characteristics of phosphopeptides. TrEnDi overcomes these challenges and increases the sensitivity of phosphopeptide analysis via permethylating analytes, rendering them permanently positively charged and eliminating the need for protonation. Synthetic phosphopeptides, FLEEpSK, FLEEpTK, and FLEEpYK loaded on strong cation exchange chromatographic resin were derivatized with diazomethane (DZM). The derivatized analytes were analyzed directly or spiked with equimolar concentrations of underivatized phosphopeptides. TrEnDi derivatization resulted in ~100% complete methylation of the acidic groups present on the phosphopeptides as indicated by a mass shift of +154 Da observed via MS, corresponding to the addition of eleven methyl groups. The complete methylation and fixed positive charges on the N-termini and lysine residues boosted the sensitivity of these phosphopeptides. TrEnDi-modified peptides containing phosphorylated serine and threonine residues rapidly lose the dimethyl phosphate group (-126 Da) upon collisional activation at low energy. MS/MS analyses of the phosphopeptides demonstrated a propensity to form highly abundant a2 fragment ions. TrEnDi modified phosphopeptides revealed a relative increase in retention by 8% and a five-fold increase in sensitivity for modified phosphopeptides compared to their unmodified counterparts. This work represents the development of a universal procedure whereby TrEnDi will increase the sensitivity of phosphopeptides in complex biological samples.

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Poster Number 23

Antimicrobial resistance of Pseudomonas aeruginosa planktonic cells to hypochlorous acid

Madison Adams1, Waleska da Cruz Nizer1, Vasily Inkovskiy1, Carole Beaulieu1, and Joerg Overhage1 1Department of Health Sciences, Carleton University, Ottawa ON K1S 5B6, Canada

P. aeruginosa is a significant Gram-negative opportunistic human pathogen well-known for causing a variety of hospital-acquired infections, particularly in immunocompromised individuals. Hypochlorous acid (HOCl) is a powerful oxidant widely utilized in industrial and clinical contexts. HOCl is also produced by the human immune system as a defence against invading pathogens. Although HOCl’s antimicrobial properties are critically important to public health, little is known about the molecular mechanisms driving microorganisms’ adaptation and resistance to oxidative stress caused by HOCl. In this study, we characterized the growth of P. aeruginosa PA14 planktonic cells in the presence of HOCl. Based on the MIC of P. aeruginosa PA14 wild-type in BM2 medium (2 μg/mL), a P. aeruginosa PA14 mutant library was screened for mutants with increased susceptibility (1 μg/mL) and resistance (8 μg/mL) against HOCl. Fifty susceptible mutants were identified, confirmed by MIC testing, and selected for further analyses. Growth curves were conducted by exposing planktonic cells to 2, 1, and 0 μg/mL of HOCl. We identified ten mutants with strong phenotypes in terms of their increased HOCl susceptibility—i.e., delayed/inhibited growth at 1 μg/mL HOCl. Of these ten mutants, three mutants showed increased HOCl susceptibility in both the PA14 and PAO1 strains. Additional analyses are ongoing focusing on identifying the roles of these mutations in P. aeruginosa’s HOCl-induced stress response. A better understanding of bacteria’s gene-specific HOCl stress responses would help determine the genes underlying antimicrobial resistance, understand host-pathogen interactions, and target specific genes to restrain bacterial colonization and spread following HOCl disinfection.

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Poster Number 24

Stress and the Truncated Tropomyosin receptor Kinase B (TRKB.T1) differentially affect behavioural phenotypes in male and female mice

Natalie Prowse, Sara Khalaf, Noora Heiratifar, Trisha Banks Wenwright, Tryston Charlton, Laura Farlam-Williams, Delenn Hills, Aditi Sankhe, Melissa Chee, Shawn Hayley (Carleton University)

Major Depressive disorder is the most common psychiatric disorder and causes tremendous disability and in the most severe cases, can lead to suicide. Current treatments for depression lack broad efficacy and often take weeks to produce a treatment response, if they are effective at all. The neurotrophin, brain-derived neurotrophic factor (BDNF) and its cognate receptor, Tropomyosin Receptor Kinase B (TrkB) are vital to recovery from depression. TrkB has several isoforms, and variations in the expression of the truncated variant, TrkB.T1 have been implicated in suicidality. Importantly, little is known about sex-based differences in TrkB.T1 signaling, or how TrkB.T1 influences gene expression in glia, such as astrocytes. Thus, we sought to understand the role this receptor plays in the context of stressor-related disorders. Using a transgenic mouse line with the TrkB.T1 receptor knocked out, we subjected both male and female mice to 35 days of chronic mild unpredictable stressors, followed by a series of behavioural tests. We found that the stressor regimen had sex-dependent effects on measures of depressive anhedonia (sucrose preference) but was unaffected by genotype. However, TrkB.T1 KO mice did display an anorexic-like phenotype and this further exacerbated by stress. Interestingly, sequencing of mRNA enriched for astrocytes in the hippocampus revealed that calcium signaling genes might be involved in the phenotypic difference observed in the TrkB.T1 KO mice. These data point to the possibility of targeting TrkB signaling for the treatment of depression and its often-comorbid eating disorders.

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Poster Number 25

Graduate Student Mental Health and Coping During the COVID-19 Pandemic

Tara Thachet - Department of Neuroscience, Carleton University Ashley Thompson - Department of Neuroscience, Carleton University Kim Hellemans - Department of Neuroscience, Carleton University

During the COVID-19 pandemic 80% of Canadian adults aged 25-44 have experienced at least one negative impact from the pandemic. Graduate students tend to fall in this young adult population and graduate student mental health has been a growing crisis for years. Despite this problem, very little research has been done looking at how graduate students were affected during the pandemic. This project aimed to investigate the following: Were Canadian graduate students and their mental health and wellbeing negatively impacted by the pandemic? How are graduate students coping during the pandemic? And finally, did the pandemic provide any positive outcomes for graduate students? The study was an online study that recruited graduate students of any discipline studying at a Canadian University (n=261). The preliminary data showed that Canadian graduate student mental health and coping were indeed affected during the pandemic. Graduate students' overall mental health was affected as 70.4% of graduate students experienced depression, 57.9% experienced anxiety, and 66.7% experienced significant psychological distress. Graduate students' stress levels, mental health, and social relationships were negatively impacted by the pandemic and their top 3 stressors were academic and course work demands, study life balance, and procrastination. To deal with these stressors and negative impacts, the top 3 coping methods used by graduate students were working, sleeping, and connecting with friends and family through video conferencing. Although the pandemic was challenging on graduate students and their mental health, spending time at home/outdoors and engaging with hobbies were the top 3 positive outcomes that came from the pandemic. Gender difference were also observed as females were more likely to report poor mental health than males and reported different negative impacts and coping strategies during the pandemic. In conclusion, Canadian graduate students are facing significant impacts to their mental health and well being as a result of the pandemic, especially female graduate students.

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Poster Number 26

Chemical Derivatization of Glufosinate via Trimethylation Enhancement Using Diazomethane (TrEnDi) for Enhanced Mass Spectrometry Analysis in Canola Samples

Krysten Sheedy1,3, Christian A. Rosales1,3, Jeffrey M. Manthorpe1,2,3, Jeffrey C. Smith1,2,3 1Department of Chemistry, Carleton University, Ottawa, Ontario, K1S 5B6 2Institute of Biochemistry, Carleton University, Ottawa, Ontario, K1S 5B6 3Carleton Mass Spectrometry Centre, Carleton University, Ottawa, Ontario, K1S 5B6

Glufosinate is the second most used herbicide worldwide behind glyphosate, being first discovered in the 1970’s in Streptomyces viridochromogenes and Streptomyces hygroscopicus. Glufosinate inhibits glutamine synthetase which results in increased ammonia levels in plants and mammals, and ingestion of an acute amount of glufosinate has been shown to cause convulsions and memory problems in humans. Glufosinate ammonium is sold under many trade names, such as Liberty®, which is commonly used on corn, soybeans, and canola crops. Due to its high polarity, low volatility, small size and lack of chromophores and fluorophores, glufosinate is difficult to detect at trace levels. Using the chemical derivatization strategy trimethylation enhancement using diazomethane (TrEnDi) glufosinate reacts with diazomethane and tetrafluoroboric acid to create a fixed permanent positive charge on the amino group by the formation of a 4° ammonium ion. When using high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS), analyte retention on the reversed-phase column is increased 3-fold and the sensitivity is increased 4.1-fold in standard solution with similar sensitivity gains seen for samples of Liberty®. TrEnDi methodology was applied to canola samples from two separate fields that were both sprayed with Liberty® in June 2021 and collected before harvest in September of 2021. Two separate canola extracts from each field were analysed, one of which contained seeds and shells, the other only seeds. The analysis showed there were trace quantities or no signal associated with unmodified glufosinate, however TrEnDi modification resulted in improved and quantifiable signals from permethylated glufosinate.

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Poster Number 27

Tipping the Motivational Scale: Examining the Role of AgRP in Ghrelin-Driven Feeding and Foraging

Frances Sherratt, Lindsay Hyland, Andrea Smith, Brena MacAulay, Andre Telfer Kaitlyn Joy, Alfonso Abizaid, Carleton University

A growing body of literature suggests that ghrelin not only increases hunger, but also initiates a spectrum of feeding-related responses. Indeed, foraging requires both increased motivational drive and suppression of fear of potential threats. The exact mechanism by which ghrelin alters these motivational states, however, has yet to be fully elucidated. We hypothesized that these effects are realized through ghrelin’s actions on agouti-related peptide (AgRP) neurons of the arcuate nucleus. To test this, we used the novelty suppressed feeding test to determine if exogenous ghrelin injected peripherally would facilitate approach to a palatable snack, and to see if Melanotan II (MTII), a synthetic analogue of -melanocyte-stimulating hormone, would block these effects. Intraperitoneal injections of either 0.1mg/mouse MTII and 0.1 mL of 0.9% saline, 0.1mg/mouse MTII and 20g/mouse ghrelin, 0.1 mL saline and 20g/mouse ghrelin, or two injections of 0.1 mL saline, were given to four separate groups of male and female C57BL/6 mice (n=18/group). 30 minutes post-injection, we recorded the latency to approach food, amount of food eaten, and the time spent in corners for a period of 10 minutes. Results show that ghrelin shortened the latency to approach and increased total food intake. In contrast, mice treated with MTII and ghrelin consumed less food than those treated with ghrelin, but spent more time eating and less time in corners than those treated with MTII. These findings suggest that ghrelin acts on AgRP neurons to suppress negative affective states associated with a novel environment and facilitate foraging and food intake.

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Poster Number 28

Demystifying Narp in the context of stroke

Georgina Lau, Meagan Milton MSc., Margarita Lui, Patrice Smith PhD.

Stroke is a growing problem for global healthcare. According to worldwide data, an estimated 13 million strokes occur every year, with 1 in 4 people over the age of 25 expected to have a stroke in their lifetime. Despite the rate at which strokes occur, there are few approved treatment options, and those that are available often have very restrictive criteria for use. The onset of cell death during ischemic stroke – the most common form of stroke – is rapid and propagating. Cells die through apoptotic and necrotic processes depending on the extent of blood flow impediment. Necrotic cell death – occurring predominantly in the core of the infarct – leads to extreme increases in levels of the excitatory neurotransmitter glutamate in the extracellular matrix. Neuronal activity-regulated pentraxin (aka Narp) is a calcium-dependent lectin and immediate early gene that is implicated in the aggregation of glutamate binding α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors at excitatory synapses – a process that is pivotal in long-term potentiation and long-term depression. The means by which Narp facilitates this process is still unknown, but the protein and/or mRNA are upregulated in some stroke and seizure rodent models, suggesting a role in excitotoxic pathologies. Herein, we consider the expression of Narp across stroke pathology using a robust and reproducible mouse model – photothrombosis. We also explore the implications of introducing Narp as a potential therapeutic option for the pathological stroke brain.