SBC 2014 National Competitor Project Descriptions

First place & Commercialization Award
Isothermal Nucleic Acid Amplification System for Point-of-Care HIV Diagnosis

Nicole Ticea, Grade 10
York House School, Burnaby, BC

Approximately 34 million people worldwide are infected with HIV, including 2.3 million children. To date, there is no accurate point-of-care HIV test for neonates or acutely infected adults. Prompt diagnosis is vital for early initiation of anti-retroviral therapy, which can significantly suppress infection and improve survival rates. This project presents a rapid isothermal nucleic acid amplification system capable of performing point-of-care HIV diagnosis from crude samples.

Elements of thermal lysis, recombinase polymerase amplification (RPA), and immunochromatographic strip (ICS) detection were combined to create a self-contained assay possessing the sensitivity necessary to diagnose the disease in its acute stage. This project outlines a strategy that is: (1) capable of performing isothermal amplification and detection of HIV DNA and RNA; (2) equipped to diagnose viral nucleic acids using raw, unprepared samples; and (3) readily adaptable for integration into a micro-device format. The RPA-ICS assay was able to amplify and detect HIV sequences, producing a robust endpoint signal. The system was shown to successfully detect HIV DNA in samples containing a 4% concentration of HIV genome-encoded cells, non-infected cells, proteins, and other whole blood constituents with minimal front-end sample preparation. For use in a point-of-care setting, a micro-device incorporating all steps of the assay was conceived using computer-assisted-design software. This device is capable of analyzing samples without external manipulation or access to specialized equipment. Through virtue of its rapid, isothermal, and self-contained nature, the system described here provides proof-of-concept results to support the development of a successful point-of-care HIV nucleic acid-based test.

Second Place
Identification of Novel Broad-Spectrum Antimicrobial Compounds in Curcuma amada

Varsha Jayasankar, Grade 12
Sir Winston Churchill Secondary School, St. Catharines, ON

Clostridium difficile and Methicillin-resistant Staphylococcus aureus (MRSA) have been the main source of nosocomial infections in North America since 2003. There is currently no effective antibiotic against both C. difficile and MRSA. Similarly, plant pathogenic bacteria such as Erwinia amylovora cause billions of dollars in crop damage. Previously, an extract (Curcuma amada Crude Extract; CACE) from mango ginger (Curcuma amada) was proven to have antimicrobial properties. In this research project, CACE was purified into four fractions using Sep-Pak filtration in order to identify antimicrobial compounds from mango ginger. These four fractions were tested for antimicrobial activity on two plant pathogenic bacteria, two human pathogenic bacteria, and two plant pathogenic fungi: Erwinia amylovora, Xanthomonas campestris, Escherichia coli, Clostridium difficile, MRSA, Verticillium dahliae, Rhizoctonia solani, respectively, and observed using fluorescent microscopy. Results proved fraction A was the most effective against fungi, while fraction D showed antibacterial activity. Since fraction D was effective against plant and human pathogens, it was investigated as a preventative and curative treatment against bacteria. Bacterial growth was significantly suppressed with both treatments. Based on these promising results, Fraction D was analyzed using Mass Spectrometry, revealing a peak at 6.8min, which contained a compound of mass 395. This compound was identified as 2,4,6-trihydroxy-3,5-diprenylhydrochalcone, the first report of this compound’s potential antibacterial activity. This compound can be developed into an antibiotic for patients suffering from nosocomial infections, a surface sanitation method in hospitals, and as an organic pesticide for crops.

Third Place
Image Processing techniques for the analysis of ultrasound stimulated bubble interactions with fibrin clots

Anoop Manjunath, Grade 11
University of Toronto Schools, Toronto, ON

Blood clots can cause cardiovascular diseases such as deep vein thrombosis, myocardial infarction and stroke. In these scenarios, the timely clearance of occlusions caused by clots is of the utmost importance and current treatments suffer from significant limitations. The ability for ultrasound stimulated microbubbles (USMBs) to potentiate blood clot lysis has been demonstrated in vitro, in vivo, and in initial clinical studies. The mechanisms, however, are not well understood. The overall purpose of this research was to gain insight into the mechanisms of action in order to guide the rational development of contrast agents (bubble sizes) and specific pulsing schemes to better perform sonothrombolysis. Within this broad goal the specific objective of the current study was to utilize image processing techniques to aid in the analysis of experimental data of the interactions between individual USMBs and fibrin clots. Pore size analysis was performed using ImageJ and Imaris to analyze 2-photon microscopy images of fibrin clots, a process which can also be applied to other fibrous networks. The two clot types were determined to have mean pore sizes of 2.97±1.02 µm and 7.32±3.03 µm in the case of ‘fine’ and ‘coarse’ clots, respectively. From fast frame images of bubbles penetrating fibrin clots the position of the bubbles over time, and thus the velocity was determined using a similar processing technique as for the pore size analysis. Greater optimization of both the preprocessing and algorithms, by for instance developing better circle detection would increase speed and reduce user intervention.

Fourth Place
Identification and Functional Characterization of Putative Phosphorylation Sites on Scleraxis

Ryan Wang, Grade 12
St. John’s-Ravenscourt School, Winnipeg, MB

Cardiac fibrosis is a significant clinical problem yet lacks effective therapies. The Czubryt laboratory has shown that the transcription factor scleraxis is a key regulator of collagen 1α2 gene expression in cardiac myofibroblasts. Here we identify the mechanism of scleraxis activation and its effect on downstream target genes. In-silico analysis of scleraxis protein sequence revealed putative phosphorylation sites which are conserved across mouse, rat and human species. Mutation of this phosphorylatable motif resulted in significant disruption of type I collagen and myofibroblast marker gene expression due to reduced binding to specific DNA sites (E boxes). A phosphorylation-mimic scleraxis mutant exhibited similar activity to the intact scleraxis. Our results show for the first time that scleraxis is constitutively phosphorylated under basal conditions by casein kinase 2 (CK2), and blockade of CK2 activity interferes with the ability of scleraxis to modulate the expression of its target genes. This is a novel mechanism by which scleraxis function is regulated, and thus could be potentially used to develop a therapeutic strategy for treating cardiac fibrosis.

Fifth Place
Inhibition of HIV-1 Replication Cycle with the CRISPR/Cas9 System

Julien Sénécal, 1st Year CEGEP
Collège Jean-de-Brébeuf, Montreal, QC

Even though there currently is an antiretroviral therapy against HIV, over 34 million individuals are infected worldwide, hence the importance of novel treatments. Over the past years, numerous technologies were developed in the domain of genetic engineering as to DNA modification in cells. The CRISPR/Cas9 system, which comes from the Streptococcus pyogenes bacteria, is one of these technologies and here is used to inhibit HIV-1 replication. The Cas9 system, guided with HIV-1 sequences (LTR, GAG, POL), is transfected into HEK 293T cells and Cas9 is expressed in the cells. Data analysis of the Western Blot validation of the system with IFITM2 inhibition, a protein that was expressed in the cells, suggests that Cas9 cleaves double-stranded DNA (as HIV-1 DNA in the infected cells). After infection of the 293T with VSV-G pseudotyped HIV-1, a decreased of the infectivity rate in the cells up to 9 fold have been observed. Based on these results, it can be said that Cas9 is a promising tool in regard of novel treatments for HIV.

Other Competitors
Development of a Novel Quantum Dot-Aptamer Bioconjugate Targeted Cancer Therapy for Precision Nanomedicine Applications

Amit Scheer, Grade 10
Colonel By Secondary School, Ottawa, ON

A novel nanoparticle for targeted cancer therapeutics is described. This research was effectuated to create a theranostic bioconjugate with an optimal effective therapeutic index, achieved by biomarker-specific targeting. Estimates show that over 14 million new cases of cancer are diagnosed annually worldwide. Aptamer-quantum dot (APT-QD) bioconjugates were synthesized by conjugating cadmium-telluride quantum dots (QDs, semiconductor nanoparticles) to aptamers (nucleic-acid based ligands), by amide crosslinking. Aptamers targeted mucin-1 (MUC1), a glycosylated surface protein overexpressed on many cancers, including MCF7 breast cancer cells, and only minimally expressed in MCF-10A non-cancerous cells. The bioconjugate and unmodified QD treatments (the control) were tested for cellular uptake and cytotoxicity in MCF7 (cancerous) and MCF-10A (comparison) cell cultures. MTT assays, which quantify cellular viability by assessing mitochondrial activity, were used for dose-response analysis at several treatment concentrations. APT-QDs caused a statistically significant decrease in viability specifically in MUC1-overexpressing cultures, suggesting cell-specific internalization by receptor-mediated endocytosis. Apoptosis and necrosis were quantified using immunofluorescence assays; bioconjugate-treated cells were early apoptotic after 4 hours, proving effective initiation of programmed cell death. Finally, confocal microscopy was used for aptamer-dependent nanoparticle internalization analysis, demonstrating that APT-QDs accumulate outside of nuclei. A fluorochrome-modified DNA complement to the aptamer was synthesized for co-localization of aptamers and QDs, proving effective endosomal escape for both components. The bioconjugate has applications in combination and theranostic treatments for cancer, and in precision medicine to diversify targeting based on patient-specific panomics analyses. The researcher created a novel bioconjugate nanoparticle and has proven numerous viable applications in cancer therapeutics.

Tumour Origami: A Three-Dimensional Visualization of Drug Resistance in Malignant Tumours

Jessica Wickware, Grade 12
Harry Ainlay High School, Edmonton, AB

The conventional method for in vitro cell culture is to grow cells on two dimensional (2D) surfaces in culture flasks. However, 2D cell culture does not accurately represent the environment of cells in vivo, as it does not replicate the chemical environment surrounding these cells inside three-dimensional (3D) tissue. In my project, I developed a 3D matrix for the culture of breast cancer cells, which mimics the environment of these cells in vivo, serving as a better model for resistance of these cells to anticancer drugs. We identified this matrix using paper arrays modified by chemicals naturally present in the extracellular matrix (peptides). After seeding the MCF-7 breast cancer cells onto these arrays, we cultured them in growth medium with or without the anticancer drug Taxol. Counting the number of cells inside each element of the array permitted the identification of peptides that support cell adhesion, growth and resistance to Taxol. From 32 tested materials (peptides), 15 supported adhesion and growth of cells and 5 supported sustained growth in the presence of a lethal dose of Taxol. Furthermore, the morphology of cells in arrays without Taxol closely resembled that of tumour cells in vivo, in that cells cultured in growth medium formed clumps (mammospheres), while cells cultured in Taxol-containing media grew as isolated cells. In summary, culturing cells on a 3D surface such as paper presents a more precise representation of in vivo tumours and subsequently a deeper understanding of drug resistance in breast cancer cells.

Identification of Leaf Rust Resistance in Wheat

Wenyu Ruan, Grade 9, & Amy Yu Ruiyun Wang, Grade 10
Walter Murray Collegiate Institute, Saskatoon, SK

Leaf rust is the most common disease in wheat, a crop which contributes $11B annually to Canada’s economy. The most effective strategy to control leaf rust has been to grow resistant varieties. There are two general types of resistance genes found in wheat: Race-specific genes confer a high-level of resistance to specific strains of leaf rust but can be easily overcome by genetic mutation in pathogen populations, while slow rusting (APR) resistance provides partial resistance to a broad spectrum of races, but is typically effective only at the adult stage of plant growth. A three-phase experiment was conducted on a doubled-haploid population derived from the cross RL4452/AC Domain to determine if the resistance of a recently discovered gene (Lr2BS) worked with other resistance genes to synergistically enhance resistance to leaf rust. Linkage and quantitative trait loci (QTL) mapping were performed by combining our new genotypic data with a previously generated genetic map for this population, then adding rust disease data from our experiment to identify genomic regions associated with leaf rust resistance. In addition, a fluorescent microscope was used to examine host-pathogen interaction on a cellular level. These experiments showed that lines carrying Lr2BS alone, and in combination with other APR genes were susceptible at the seedling stage, which suggests that Lr2BS is an adult plant gene. It appears that the synergistic effect of some multiple gene combinations, including Lr2BS, enhances leaf rust resistance. Furthermore, QTL mapping identified an uncharacterized resistance gene (LrUsw4B) that conferred resistance at the seedling stage.

Can Popular Acne Treatments Stimulate or Exacerbate Cancer Growth?

Mark Hewitt, Grade 12
Bishops College High School, St. John’s, NFLD

Acne is the most common skin disease in the world. 85% of all people will experience acne, and 60% of these people will treat it. Benzoyl Peroxide and Retinoic Acid have been identified as the two most common acne fighting regimens used today. Approximately 3.9 billion people are actively using these compounds to treat their acne. Benzoyl Peroxide and Retinoic Acid have the ability to form free radical compounds upon contact with the body. Free radicals are highly reactive chemicals that have the potential to harm cells by means of damaging cellular DNA, proteins and the cell membrane. Damage to cellular DNA specifically, may play an important role in the development of cancer. However, these warnings do not appear once on the bottles of these acne treatments. Cancer is a huge public health problem. There are over 200 different types of cancer that affect humans today, making cancer the second leading cause of human death. Two different principal pathways of malignancy were examined in this project: • Cell Proliferation (cell growth) • Invasiveness (through protein expression) After 8 very meticulous months in the lab, it was shown that these compounds increased cellular proliferation and invasiveness in normal human cells, cells from a non- invasive cancer and cells from an invasive cancer. This project yields results that are incredibly important in the biotechnology field. If something that people use every day is found to stimulate cancer growth, it can eliminated from society. This is already half the battle of fighting cancer.