bluuebunny/crossref_metadata_2025 huggingface dataset:
INVESTIGATING THE ROLE AND REGULATION OF THE SPECIFIC ANDGENERAL PRB-E2F1 INTERACTIONS
Authors: Cecchini Matthew J , Dick Frederick A
Published: May 2017

Background/Purpose. The retinoblastoma tumor suppressor protein (pRB) plays a central role in proliferative control and is a frequent target for inactivation in cancer. The G1-Sphase transition of the cell cycle is regulated by pRB, which is capable of interacting with E2F family members and inhibiting the transcription of genesrequired to progress into S-phase. E2F1 is unique from other E2F family members as it can induce both apoptosis and proliferation. To control these contrasting functions of E2F1, a second E2F1 binding site exists in the C-terminus of pRB that can control apoptosis separately from proliferation. This anti-apoptotic function of pRBcan in some circumstances promote tumorigenesis, which leads to the question; is pRB a tumor suppressor or an oncogene?

Methods. To investigate this, a gene-targeted mouse model is being engineered to selectively disrupt the ability of pRB to control proliferation through the general E2F binding site while still retaining the ability tocontrol apoptosis through the specific E2F1 site.

Results. A series of novel mutants were engineered to selectively disrupt the binding of E2Fs at the general site, and prevent pRB from controlling proliferation. The mutants retain the ability to bind E2F1 and control apoptosis through thespecific binding site, which is not disrupted.

Conclusions. By separating the ability of pRB to control proliferation and apoptosis under in vivo conditions, a better understanding into the significance of these two functions in development and tumorigenesis can be gained.
DISSECTING THE SIGNALLING PATHWAYS INVOLVED IN THEANTI-HYPERTROPHIC EFFECTS OF RESVERATROL
Authors: Chan Anita YM , Dolinsky Vernon W , Soltys Carrie-Lynn M , Dyck Jason RB
Published: May 2017

Background: Pathological left ventricular hypertrophy is associated with all-cause mortality; however, effective treatment for this condition is currently lacking. We have shown that activation of AMP-activated protein kinase (AMPK) by resveratrol can inhibit myocardial hypertrophy by decreasing protein synthesis and suppressing nuclear factor ofactivated T-cells (NFAT) activation. However, the mechanism by which resveratrol affects AMPK isunknown. Since LKB1 is the upstream kinase of AMPK, we hypothesize that resveratrol signals via LKB1 toactivate AMPK and it is this signalling pathway that contributes to the anti-hypertrophic effects of resveratrol.

Methods: Wildtype (WT), LKB1 null, and AMPK null mouseembryonic fibroblasts (MEFs) were treated with vehicle or 100?M resveratrol for 1 h. Cell lysates were subjected to immunoblot analysis to examine the phosphorylation status of the proteins of interest. NFAT-dependent transcription was also measured in these MEFs using a NFAT-luciferase reporter transgene.

Results: While resveratrol treatment increased AMPK phosphorylation in WT MEFs, resveratrol was unable to activate AMPK in LKB1 null MEFs. In addition, resveratrol suppressed NFAT-dependent transcription in WT MEFs, yet failed to inhibit NFAT activity in AMPK null MEFs.

Conclusion: These data combined with our previous data suggest that resveratrol signals through LKB1 to activate AMPK and that this activation results in suppressed protein synthesis and reduced NFAT activation. As the development of pathologicalcardiac hypertrophy is dependent on protein synthesis and NFAT activation, inhibition of these two pathways by resveratrol may be an exciting new approach for the treatment of pathological cardiac hypertrophy.
CHEMICAL GENETICS REVEALS A COMPLEX FUNCTIONAL GROUND STATEOF NEURAL STEM CELLS
Authors: Diamandis Phedias , Wildenhain Jan , Clarke Ian D , Sacher Adrian G , Graham Jeremy , Bellows David S , Ling Erick KM , Ward Ryan J , Jamieson Leanne G , Tyers Mike , Dirks Peter B
Published: May 2017

The identification of self-renewing and multipotent neural stem cells (NSCs) in the mammalian brain holds promise for the treatment of neurological diseases and has yielded newinsight into brain cancer. However, the complete repertoire of signaling pathways that governs the proliferation and self-renewal of NSCs, which we refer to as the ‘ground state’, remains largely uncharacterized. Although the candidate gene approach has uncovered vital pathways in NSC biology, so far only a few highly studied pathways have been investigated. Based on the intimate relationship between NSC self-renewal and neurosphere proliferation,we undertook a chemical genetic screen for inhibitors of neurosphere proliferation in order to probe the operational circuitry of the NSC. The screen recovered small molecules known to affect neurotransmission pathways previously thought to operate primarily in the mature central nervous system; these compounds also had potent inhibitory effects on cultures enriched for brain cancer stem cells. These results suggest that clinically approved neuromodulators may remodel the mature central nervous system and find application in the treatment of brain cancer.

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GENERATION OF AN EFFECTIVE IN VIVO CTL RESPONSE USINGVACCINE APPROACHES IN A MODEL OF AUTOIMMUNITY
Authors: Dissanayake D T , Ohashi P S
Published: May 2017

Background: Various interactions between tumours and their environment make it difficult to determine the factors that are essential to the induction of a robust CTL response by immunotherapeutic strategies against tissue antigens. We therefore sought to develop a model of autoimmunity, in which vaccine strategies could be evaluated for their ability to elicit CTL activity towards a model antigen.

Methods: Transgenic mice expressing lymphocytic choriomeningitis virus glycoprotein (LCMV-gp) specifically in the insulin-producing beta-islets of the pancreas allowed anti-beta islet CTL responses to be measured by blood glucose levels, tetramer and intracellular cytokine staining. Peptide vaccines consisted of i.v. administration of the known immunodominant epitopesof LCMV-gp alongside dendritic cell (DC) maturation stimuli. DC vaccines consisted of i.v.administration of 2x10^6 mature LCMV-gp peptide-pulsed bone-marrow-derived DC.

Results: While administration of antigenic peptides with adjuvants and/or costimulatory molecules were ineffective, DC vaccination induced hyperglycemia in approximately 70% of mice. Interestingly, while a class II epitope was not required for induction of autoimmunity, pulsing with a single class I epitope was insufficient. Furthermore, while tetramer-positive populations could be seen in the blood of peptide-vaccinated mice, no such population is seen in DC-treated mice.

Conclusions: The lack of correlation between presence of tetramer-positive populations in the blood and effective beta-islet cell cytolysis underlines the requirement for better markers of CTL activity inclinical trials. These results demonstrate the effectiveness of DC vaccination in this model and allow for further investigation of the factors that direct a strong CD8+ T-cell response towards tissue antigens.
THE P53 FAMILY MEMBER, P63, REGULATES NEURAL PRECURSOR CELL SURVIVAL DURING CORTICAL DEVELOPMENT
Authors: Dugani Sagar , Paquin Annie , Fujitani Masashi , Kaplan David R , Miller Freda D
Published: May 2017

Background: p63, a member of the p53 family of proteins, is involved in the regulation of naturally-occurring apoptosis in sympathetic neurons of the peripheral nervous system. Since data from our laboratory indicated that p63 is also expressed in stem cells and neurons within the developing brain, we hypothesized that p63 is involved in regulating the genesis and survival of developing neurons.

Methods: As cortical neurogenesis is initiated at embryonic day 12, we knocked-down p63 levels in isolated murine cortical precursors byusing shRNA against p63 or by transfecting floxed-p63 precursors with Cre recombinase. We performed similar studies in vivo using in uteroelectroporation to express either p63 shRNA or Cre recombinase to acutely knockdown or genetically ablate p63. We then performed immunofluorescence for known markers of apoptosis, cell-division, and differentiation to assess the level of cell death, proliferation and neurogenesis.

Results: Knock-down of p63 in vitro resulted in a 2-foldincrease in the death of precursors and neurons, associated with blunted neurogenesis but unaltered precursor proliferation. Coincident knock-down of p63 family members, p53, but not p73, rescued the elevated death suggesting that p63 and p53 antagonize each other to promote survival. Similar results were observed in vivo, where knockdown of p63 caused cell death and a decrease in the proportionof neurons in the cortical plate.

Conclusions: These experiments indicate that p63 is required forthe survival of neural precursors and newly-born neurons, and for normal cortical development. Ongoing work will explore the environmental cues that regulate p63 during neurogenesis.
OPPOSING FUNCTIONS FOR A PROTEIN KINASE: A JNK1 DEPENDENT SWITCH DETERMINES THE ONCOGENIC OR TUMOR SUPPRESSIVE ACTIVITY OF ILK INRHABDOMYOSARCOMA
Authors: Durbin Adam D , Somers Gino R , Forrester Michael , Hannigan Gregory E , Malkin David
Published: May 2017

Background:The integrin-linked kinase (ILK) is a protein kinase involved in the regulation of pathogenic cancer cell behaviours, such as proliferation, survival and invasion. ILK appears to be pro-oncogenic in vitro and in vivo models of tumorigenesis. Rhabdomyosarcoma (RMS) is a primitive mesenchyme-derived tumor and is subclassified into primarily embryonal (ERMS) and alveolar (ARMS) variants. Patients who present with metastatic RMS tumors have a less than 20% chance of cure, suggesting a need to define novel targets for chemotherapeutic intervention.

Methods: We used cell culture, murine xenografts and primary human tumors to examine ILK expression and functionality. RNAi and adenoviruses were used to knock down or over expressproteins, and SP600125 was used to inhibit JNK kinase activity. ERMS cells stablye xpressing PAX3-FOXO1A we regenerated using pcDNA3.1 with the full length PAX3-FOXO1A cDNA insert.

Results: RNAi-mediated ablation of ILK induced stimulation of ERMS and inhibition of ARMS cell growth in vitro and in vivo. Overexpression of ILK, but not the ILK-R211A mutant reversed these effects. High-throughput screening of multiple tumor cell lines and mesenchymal progenitor cells demonstrated similar ILK anti-growth effects. Consistent with these results, clinical correlations made between ILK immunohistochemical staining intensity and patterns on an ERMS tumor tissue microarray revealed downregulation of ILK in stage III/IV primary tumors. Mechanistically, ILK silencing induced selective phosphorylation of the c-jun amino terminal kinase (JNK) and its target c-Jun in ERMS cells with attenuated phosphorylation in ARMS cells. ERMS cells express higher levels of JNK1 isoforms than ARMS cells. Introduction of the ARMS-associated PAX3-FOXO1A fusion gene into ERMS cells restored the oncogenic function of ILK and downregulated of JNK1. Coupling ILK siRNA with inhibition of the JNK-c-Jun signaling pathway in ERMS cells resulted in growth reductions and apoptotic induction. In contrast, coupling ILK knockdown with overexpression of JNK1 in ARMS cells resulted in growth and c-jun phosphorylation.

Conclusion: In summary, these data suggest a model whereby the effect of ILK as an oncogene or tumor suppressor is determined by JNK1. Finally, this data suggests that ILK kinase inhibition may be warranted in ARMS tumors, and may be contraindicated in ERMS.
EPIGENETIC ALTERATIONS ASSOCIATED WITH PREMATURE OVARIAN FAILURE
Authors: Ghahremani Manda , Hannah Courtney W , Peneherrera Maria , Bretherick Karla L , Fluker Margo R , Robinson Wendy P
Published: May 2017

Background/Purpose: Premature ovarian failure (POF) affects 1% of women with a largely idiopathic and poorly understood etiology. The objective of this study was to identify specific epigenetic alterations by measuring DNA methylation of gene regulatory regions in women with POF vs. controls.

Methods: Blood samples were collected from idiopathic POFpatients (Amenorrhea for at least 3 months and 2 serum FSH levels of > 40mIU/ml obtained > 1 month apart prior to age 40) and control women (CW) (healthy pregnancy after age 37 with out a pregnancy loss). Genomic DNA was extracted from EDTA anticoagulated blood and bisulfite converted for analysis using the Illumina Golden Gate Methylation Panel which measures DNA methylation at 1506 CpG sites in the promoter regions of 807 genes in 10 POF and 12 CW. Candidate genes with altered epigenetic marks between POF and CW at a nominal P-value < 0.05 were identified using a t-testcomparison within the Illumina bead studio software. Genes of interest were further analyzed for quantitative methylation at specific CpG sites using pyrosequencing in 30 POF and 30 CW.

Results: Comparison of DNA methylation profiles of our initial POF and CW groups identified several genes with statistically significanthyper- or hypo- methylation in the POF group (P < 0.05), including the Androgen Receptor (AR)promoter region, which was significantly hypermethylated. To further validate these results, DNA methylation of the AR gene promoter was quantified bypryosequencing in a larger group of POF and CW. Pyrosequencing further confirmed a significantly higher DNA methylation of the AR promoter region inPOF vs. CW (P=0.007).

Conclusions: This is a novel study identifying epigenetic alterations in POF. The hypermethylation of the AR gene in POF patients may cause decreased level of the AR in these women. This is especially interesting given a recent report of induced POF in AR deficient mice^1. Specific epigenetic markers, as identified by DNA methylation array profiling in blood, may serve as useful biomarkers for POF and other fertility disorders. However, it will need to be determined if these methylation changes are present prior to diagnosis, or are a consequence of menopause itself.

Reference:

1.Hiroko S. et al. Premature ovarian failure in androgenreceptor deficient mice. PNAS;103:224-9
MUNC13 REGULATES SECRETION AT THE GOLGI IN A LOCALIZATION-DEPENDENT MANNER
Authors: Goldenberg Neil M , Silverman Mel
Published: May 2017

Background: Constitutive secretion is critical for the maintenance of eukaryotic cell structure and function. Our lab has shown that Rab34 is required for secretion at the Golgi^1, and that the C1 domain-containing protein, Munc13, is an effector of Rab34^2. Current studies seek to elucidate potential roles for Munc13in secretion at the Golgi.

Methods: Using a temperature-sensitive mutant of the Vesicular Stomatitis G-protein fused to GFP (VSVG-GFP) to monitor secretion, we examinedthe role of Munc13 in secretion in HeLa cells. Cells transfected with VSVG-GFP were treated with Munc13, amutant lacking the C1 domain (C1-less), and the phorbol esters TPA andPDBu. The rate of VSVG-GFP secretion was monitored using surface labelling of plasmalemmal VSVG-GFP and spinning disc confocal microscopy.

Results: TPA treatment resulted in an increase in the rate of VSVG-GFP appearance at the plasma membrane. Co-transfection of either Munc13 or C1-less alone also resulted in an increased rate of VSVG-GFP transport. Transfection of Munc13 plus TPA treatment resulted in amarked decrease in the rate of VSVG-GFP transport. Since TPA treatment relocalizes Munc13 to the plasma membrane, this result suggests that the availability of Munc13 in the cytosol is required for its effect on VSVG-GFP secretion.

Conclusions: Munc13 over-expression increases the rate of VSVG-GFP secretion to the plasma membrane. Sequestration of Munc13 at the plasma membrane with TPA abrogates thiseffect, and reduces the rate of VSVG-GFP secretion. We propose that Munc13 effects VSVG-GFP secretion via its interaction with Rab34 at the Golgi.

References:

1. Goldenberg, NM, S. Grinstein, M. Silverman. Golgi-bound Rab34 is a Novel Member ofthe Secretory Pathway. Mol BiolCell. 18(12):4762-4771 (2007).

2. Speight, P, M. Silverman.Diacylglycerol-Activated Hmunc13 Serves as an Effector of the GTPaseRab34. Traffic.6(10):858-865 (2005).
EFFECTS OF LOW NEIGHBOURHOOD SOCIOECONOMIC STATUS ON INFLAMMATION, OXIDATIVE STRESS, AND RISK OF MORTALITY IN PATIENTS WITH CORONARY ARTERY DISEASE
Authors: Heslop C L , Miller G , Hill J S
Published: May 2017

Background: Socioeconomicstatus (SES) is an influential determinant of prognosis in coronary artery disease (CAD). Patient neighbourhood SES may contribute to CAD outcomes, beyond effects of personal SES.

Methods: Following 485 CAD patients for > 10 years, we examine the effect of neighbourhood income, education, and unemployment on survival, and investigate relationships between SES and markers of inflammation and oxidative stress.

Results: SES was associated significantly with risk of mortality, however this relationship was not observed for cardiovascular death. Each one quintile decrease in income, education, and employment was associated with a 32%, 40% and 45% greater risk of non-cardiovascular mortality, respectively. Inflammatory and oxidative stress markers correlate with income, but do not diminish associations between neighbourhood SES and mortality.

Conclusions: Significant disparities in non-cardiovascular mortality related to neighbourhood SES were observed in this study, which argues for greater attention to socioeconomic factors in chronic disease prevention and health care delivery.

C.L.H. is supported bya Providence Health Research Institute & Canadian Institutes of HealthResearch MD/PhD Studentship Award, and a Michael Smith Foundation for HealthResearch Trainee Award.

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TUMOUR PATHOLOGY PREDICTS MICROSATELLITE INSTABILITY IN A POPULATION-BASED SERIES OF COLORECTAL CANCER CASES
Authors: Hyde A J , Fontaine D , Green R C , Simms M , Parfrey P S , Younghusband H B
Published: May 2017

Background: Lynch Syndrome is an autosomal dominant trait that accounts forapproximately 3% of all cases of colorectal cancer (CRC). It is caused by mutations in DNA mismatch repair (MMR) genes, most commonly MLH1 or MSH2. These MMR defects cause high levels of microsatellite instability (MSI-H) in the tumours. MSI testing of all CRCs to identify potential Lynch Syndrome cases is not practical, so the Bethesda Guidelines, which use clinical and pathological features, were created to identify those tumours most likely to be MSI-H^1. In 2007 Jenkins et. al. created MsPath, a tool based on the pathological features described in the rarely used 3^rd Bethesda criterion, to improve prediction of MSI-H tumours among CRC cases diagnosed before age 60 years^2.

Methods: We collected a population-based cohort of 716 CRC cases diagnosed before age 75 years in Newfoundland. For each of these cases we collected family history, performed MSI analysis, and scored a number of pathological features for the purpose of evaluating the accuracy of the Bethesda Criteria and MsPath at predicting MSI-H tumours.

Results: Our work validates the MsPath tool in the Newfoundland population for the same age group used to create the tool. We found it identified MSI-H cases with a sensitivity of 95% and specificity of 35% in our population of CRCcases diagnosed before age 60 years (n=290). We also tested this tool on our older population of CRCcases, diagnosed at ages 60 to 74 years (n=426). We found it to be at least as predictive in this population,with a sensitivity of 95% and a specificity of 42%. We then used our entire cohort (N=716) to compare MsPath with the other Bethesda criteria.Bethesda criteria 1, 2, 4 and 5 together predicted MSI-H cases with a sensitivity of 67% and a specificity of 51%. MsPath was better at identifying these cases, with a sensitivity of 95% and a specificity of 39%.

Conclusions: We conclude that MsPath can be extended to include patients diagnosed with CRC before age 75 years. As well, we have found that MsPath is a better predictive tool than the Revised Bethesda Guidelines for identifying MSI-H cases within a population-based setting of colorectal cancer.

References:

1. Umar, A. et. al. J Natl Cancer Inst 2004;96:261-8

2.Jenkins, M.A. et. al. Gastroenterology 2007;133:48-56
PLASMACYTOID DENDRITIC CELLS FUNCTION IN HIV INFECTION
Authors: Hyrcza Martin , Ostrowski Mario , Der Sandy
Published: May 2017

Plasmacytoid dendritic cells (pDCs) are innate immune cells able to produce large quantities of type I interferons (IFN) when activated. Human immunodeficiency virus (HIV)-infected patients show generalized immune dysfunction characterized in part by chronic interferon response. In this study we investigated the role of dendritic cells inactivating and maintaining this response. Specifically we compared the IFN geneactivity in pDCs in response to several viruses and TLR agonists. We hypothesized that 1) the pattern of IFN gene transcription would differ in pDCs treated with HIV than with other agents, and 2) that pDCs from patients from different stages of disease would respond differently to the stimulations. To test these hypotheses, we obtained pDCs from 15 HIV-infected and uninfected individuals and treated freshly isolated pDCs with either HIV (BAL strain), influenza virus (A/PR/8/34), Sendai virus (Cantell strain), TLR7 agonist(imiquimod), or TLR9 agonist (CpG-ODN) for 6h. Type I IFN gene transcription was monitored by real time qPCRfor IFNA1, A2, A5, A6, A8,A17, B1, and E1, and cytokine levels were assayed by Cytometric Bead Arrays forTNF?, IL6, IL8, IL10, IL1?, and IL12p70. pDC function as determined by these two assays showed no difference between HIV-infected and uninfected patients or between patients with early or chronic infection. Specifically, HIV did notinduce type I IFN gene expression, whereas influenza virus, Sendai virus and imiquimod did. Similarly, HIV failed to induce any cytokine release from pDCs in contrast to influenza virus, Sendai virus and imiquimod, which stimulatedrelease of TNF?, IL6, or IL8. Together these results suggest that the reaction of pDCs to HIV virus is quantitatively different from the response to agents such as virus, Sendai virus, and imiquimod. In addition, pDCs from HIV-infected persons have responses similar to pDCs from uninfected donors, suggesting, that the DC function may not be affected by HIV infection.
THE ROLE OF THE SRC-LIKE ADAPTOR PROTEINS, SLAP AND SLAP2,IN FLT3 RECEPTOR TYROSINE KINASE SIGNALING
Authors: Liontos Larissa , McGlade Jane
Published: May 2017

Introduction: The Src-like Adaptor Proteins, SLAP and SLAP2, are primarily expressed in hematopoietic cells and have an established role in the negative regulation of T-cell signaling. SLAP and SLAP2 act by recruiting the E3 ubiquitin ligase, c-Cbl, to components of the T-cell receptor leading to receptor ubiquitination and down-regulation. In addition to having a role in the negative regulation of T cell signaling, our lab has previously shown that SLAP and SLAP2 interact with the CSF-1 receptor, Fms, and interfere with CSF-1R function. The CSF-1 receptor is part of the type III family of receptor tyrosine kinases (RTKs) which includes the PDGF, c-Kit and Flt3 receptors. The Flt3 receptor plays an important role in both B-cell and Dendritic cell development and is commonly deregulated in acute myeloid leukemia (AML). The objective of this study is to determine whether SLAP and SLAP2 are involved in the negative regulation of Flt3 signaling.

Methods: To study Flt3 signaling in vivo, murine Dendritic cells(DCs) were utilized. We first analyzed the splenic DC populations in SLAP1/2-/- double knock-out mice and determinedthat there is a defect in the myeloid DC population (CD11b/CD11c+). To study receptor signaling, we generated DCs in vitro from the bone marrow of these mice using Flt3 ligand(Flt3L). SLAP1/2-/- mice producedless BM-DCs than WT mice and the functionality of these DCs is currently being investigated.

Results: SLAP and SLAP2 interact with the Flt3 receptor in vitro and bind to the receptor within the juxtamembrane region. BaF3/Flt3 cells that stably express SLAP, SLAP2 and SH2 mutant forms of these proteins were generated. The expression of SLAP2 in these lines resulted in a change in the phosphorylation kinetics of the receptor. In addition, growth of SLAP2-expressingcells in response to Flt3 ligand was enhanced in comparison to cells expressinga GFP vector alone.

Conclusions: SLAP and SLAP2 may be involved in Flt3 regulation and appear to play arole in Dendritic cell differenation, a process that is dependent on Flt3 signalling.
GENOMIC AND TRANSCRIPTOME PROFILING OF EPITHELIAL OVARIAN CANCER
Authors: Menzies Rebecca JZ , Bukhman Yury V , Ng Nancy F , Shaw Patricia A , Mak Tak W
Published: May 2017

Background: Epithelial ovarian cancer is the leading cause of death by gynecological malignancy. Due to inadequate screening modalities, a lack of characteristic presenting symptoms, limited treatments and a poor understanding of the molecular underpinnings of the disease, only 25% of ovarian cancers are diagnosed at an early stage. Current 5-year survival rates range from 80%, for disease diagnosed in Stage I to as low as 13% for Stage IV. Current screening for ovarian cancer involves measuring CA-125 levels. However, CA-125 testing has low sensitivity since it can be elevated in a variety of other gynecological diseases. Numerous studies have found molecular heterogeneity between the four histological subtypes of ovarian cancer (serous, endometrioid, clear cell and mucinous). However, treatments remain the same for all subtypes regardless of molecular heterogeneity. Thus, better treatment targets and biomarkers must be found for this disease.

Methods:In our study 300 ovarian tumors will be genomically profiled using the Affymetrix Genome-Wide SNP Array 6.0 to identify loci and genes implicated in ovarian cancer. To date, 51 ovarian tumors have been analyzed using the SNP array.

Results:Preliminary analysis of copy number variation in these tumors using Partek software has revealed a total of 978 loci. Known amplifications derived from the literature were seen at CCNE1 and ERBB2. Similarly, well known deletions ofp53 and RB1 in ovarian cancer were detected. Novel amplified loci at 18q11.2 and 4q33 were also detected. Novel deletions were detected at 7p13 and 8q22.2.

Conclusion: Future work will include running the remaining 249 tumor samples on the SNP array and analyzing the complete dataset using Partek software. Future validation of identified genes in vitro and in vivo may provide insight and possible biomarkers that may be used clinically to benefit the ovarian cancer patient.
INVESTIGATING CRMP4 FUNCTION IN NERVE REGENERATION
Authors: Tone S Ong , Alabed Y Z , Di Polo A , Fournier A E
Published: May 2017

Background: The failure of CNS neurons to spontaneously regenerate following injury can be partially attributed to the expression of neurite outgrowth inhibitory myelin associated inhibitors (MAIs). MAIs signal through a tripartite receptor complex to activate the cytosolic protein RhoA and influence cytoskeletal dynamics. RhoA antagonists promote neuronal survival and regeneration in animal models of nerve injury. However, RhoA's potential as a therapeutic target may be limited by its widespread roles in multiple cellular processes and cell types. In an attempt to discover more specific therapeutic targets to promote nerve regeneration, our lab identified the cytosolic phosphoprotein CRMP4b (Collapsin Response Mediator Protein 4b) as a protein that functionally interacts with RhoA to mediate neurite outgrowth inhibition. Blockade of the RhoA-CRMP4b interaction with a competitive peptide (C4RIP) attenuates myelin-dependent neurite outgrowth inhibition.

Methods: We are currently investigating the in vivo roles of CRMP4in regeneration in an optic nerve injury model by developing a readily deliverable version of C4RIP.

Results: Preliminary results suggest that overexpression of C4RIP in retinal ganglion cells by adeno-associated virus does not promote regeneration. However, studies investigating the ability of C4RIP to promote nerve regeneration into the optic nerve following stimulation of neurons into anactive growth state are currently in progress.

Conclusion: Elucidating the role of CRMP4 in nerve regeneration may provide insight into the molecular mechanisms following nervous system injury and lead to the development of more specific therapeutic interventions.

Reference:

Alabed YZ, Pool M, Ong Tone S, Fournier AE.Identification of CRMP4 as a convergent regulator of axon outgrowth inhibition. J Neurosci 2007;27:1702-11.
CONTRIBUTION OF DNA COPY-NUMBER VARIATION (CNV) TO CANCER SUSCEPTIBILITY AND LARGE-SCALE GENOME ALTERATIONS IN OSTEOSARCOMA (OS)
Authors: Pasic I , Shlien A , Novokmet A , Zhang C , Tabori U , Pinto D , Scherer S W , Malkin D
Published: May 2017

Introduction: OS, a common Li-Fraumeni syndrome (LFS)-associated neoplasm, is a common bone malignancy of children and adolescents. Sporadic OS is also characterized by young age of onset and high genomic instability, suggesting a genetic contribution to disease. This study examined the contribution of novel DNA structural variation elements, CNVs, to OS susceptibility. Given our finding of excessive constitutional DNA CNV in LFS patients, which often coincide with cancer-related genes, we hypothesized that constitutional CNV may also provide clues about the aetiology of LFS-related sporadic neoplasms like OS.

Methods: CNV in blood DNA of 26 patients with sporadic OS was compared to that of 263 normal control samples from the International HapMap project, as well as 62 local controls. Analysis was performed on DNA hybridized to Affymetrix genome-wide human SNP array 6.0 by Partek Genomic Suite.

Results: There was no detectable difference in average number of CNVs, CNV length, and total structural variation (product of average CNV number and length) between individuals with OS and controls. While this data is preliminary (small sample size), it argues against the presence of constitutional genomic instability in individuals with sporadic OS.

Conclusion: We found that the majority of tumours from patients with sporadic OS show CN loss at chr3q13.31, raising the possibility that chr3q13.31 may represent a “driver” region in OS aetiology. In at least one OS tumour, which displays CN loss at chr3q13.31, we demonstrate decreased expression of a known tumour suppressor gene located at chr3q13.31. We are investigating the role ofchr3q13.31 in development of OS.
GAIT ABNORMALITIES AND SENSORY CHANGES ACCOMPANY NEURO-INFLAMMATION IN ANIMAL MODELS OF DISC HERNIATION RADICULOPATHY
Authors: Shamji Mohammed F , Allen Kyle D , Adams Samuel B , So Stephen , Jing Liufang , Chen Jun , Richardson William J , Setton Lori A
Published: May 2017

Objective: The goal of this study was to evaluate gait and behavioral changes in an animal model of disc-herniation induced radiculopathy. A second objective was to correlate these functional changes to evidence of neuroinflammation and autoreactive lymphocyte immune activation.

Methods: The animal model of radiculopathy involved harvesting autologous nucleus pulposus (NP) from a tail intervertebral disc, and then exposing the L5 dorsal root ganglion (DRG) by hemilaminectomy and partial facetectomy. Experimental animals (n = 16) received NP placement onto the DRG andcontrol animals (n = 16) underwent exposure only. At weekly time points, animals were evaluated for mechanical allodynia by Von Frey testing and for gait symmetry by digitized video analysis. At sacrifice, serum was evaluated for inflammatory cytokine content. The L5 DRGs were then evaluated by immunohistochemistry for mediators of inflammation and immune activation. Statistical analyses were at the ? = 0.05 level of significance, with Bonferroni corrections for multiple comparisons when appropriate.

Results: Sensory testing revealed persistent mechanical allodynia in rats subjected to NP stimulus compared with the sham surgery group (Von Frey testing, p < 0.01). Gait analysis reflected the functional consequence of this altered sensation revealing marked asymmetry and apreference to place load on the contralateral limb (symmetry index, p < 0.01). Serum cytokine expressionwas equivalent between groups, reaffirming that the sensation and behavioral changes observed in these animals results primarily from local inflammatory changes. Immunohistochemicalanalysis of the sectioned DRGs after sacrifice revealed equivalent post-surgical inflammatory activation (IL23, p = 0.47), but substantial immuneactivation in the NP group (IL17, p = 0.01).

Conclusion: This model of radiculopathy provides the first evidence of altered gait symmetry and locomotor ability in animals subjected to non-compressive placement of NP tissue. Systemic inflammation was absent as expected, but mechanical allodynia, local inflammation, and autoreactive immune activation were observed. Future work involves therapeutic interventions targeting these changes to rescue animals from the phenotype of inflammatory radiculopathy.
THE ASSOCIATION OF LOBULAR BREAST CANCER WITH GERMLINE MUTATIONS OF CDH1
Authors: Schrader K , Masciari S , Boyd N , Senz J , Kaurah P , Terry M B , John E , Andrulis I L , Knight J , O'Malley F P , Daly M , Bender P , Southey M C , Hopper J L , Garber J , Huntsman D G
Published: May 2017

Background: CDH1 encodes the cell-cell adhesion molecule, E-cadherin, for which loss of expression facilitates the infiltrative and metastatic potential of cancers. Germline mutations in CDH1 are associated with hereditary diffuse gastric cancer (HDGC), and in this setting female carriers have been estimated to have a 39-50% risk of lobular breast cancer (LBC) by age 80 years.

Aim: To determine the frequency of CDH1 germline mutations inindividuals with early-onset LBC or those with LBC and a family history of multiple breast cancers but no gastric cancers.

Methods: Germline DNA analysis of CDH1 in women with LBC, for whom germline BRCA1 and BRCA2 mutations have been excluded, who have been (1) diagnosed before the age of 45 years or (2) diagnosed at any age and have a family history of breast cancer.

Results: Analysis of 194 LBC cases has thus far revealed two novel missense mutations predicted to affect protein function. Functional assays to assess their pathogenicity along with germline analyses of the remaining 200 cases are currently underway. Several unreported silent changes have also been identified and will be measured in a case- control sample to assess whether they are associated with LBC risk.

Conclusion: Germline CDH1 mutations may cause a small proportion of familial and early onset LBC.
ROLE OF THE CYSTIC FIBROSIS TRANSMEMBRANE REGULATOR (CFTR) CHLORIDE CHANNEL IN MACROPHAGE LYSOSOME ACIDIFICATION
Authors: Steinberg Benjamin E , Brodovitch Alexandre , Huynh Kassidy K , Grinstein Sergio
Published: May 2017

Background: Lysosome acidification is the result of proton pumping by thevacuolar-type ATPase (V-ATPase). Because the V-ATPase is electrogenic, a substantial lysosomal membrane potential can develop if left uncompensated by counterions. An increasing membrane potential will oppose further proton pumping, limiting the acidification. It has generally been assumed that a parallel anion influx accompanies proton pumping to enable acidification. Indeed, defective anion channel function in cystic fibrosis (CF) has been suggested as the mechanism underlying attenuated lysosomal acidification and impaired microbial killing in the lung (Di A, et al., 2006, Nature Cell Biol. 8, 933-944). Chronic lung inflammation and infection represent the major source of morbidity and mortality in CF, and understanding the mechanism underlying the disease will therefore have far-reaching therapeutic implications. As such, it is important to accurately evaluate the reported role of CFTR in lysosome acidification.

Methods: To assess the individual contribution of counterions to acidification, we carried out cytosolic and lysosomal ion substitution experiments in intact cells while monitoring lysosomal pH by ratiometric imaging.

Results: Replacement of cytosolic Cl^- with impermeant anions did not alter the rate or extent of proton pumping. In contrast, permeant luminalcations were required for normal acidification. Because cations are the main counterion for lysosomal proton uptake, defects in the lysosomal pH are not anticipated in CF cells. Accordingly, the lysosomes of CFTR-deficient alveolar macrophages were found to acidify normally.

Conclusion: We conclude that cations are the primary counterions responsible for lysosomal acidification and that defects in lysosomal anion conductance cannot explain the impaired microbicidal capacity of CF phagocytes.
SHCA PHOSPHOTYROSINE DERIVED SIGNALING IS REQUIRED FOR THE MAINTENANCE OF CARDIAC FUNCTION
Authors: Vanderlaan Rachel , Hardy Rod , Kabir Golam , Back Peter , Pawson A J
Published: May 2017

Background: ShcA, a scaffolding protein, generates signalspecificity by docking to activated tyrosine kinases through distinct phosphotyrosine recognition motifs, while mediating signal complexity through formation of diverse downstream phosphotyrosine complexes. Mammalian ShcA encodes 3 isoforms having a modular architecture of a PTB domain and SH2 domain, separated by a CH1 region containing tyrosine phosphorylation sites important in Ras-MAPK activation.

Objective and Methods: ShcA has a necessary role in cardiovascular development^1,2. However, the role of ShcA in the adult myocardium is largely unknown, also unclear, is how ShcA uses its signaling modules to mediate downstream signaling. To this end, cre/loxP technology was employed to generate a conditional ShcA allele series. The myocardial specific ShcA KO (ShcA CKO) and myocardial restricted domain mutant KI mice were generated using cre expressed from the mlc2v locus^3 coupled with the ShcA floxed allele and in combination with the individual ShcA domain mutant KI alleles^2.

Results: ShcACKO mice develop a dilated cardiomyopathy phenotype by 3 months of life, typified by depressed cardiac function and enlarged chamber dimensions. Isolated cardiomyocytes from ShcA CKO mice have preserved contractility indicating an uncoupling between global heart function and single myocyte contractile mechanics. Force-length experiments suggest that the loss of shcAmediates the uncoupling through deregulation of extracellular matrix interactions. Subsequent, analysis of the ShcA myocardial restricted domain mutant KImice suggests that ShcA requires PTB domain docking to upstream tyrosine kinases and subsequent phosphorylation of the CH1 tyrosines important for downstream signaling.

Conclusion: ShcA is required for proper maintenance of cardiac function, possibly regulation of extracellular matrix interactions.

References:

1. Lai KV, Pawson AJ. The ShcA phosphotyrosine docking protein sensitizescardiovascular signaling in the mouse embryo. Genes and Dev 2000;14:1132-45.

2. Hardy WR. et al. Combinatorial ShcA docking interactions supportdiversity in tissue morphogenesis. Science2007;317:251-6.

3.Minamisawa, s. et al. A post-transcriptional compensatory pathway inheterozygous ventricular myosin light chain 2-deficient mice results in lack ofgene dosage effect during normal cardiac growth or hypertrophy. J Biol Chem 1999;274:10066-70.
QUANTITATIVE EXAMINATION OF A NOVEL CLUSTERING METHOD USING MAGNETIC RESONANCE DIFFUSION TENSOR TRACTOGRAPHY
Authors: Voineskos Aristotle N , O’Donnell L J , Lobaugh N J , Markant D , Niethammer M , Mulsant B H , Pollock B G , Kennedy J L , Westin C F , Shenton M E
Published: May 2017

Introduction: MR diffusion tensor imaging (DTI) is the most powerful and currentlythe only way to visualize the organization of white matter fiber tracts in vivo. As this is a relatively newimaging technique, new tools are developed for quantifying fiber tracts, andrequire evaluation. We examined scalar indices of the diffusion tensor with two different tractography methods. We compared a novel clustering approach with a multiple region of interest (MROI) approach in a healthy and disease (schizophrenia) population.

Methods: DTI images were acquired in 12 participants (n=6 patients withschizophrenia: 58 ± 12 years; n=6 controls: 57 ± 21 years) on a 1.5 Tesla GE system with diffusion gradients applied in 23 non-collinear directions, repeated three times. Tractography andfiber tract creation was performed using 3D Slicer software. Interraterreliability of the clustering approach and its similarity to the MROI methodwere evaluated.

Results: The clustering approach was reliable both quantitatively and spatially (k > 0.8 for all tracts). There was high spatial(voxel-based) agreement between the clustering and MROI methods. Fractionalan isotropy and trace values were highly correlated between the clustering and MROI methods (p < 0.001 for all tracts).

Discussion: Our clustering method has excellent interrater reliability and thereis a high level of agreement between our clustering method and the MROI method, both quantitatively and spatially. The clustering method is less susceptible touser bias. Moreover, not limited by a priori predictions, our clustering method may be a more robust and efficient way to identify and measure fiber tracts of interest.

(colour figure available in PDF version)
ENDOTHELIAL NO-SYNTHASE GENE TRANSFER RESTORES REGENERATIVE CAPACITY OF ENDOTHELIAL PROGENITOR CELLS FROM PATIENTS WITH CORONARY ARTERY DISEASE
Authors: Ward Michael R , Isaac Kathryn , Thompson Kathleen , Vecchiarelli Jonathan , Kutryk Michael JB , Stewart Duncan J
Published: May 2017

Background: Endothelial progenitor cells (EPCs) from patients with coronary arterydisease (CAD) or CAD risk factors exhibit greatly reduced regenerativecapacity, which likely contributes to the relatively modest nature of the benefit seen in recent clinical trials of autologous cell therapy postmyocardial infarction. We hypothesized that eNOS overexpression will improve the functional capacity of EPCs from these patients.

Methods and Results: EPCs were isolated from the peripheral blood ofpatients with high Framingham risk scores (FRS > 15%) and were transducedusing lentiviral vectors containing either eNOS or GFP (sham). We observed that eNOS-transduction significantly improved migration toward chemotactic factors(VEGF and SDF-1) compared to sham-transduced cells. EPCs were co-cultured witha mature endothelial cell (EC) line on Matrigel to measure their pro-angiogenicfunction in vitro. eNOS-transduced EPCs induced longer angiogenic tubes withmore branch points compared to sham-transduced cells, and exhibited higherassociation with EC tubes. We did not observe a significant difference in the adhesion of EPCs to an EC layer pre-activated with TNF-?, suggesting that the association to angiogenictubes is likely through a different mechanism. In immunodeficient mice, eNOS-transduced EPCs resulted in significant improvement in ischemic hindlimbperfusion compared to sham-transduced cells. PCR arrays revealed changes in angiogenic and pro-survivalgene expression in response to eNOS overexpression, providing preliminaryinsight into the mechanisms underlying its beneficial actions.

Conclusions: The present data show that reduced regenerative activity of EPCsisolated from CAD patients can be significantly improved by the overexpressionof eNOS. The combination of cell and gene therapy may improve the efficacy ofautologous cell therapies for cardiovascular disease.
IN UTERO GENE DELIVERY USING CHITOSAN-DNA NANOPARTICLES IN MICE
Authors: Yang P T , Jia W , Skarsgard E D
Published: May 2017

Background:In utero gene therapy is a noveltherapy for monogenic disorders of the fetus. Viral vector-mediated gene transfer risks endogenous viral recombination, and random transgene insertion causing insertional mutagenesis. Enhanced biosafety alternatives include non-viral vectors such as the complex polymer, chitosan. The purpose of this study was toevaluate chitosan-mediated transfection in a murine model of fetal genetherapy.

Methods:
1. Chitosan colloidal suspensions wereprepared, and particle size analysis in amniotic fluid (AF) was performed using a zetasizer.
2. Plasmid enhanced green fluorescent protein (eGFP)-chitosan constructs were prepared and protection from endogenous digestion in AF was analyzed by gel electrophoresis.
3. 0.25x105 HEK293T cells were transfected over 2 hours with 0.6 ?g of chitosan-peGFP in varying proportions of medium and AF. After 48 h, cells were directly imaged by fluorescence microscopy for eGFP expression and fluorescence activated cell sorting (FACS) sorting was done to determine transfection efficiency.
4. Amniotic sacs of time-mated CD-1 mice were injected with 30
?L chitosan-peGFP (12.5 ug DNA) on G17. Following natural birth, pupswere sacrificed and tissues were examined for eGFP DNA and mRNA by DNA PCR, RT-PCR, and fluorescence microscopy.

Results: Chitosan forms aggregates in AF, and although in vitro transfection efficiency is decreased by AF, eGFP-chitosan delivery into AF achieves transfection and transgene expression in lung and intestine of mice after birth.

Conclusions: In utero delivery of eGFP plasmid by chitosan results in postnatal gene expression, and shows promise for non-viral gene transfer in animal models of fetal gene therapy.

P.T.P.Y. is supported by a Child and Family–UBCMD/PhD Studentship Award.
REGULATION OF HUNTINGTIN PALITOYLATION AND ITS ROLE IN HUNTINGTON DISEASE
Authors: Young Fiona BJ , Deng Deborah Yu , Singaraja Roshni R , Hayden Michael R
Published: May 2017

Huntington Disease(HD) is an inherited and ultimately fatal neurodegenerative disease demonstrating both neurological and psychiatric symptoms. The protein huntingtin (htt) undergoes many post-translational modifications, such as phosphorylation, palmitoylation, and proteolysis. Palmitoylation, the process by which a 16-carbon fatty acid forms a thioester bond with cysteine residues, is a reversible modification known to influence protein trafficking and function.

Huntingtin Interacting Protein 14 (HIP14) was identified as a major palmitoyl acyl transferase (PAT) that interacts robustly with wild-type htt, but has significantly reduced interaction with mutant polyglutamine expanded htt. HIP14is a major PAT for htt and palmitoylation of mutant htt by HIP14 is significantly reduced. Down regulation of HIP14 by siRNA in vitro results in increased cell death in neurons, whereas co-transfection with htt and HIP14 results in enhanced palmitoylation and reduction in number of inclusions. Our laboratory has developed a HIP14 knockout mouse (HIP14-/-), which demonstrates many HD-like features similar to those seen in the YAC128 transgenic mouse model of HD. Notably, these mice demonstrate a much earlier and more severe phenotype as compared to the YAC128 mice, suggesting a critical role for HIP14 in HD pathogenesis.

The overarching goal of these studies is to explore the role of HIP14 and palmitoylation in the pathogenesis of HD using in vitro, in vivo, and ex vivo methods. A human Bacterial Artificial Chromosome (BAC) containing HIP14 was identified, prepared, and submitted for microinjection, and we have now generated HIP14-overexpressing transgenic mice. These mice are currently undergoing preliminary analyses, and a subset of founders will be selected to undergo further characterization. These human HIP14 BAC transgenic mice will represent the first mammalian model of PAT overexpression, and will significantly further our understanding of PAT activity in vivo.

The YAC transgenic model for HD, developed previously in our laboratory, recapitulates many aspects of HD as seen in humans. After preliminary characterization of the human HIP14 BAC transgenic mice, these mice will be crossed to the YAC model of HD. We anticipate that overexpression of HIP14 in vivo will delay and/or ameliorate the features of HD observed in the YAC128 mouse, providing validation of this pathway as a potential therapeutic target for HD.

F.B.J.Y. is supported by a Canadian Institutes of Health Research Walter and Jessie Boyd & Charles Scriver and Child & Family Research Institute –UBC MD/PhDStudentship Award. She also receives funding from the Michael Smith Foundation for Health Research as a Junior Trainee.