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Session #3: Basic Science Srishti Ahuja Claire Cheung Erin Tanaka Catalina Ionescu Jessica Koe
Phuong Nguyen Sara Niyyati Ethan Elliot Rajkumar Amardeep Singh Sekhon Nima Toussi
Judge: Maryam Rahimi-Balaei
Srishti Ahuja
#25
Undergraduate Student, University of Waterloo Supervisors: Michael Kobor & Fizza Fatima Healthy Starts mQTLHub: A database to explore mQTLs in human buccal epithelial cells
Abstract & Poster - https://bcchr.ca/posterday
University of British Columbia, Kobor Lab
mQTLhub: A database to explore mQTLs in human buccal epithelial cells Srishti Ahuja, Fizza Fatima, Dr. Chaini Konwar, Dr. Michael Kobor
Summary
Results
Results (Continued)
Methylation quantitative trait loci (mQTLs) are genetic variants that influence DNA methylation patterns of CpG sites (1). I created, mQTLhub, a publicly available database to catalogue mQTLs characterized in human buccal tissue. mQTLhub is an interactive and user-friendly database that allows users to filter, visualize, and download mQTLs based on the ID/location of associated CpG and SNP sites. All in all, mQTLhub allows quick access to mQTLs of interest and will help in understanding the genetic drivers of DNAm.
Background
Further Directions
Methods The objective of mQTLhub is to catalogue mQTLs identified in samples (>2000) from multiple cohorts of different age groups, geographical locations and ethnic backgrounds. The plot is a typical example of an mQTL which illustrates that different genotypes in the SNP loci have various levels of DNA methylation. AA homozygous samples have lower DNA methylation level, TT homozygous have increased and as expected the AT heterozygotes exhibit intermediate.
mQTLhub is created with R Shiny Web Apps, with a sample mQTL dataset from mqtldb.org. Currently filtering options in mQTLhub include genetic distance, p-value and effect size.
Created and Presented by:
Srishti Ahuja
Supervisors:
Fizza Fatima Dr. Michael Kobor
References
The database will further be expanded to include mQTLs reported in blood, cord and brain tissues. Advanced Filtering options for timepoint and genetic ancestry will be added to allow users to explore the stability of mQTLs over time (2) and across different ethnic groups (3).
1)
Abundant Quantitative Trait Loci Exist for DNA Methylation and Gene Expression in Human Brain Gibbs JR, van der Brug MP, Hernandez DG, Traynor BJ, Nalls MA, et al. (2010) Abundant Quantitative Trait Loci Exist for DNA Methylation and Gene Expression in Human Brain. PLOS Genetics 6(5): e1000952. https://doi.org/10.1371/journal.pgen.1000952
2)
Gaunt, T.R., Shihab, H.A., Hemani, G. et al. Systematic identification of genetic influences on methylation across the human life course. Genome Biol 17, 61 (2016). https://doi.org/10.1186/s13059-0160926-z
3)
Irfahan Kassam, Sili Tan, Fei Fei Gan, Woei-Yuh Saw, Linda Wei-Lin Tan, Don Kyin Nwe Moong, Richie Soong, Yik-Ying Teo, Marie Loh, Genome-wide identification of cis DNA methylation quantitative trait loci in three Southeast Asian Populations, Human Molecular Genetics, Volume 30, Issue 7, 1 April 2021, Pages 603–618, https://doi.org/10.1093/hmg/ddab038
Claire Cheung
#26
Undergraduate Student, University of British Columbia Supervisor: Pascal Lavoie, Healthy Starts The mTOR inhibitor DDIT4L decreases protein synthesis in monocytes
Abstract & Poster - https://bcchr.ca/posterday
The mTOR inhibitor DDIT4L decreases global protein synthesis in monocytes C Cheung1,2, C Michalski1,2, E Ackermann1, MA Prusinkiewicz1, CR Popescu1,2, PC Orban1,3, PM Lavoie1,4 1) BC Children’s Hospital Research Institute; 2) Department of Medicine; 3) Surgery or 4) Pediatrics, University of British Columbia
Previously, we found dampened mTOR activity in preterm monocytes, in concomitance with an upregulation of DNA-Damage-Inducible-Transcript-4Like (DDIT4L). 2
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DDIT4L REDUCES PROTEIN AND CYTOKINE SYNTHESIS
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Figure 2. DDIT4L overexpression decreases (A) protein synthesis and (B) IL-8 cytokine production. Data represented as percentage of the vehicle condition; one sample t tests.
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Figure 1. DDIT4L decreases levels of activated mTOR and its downstream target S6. MFI = median fluorescence intensity. Paired t test comparing vehicle control to doxycycline (dox) treated cells (* = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001). Only significant comparisons are shown.
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PROTEIN SYNTHESIS Clones were incubated overnight with DMSO, rapamycin, or doxycycline. After methionine depletion, the methionine analogue HPG was added for incorporation into nascent proteins and detected by flow cytometry after reaction with a fluorescent azide.
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CELL LINE MODEL U937 monocytes were transduced with either an empty (EV) or a doxycycline-inducible, DDIT4Lcontaining lentivirus; clones were generated through single-cell sorting. Select EV and DDIT4L clones were then used for downstream experiments.
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We found that DDIT4L overexpression resulted in reduced activation of both mTOR and its downstream target S6, confirming that DDIT4L serves as a negative regulator of mTOR activity. Overexpression of DDIT4L also decreased global protein synthesis, as well as moderately decreased production of the proinflammatory cytokine IL-8. Preliminary data also shows that DDIT4L remodels cellular metabolism by limiting mitochondrial capacity. Together, these results suggest a potential mechanism for the reduced responsiveness of preterm monocytes.
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Prematurely-born infants have a particularly high risk of developing severe infections during the neonatal period; this is thought to be due to the infant still possessing an immune system that is optimized for fetal life. 1 Accordingly, monocytes isolated from preterm cord blood have been reported to be less responsive to bacterial stimulation.
DDIT4L INHIBITS mTOR ACTIVATION AND FUNCTION
DDIT4L (MFI)
BACKGROUND AND OBJECTIVES
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Figure 3. DDIT4L overexpression reduces maximal respiration of mitochondria. Two-way ANOVA with an uncorrected Fisher’s LSD.
Limitations of these experiments include the use of a cell line model; the metabolism and cellular function of these clones do not completely replicate those of primary cells. REFERENCES
1. Melville JM, Moss TJM. The immune consequences of preterm birth. Frontiers in Neuroscience. 2013. 2. Kan B, Michalski C et al. Cellular metabolism constrains innate immune responses in early human ontogeny. Nature Communications. 2018. 3. Saxton RA, Sabatini DM. mTOR signalling in growth, metabolism, and disease. Cell. 2017. ACKNOWLEDGEMENTS
This project is funded by a CIHR Project Grant. CC was funded by the UBC FoM and BCCHR SSRPs, and BioTalent Canada; CM was funded by a BCCHR Graduate Studentship; PML is funded by a ClinicianInvestigator Grant Award from the BCCH Foundation.
Erin Tanaka
#27
Master’s Student, University of British Columbia Supervisor: Ramon Klein Geltink, Childhood Diseases Uncovering the regulation and function of the glycolytic enzyme triosephosphate isomerase in CD8+ T cells
Abstract & Poster - https://bcchr.ca/posterday
Uncovering the Regulation and Function of the Glycolytic Enzyme Erin Tanaka, Annette Patterson, Ramon Klein Geltink Triosephosphate Isomerase in CD8+ T Cells Introduction
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cellular energy
metabolic enzymes
Glycolysis biomass
Although glycolysis has been studied for over a century2, the molecular mechanisms of regulation of some of its enzymes remains poorly understood.
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Figure 1. TPI, GPD1, and GPD2 have diff erent levels of protein expresion during expansion and diff erentiation of mouse CD8+ T cells. One representative sample of three biological replicates shown.
TPI has the highest activity in glucose-fed effector cells
G3P Pyruvate
Figure 2. Mouse TPI activity as measured by rate of conversion from DHAP to GAP per minute per ml of cell lysate. Data shows mean±SEM for two biological replicates. *p<0.05
References activation, expansion and treatment CD8+ T cell isolation data acquisition
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Methods
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We hypothesize that different cellular environments drive triosephosphate isomerase GPD1 (TPI) regulation in T cells subsets, which influences their metabolism and function.
human PBMC
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treatment 10mM 1mM IL-15
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relative TPI activity /min/mL
nutrient availability
TPI inhibition results in marked changes to cellular metabolic activity
% Spare respiratory capacity
Immune cells rely on different metabolic pathways for function1, which is extensively studied in the context of anti-tumour activity of CD8+ T cells. One of the most studied pathway associated with the tumour-killing activity of CD8+ T cells is glycolysis.
TPI expression changes during expansion and differentiation
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Mitochondrial function can be assessed through spare respiratory capacity (SRC), which is an energy reserve of the cell that can be used during times of work or stress. SRC is associated with enhanced in vivo antitumour activity. Basal metabolic activity was determined by comparing the preference of cells to run mitochondrial or glycolytic metabolism. A higher preference for oxidative metabolism is linked with benefi cial in vivo survival. Figure 3. The TPI inhibitor 2PG (A) decreases spare respiratory capacity in both fed and glucose-starved conditions and (B) increases mitochondrial metabolism in human CD8+ T cells. Data shows mean±SEM for two biological replicates. *p<0.001
Conclusions These results suggest that the essential metabolic enzyme TPI is under complex environmental regulation and might play a role during growth, differentiation and during adaptations to environmental conditions of T cells.
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1. Klein Geltink et al. 2018. Annu.Rev. Immunol. 36,461– 488. 2.Warburg, O. 1925. Cancer Res. 9(1), 148–163. This research was funded by the UBC Work Learn program, UBC Pathology and Laboratory Medicine, BC Children's Hospital Foundation, and the Natural Sciences and Engineering Research Council of Canada.
This opens up the possibility to manipulate these pathways to further improve cellular therapy of cancer by improving T cell metabolism.
Catalina Ionescu
#28
Undergraduate Student, University of British Columbia Supervisor: Michael Anglesio, Healthy Starts Somatic activating KRAS G12 mutations in endometriosis
Abstract & Poster - https://bcchr.ca/posterday
University of British Columbia, Department of Obstetrics & Gynaecology
Somatic Activating KRAS G12 Mutations in Endometriosis Catalina L Ionescu; Natasha L Orr, MSc; Julie Hong; Amy Lum, BSc; Paul J Yong, MD, PhD; Michael Anglesio, PhD
Background
Methods
Discussions
• Endometriosis affects ~10% of people born with a uterus and is characterized by growth of endometrial-type tissue outside of the uterus. 1,2
Design: Prospective analysis of 123 people with endometriosis between 2014- 2017.
Strengths: • Enrichment of endometriosis 2,3 • The use of proven high sensitivity 2,3 methods
• Activating mutation in the Kristen rat sarcoma (KRAS) viral oncogene homologue had been found in endometriosis without risk of cancer 1,2
• KRAS is part of the growth factor receptor tyrosine kinase pathways and mutations typically in codon 12 are associated with transformation, proliferation, and invasion. 2
Objectives
Setting: BC Women’s Centre for Pelvic Pain and Endometriosis, BC Cancer Research Center, Robert H. N Ho Research Centre
Results Inclusion: People ages 18-50 with surgically confirmed endometriosis Exclusion: Concurrent cancer
Limitations: • Manual needle microdissection can result in surrounding tissue collection not associated with endometriosis2,3
Conclusions
Experimental Methods: Enrichment was done through manual needle microdissection or laser capture microdissection. Molecular testing was assayed by droplet digital PCR.
A higher prevalence of KRAS mutations was observed in the endometrioma (OMA) types compared to the deep infiltrating (DIE) and superficial peritoneal (SUP) types, similar to 2 previous literature. Although there was no significant difference between the types (p=0.13).
Statistical Analysis: Bivariate associations. Significance is p<0.05.
The analysis of the clinical data is ongoing.
1. Chen I, Thavorn K, Yong P, Choudhry AJ, Allaire C. (2019) Journal of Minimally Invasive Gynecology pii: S1553-4650(19)31182-3. doi: 10.1016/j.jmig.2019.09.771 2. Praetorius, T., Lac, V., Tessier-Cloutier, B., Senz, J., Nazeran, T., & Köbel, M. et al. (2021). Is endometriosis metastasizing? Shared somatic alterations suggest common origins across endometriotic lesions. https://doi.org/10.1101/2021.04.12.21255355 3. 3. Yong,P.J., Talhouk, A., and Anglesio, M.S. (2021). Somatic genomic events in endometriosis: review of the literature and approach to phenotyping. Reproductive sciences epub ahead of print, x.
The objective of this study is to determine the prevalence of codon 12 KRAS mutations between the three types of endometriosis and evaluate their association with pain We hypothesize that anatomical types of endometriosis will have differing prevalence of KRAS mutations and the group of people with KRAS mutations will have an increased severity of chronic pelvic pain.
Figure 3. Example of laser capture microdissection showing H&E slide (left), the before dissection (middle), and after dissection (right) images
Table 1. The prevalence of one or more somatic KRAS mutation(s) in the three types of endometriosis
Figure 1. H&E slide image
Type of One or more Somatic Endometriosis KRAS mutation prevalence
DIE
Figure 2. Example of manual needle microdissection showing the before dissection (left), and after dissection (right) images
Partners
In summary, by determining cancerassociated mutations in a noncancerous condition, it may suggest that treatments targeting molecular pathways could be considered for endometriosis.
Reference
42% (21/50)
Acknowledgement
OMA
56% (14/25)
SUP
23% (24/72)
Thank you to the Faculty of Medicine for funding my work in this project through the SSRP Award. Thank you Dr. Michael Anglesio, Dr. Paul Yong, and Natasha Orr for being phenomenal supervisors and mentors.
Jessica Koe
#29
Undergraduate Student, University of British Columbia Supervisor: Seth Parker Elucidating how post-translational modifications regulate SLC38A2/SNAT2 localization
Abstract & Poster - https://bcchr.ca/posterday
Elucidating how post-translational modifications regulate SLC38A2/SNAT2 localization Jessica Koe, Seth Parker BC Children’s Hospital Research Institute; Dept. of Biochemistry and Molecular Biology, University of British Columbia Highlights
Objective
Ø PDAC upregulates SLC38A2 at the plasma membrane to uptake alanine Ø Non-malignant cells display low levels of SLC38A2 within the cell, suggested to be stored in endosomes Ø SLC38A2 is highly post-translationally modified Ø Phosphorylation may be involved in the localization mechanism between the plasma membrane and endosomes Ø Three potential phosphorylation sites at N-terminus: Y20, Y28, Y41
To determine if phosphorylation of tyrosines Y20, Y28, and Y41 drives the localization of SLC38A2 from the plasma membrane to intracellular endosomes.
Background Role of Alanine in Pancreatic Cancer Pancreatic ductal adenocarcinoma (PDAC), the major type of pancreatic cancer, is a threat due to the lack of effective treatments. Pancreatic stellate cells (PSCs) play a role in supporting tumour growth and therapeutic resistance by secreting alanine to compensate for insufficient nutrient availability in the tumour microenvironment (1, 2). Pancreatic cancer cells exploit the alanine as their preferred carbon source to maintain biosynthesis and energy metabolism (1). Figure 1: Fate of Alanine in PDAC. Alanine (A) enters the cell through SLC38A2. In mitochondria, alanine is converted to pyruvate, which can then be converted to acetyl CoA (3). Acetyl CoA fuels the tricarboxylic acid (TCA) cycle, producing energy and biosynthetic precursors (3, 1). These cells can reserve glucose and glutamine for aerobic glycolysis and the production of biosynthetic precursors (1, 2). Image created with Biorender.com
Methods Two mutants: 1) SLC38A2-Y20F/Y28F/Y41F Ø Phosphoresistant – inhibited phosphorylation 2) SLC38A2-Y20E/Y28E/Y41E Ø Phosphomimetic – mimics phosphorylation
Table 1: Expected Localization of SLC38A2/SNAT2 for each mutant Condition Expected Localization Wild type (Control) Plasma membrane, some at endosomes SLC38A2-EGFP SLC38A2-Y20F/Y28F/Y41F Plasma membrane SLC38A2-Y20E/Y28E/Y41E A
Endosomes
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Workflow 1. Designed primers containing desired mutations, linker sequence, and/or attB sites
2. Created SLC38A2-EGFP wild-type construct by performing PCR on SLC38A2 and EGFP. Gibson Assembly to fuse PCR products, Gateway Cloning to insert wild-type sequence into vector.
Figure 4: Confocal Microscopy Images of SLC7A11 Localization from a Similar Study (5). Green represents SLC7A11, red represents acidic lysosomes and endosomes, and yellow produced as a result of co-localization. (A) Wild-type SLC7A11 (B) SLC7A11-S26A (C) SLC7A11-S26E.
Significance and Future Directions Figure 3: Diagram of Constructing Mutant SLC38A2-EGFP. 3. Created mutants by PCR on wild-type SLC38A2. Gibson Assembly to fuse PCR products, introducing mutations via primers. Gateway Cloning to insert SLC38A2-EGFP mutants into vector. Image created with Biorender.com
If results successfully shows that phosphorylation regulates SLC38A2 localization, this project would allow for follow-up studies: Ø Identify protein tyrosine kinase(s)/phosphatase(s) involved in phosphorylation/dephosphorylation mechanisms Ø Characterize activity of SLC38A2 at the endosomes Ø Discover novel strategies for targeting SLC38A2, leading to anti-cancer drugs
References 1. Sousa, C. M., Biancur, D. E., Wang, X., Halbrook, C. J., Sherman, M. H., Zhang, L., Kremer, D., Hwang, R. F., Witkiewicz, A. K., Ying, H., Asara, J. M., Evans, R. M., Cantley, L. C., Lyssiotis, C. A., and Kimmelman, A. C. (2016) Pancreatic stellate cells support tumour metabolism through autophagic alanine secretion. Nature. 536, 479-483T 2. Parker, S. J., Amendola, C. R., Hollinshead, K. E. R., Yu, Q., Yamamoto, K., EncarnaciónRosado, J., Rose, R. E., LaRue, M. M., Sohn, A. S. W., Biancur, D. E., Paulo, J. A., Gygi, S. P., Jones, D. R., Wang, H., Philips, M. R., Bar-Sagi, D., Mancias, J. D., and Kimmelman, A. C. (2020) Selective Alanine Transporter Utilization Creates a Targetable Metabolic Niche in Pancreatic Cancer. Cancer Discov. 10, 1018–1037 3. MetaCyc L-alanine degradation III. (n.d.). Retrieved July 25, 2021, from https://biocyc.org/META/NEW-IMAGE?type=PATHWAY&object=ALANINE-DEG3PWY&&ENZORG=TAX-9606&detail-level=3 4. SLC38A2 (human). (n.d.). Retrieved July 27, 2021, from https://www.phosphosite.org/proteinAction.action?id=7181&showAllSites=true 5. Mukhopadhyay, S., Biancur, D. E., Parker, S. J., Yamamoto, K., Banh, R. S., Paulo, J. A., Mancias, J. D., & Kimmelman, A. C. (2021). Autophagy is required for proper cysteine homeostasis in pancreatic cancer through regulation of SLC7A11. Proceedings of the National Academy of Sciences, 118(6). https://doi.org/10.1073/pnas.2021475118
Regulation of SLC38A2 (SNAT2) PDAC localizes SLC38A2 at the plasma membrane to co-transport sodium ions and alanine provided by PSCs from the extracellular space into the cell.
4. Transfected pancreatic cancer cells and stained cells with LysoTracker Red Figure 2: High Prevalence Post-Translational Modifications in SLC38A2 from Phosphosite (4). Proteomic data shows that SLC38A2 is highly modified at N-terminal Y20, Y28, and Y41.
Expected Results
5. Imaged using live-cell microscopy to measure co-localization of SLC38A2 and acidic organelles (endosomes/lysosomes)
Acknowledgements Ø Parker Research Team Ø Amritpal Johal Ø Siwoo Lee Ø Keeley Hewton Ø Seth Parker Ø Nisha Johal Ø Funding: UBC Faculty of Medicine Summer Studentship
Phuong Nguyen
#30
Undergraduate Student, University of British Columbia Supervisor: Christopher Maxwell, Childhood Diseases Control of the cell division axis and daughter cell size through Hyaluronan Mediated Motility Receptor (HMMR)
Abstract & Poster - https://bcchr.ca/posterday
Control of the cell division axis and daughter cell size through Hyaluronan Mediated Motility Receptor (HMMR) Phuong
(1) Nguyen ,
1 Department
Lin
(1) Mei ,
and Christopher A. Maxwell
(1) (PhD) .
of Pediatrics, BC Children’s Hospital Research Institute, University of British Columbia, Vancouver, Canada
OBJECTIVES To generate and characterize HMMR rs299290 SNP, which is associated with elevated breast cancer risk and HMMR overexpression.
1. Generation of rs299290 clones
2. Characterization of rs299290 clones Spindle position Daughter cell sizes
A
INTRODUCTION Mitotic spindle orientation is an important process that contributes to daughter cell size and fate. Spindle disorientation can lead to tumorigenesis. Hyaluronan mediated motor receptor (HMMR) has been reported to control spindle orientation by binding to CHICA and dynein light chain 1 (DYNLL1) [1][2]. Overexpression of HMMR has been found in many cancer types [3]. The mechanism and extent to which different single nucleotide polymorphisms (SNPs) in HMMR affect cell division, spindle orientation and tumorigenesis, are currently unknown.
B
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HYPOTHESIS: HMMR rs299290 SNP is associated with elevated breast cancer [4][5]. Since rs299290 occurs in the CHICA binding region, I hypothesise that rs299290 disrupts a specific protein interaction (CHICA-DYNLL1) that orients the mitotic spindle.
PRIME EDITING WORKFLOW
Figure 3. Proposed results of the characterization of rs299290 clones. Mitotic spindle orientation of rs299290 clones, are expected to misalign, resulting in unequal daughter cell sizes.
FUTURE DIRECTIONS After validating the genome edit, characterization of rs299290 clones will be carried out. Concentration of HMMR-CHICA protein complex, DYNLL1 localization, mitotic spindle orientation, and daughter cell size of rs299290 edits and WT will be compared.
SIGNIFICANCE
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Variation in cell size can affect homeostasis, development, and tumorigenesis. Findings from this study could elucidate the role of HMMR rs299290 in genomic instability and potentially, tumorigenesis.
ACKNOWLEDGEMENTS
REFERENCES
Figure 1. Prime editing general workflow.
Figure 2. (A) A prime editing complex consists of a DNA-nicking protein Cas9 nickase, fused with a Reverse Transcriptase and pegRNA [6]. (B) Sequencing results of rs299290 prime editing design 1 and 2 of wildtype (top) and rs299290 clones (bottom). Both prime editing complexes were unsuccessful in editing MCF10A cell line.
[1]Connell, Marisa, et al. “HMMR Acts in the PLK1-Dependent Spindle Positioning Pathway and Supports Neural Development.” ELife, 2017,doi:10.7554/eLife.28672. [2] Dunsch, Anja K., et al. “Dynein Light Chain 1 and a Spindle-Associated Adaptor Promote Dynein Asymmetry and Spindle Orientation.” Journal of Cell Biology, 2012, doi:10.1083/jcb.201202112. [3] He, Zhengcheng, et al. “Hyaluronan Mediated Motility Receptor (HMMR) Encodes an Evolutionarily Conserved Homeostasis, Mitosis, and Meiosis Regulator Rather than a Hyaluronan Receptor.” Cells, 2020, doi:10.3390/cells9040819. [4] Maxwell, Christopher A., et al. “Interplay between BRCA1 and RHAMM Regulates Epithelial Apicobasal Polarization and May Influence Risk of Breast Cancer.” PLoS Biology, 2011, doi:10.1371/journal.pbio.1001199. [5] Pujana, Miguel Angel, et al. “Network Modeling Links Breast Cancer Susceptibility and Centrosome Dysfunction.” Nature Genetics, 2007, doi:10.1038/ng.2007.2. [6] Anzalone, Andrew V et al. “Search-and-replace genome editing without double-strand breaks or donor DNA.” Nature vol. 576,7785 (2019): 149-157.
Sara Niyyati
#31
Undergraduate Student, University of British Columbia Supervisor: Ramon Klein Geltink, Childhood Diseases Assessing and Improving the Metabolic Fitness of Expanded Human CD8+ T-cells
Abstract & Poster - https://bcchr.ca/posterday
Metabolic Re-programming of Expanded Human CD8+ T-cells for Adoptive Cellular Therapy Sara Niyyati1,2, Erin Tanaka1,2, Annette Patterson1,2, Suzanne Vercauteren1,2, Ramon Klein Geltink1,2 1 BC
Children’s Hospital Research Institute. 2Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada.
Introduction
Results
CD8 expressing T-cells are a crucial part of our immune system and can provide protection against viral infections and the growth of cancer. The success of T-cell infusions as a therapeutic strategy requires large numbers of cells to be generated in the lab.
The oxygen consumption rate (OCR) of cells is an indicator of mitochondrial respiration, as oxygen is consumed as the ultimate electron acceptor at the end of the electron transport chain during oxidative phosphorylation (OXPHOS). The extracellular acidification rate (ECAR) is a measure of the pH in the extracellular media, and it is an indicator of glycolytic metabolism as lactate is produced during glycolysis.(2) The OCR:ECAR ratio is an indicator of the metabolic profile of each cell culture: a higher ratio indicates relatively more mitochondrial metabolism whereas a lower ratio signifies relatively more glycolytic metabolism.(2) The spare respiratory capacity (SRC) is a term used to measure the excess ATP the mitochondria can produce, above regular baseline levels, in the case of a sudden increase in energy demand and it acts as an indicator of mitochondrial fitness.(3) A
B
Figure 1. 24-hour starved human CD8+ T-cells exhibit more mitochondrial metabolism and greater fitness, relative to control cells at 1-week post-activation. (A) The starved cells from a representative donor demonstrate a significant increase in OCR:ECAR 1-week post-activation, indicating that the glucose-restricted cells exhibit relatively more mitochondrial metabolism, in comparison to the non-glucose-restricted cells. n=4 technical replicates, ****p<0.0001 (B) The starved cells from a representative donor demonstrate a significant increase in SRC, indicating that they are more metabolically fit, in comparison to the control cells. n=4 technical replicates, ****p<0.0001
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Figure 2. 24-hour and 48-hour glucose-restricted human CD8+ T-cells exhibit more glycolytic metabolism and reduced fitness, relative to un-starved cells at 2-weeks post-activation. (A) The starved cells (24h and 48h) from a representative donor demonstrate a significant decrease in OCR:ECAR 1-week post-activation, indicating that the non-glucoserestricted cells exhibit relatively more mitochondrial metabolism, in comparison to the glucose-restricted cells. n=4 technical replicates, *p<0.05, **p<0.01, ns: non-significant. (B) There is no significant difference in SRC between the starved (24h/48h) and control treatments. n=4 technical replicates, ns: non-significant.
The rapid expansion of T-cells is very demanding on cellular resources for both energy and biomass production. Therefore, cellular metabolism plays an important role in the T-cell mediated immune response. CD8+ T-cells have two main metabolic processes: 1) Glycolysis (glycolytic metabolism) 2) TCA cycle/oxidative phosphorylation __.(mitochondrial metabolism)
Previous studies using mouse models of cancer found that temporarily reducing glucose metabolism and enhancing mitochondrial metabolism via glucose restriction was associated with better anti-tumor immune function.(1) This finding could be applied to cancer therapy, however, the current procedure and medium used for clinical human T-cell product expansions promotes enhanced glucose metabolism as the cells use glucose metabolism to grow rapidly – a characteristic that leads to reduced in-vivo function. To translate the previous finding to a clinically relevant context, we hypothesized that expanded human T-cell products would strongly benefit from a protocol by which mitochondrial metabolism is increased by glucose restriction before infusion.
Methods
Conclusions and Future Directions CD8+ T-cells were isolated and activated using anti-CD2/3/28 Tetramers and expanded in immunocult media (StemCell Technologies). 1 and 2 weeks post-activation, expanded CD8+ T-cells were exposed to 1mM glucose (Starved) or 25mM glucose (Control) for 24-48 hours and harvested for Seahorse assay (Agilent) to assess relative changes in glucose and mitochondrial metabolism.
The results demonstrated that the glucose restricted cells alter their metabolism to exhibit more mitochondrial metabolism and become less glucose dependent 1-week post-activation, relative to the control non-glucose restricted cells. The starved cells also exhibited greater mitochondrial fitness at week 1, in comparison to the control cells. These effects were no longer achieved at the 2-week timepoint. As such, like mouse cells, glucose-restricted human CD8+ T-cells increase mitochondrial metabolism, previously associated with improved in-vivo function. Based on these data, starvation protocols are achievable 1-week postactivation. We will next test if our protocols can be applied to T-cell expansions for clinical use and further optimize the timing of starvation intervention.
References and Acknowledgements 1.
Klein Geltink, R.I., Edwards-Hicks, J., Apostolova, P. et al. Metabolic conditioning of CD8+ effector T cells for adoptive cell therapy. Nat Metab 2, 703– 716 (2020). https://doi.org/10.1038/s42255-020-0256-z Agilent. (n.d.). https://www.agilent.com/en/products/cell-analysis/how-seahorse-xf-analyzers-work. Claus Desler, Thomas Lau Hansen, Jane Bruun Frederiksen, Maiken Lise Marcker, Keshav K. Singh, Lene Juel Rasmussen, "Is There a Link between Mitochondrial Reserve Respiratory Capacity and Aging?", Journal of Aging Research, vol. 2012, Article ID 192503, 9 pages, 2012. https://doi.org/10.1155/2012/192503 Figures created using Biorender.com 2. 3.
My work in this project was funded by a BC Children’s Hospital Summer Studentship
#32
Ethan Elliot Rajkumar
Undergraduate Student, University of British Columbia Supervisor: Bruce Vallance, Childhood Diseases A Closer Examination into Innate Epithelial Response Regulation by Gut Microbiota Against Enteric Bacterial Pathogens
Abstract & Poster - https://bcchr.ca/posterday
A Closer Examination into Innate Epithelial Response Regulation by Gut Microbiota Against Enteric Pathogens
Ethan Elliot Rajkumar1 , Joannie Allaire1 , Yan Chen1 , Larissa Celiberto1 , Mariana Hill1 , Elena Verdu2 , Bruce Vallance1 1
2
: Department of Pediatrics, University of British Columbia, BC Children′ s Hospital Research Institute
METHODS
: Department of Medicine, McMaster University
In vitro: Primary intestinal epithelial organoid monolayers derived from mouse are infected with C. rodentium, pre-induced with DMEM cell culture media or not pre-induced. TUNEL staining and LDH assay was used to determine the extent of cell death.
INTRODUCTION
Intestinal Epithelial Cells (IEC) are a single layer of cells that preserve gut homeostasis. They can activate the innate immune response by promoting proinflammatory mechanisms. [1]
SPECIFIC PATHOGENIC FREE (SPF)
Certain enteric pathogens such as Enterhemmoragic Escherichia coli (EHEC) and Citrobacter rodentium (mouse model analog), can circumvent these proinflammatory mechanisms by forming footholds. [2] Little is known about how the gut microbiota can influence the innate immune response against these pathogens. [3]
HYPOTHESIS
OBJECTIVE
Proper exposure to the gut microbiota exerts important influences on the early innate immune response by IEC to C. rodentium.
Develop an in vitro model of infection using C. rodentium and primary IEC monolayers. Evaluate in vivo IEC innate immune responses of germ-free (GF) and specific-pathogen-free (SPF) mice following C. rodentium infection.
Figure 1: Enteric Pathogen adhering to an intestinal epithelial cell by forming a foothold/pedestal REFERENCES
GERM FREE (GF)
In vivo: SPF and GF mice were infected with C. Rodentium for 1 to 3 days. After mice tissues were extracted, gene expression analysis and pathology score calculations were obtained to determine cell integrity, death and bacterial adherence.
IN VITRO
IN VIVO
CONCLUSIONS: qPCR
ORGANOID GROWTH
HISTOPATHOLOGY
MONOLAYER INFECTION
IMMUNOSTAINING
LDH CELL DEATH ASSAY
The use of pre-induced bacteria greatly increased the infection levels which correlated with more cell death observed on infected monolayer than the non-infected one. In vivo, histological staining showed that infected germ-free mice have increased cell death as early as day 3 post-infection whereas little to no cell death was present in infected SPF mice.
IMPLICATIONS:
This in vitro infection model can serve as a standard method to determine how the microbiota is impacting IEC inflammatory response and cell death when facing Citrobacter. It will help to understand this relationship. This can also aid in creating for alternative therapies where certain antibiotic treatments fail.
[1] Allaire JM, et. al, Trends Immunol. 2018 [2] Campellone, K. Current Opinion in Microbiology 2003 [3] Rakoff-Nahoum et. al, 2004. Cell, Photography: Woodward et. Al, Journal of Comparative Physiology (2005), all images made with BioRender and Prism
LDH Assay Results
IN VIVO RESULTS % LDH VS NI
IN VITRO RESULTS
qPCR
60
GF Figure 2: Gene expression analysis on cecum tissues of C. rodentium infected SPF and GF mice. *P> 0.05, MannWhitney and Welch’s t-tests n=3.
40
20
NI 6H
A
NI
C
WT 6H
NI 6H
Non SPF Induced
WT 6H
WT 8H
Pre-Induced
Figure 4 A , B, C: Monolayer infected with overnight culture of C. rodentium for 6 hour (A). Monolayer infected for 6 and 8 hours using a pre-induced culture of C. rodentium (B). Cell death ratio of the represented monolayer (shown as # TUNEL + cells / total # cells, NI means noninfected) (C).
6 HR
B
6 HR
8 HR
B
Type of Infection
NO SPIN Figure 3: H&E staining (A) and histological scoring graphs (B) of cecum from C. rodentium infected SPF and GF mice at Day 1 and Day 3.
DAPI TUNEL LPS PHALLODIN
SPIN WT D1
WT D3
WT
SPF
0
GF
A
Figure 5: Lactate Dehydrogenase (LDH) assay used to evaluate cell death on the supernatant of C. rodentium infected monolayer, represented as percentage over noninfected (NI).
#33 Amardeep Singh Sekhon
Undergraduate Student, University of British Columbia Supervisor: Bruce Verchere, Childhood Diseases The role of islet amyloid polypeptide in islet function and beta-cell mass
Abstract & Poster - https://bcchr.ca/posterday
Role of islet amyloid polypeptide in pancreatic islet function & β-cell mass Amardeep S.
1,2 Sekhon
, Austin J.
3 Taylor ,
C. Bruce
3,4 Verchere
(1) Department
of Anesthesiology, Pharmacology and Therapeutics, Faculty of Medicine, University of British Columbia, (2) Canucks for Kids Fund Childhood Diabetes Laboratories Summer Studentship (3) Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of British Columbia, (4) Department of Surgery, Faculty of Medicine, University of British Columbia
Study Background and Objectives
Previous Results
• Islet amyloid polypeptide (IAPP) is co-stored and cosecreted with insulin and is the 2nd most abundant peptide in β cells. • IAPP may induce satiety and slow gastric emptying (Figure 1), but the biological role of IAPP is not fully understood.
A
B
• Elevated fasting glycemia in IAPP-/- mice may be due to altered islet function
Significance Figures 2A-2C. Body weight, blood glucose (random), and pancreas weights from both IAPP-null (KO) and wild-type (WT) mice fed a control diet and high-fat diet at 12months-old.
Figure 1. Diagram summarizing the roles of islet amyloid polypeptide (IAPP) in the context of body weight. Biological actions for IAPP have been found in the induction of satiety and slowing of gastric emptying, but its actions remain poorly understood in regulating glycemia.
• β-cell mass and function are increased in insulin resistance and obesity.
Results
B
A
• β-cell mass was calculated by (pancreas mass X insulinpositive area) / pancreas area.
1. Mietlicki-Baase EG. Amylin-mediated control of glycemia, energy balance, and cognition. Physiology & Behavior. 2016;162:130-40.
Acknowledgement
+/+ IAPP
C
-/IAPP
beta-cell mass Figure 3. (A,B) Stained pancreas sections of +/+ IAPP islet amyloid polypeptide (IAPP) knockout -/-) mice, -/(IAPPIAPP wild-type (IAPP+/+) mice. (C) βcell mass of IAPP knockout (KO; IAPP-/-) mice, and wild-type (WT; IAPP+/+) mice at 12months-old.
6
Beta-cell mass (mg)
• Pancreas sections (3 per animal) were analyzed by immunohistochemistry for insulin, and whole pancreas sections were analyzed by tiled brightfield imaging.
• Further studies are needed on the use of exogenous IAPP analogues as replacement therapies to improve obesity and glycemic control in T1D and T2D.
2. Inaishi J, Saisho Y. Beta-Cell Mass in Obesity and Type 2 Diabetes, and Its Relation to Pancreas Fat: A Mini-Review. Nutrients. 2020;12(12):3846.
• To determine whether β-cell mass, granule morphology and islet architecture is altered in IAPP-deficient mice on a standard diet.
• Pancreases from IAPP-/- and IAPP+/+ mice were dissected at 12-months old and weighed (n=5-18). Mice were fed either a control diet (10% kcal from fat) or high-fat diet (45% kcal from fat).
• Loss of IAPP alongside loss of insulin in both type 1 and type 2 diabetes may further impact glycemic control and regulation of food intake.
References
Objective
Methods
• Based on our previous data, we hypothesized that IAPP-/- mice will have increased β-cell mass compared to IAPP+/+ (both control diet-fed) • β-cell mass is not significantly different between IAPP-/- and IAPP+/+ male mice.
C
• Preliminary results found that male IAPP-null (IAPP-/-) fed a normal diet tended to have higher body weight and elevated fasting blood glucose levels compared to wild-type (WT) mice (Figure 2).
Conclusions
4
2
0
IAPP:
WT
KO
• I would like to extend my huge gratitude towards the Canucks for Kids Fund Childhood Diabetes Laboratories Summer Studentship for funding my work over the summer. • I would also like to give a special thanks to Austin J. Taylor for supplying the in vivo data used in this project. • Last, but not least, I would like to thank Dr. Bruce Verchere for taking me on as a student within his lab and his ongoing support as a supervisor. Funding
Nima Toussi
#34
Undergraduate Student, University of British Columbia Supervisor: Todd Woodward, Brain, Behaviour & Development Functional Assessment of the fMRI-derived Extraction of Meaning Network
Abstract & Poster - https://bcchr.ca/posterday
Department of Psychiatry, University of British Columbia
Functional Assessment of the fMRI-derived Extraction of Meaning Network Nima Toussi1,2, Chantal Percival1,2, Todd Woodward1,2 1. Department of Psychiatry, University of British Columbia, Vancouver, BC
2. BC Mental Health & Substance Use Services, Vancouver, BC
Introduction
Metrical Stress Task [2]
Task Switching Task [3]
Facial Discrimination Task [4]
The Extraction of Meaning Network (LANG) is one of the twelve fMRI-derived functional brain networks identified through Task-based Functional Magnetic Resonance Imaging (fMRI) and Constrained Principal Component Analysis (CPCA) [1]. The LANG network is hypothesized to be recruited for the generation of semantic associations
•
•
• Participants (n=70) were presented with facial discrimination conditions and asked to respond “target” or “nontarget” with button presses. • Facial discrimination conditions included: Angry, Fear, Happy, Sad, and Age. • Significant effect of target condition, demonstrating that the LANG network responds to non-lexical stimuli
Objectives
•
Determine the function of the LANG network through analyzing the influences of task conditions on network recruitment
• • •
Participants (n=79) tasked with evaluating auditory and semantic characteristics of words Two conditions: Phonological and Semantic: In Phonological condition, participants had to judge which syllable stress was placed on. In the Semantic Condition, participants had to evaluate if the presented word was positive or negative No statistical significance between the two conditions, demonstrating that LANG network responds to both phonological and semantic mediums of lexical stimuli
•
•
Participants (n=44) tasked with performing 3 discrete tasks in alteration: (1) judging whether shapes are blue or red, (2) judging whether numbers are odd or even, and (3) judging whether letters are uppercase or lowercase. Manipulated variable is that the judged-stimuli can contain one, two or all three task dimensions, termed as stimulus valency No statistical significance of stimulus valency on response, demonstrating that LANG network is not influenced by volitional attention
Methods •
•
Previous studies extracted functional brain networks through fMRI-CPCA [2,3,4], and we compiled all the cognitive tasks that showed recruitment of the LANG; three are presented here. LANG networks were identified by characteristic activation and suppression patterns. Corresponding estimated HDRs were created and examined for condition effects and interactions using IBM SPSS.
Significance Identification of the differences in activation and deactivation of the LANG network in patients with schizophrenia and healthy controls will increase our understanding of functional neural connectivity in schizophrenia patients and contribute to the future use of neuromodulation as a possible treatment option for schizophrenia.
Reference / Bibliography 1. Percival, C. M., Zahid, H. B., Woodward, T. S. (2020). Task-Based Brain Networks Detectable with fMRI [fMRI image]. Github.com. https://github.com/CNoSLab/Woodward_Atlas/tree/main/Network_Images 2. Sanford, N. (n.d.).TSI-TGT. University of British Columbia. 3. Metzak, P., Sanford, N., Percival, C.M., Hsiao, H., and Woodward, T S. (n.d.). Functional Brain Networks involved in Attentional Biasing in Schizophrenia. University of British Columbia 4. Ghogari, V.M., Sanford, N., Spilka, M. J., and Woodward, T.S. (2017). Task-related functional connectivity analysis of emotion discrimination in a family study of Schizophrenia. Schizophrenia
Conclusion LANG network is recruited for the cognitive ‘super-process’ of semantic processing, regardless of type of stimulus
Bulletin, 43(6): 1348-1362. https://doi:10.1093/schbul/sbx004
Acknowledgements This project was funded by the funding from the NSERC Undergraduate Student Research Award