Background: Acute kidney injury (AKI) during pediatric intensive care unit (PICU) admission may be a risk factor for chronic kidney disease (CKD) and hypertension development. We determined 1) CKD and elevated BP (eBP) prevalence 11 years after PICU; 2) If PICU-AKI is associated with worsening CKD and BP from 6-11 years post-PICU.
Methods: Follow-up study (≥9.5 years post-PICU) of children previously studied at 6 years post-PICU. Protocol: Children were contacted for a study visit 11±1.5 years post-PICU; blood (serum creatinine) and urine (albumin/creatinine [ACR]) were collected and height/weight/BP measured. Exposure: AKI during PICU (KDIGO definition). Outcomes: CKD [estimated glomerular filtration rate (eGFR) < 90ml/min/1.73m2 or ACR ≥ 3mg/mmol]; eBP: BP ≥ 90th percentile or ≥120/80mmHg in adults). 6 vs. 11 years outcome prevalences and AKI vs. non-AKI change in eGFR, ACR and BP percentile were compared (univariate tests).
Results: Data were available for n=71 (4.5±5.3 years old at PICU; 55% male). Prevalences of low eGFR, high ACR, CKD and eBP remained high at 11 years post-PICU, similar to 6 years post-PICU (Table). There was a statistically significant decrease in eGFR and diastolic BP from 6 years to 11 years follow-up (p < 0.05), but no significant change in ACR or systolic BP (Table). AKI during PICU was not significantly associated with a change in eGFR, ACR or BP percentile from 6 years to 11 years follow-up (Table, all p > 0.05).
Conclusion: High prevalence of CKD and eBP persists at 11 years after PICU admission. We did not find that PICU AKI impacts on CKD or eBP progression over time. Interventions are needed to reduce long-term cardiovascular risk in PICU patients. Risk factors for persistent or progressive CKD and eBP must be elucidated.
Patient Reported Outcomes (PROs) are used by researchers and clinicians to evaluate patient symptoms and improve treatment outcomes. When patients answer PRO questionnaires and have their answers acted upon, they experience a higher quality of life during and after their cancer treatment. Opal (opalmedapps.com), a patient-centered app for smartphones developed by the Opal Health Informatics Group at the MUHC, provides an excellent medium for implementing customizable PROs.
This research project involved the design and implementation of a comprehensive PRO system for Opal, which supports many standard and novel features. Researchers and clinicians can define new questionnaires, based on pre-existing questions, or by defining new questions, and they can set up rules to determine which patients should receive which PRO questionnaires and when. A wide variety of question types and visualizations are provided, with each question’s visualization carefully tailored to properly display on a mobile phone screen. Furthermore, visualizations to show the historical trends in answers have been developed, with the intent to provide these to both the patient and the clinician in order to support meaningful discussions between patients and their treating team. A novel patient-oriented feature is the ability of patients to provide feedback on questions, and a star-rating system for questionnaires. This should aid in improving questions and questionnaires so that they are better accepted by patients.
At the heart of Opal’s questionnaire system is a carefully designed database, which stores both the definition of questions and questionnaires, as well as patients’ responses. Both the researcher/clinician tools and the patients’ apps automatically interact with this database. The overall system will provide new opportunities for researchers/clinicians to define new PROs, and a more patient-centered and customizable experience for patients.
Axonal degeneration of retinal ganglion cells (RGCs) is a feature shared by all optic neuropathies. Axonal injury of RGCs induced by transection leads to dimerization of proteolipid protein (PLP) and eventually to axonal degeneration. PLP is the predominant protein of central nervous system myelin and is important for the maintenance of the multilamellar structure of myelin. Moreover, PLP remains mostly as a monomer under healthy conditions. Therefore, increased dimerization of PLP presumably could disrupt myelin morphology, and neurons with damaged myelin could become susceptible to degeneration. To understand the relationship between axonal degeneration and PLP dimerization in optic nerves (ONs) following transection, we will use three groups of wild-type rats: intact ON, transected ON of eyes injected with bis (3‐propionic acid methyl ester) phenylphosphine borane complex 1 (PB1), and transected ON of eyes injected with vehicle. PB1 is a cell-permeable disulfide reducing agent, and should reduce the disulfide bonds of PLP dimers. The RGCs will be imaged to examine their survival rates and the optic nerves will be collected to determine the ratios of PLP dimerization with Western blot.
We hypothesize that PB1 will chemically reduce PLP dimers, thereby restoring myelin integrity and decreasing RGC axonal degeneration. The results of this project could contribute to discovering new treatments for optic neuropathies and prevent the permanent loss of neuronal connections in the central nervous system.
Long-acting injectables (LAI) have been used for convenience and to promote adherence to antipsychotics, however, the patients’ perspective in changing from a monthly paliperidone palmitate LAI (PP1M) to a 3-monthly (PP3M) has not been documented. Our objective is to investigate patients’ perception and attitude towards changing between monthly and less frequently administered LAIs.
14 patients were switched from PP1M (Invega Sustenna) to PP3M (Invega Trinza) who were diagnosed with schizophrenia or schizoaffective disorder. Patients were encouraged to give their opinions both prior and after medication change, and all remarks were recorded during the clinical interview.
All patients reported a positive or neutral experience when switching from PP1M to PP3M. 13 out of the 14 (92.85%) patients reported a neutral outcome and that the switch had “no difference” or did not feel a significant change between PP1M and PP3M. Patients initially reported feeling “anxious” from the needle or concerned about medication cost, but later commented that the “change is more beneficial”, medication is “more convenient” and that they felt “more relaxed” afterwards. One patient highlighted that PP3M is much better since it made her “forget [she] had a problem”.
All patients were open to trying PP3M and reported a neutral or favourable outcome after switching from PP1M.
Purpose: Prostate cancer patients undergoing radiotherapy may experience side effects during and after their treatment such as diarrhea and urinary issues. These side effects may be related to anatomical/physiological changes during radiotherapy that reduce the ability to deliver treatment as planned. We hypothesized that changes to the gas content of the rectum can change its volume and shape over the course of a 20-day treatment, causing the original treatment plan, created for the patient using a single “planning CT” acquired before treatment, to become inaccurate during treatment. We sought to prove this by using radiomics techniques to search for a correlation between the amount of gas in the rectum and changes to its shape and size during treatment.
Methods: Each day, the prostate position, and rectal and bladder volumes are obtained prior to the radiotherapy by a cone beam computed tomography (CBCT) scan to image-guide treatment delivery. Previously, software was written to extract information contained in a CBCT and convert it into a measure of gas in the rectum. This was achieved by performing a linear transformation from grayscale CBCT voxels to Hounsfield Units (HU), which measure the radiodensity of materials. Voxels registering an HU of between -1100 and -340 were found to correspond to gas in the rectum. In the present study, software was developed to extract additional information from a patient database, including planned and delivered radiation doses, planned and actual rectal volumes, and planned and actual rectal lengths at each treatment. The differences between planned and actual quantities (lengths or volumes) were plotted against the deviation of rectal gas between planning and delivery and a linear regression was performed. A cohort of 12 patients, corresponding to a total of 252 (CB)CT scans, was used to perform this data analysis.
Results: A clear correlation between the difference in planned and actual rectal volumes and the deviation of rectal gas from planned was found. The linear regression gave a fit of y = 1.27 +/- 0.06 x + 5 +/- 1 with a coefficient of determination of R^2 = 0.641.
Conclusion: This correlation supports the hypothesis that the presence of gas in the rectum alters its shape and volume during a course of radiotherapy, modifying the original radiotherapy plan and organs-at-risk constraints. These findings stimulate further studies into the effects that inaccurate treatment delivery may have on the side effects experienced by prostate cancer patients.
Intraocular and orbital metastases are rare and indicate
hematagenous dissemination of a primary cancer. The orbit and
the choroid are the most common sites for ocular involvement for
disseminating malignancies. Immunohistochemistry using a panel
of antibodies is useful to determine the nature of the metastatic
tumor even in patients with no known primary cancer. The
objective of this study is to report twelve unusual metastases to the
eye and the orbit diagnosed using immunohistochemical methods.
Over a ten year period (2007-2017), 30 metastatic
tumors to the eye and orbit were retrieved from the MUHC-McGill
University Ocular Pathology Laboratory. In total, 12 of these 30
cases were selected based on unusual presentation, complete
clinical history and imaging, availability of both primary and
metastatic tissue, as well as a comprehensive
immunohistochemical profile. The immunomarkers included: CD4,
CD20, CK-8/18, CK-7, HMB45, S-100, Melan-A, CD138,
synaptophysin, chromogranin, vimentin, desmin, estrogen,
progesterone, and BRST-1 and -2.
Of the 12 cases, nine were orbital and three were
intraocular (choroidal). Six of the orbital cases were ductal
carcinoma of the breast metastatic to the orbit. Five of these six
were BRST-1 and -2 positive, while the other case was CK-7,
estrogen, and progesterone positive. The primary tumor of this
latter patient was BRST-1 and -2 positive. Three of these six
presented as bilateral proptosis while the other three had unilateral
orbital involvement. The remaining three orbital cases were
diagnosed as a conjunctival malignant melanoma, cutaneous
melanoma, and alveolar soft part sarcoma. The
immunohistochemical profile of melanoma cases were HMB45 and
Melan-A positive. The alveolar sarcoma was positive for vimentin
and desmin. The three intraocular uveal tumors included a
metastatic carcinoid, plasmacytoma (multiple myeloma), and T-cell
lymphoma. Chromogranin and synaptophysin were positive for the
metastatic carcinoid, CD-138 was positive in the plasmacytoma,
and the T-cell lymphoma was positive for CD4.
Even though metastasis to the eye or orbit is rare, an
accurate diagnosis is crucial for patient treatment.
Immunohistochemical evaluation of the metastasis is the gold
standard to establish a correct diagnosis. A panel of
immunohistochemical markers is always required as metastatic
tumors may have a different profile compared to the primary tumor.
Patients of reproductive age undergoing treatment for cancer may become infertile as a result of their chemotherapy or radiotherapy. A program for fertility preservation (egg or sperm banking) exists at the McGill University Health Centre (MUHC). However, it is not well-advertised to cancer patients and an effort, led by the Rossy Cancer Network, is underway to improve this situation.
The goal of this project was to develop a solution in the Opal patient portal (opalmedapps.com), to automatically distribute fertility preservation information and monitor its uptake by patients.
This project involved the design and implementation of new features within both the Opal frontend (i.e. patient facing) app and the Opal backend (staff-facing) software. After consultation with the Fertility Preservation team and the Rossy Cancer Network, and by studying Opal backend and frontend software, we decided to add new features on the existing code base. New interfaces and corresponding backend functionalities were necessary to examine patient uptake.
After a thoughtful discussion about database design, a new feature called “Opal Education Package” (OEP) was developed to bundle education materials and send them to patients. We carefully designed and implemented a multi-layered logging system to monitor patient uptake. With this system, researchers are able to get information on when a patient opened a document, the time that they spent reading the document, and whether or not they scrolled to the bottom of it.
Our improved Opal education material system provides data regarding patient uptake and allows clinicians to send education material to patients efficiently. Future work will be needed to automatically provide questionnaires and notifications to patients based on their uptake or lack of uptake and to visualize logs.
Introduction: The response of epithelial cells to infection leads to a series of intracellular events. Among them, ubiquitination is a process that modulates signaling pathways by activating or inhibiting specific proteins. Thus, ubiquitins are polymerized with lysines or methionines. In order to identify new signaling components, we therefore carried out a large-scale screening of ubiquitinated proteins after infection with Pseudomonas aeruginosa (Psa), a common pathogen in cystic fibrosis.
Methods: Bronchial epithelial cells (Beas-2B) were stimulated with diffusible Psa material (PsaDM) and lysed with non-denaturing buffer. Protein extracts were sequentially purified using affinity columns, applying the eluate from one column to the other (K48→M1→K63→Ub). The proteins bound to the columns were then analyzed by mass spectrometry (LC-MS / MS).
Results: Following PsaDM stimulation and LC-MS / MS analysis, 6 proteins were isolated from the K48 column, 4 from the M1 column, 22 from the K63 column and 10 from the general ubiquitin column. Among the results, the Alix protein proved to be particularly interesting in view of its functions of cytokinogenesis and the control of apoptosis. Inactivation of ALIX by CRISPR-Cas9 causes loss of response of CXCL8 to PsaDM and live bacteria. Infection of Beas-2B cells with live bacteria causes internalization and cytoplasmic localization of the latter while they are found at the nucleus in Beas-2B ALIX-KO cells.
Conclusion: Our screening results suggest that ALIX binds K63 polymers in response to infection. In addition, ALIX seems directly related to the strength of the innate immune response of lung epithelial cells. Functionality and characterization studies are needed to fully understand the role of ALIX in the host-pathogen interaction
Alveolar macrophages (AM) are tissue resident macrophages that reside extra-epithelially and are involved in maintaining pulmonary homeostasis such as surfactant homeostasis and gas exchange. AM are also the first immune cells encountered by airway pathogens. Eicosanoids, the host bioactive lipid mediators, play a key role in pulmonary homeostasis and inflammation. Interestingly, AM have been shown to be a major source of various eicosanoids, including the 15-lipoxygenase-derived lipoxin A4 (LXA4), a specialized pro-resolving mediator. This study aimed to investigate the potential contribution of LXA4 on maturation and self-renewal capacities of AM. Age and gender-matched C57Bl/6J wild type (WT) and 15-lipoxygenase knockout (Alox15-/-) mice are used in this study. The number of AM were significantly lower in both broncho-alveolar lavage (BAL) and lungs of Alox15-/- mice compared to WT mice (6-8 weeks old), phenotype specific to AM only. Additionally, we did not find difference in other tissue-resident macrophages populations from brain, spleen, liver or peritoneum. Most surprisingly, we did not find differences in AM population at post-natal day (PND) 3, suggesting a defect in proliferation rather than maturation. In line with this finding, Alox15-/- AM have reduced expression of Ki67 compared to WT mice. Surprisingly, GM-CSF and TGF-β1 levels were similar between genotypes. However, BAL LXA4 levels were significantly reduced in Alox15-/- mice compared to WT mice. Our findings suggest that the proliferation capacity of AM is impaired after birth in Alox15-/- mice and 15-lipoxygenase pathway plays pivotal role in AM function and homeostasis by regulating AM proliferation.
Asthma is an inflammatory disease characterized by bronchial hyperresponsiveness and airway obstruction. The airways of asthmatic subjects are often referred to as being twitchy. We previously performed mechanics measurements on airway smooth muscle (ASM) from human asthmatic and normal donors and observed spontaneous oscillations on the force traces during methacholine induced contraction. To improve our understanding of the twitchiness of asthmatic airways, we quantified and compared the amplitude and frequency of these spontaneous oscillations.
A computer program was created using Matlab to analyze ASM data from three sites (trachea, main bronchi, and intra-pulmonary bronchi) obtained from asthmatic and normal lungs. These files contained signals from the tissue equilibration periods, time during which muscle strips were stimulated to contract with methacholine to allow them to recover from potential damage that could have occurred during dissection and manipulation. Signals were separated in 5 segments, representing the baseline, rise, peak, decrease and the recovery phases of the contraction of the muscle when stimulated. For each segment, a moving average was used to remove the baseline drift induced by the contraction. Then, the oscillation peaks were found by computing the local maxima. The average period between each peak was calculated to obtain the frequency (1/period) of the oscillations. The root mean square of the drift corrected signal was then calculated to determine the average amplitude of the oscillations. All data obtained in one given lung were averaged to yield 1 value per site for each of the 5 segments.
Our preliminary results showed that asthmatic tissues tend to have oscillations with higher frequencies than those of controls. The oscillations of asthmatics spread from 0 to 2 Hz whereas those of controls were mostly centered around 0.4 Hz. The high oscillation frequencies in asthmatics were mostly observed during the baseline and recovery segments whereas they were more uniform throughout the contraction in the controls.
Analysis of the amplitude of the oscillations showed that, asthmatics tissues had smaller oscillations than control for the trachea and main bronchi tissues. However, this trend was markedly different in the intra-pulmonary airways where the average amplitude of the oscillations was approximately 0.002 mN in control whereas the asthmatic tissues showed several oscillations ranging from 0.005 and 0.017 mN.
These data suggest that a greater oscillation frequency and amplitude may be associated with the twitchiness of intra-pulmonary ASM in asthma.
Background: A recent study of kidney transplant recipients revealed important sex differences in graft outcomes, which varied in magnitude and direction by age and by donor sex. However, relationships observed in kidney transplant may not be the same across all organ types.
Hypothesis: We hypothesize that girls and young women have a higher risk of liver graft failure than boys and young men, whereas graft failure risk does not differ by sex among older individuals. We expect that donor sex will modify the relationship between recipient sex and graft failure risk.
Methods: We used time-varying Cox models to estimate the association between recipient sex and graft failure among first deceased donor liver transplant recipients recorded in the Scientific Registry of Transplant Recipients 1998-2018. Graft failure was defined as death following graft failure or retransplantation. We considered the possibility that the association between recipient sex and graft failure risk differs by recipient age and by donor sex. Recipient age was treated as a time-dependent variable. Models were adjusted for primary liver disease, race, donor age, era of transplant and insurer.
Results: The cohort included 133,757 liver transplant recipients, of whom 21, 608 experienced graft failure (9743 retransplants, 11, 501 deaths folllowing graft failure). Preliminary analyses, stratified by donor sex, showed a similar pattern to that seen in kidney transplant, but the magnitudes of the hazards ratios were smaller and differences were not statistically significant. When the donor was female, there were no material or statistically significant differences in the outcomes of female versus male recipients, except among those >45 years in whom females had significantly lower risks of graft failure than males (HR 0.90 [95% CI 0.84, 0.97]). When the donor was male, the risks of graft failure were higher in female than male recipients of all ages, but the differences were not statistically significant (0-12y HR 1.31 [0.98 to 1.76]; 13-29y HR 1.16 [0.92 to 1.46]; 30-44y HR 1.06 [0.87-1.28]; ³45y HR 1.02 [0.96 to 1.09].
Conclusions: There were no significant differences in the risk of graft failure by sex among liver transplant recipients. However, the overall patterns observed suggested similar sex differences to those observed in kidney transplant, but of attenuated magnitude, possibly in keeping with the lower immunologic risk in liver transplantation.
Clostridium difficile is an anaerobic gram-positive bacillus bacterium. Its other characteristics include spore formation and production of two main toxins; toxin A (TcdA) and toxin B (TcdB). These toxins are responsible for the pathology observed during C. difficile infection (CDI). In the early 2000s, hospitals began to report cases of CDI with increased severity. These infections were due to a new strain of C. difficile that became known as the BI/NAP1/027 strain (NAP1). It was found that infections caused by NAP1 were three times more virulent than other known C. difficile strains. Lanis et al (2013) found that NAP1 TcdB is responsible for the increased virulence. The greatest variation between NAP1 TcdB and historical TcdB is observed in the C-terminus domain (CTD) of the toxin. This region is highly immunogenic and commonly targeted by C. difficile vaccines. Unfortunately, studies have shown that vaccines targeting the CTD region of a historical strain of C. difficile cannot protect against NAP1. On the contrary, the glucosyltransferase domain (GTD) on the N terminus of TcdB is highly conserved between NAP1 and historical strains of C. difficile. This region was shown to be immunogenic by Jin et al (2013) and can be neutralized when targeted by a monoclonal antibody as described by Kroh et al (2018). Thus, this project will develop a candidate vaccine against the NAP1 strain by cloning the GTD of the NAP1 TcdB for expression in an attenuated strain of Salmonella typhimurium, YS1646. YS1646 vector is delivered orally to trigger a local and systemic immune response against mucosal infections. Chen et al (2011) have shown that this S. typhimurium strain can be used to successfully secrete heterologous antigen. First, the GTD of the NAP1 TcdB will be cloned into pQE30 plasmids and sequenced. These plasmids are designed to exploit different S. typhimurium secretions systems and are in current use in the laboratory as candidate vaccines for a historical strain of C. difficile. Plasmids with the proper insertion of the GTD sequence will be used to transform S. typhimurium, so that the NAP1 TcdB GTD can be expressed. We will examine expression of the TcdB GTD in S. typhimurium, in both axenic culture and in vitro macrophage infections using western blots. Successful expression of the NAP1 GTD will allow immunization of mice. Following vaccination, we will examine the humoral and mucosal immune response that is directed towards the NAP1 GTD of TcdB
Hypertension is a leading risk factor for death and cardiovascular diseases such as heart failure, myocardial infarction (MI) and stroke. Women older than 65 are more likely than men to have uncontrolled hypertension and suffer from these consequences. However, this increased prevalence in women only presents after menopause. Studies investigating the relationship between menopause and hypertension have failed to show a clear relationship. Blood pressure (BP) alone may not capture the vascular changes that lead to these poor outcomes. Recently, blood pressure variability (BPV) has emerged as a new measure that can be used to predict MI, stroke and even dementia. A study in hypertensive women was able to determine that menopause contributes to BPV. BRAVE aims to build upon this study by investigating BPV of healthy women around menopause and correlating it with measures of vascular health such as endothelial function, arterial stiffness and carotid wall thickness, thus generating a more complete picture of the changes to the vascular system around menopause.
Fifty healthy women were recruited from the community in Montreal. Three BP measures were taken: automated office BP, 24 hour ambulatory BP and home BP over 1 week. BPV was measured through coefficient of variation of readings. Blood tests (serum FSH levels) and questionnaires were used to determine menopausal status. Information was also collected about other cardiac risk factors (e.g. smoking, obesity) and more recently characterized gender related risk factors (e.g. gender identity and gender roles). Arterial stiffness was measured using carotid femoral pulse wave velocity (cfPWV). Carotid intimal medial thickness (cIMT) was measured using ultrasound and endothelial function measured using finger plethysmography (EndoPAT). Univariable and multivariable linear and logistic regressions will also be used to assess for the effect of menopausal status on BPV and the effect of BPV on cfPWV, cIMT and EndoPAT.
[Analyses currently underway, and results will be ready to present by August 13th]
Understanding the changes in vascular health around menopause will allow us to better predict which women are more likely to suffer from hypertension, and better tailor management to improve outcomes.
Cystic Fibrosis (CF) is an inherited genetic disease leading to the dysfunction of the cystic fibrosis transmembrane conductance regulator, a chloride ion channel. In CF patients, Pseudomonas aeruginosa is the first opportunistic pathogen responsible for chronic airway infection, which determines the outcome of the disease. Why P. aeruginosa persists despite antimicrobial therapy and activation of immune responses remains incompletely understood. To date, the prevailing view suggests that impaired muco-ciliary clearance and P. aeruginosa biofilm lifestyle are the primary mechanisms contributing to the persistence in CF lung. Recent work from our laboratory has highlighted that P. aeruginosa can establish intracellular infection, in addition to extracellular persistence. In this study, we will characterize and compare the swimming motility, the biofilm formation, the induced-cytotoxicity, and the antimicrobial profile of intracellular and extracellular isolates collected from CF lungs from patients who underwent transplantation for end-stage disease.
P. aeruginosa clinical isolates were collected between 2016 and 2018 from CF lung explants. Extracellular isolates were collected from supernatants of the lung samples. Thereafter, the explants were treated with aminoglycosides to clear extracellular pathogens. Intracellular isolates were sampled after homogenization of the lung tissue in a solution containing 0,5% triton. Assessment of swimming motility was measured on 0.3% LB agar after 8 hours incubation at 37°C. PAO1 and non-motile mutant strain PAO1∆FliC, lacking its flagellum, were used as reference strains to compare clinical isolates. Biofilm formation was measured with a crystal violet assay after 24 hours incubation at 37°C. P. aeruginosa-induced cytotoxicity was realized in Beas-2B cells, an immortalized human bronchial epithelial cell lineage. Lactate dehydrogenase release in the supernatant was measured after 6 hours infection at a multiplicity of infection of 1. The antimicrobial profile was assessed by growth in serial dilution in 96 well plates.
Fifty-three Pseudomonas aeruginosa strains were isolated from 8 different clinical CF lung. Among these, 30 were identified as intracellular and 23 as extracellular. Sixteen strains from 3 patients were chosen for phenotypic characterization. There was no difference in the swimming motility between intracellular and extracellular strains in the three patients (intracellular motility (mean±SD) 3.05±0.7 cm, 0.67±0.3 cm, 2.15±2.0 cm and extracellular 2.72±0.72 cm, 0.46 ±0.13 cm, and 2.53 ±0.47 cm). Biofilm, cytotoxicity and antimicrobial profile assessments are currently in progress.
P. aeruginosa intracellular persistence is a newly-found lifestyle in the airways and their characterisation may prove to be helpful for the understanding of underlying mechanisms behind the host-pathogen interactions.
Congenital Anomalies in the Kidney and Urinary Tract (CAKUT) are defects that affect the formation of the kidneys and the urinary tracts. These organs develop from the nephric ducts, bilateral tubes that differentiate from the intermediate mesoderm, become epithelial and then elongate with the growth of the embryo. Tight junctions are cell-cell junctions between epithelial cells that contain specialized proteins including claudins, a family of proteins that form pores within the paracellular space. As CAKUT may have a genetic origin, claudin genes were sequenced in 95 children with these defects. 37 non-synonymous variants were identified, including two rare variants Claudin-8 A94V and Claudin-14 S58R, and many common variants including Claudin-8 S151P. These variants were selected because both claudins are expressed in the developing mouse kidney. My aim is to determine the effect of these variants on nephric duct formation in the chick. I hypothesized that the presence of rare variants would impair nephric duct elongation and result in a CAKUT phenotype, while the common variant would not have any effect. A retroviral vector called RCAS (Replication Competent ALV LTR with a Splice acceptor) containing each of the three variants was injected in the precursor of the nephric duct on one side of the embryos, then the embryos were cultured on plates for 24 hours to assess nephric duct elongation. A major aspect of my project was to establish a protocol that permits the simultaneous assessment of nephric duct elongation and retroviral infection of the target tissue. To this end, a two-color double in situ hybridization assay will be performed. To visualize the nephric duct, an antisense probe was used to detect Lim1 mRNA, a transcription factor that is a marker of the nephric duct. The RCAS-injected nephric duct was compared to the uninjected side to assess if there were differences in the morphology of the duct. This was performed for the common variant CLDN8 S151P and there was no phenotypic difference between the two nephric ducts based on their morphology (N=23 embryos). To determine if the retrovirus has infected the nephric duct resulting in overexpression of the claudin variant, a probe targeting the envelope gene env mRNA transcript will be used. When the protocol will be optimized, I will use it to test the effect of CLDN8 A94V and CLDN14 S58R variants found in children with CAKUT.
Characterization of the secreted glycoprotein mesh during neural tube closure in chick embryos.
The neural tube is one of the first structures to form during embryonic development. Its formation can be summarized in a few steps. First, the dorsal ectoderm thickens to form the neural plate. It then bends in a U-shape so that the neural folds converge towards each other, until they finally fuse to form a closed neural tube overlain by a non-neural ectoderm sheet. During the last steps of neural tube closure, a glycoprotein mesh-like network has been discovered between the two neural folds. This mesh is thought to be a temporary adhesive that holds the apposing neural folds together prior to the epithelial remodeling events that establish a strong and stable union between apposing neural folds.
Tight junctions are the most apical intercellular junctions and they are composed of different members of the claudin family of transmembrane proteins. Previous members of our lab discovered that Cldn-3 depleted embryos have neural tube defects due to a failure of neural fold fusion. Furthermore, scanning electron microscopy revealed that these Cldn-3 depleted embryos are missing the glycoprotein mesh. In order to better characterize the glycoprotein mesh, I am performing lectin staining. Lectins are agglutinin proteins with specific affinities for carbohydrates and allow the localization and identification of glycoproteins and glycolipids in tissues. I am using Concanavalin A (ConA; recognizes a-D-Glc ; a-D-Man), Peanut agglutinin (PNA; recognizes D-Galactose and b-N-acetylgalactosamine) and Soybean agglutinin (SBA; recognizes a or b-N-acetylgalactosamine) on wild type chick embryos at different developmental stages. All three lectins are fluorescently-tagged by either Alexa Fluor 488, 584 or 647. My preliminary data showed that SBA primarily recognizes sugars in the somite area, whereas ConA’s preferred sugars are found at the edge of the head and at the closure point in the anterior region of the neural tube. Glycans recognized by PNA are found at the closure point in anterior/posterior region and at the edge of the head. I will also present data showing that lectin staining is altered in the claudin-depleted embryos. If changes are found in the glycoprotein composition, we will determine if these changes are due to defective trafficking or secretion of carbohydrates.
Community-Academic Partnerships : Collaboration between academic institutions and their community partners according to the Authentic Partnership Framework
Geneviève Laporte, Diana Gausden, Françoise Fillion, Mélanie Lavoie-Tremblay
Background : Healthcare challenges of the 21stcentury require nurses to learn beyond the scope of hospital-based care and further explore their role in health promotion and illness prevention for underserved and vulnerable populations. For nursing schools, access to these populations requires partnership with local community-benefit organizations (CBO). However, many gaps remain in our knowledge pertaining to community-academic partnerships, because they are not as commonly described in the literature as those within hospitals and clinical settings (Granger et al., 2012)(Schaffer, A., Schoon, & Brueshoff, 2017)Furthermore, despite their altruistic goals to address community health problems and engage vulnerable populations in research or intervention projects, these partnerships often struggle with fundamental issues such as effectiveness and sustainability. (Brush, Baiardi, & Lapides, 2011).
Method :As part of a larger study investigating the impact of CBO partnerships, this poster will describe and analyze community projects (N = 133) implemented by nursing students over a 3-year partnership between the Ingram School of Nursing (McGill University) and 20 urban community-benefit organizations. The analysis examines the determinants of health addressed by these projects as well as targeted priority issues and sustainability strategies. In addition, using the lens of the Authentic Partnership Framework, the meaningfulness of the student projects was analyzed by comparing the CBO’s mandate and the aim of the implemented projects to assess the relevance of student projects for the local community partners.
Results:Amongst the determinants of health tackled by the projects, the three most common were education (74%), personal health practices & coping skills (72%) and social support networks (44%). In terms of priority issues, equal proportions (28%) of the projects listed health education or psychosocial education as a priority. Several types of sustainability strategies were implemented, from posters and pamphlets to donation of materials and ongoing sponsorship to maintain the project in place at the CBO. When assessing meaningfulness, 57 % of projects were developed in accordance with the CBO’s mission.
Conclusion:In light of these results, the high level of relevance suggests that effectiveness and sustainability are within reach in the partnership between the Ingram School of Nursing and 20 urban community-benefit organizations, thus creating an Authentic Collaborative Partnership.
Purpose: Radiomics is the extraction and analysis of large numbers of numerical quantities (referred to as ‘features’, e.g. tumor size, gray-level co-occurrence matrix) from medical images. It is cost-effective, and betters the precision of disease diagnosis and prognosis. In our study, Dual-Energy Computed Tomography (DECT) enables data to be acquired in 21 energy levels, ranging between 40 keV and 140 keV. Subsequently, feature values could be extracted from these scans in both 2D and 3D. 2D analysis is performed on the central slice of the tumor whereas 3D analysis is done on the entire tumor. Though 3D contouring (i.e. outlining the tumor at each slice) of CT images may deliver “more” information, its process is significantly more time-consuming than that of 2D. Thus, a radiomics analysis is implemented to reveal if 3D features necessarily outperform 2D features in terms of their predictability for lymphadenopathy, a disease of abnormal lymph nodes in their size, number or consistency.
Methods: There were 87 patients' head and neck DECT scans obtained from a single institution whose image data were converted into 2D and 3D feature values. The datasets were split into two sets in a 2:1 ratio: the teaching and testing sets. There were 6092 features gathered for every patient for all 21 energy levels. Features with an absolute correlation coefficient with the outcome of > 0.3 were deemed promising for accurate lymphadenopathy prediction. Outcome prediction capabilities were quantified by AUC values of a ROC (Receiver Operating Characteristic) curve.
Results: Pearson correlation coefficients (R) between feature values and the outcomes are computed for all features of each patient, but only done on the teaching set. This is to ensure the testing set is independent of the lymphadenopathy outcomes. The R values are systematically higher in 2D than that of 3D for every energy level. A metric quantifying this improvement in correlation of 2D features is called NIF (Net Improvement Fraction). NIF = 0 indicates no global improvement over all promising features, and NIF =1 implies an enhancement for all promising features. Currently, our NIF spans from 5.6% to 23.6% for various energy levels illustrating a better prediction performance in lymphadenopathy with 2D features compared to 3D features.
Conclusion: Extracted 2D features from DECT scans at differential energy levels may have a stronger power for predicting lymphadenopathy than that of 3D features. Investigation for the reasons behind this preliminary result is being further executed.
Introduction: Previous research suggests that mutations in Uroplakins cause Congenital Anomalies of the Kidneys and the Urinary Tract which are the leading cause of renal failure in children. Many of these defects are diagnosed based on ultrasound images that reveal the presence of a dilated renal pelvis defined as hydronephrosis. Uroplakins (UpkIa, UpkIb, UpkII and UpkIII) are integral membrane proteins secreted by the urothelial lining. They interact to form plaques which line the apical surface of the urinary tract rendering it impermeable and they also regulate cell growth.
UpkIII has two isoforms: UpkIIIa and a minor isoform, UpkIIIb. When UpkIIIa is ablated from mice, reduced urothelial plaque size, compromised urothelium permeability, and hydronephrosis are observed. Phenotypes induced by UpkIIIb ablation have not been characterized. Also, the timing of hydronephrosis development in UpkIIIa-/-mice is unknown. Our objectives are 1) to determine if KO mice for UpkIIIb will also develop hydronephrosis and 2) to compare the urinary tract of UpkIIIb-/-and UpkIIIa-/- mice by ultrasound.
Methods: Ultrasounds were performed on UpkIIIb-/-mice (n=9: 5M, 4F), UpkIIIa-/-(n=3 pups) and wildtype controls (n=4 mice: 1F, 3M and n=4 pups) at birth, 1 month and 2 months. Measurements of the longitudinal kidney length, the renal papilla width, the renal pelvis diameter, and the transverse renal width were taken to measure the proportion of hypoechogenic space that demarcates the distension of the renal pelvis within the parenchyma of the kidney. The severity of hydronephrosis was defined as percentage of renal parenchyma 100% = absent, >67%= mild, 34-66%= moderate, <33%=severe.
Results: Only 1 of 5 male UpkIIIb-/- mice exhibited mild unilateral right-sided hydronephrosis at the 2 month image. None of the females showed hydronephrosis. UpkIIIa-/- pups (n=3) died neonatally. Two exhibited hydronephrosis at birth. One had mild unilateral hydronephrosis on the right kidney and one had bilateral hydronephrosis: moderate on the left and mild on the right kidneys, respectively. The pups remained small and only one survived. This male was imaged and euthanized at 21 days. Its’ mild unilateral hydronephrosis progressed to affect both kidneys with moderate severity. One male, UpkIIIa-/-, used to generate the litters, was discovered with severe hydronephrosis at 7 months of age.
Conclusion: UpkIIIa-/-mice died neonatally suggesting that mutations in UpkIIIa might not be well tolerated. UpkIIIb-/-mice appeared to have no significant hydronephrosis. UPKIIIa appears to rescue plaque formation in the absence of UPKIIIb. In contrast, UPKIIIb protein does not appear to compensate for the loss of UPKIIIa.
Background: The etiology of murine malocclusion is not well understood. A malocclusion presents as an overgrowth of the lower or upper incisors causes inability of the jaw to occlude leading to feeding impairment and thin, dehydrated mice. Malocclusion occurs in all murine species but at low frequency (<0.1%). Much is still unknown about root dentin deposition and resorption. Mice constitutively null for menin, the product of the multiple endocrine neoplasia type 1 (MEN1) tumor suppressor gene, die at midgestation with development defects including craniofacial defects. Adult mice in which menin is deleted in the pre-osteoblast and osteoblast lineage exhibit low bone mass and mineral density and bone strength. The relationship between menin and tooth morphogenesis has not been investigated previously.
Aim: To document the increased frequency of malocclusion in mice with conditional knockout of deletion of menin in the osteoblast and odontogenic lineages.
Methods: Conditional knockout mice in which the Men1 gene is specifically deleted early in the osteoblast lineage were generated by using the Cre-LoxP recombination system. Osx-Cre; Men1f/f mice represent knockout of the Men1 gene at the level of the preosteoblast. Incidence of the malocclusion phenotype was recorded in our Osx-Cre;Men1f/f mouse colony. Osx-Cre; Men1f/f (n=5) and Men1f/f (n=4) mice at 9 months of age were selected for dissection of the alveolar region of the mandible. Samples were collected aseptically and stripped of all muscle and connective tissue. DNA was isolated for Men1 gene recombination analysis via polymerase chain reaction (PCR). Menin, osteocalcin and alkaline phosphatase gene expression were assessed by RT-qPCR RNA using extracted RNA samples.
Results: The frequency of the malocclusion phenotype in the Osx-Cre Men1f/f (9 months or older) was significantly increased (Chi-square value = 18.77, p < 0.001) compared to the Men1f/f control mice. PCR amplification of mandibular genomic DNA confirmed that Cre-mediated recombination occurred specifically in the Osx-Cre;Men1f/f mice, as opposed to the Men1f/f control group. The gene expression profiles of menin, osteocalcin and alkaline phosphatase in the mandibular RNA of the Osx-Cre;Men1f/f experimental group were altered by -42.0%±8.2% (p<0.01), -45.7%±16.7% (p<0.05) and +53.8%±31.0% (p=0.13) compared to Men1f/f mice, respectively.
Conclusions: We have demonstrated that mice with conditional knockout of deletion of menin in the osteoblast and odontogenic lineages have an increased frequency of malocclusion. Further studies are ongoing to elucidate mechanisms.
Asthma is a heterogeneous, inflammatory disease that has long been associated with airway hyperresponsiveness (AHR), an exaggerated response of the airways to contractile stimuli. However, the causes of AHR remain unclear. Airway remodelling, characterized by an increase in airway smooth muscle (ASM) mass and a hypercontractile ASM cell (ASMC) phenotype, has been proposed to be responsible for this phenomenon. Both changes have been hypothesized to promote AHR by mediating excessive narrowing. CD4+ T helper cells have been implicated as drivers of airway remodelling. Studies using rat models of asthma have demonstrated that CD4+ T cell migrate to the airways and adhere to ASM, causing remodelling. Despite these observations, little is known about bidirectional effects mediated by cellular interactions between these two cell types. This project aims to determine how CD4+ T cell drive ASM remodelling and whether T cells themselves are also affected by their interaction with ASM.
CD4+ T cells induce a proliferative phenotype in ASMC resulting in increased ASMC proliferation and decreased contractility.
Peripheral blood mononuclear cells (PBMCs) of healthy volunteers were activated and CD4+ T cells then isolated. Human ASMCs were isolated from healthy donor lung tissue and cultured in starvation medium, before being seeded in a Transwell system or in direct co-culture with T cells. mRNA isolated from both cell types was used for quantitative PCR to assess cytokine expression levels. Calcium assays used to assess ASMC contractility were performed using Fura-2 AM dye to obtain intracellular calcium transients and histamine was used to stimulate calcium release. ASMC proliferation was assessed using bromodeoxyuridine (BrdU) incorporation, which was added to the culture system 18 hours before cell harvest. Cells were stained with the efluor780 viability dye, anti-BrdU and anti-CD4 antibodies. BrdU incorporation was analyzed using flow cytometry.
After 48 hours in coculture, ASMC demonstrate increased mRNA expression of IFN-g and TNF-a, but these results were not observed in the Transwell system. In contrast, CD4+ T cells have downregulated expression of these cytokines. ASMC demonstrated decreased peak calcium responses to histamine after coculture. There was a decrease in expression of calponin mRNA 48 hours after direct coculture compared with the Transwell condition and ASMC-only control. Contrarily, cells demonstrated increased a-actin mRNA expression. BrdU incorporation was increased in ASMC after coculture.
CD4+ T cells induce an inflammatory phenotype in ASMC which may be responsible for inducing a proliferative phenotype. Inversely, this interaction reduced CD4+ T cell cytokine production.
Educational videos offer a unique method of conveying information, perspective, and images to patients, hospital newcomers, and outsiders. By selecting specific topics and presenting them concisely and transparently, viewers can become accustomed to an otherwise unfamiliar subject in a matter of minutes. This research project involved identifying aspects of both the Opal app (opalmedapps.com) and the MUHC hospital experience that could provide difficulty for patients and creating videos to explain these aspects.
To demonstrate the various aspects of the Opal app, our videos incorporate animations, transitions, and subtitles. Animations of the Opal app demonstrate to viewers how to access certain functions. Custom transitions, including animated wipes with masking and dissolves, make videos smoother and more watchable for the viewer. Custom animated subtitles with highlighted text mean the viewer does not need to pay complete attention to voice-over narration. We produced videos in both French and English.
A focus on the patient perspective during video production process means that this project offers a unique opportunity to cater to the true patient experience. Many aspects of the patient experience are difficult to explain verbally, and videos can provide much-needed first-hand information. For instance, we created a first-person perspective video of radiotherapy treatment (by having a patient wear a GoPro camera during treatment) so that patients have a good understanding of what will happen during their own treatment, alleviating first-time anxiety.
Posted on YouTube and distributed as Opal educational material, the educational videos can also easily be shared to new viewers in a variety of ways and viewed on nearly any device. This means that videos can be useful in the waiting room (playing on a television), at home (playing on personal computer), or nearly anywhere else (playing on mobile devices). As a result, the utility of educational videos extends both within and outside the hospital environment.
Video subjects include navigating the hospital and the Cedars Cancer Centre (CCC), understanding treatments before undertaking them, and using and understanding the Opal app. Already-made videos include an introduction to the Opal app, an explanation of the role of the Cancer Mission Patients’ Committee, a first-person radiotherapy video, and a navigational video on reaching the CCC from the Vendôme metro. We plan on creating a tutorial on how to navigate the CCC, an explanation of the hospital’s parking system, a video of the chemotherapy process, and animations of specific Opal app features.
Brain-computer interfaces (BCI) are technologies that read electrical brain activity and translate these signals into computer input. These technologies are projected to enable individuals with severe motor impairments to interact and communicate with the world around them by bypassing the need for motor and behavioral control. However, the adoption of BCIs by the target population has been hindered by several limitations, including lengthy training periods. We hypothesize that the training period to master BCI performance can be reduced by the application of a neuromodulatory technique: transcranial direct current stimulation (tDCS). We recruited 20 healthy participants (ages x to y) to learn to control a custom-built BCI over 10 sessions. Participants were tasked with controlling the left-right movement of an onscreen ball using motor imagery to move the ball to the right and rest to move the ball to the left. Using a single-blinded sham-controlled study design, we applied tDCS for 20 minutes over the motor cortex of the experimental group (n = 10), and sham-stimulation to the control group (n = 10). The effect of tDCS will be assessed by testing the null hypothesis that there is no difference in the rate of change in either performance accuracy, or the power of the sensorimotor rhythms of the M1 area, across the 10 sessions. The results of this study will contribute to the development of more efficient training techniques for BCIs, which may facilitate adoption of BCIs in target populations in their daily lives.
Key words: tDCS, BCI, motor imagery, primary motor cortex, EEG
Sleep complaints are one of the most common symptoms related to the menopausal transition, affecting 40-60% of women. However, it is difficult to disentangle changes in sleep that are due to aging from those that are due to menopause-related changes. The aim of this study was to compare different types of sleep disorders in menopausal and pre/peri-menopausal women of similar age in a large population-based study
Material and Method:
Women aged between 45-60 years were selected from the database of the Canadian Longitudinal Study of Aging, a 50,000-participant population-based study. All women who self-reported menopausal status were included, excluding those with history of hysterectomy. Participants completed symptom screens for overall sleep satisfaction, hours of sleep per day, sleep-onset insomnia, sleep-maintenance insomnia, daytime somnolence, rapid eye movement sleep behavior disorder (RBD), restless leg syndrome (RLS) and obstructive sleep apnea (OSA). Odds Ratio for each sleep variable was compared between post-menopausal and premenopausal women using multivariate regression, adjusting for age, propensity score for menopause, anxiety, depression, psychological disorders, hypertension and thyroid dysfunctions.
Among 6179 women included in the analyses, 3713 (60.1%; mean age=55.7±3.3) were post-menopausal and 2466 (39.1%) were pre/peri-menopausal (mean age: 49.80±3.1). As compared to pre/peri menopausal women, post-menopausal women were more likely to report requiring ≥30 minutes to fall asleep (20.4% vs. 15.5%, Adjusted OR=1.40, 95%CI=1.22-1.60) and were more likely to meet criteria for clinical sleep-onset insomnia (10.8% vs. 7.3%, OR= 1.54 [1.25-1.90]). When symptom onset was plotted according to menopause onset, there was a clear peak in insomnia onset in the 2 years before and after menopause onset. Menopausal women were also 1.4 times more likely to screen positive for OSA (14.6% vs. 10.4%, Adjusted 95%CI 1.26-1.74).
The two groups did not differ on overall sleep satisfaction, with over 50% expressing good sleep satisfaction. Similarly, there was no group difference in clinically-significant daytime somnolence (1.6% vs. 1.3%), sleep maintenance insomnia (17% vs. 14.5%), RLS (23.5% vs. 20.9%) or possible RBD (3.9% vs 4.0%).
Compared to pre/peri menopausal women of similar age, menopausal women have increased insomnia, which appears to be specific to sleep-onset difficulties, and with an onset of symptoms that is commonly timed to the onset of menopause. Menopausal women also are more likely to screen positive for OSA. However, they do not differ on sleep maintenance, somnolence, RLS, or possible REM sleep behavior disorder.
Keywords: Menopause, Sleep, Insomnia, Hypersomnolence, RBD, RLS, OSA
Intestinal helminths infect an estimated 2 billion people across the world, leading to impaired cognitive development and significant morbidity. These parasitic worms often cause chronic infections and are able to persist due to their effective modulation of the host immune system. Many helminths establish infection in the small intestine where they intimately interact with the microbiota – a collection of microbes (mostly bacteria) that populate the gut and provide critical signals for immune system development, nutrient absorption and host defense. Due to this close proximity, helminths have evolved to affect the host immune system, often doing so through altering the composition and behaviour of the microbiota in which they are coated. To understand this double-edged nature of how helminths modulate host immunity, we must decipher which mechanisms are intrinsic to helminths and which are an indirect result of a change in the gut microbiota. To this end, we have developed a method for rearing germ-free larvae of Heligmosomoides polygyrus bakeri - a natural mouse parasite used to model human roundworm infection. Using in vitro and in vivo quantitative assessment of bacterial 16S rDNA and worm abundance, we describe a method to generate infectious, sterile Hpb larvae. Development of this methodology sets the stage for understanding the impact of helminth infection on host fitness and immunity in the absence of a commensal microbiota.
Evaluation of the immune landscape in Esophago-Gastric Adenocarcinoma
Megan Liu3,4, Duc-Vinh Thai2, Dongmei Zuo1, Dan Moldoveanu1, Emma Lee3,4, Victoria Marcus2, Morag Park1,2,4 , Lorenzo Ferri3,4, Veena Sangwan3,4
1Goodman Cancer Research Center; 2Department of Pathology; 3Department of Surgery, McGill University; 4RI-McGill University Health Center
Summary. Adenocarcinomas of the distal esophagus and proximal stomach are the fastest rising malignancies in North America. Emerging mutational analysis data demonstrate that proximal gastric adenocarcinoma and distal esophageal adenocarcinoma are genomically identical, and thus should be considered one disease entity – Esophago-Gastric Adenocarcinoma (EGA) – and treated similarly. The five-year survival rate for EGA is about 20%, as the majority of patients present with advanced disease.
Although chemotherapy improves outcomes, not all patients benefit and response is unpredictable, emphasizing the need to understand the mechanisms determining poor outcome and response to therapy. Recent studies have demonstrated that an activated immune system is linked to good outcome, and immune checkpoint inhibitors are in the clinic.
Objectives: To explore the impact of the immune landscape in esophago-gastric cancer.
Methodology: A tissue microarray containing formalin fixed paraffin embedded tumor and normal tissue from 26 esophago-gastric adenocarcinoma patients. Tumor cores were evaluated for presence of CD8, FoxP3, CD4, CD20, CD68, pancytokeratin and DAPI. TMA cores were scanned using a confocal microscope (Zeiss LSM710) and images were quantitatively analysed using the HALO software (Indica Labs). Percent positive cells in each core were counted and the ratio between different cell types were compared to clinical recurrence of cancer.
Results: Patients were subdivided into “immune cold” (8 patients), “fully inflamed” (8 patients) and “margin restricted” (10 patients) based on the presence of CD8+ cells in the tumor. Disease free survival <692 days was observed in 13/18 immune cold and margin restricted groups, and >692 days in 5/18 patients from these groups. For patients with fully inflamed tumors, 3/8 had disease free survival <692 days, and 5/8 of >692 days.
Conclusion: Presence of higher number of CD8+ immunoreactive T-cells in the tumor bed is a favourable prognostic marker for disease free survival in esophago-gastric adenocarcinomas.
Background: Mentorship has been identified as one of the main ways to decrease the critical shortage and high rate of premature departure amongst nursing faculty. Although faculty mentorship programs in nursing primarily focus on the needs of new faculty members, the literature shows that more-experienced faculty also have mentoring needs. Isolation, stress, burnout and turnover are serious issues in nursing programs where mentoring is not offered, regardless of how experienced the faculty are. Therefore, midlevel and senior faculty members could also benefit from a mentorship program. In light of this fact, in June 2017 the Ingram School of Nursing Faculty Mentorship Program (ISoN-FMP) implemented a program that would be available to every faculty member, even those with significant levels of experience. This paper describes how the ISoN-FMP was developed, implemented and evaluated. It will also discuss the results of a survey used to evaluate the satisfaction of the participants, as well as the specific needs of the faculty members at different levels of seniority.
Method: The ISoN-FMP matched 25 dyads based on shared areas of interest, perceived mentorship needs, and personal preferences. One-year post implementation, participants responded to a descriptive, cross-sectional evaluation survey. The survey addressed three main themes: the primary goals of the mentoring partnership, overall level of satisfaction with the ISoN-FMP (Mentorship Effectiveness Scale) and the characteristics of each dyad’s mentoring agreement (strategies used to communicate, where the dyads meet, etc.). Analysis was composed of descriptive statistics and quantitative analysis of participants’ textual responses to open-ended questions
Results: A total of 19 mentees (RR 79.17%) and 15 mentors (RR 71.43%) completed the survey, with both groups reporting high satisfaction (79% of mentees, 87% of mentors) with the overall ISoN-FMP. Also, the ISoN-FMP was judged useful by 89% of mentees and 93% of mentors. Furthermore, the survey revealed that mentees with five years or less of experience seek guidance on teaching, career-planning, and developing leadership and programs, and that they are hoping to be able to share knowledge and resources with their mentor. Mentees with more than five years of experience hope to get guidance on teaching and career-planning as well, but they also hope to be mentored about research and publications.
Conclusion: Even though specific mentoring needs differed according to seniority, the ISoN-FMP was shown to be beneficial. The program can also be judged as being effective, since it received overwhelmingly positive feedback from the participants.
Methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) is a homodimeric cytoplasmic enzyme that plays key role in cytosolic folate-mediated one-carbon metabolism. It is a trifunctional protein, comprising 10-formylTHF synthetase, 5,10-methenylTHF cyclohydrolase, and 5,10-methyleneTHF dehydrogenase activities. This protein is encoded by the MTHFD1 gene, and is important for maintaining the different oxidation states of one-carbon tetrahydrofolates (THFs). These are required for de novo purine synthesis, synthesis of thymidylate, and remethylation of homocysteine to methionine by methionine synthase.
Seven patients have been described with biallelic mutations in MTHFD1. This resulted in a clinical diagnosis of MTHFD1 deficiency, which is associated with severe combined immunodeficiency (SCID), and megaloblastic anemia. The first patient to be described was found to have one mutation (c.727+1G>A) affecting a splice acceptor site and a second mutation (c.517C>T) in a critical arginine residue in the NADP-binding site of the protein. When the enzyme’s dehydrogenase function was measured in cultured patient fibroblasts, no activity was detected.
A patient was referred to our laboratory with suspected MTHFD1 deficiency on clinical grounds. Examination of cDNA from this patient confirmed a single novel variant in MTHFD1 (c.52C>T/ p.A18V, a missense mutation in exon 2), as well as the expression of both MTHFD1 alleles. A second mutation has not been identified.
MTHFD1 dehydrogenase function was analyzed by measuring its activity in extracts of cultured fibroblasts from the referred patient and a control cell line. In contrast to cell extracts from the original patient which had no detectable activity, extracts from this patient had 24% of the specific activity seen in the control extract.
Currently, dehydrogenase activity is being measured in fibroblast extracts from seven patients: the original patient with no detectable activity; the referred patient with 24% activity; this patient’s brother, who is heterozygous for the same mutation; his mother who does not carry the mutation; members of two families with MTHFD1 deficiency diagnosed in the laboratory of Matthias Baumgartner in Zurich.
Background: Dynamic knee extensor (KE) exercise has been suggested as an alternative training modality to conventional 2-leg cycling for adults with chronic obstructive pulmonary disease (COPD) on the theoretical basis that isolating a smaller muscle mass lowers intensity ratings of breathlessness by lowering metabolic and ventilatory demands. The purpose of this study was to further characterize the physiological and perceptual response of small vs. large muscle mass exercise in COPD, most notably on the inter-relationships between ventilation, breathing pattern, dynamic operating lung volumes and intensity ratings of breathlessness.
Methods: Using a randomized, cross-over study design, seven men with moderate-to-severe COPD (percentage of predicted forced expiratory volume in 1-sec, 60% predicted) completed a steady-state cycle exercise test as well as a steady-state 1-leg and 2-leg KE exercise test at 65% of modality-specific peak power output. O2 consumption (VO2), ventilation (VE), breathing pattern, inspiratory capacity, inspiratory reserve volume (IRV) and breathlessness responses were collected at rest and during exercise.
Results: Compared to cycling, 2-leg and 1-leg KE exercise elicited markedly lower absolute levels of VO2 (by ~0.20 and 0.40 L/min, respectively) and VE (by ~13 and 18 L/min, respectively). Despite these differences, intensity ratings of breathlessness were comparable across exercise modalities; that is, breathlessness intensity-VE slopes were higher during KE vs. cycle exercise. Compared to cycling, 2-leg and 1-leg KE exercise resulted in a more rapid and shallow breathing pattern, which, in the setting of a greater rate of dynamic lung hyperinflation during KE vs. cycle exercise, helped maintain IRV-VE relationships across exercise modalities.
Conclusion: The physiological response to small vs. large muscle mass exercise in COPD was characterized by lower metabolic and ventilatory demands, adoption of a rapid and shallow breathing pattern, a greater rate of dynamic lung hyperinflation, and a higher breathlessness-ventilation relationship, which could not be explained by differences in the behaviour of dynamic IRV.
TMED2, or p24β1, is a member of the transmembrane emp24 domain-containing (TMED) cargo receptors involved in trafficking properly folded proteins within the secretory pathway. It has important functions in this process from cargo selection, quality control processing, and vesicle biogenesis. At the organismal level, TMED2 is also shown to have a role in the developing mouse embryo, placenta and liver. We hypothesize that TMED2 interacts with cargo proteins in the secretory pathway to regulate transport. To identify TMED2 interacting proteins in the early secretory pathway, interactions on the cytosolic, and luminal domains will be analyzed in Human Embryonic Kidney cells (HEK293 TRE x Flp). Flp recombinase-mediated transfection of plasmid constructs containing biotin ligase tag (BirA*) cytosolic and luminal domain of TMED2 will be used for proximity-dependent biotin identification. Preliminary experiments from liver proteomic studies, proximity-dependent biotin identification and yeast-two hybrid data has revealed approximately ten ER-resident proteins that interact with TMED2. These proteins are important in functions such as glycosylation and GPI anchor remodelling. Western Blot analysis of five out of the eleven proteins showed that three of these proteins are present in HEK293 TRE x Flp cells. Antibodies will be identified for the remaining proteins to confirm that they are present in HEK293 TRE x Flp cells. Proteins present in these cells will be used for co-immunoprecipitation in tissue lysates to determine if they are found in TMED2 complexes. Cellular pathway regulated by these candidates will be analyzed in Tmed2 mutant embryos and organs.
Prostate cancer is the most common cancer in men and a leading cause of cancer-related mortality. Evading immune destruction has been identified as an emerging cancer hallmark. This lends weight to the concept of immune modulation as a relevant area of study in the fight against cancer. In fact, immunotherapy has transformed treatment for many cancer types in the past decade, but progress has been slower for the treatment of prostate cancer. This study will focus on the characterization of immune checkpoint ligand expression in prostate cancer.
Firstly, the study will determine the basal level of expression of checkpoint ligands on prostate cancer cell lines using multicolor flow cytometry. Selected checkpoint markers are Galectin 9, CD40, CD80, CD86, PD-L1, PD-L2 and OX40L. Major histocompatibility complex (MHC) - class I and II expression will also be measured (I-A/I-E, H-2Kb, H-2Db). We will use a selection of murine tumor cell lines representative of the genetic alterations commonly observed in prostate cancer such as the overexpression of the oncogene c-Myc or the loss of tumor suppressor gene Pten. Secondly, the antitumoral immune response is known to be highly driven by intratumoral IFN- levels, which in turn can modulate checkpoint ligand expression. Therefore, we will measure the expression of checkpoint ligands following IFN- treatment across our cell lines. Finally, we will determine whether our prostate cancer cell line models maintain their expression profile of checkpoint ligand molecules when grown in vivo as xenografts in immunocompetent animals.
These data will allow us to determine the molecular basis to the expression of checkpoint ligands in prostate cancer. The overarching objective of this study is, through genetic testing, to identify prostate cancer patients likely to benefit from clinically available immune checkpoint therapies.
Introduction: During treatment, many patients are accompanied and supported by their families, friends and other informal caregivers who care about their health and well-being. Therefore, it is beneficial if patients can share their data with their caregivers easily and securely. To achieve this objective, we decided to expand the functionalities of the Opal patient portal system (opalmedapps.com), which allows patients to access their medical data at the MUHC.
Objective: To design and implement an Opal caregiver module to enable patients to share selected data with their caregivers and enable caregivers to view their patient’s or patients’ data and to educate themselves for better caregiving.
Methods: A prototype caregiver module for Opal, completed in summer 2017, provides patients with control of their data. Patients can choose to give their caregivers full access to certain categories of data or to individually share specific data elements with them. After a thorough investigation of the prototype, we decided that refactoring the code base and a new user interface were necessary in order to make the caregiver module more compatible with the current version of Opal and to improve its performance. The Moqup software suite was used to visualize new designs and to compare their pros and cons.
Results: Building on the prototype, a new caregiver user interface, consisting of a carousel of user/patient icons on the home screen to represent the main user and his/her patients, was implemented. This allows the user to clearly distinguish between the profiles of the caregiver and each patient. When the user slides to a different icon, the color theme of the entire app is changed and all visible contents of the corresponding patient are shown on the screen. Without changing Opal’s layout and functionality, the caregiver module allows patients to easily add or remove their caregivers and fully control each caregiver’s accessibility. It also gives caregivers a clear mechanism to switch between their patients’ profiles and themselves. Furthermore, caregivers may be provided with targeted educational materials and questionnaires to help them gain knowledge about the disease(s) and caregiving of their patient(s).
Discussion: The caregiver module fits well into the existing Opal app and allows patients to share their data with their caregivers. Further features may be implemented to allow patients to notify all their caregivers in the event of an emergency.
Radioembolization is a specialized brachytherapy treatment that works with 90Y filled resin or glass microspheres. Injected through a catheter, microspheres are selectively deposited and permanently lodged within the hepatic arteries to preferentially irradiate hepatic tumors. Multi-disciplinary in nature, radioembolization has numerous treatment steps that include a clinical workup, an angiography, pre-treatment SPECT/CT imaging, the treatment itself, and post-treatment SPECT/CT or PET/CT. The literature contains many improvements on radioembolization’s treatment effectiveness; however, dosimetry remains poor and simple. There is need much improvement. My project aims to develop a Monte Carlo based dose calculation software that will improve upon the current dosimetry. By taking post-treatment SPECT/CT or PET/CT co-registered images, the software will calculate the “true” therapeutic dose of the treatment. The end goal is to provide an easily implementable, accurate, and simple tool to improve on the dosimetric efficacy of radioemoblization.
BACKGROUND: Quality healthcare cannot be delivered with only medicines. Patients need access to early and accurate diagnosis. Forty years after the first Essential Medicines List (EML) was published, the World Health Organization (WHO) released its first “Essential In Vitro Diagnostics List” (EDL) on May 16, 2018, further fostering universal health coverage and providing a supporting scheme for the EML. The EDL stresses the importance of diagnosis within low-and-middle-income countries (LMICs), kick-starting a shift in their healthcare systems from the usual practice of “treat and test” to “test and treat”. A major impact of the EDL will be felt by the diagnostics industry, whose role is pivotal in developing and supplying in vitro diagnostics. So, it is important to engage the industry and get their perspectives on the EDL and how it can be improved.
METHODS: To solicit the opinions of the industry, we anonymously surveyed 27 representatives from 23 different diagnostic companies. An online survey was created to collect data. A total of 35 company representatives were approached; 17 surveys were completed in person during the 2018 Summer Institute hosted by McGill University and 10 surveys via e-mail (total n = 27 respondents).
RESULTS: Most industry representatives (16/27, 96%) were aware the WHO published an EDL and believed it would be beneficial for the diagnostics field. The main positive outcome is that the EDL serves as a compendium and prioritizes diagnostics, a statement 59% (16/27) of the respondents endorsed. Many industry members (23/27, 88%) agreed that omitting specific brand names on the EDL was a good strategy. Others (20/27, 74%) were hopeful that the EDL will improve access to diagnostics as the EML did for medicines. Yet, 59% (16/27) agreed that the EDL might have some negative consequences. Common concerns raised, in decreasing order, include: 1) Price capping (11/27, 40%); 2) EDL revisions not keeping pace with new developments (6/27, 22%); 3) Fear of specific companies’ products not making the list (3/27, 11%).
CONCLUSION: Overall, our survey suggests that the industry welcomes the WHO EDL initiative, which reinforces the importance of diagnostics within universal health coverage. However, industry representatives expressed some concern about price controls as well as the need to keep up with new technological advances. WHO will need to find a mechanism to solicit input from industry stakeholders, since they are key players in developing new technologies and in providing better diagnostics.
RI-MUHC Summer Student Research Day
Title: Innovative model of nursing care in oncology: Feasibility study of an uncertainty management telephone intervention for women attending a rapid diagnostic clinic for a suspicious breast abnormality
Student: Robin Grantner
Supervisor: Christine Maheu, RN, PhD
The time period between receiving news of a suspicious breast abnormality and receiving confirmed results of further testing is one of a great deal of uncertainty and anxiety among women undergoing this experience. The literature shows that anxiety can weaken individual coping skills and worsen quality of life during and after this pre-diagnostic phase. To shorten this phase of uncertainty, some centres have introduced rapid diagnostic clinics (RDCs) to provide testing and results within a shorter time frame. These RDCs effectively shorten women’s waiting time to diagnosis, though it is unknown whether women attending these clinics would still benefit from psychosocial interventions to help manage their anxiety before their results are confirmed. To address this gap in the literature, this study tests the feasibility and acceptability of a telephone intervention given by nurses to women attending a RDC to provide tools for coping with uncertainty and anxiety in the pre-diagnostic phase.
Six women attending the Gattuso Rapid Diagnostic Clinic at Princess Margaret Cancer Centre were recruited for the study. They participated in two psychosocial telephone interventions, one before attending the clinic and one two days after receiving their test results. Their experience will be evaluated based on a questionnaire distributed to each participant before, three days after, and three weeks after testing. The questionnaire will measure their level of anxiety, level of uncertainty, reactions to the stressful event, and coping skills.
The data collected will be analyzed to measure the effect of the telephone interventions on the participants’ psychological state and coping skills in the pre-diagnostic phase. The study will also be evaluated to consider recruitment issues and intervention feasibility considering the short timeframe between participants’ referral to the clinic and reception of results. The final results will be compared with evidence from an updated review of the literature concerning the stress associated with uncertainty around potential breast cancer diagnoses and psychosocial interventions for management of this change in psychological status.
Background: At the Ingram School of Nursing (ISoN), a new model of learning has been launched in the Fall of 2017; Inquiry-Based Learning (IBL). Learning objectives are achieved through the exploration of complex scenarios in groups and individually. Knowing the strengths and characteristics of each generation of nursing students, and by understanding generational diversity, this knowledge can be an effective teaching tool to improve students’ learning experiences during IBL sessions. Several studies highlighted the benefits of IBL for university students (Hugerat & Kortam, 2014; Laursen, Hassi, Kogan, & Weston, 2014). However, few studies describe the experience of first-year nursing students introduced to IBL. Thus, our aim is to describe the experience of first-year nursing students with IBL at McGill University.
Method: This study is a descriptive qualitative design with two focus groups conducted in June 2018 with first-year nursing students (Focus group 1 N=5, Focus group 2 N=3). The qualitative data were subjected to a content analysis using the method proposed by Miles and Huberman.
Results: Participants experienced different phases in accepting IBL during their first year: Resistance (unwillingness to use IBL, lack of motivation, minimum effort); Search for guidance/structure (willingness to do extra work, confusion with learning objectives, overwhelmed and anxiety) and Acceptance (begins making meaningful links, understand learning objectives). At this last stage, students were able to identify some benefits (teamwork, communication, unique learning).
Discussion: The current generation of students, Generation Y, are technically savvy, fast-learning multitaskers and expect immediate access to information electronically, as they have grown up in a world with massive amounts of information being available (Chung & Fitzsimons, 2013; Weingarten, 2009). However, IBL is a self-directed learning which focuses more on the inquiry process than on immediate and concrete outcomes. IBL requires more engagement from the students and has fewer immediate rewards than the traditional learning. IBL is a new approach for both students and professors. It is important to acknowledge that the IBL experience of the professor may impact the transition phases of first-year nursing students.
Conclusion: In light of these results, for first-year students, the goal shouldn’t be the adoption of IBL, but working towards the acceptance of IBL. In addition, efforts should be made to have a progression; meaning slowly transitioning from the traditional approach toward the IBL approach.
Background: Assessment of male infertility has mainly been based on the World Health Organization (WHO) manual-based conventional semen parameters such as sperm concentration, motility and morphology. Limitations with using conventional semen parameters analysis as a diagnostic tool include their high intra-individual variation and weak predictive values in reproductive success or failure. Current literature supports that determination of sperm nuclear DNA integrity, as measured for example by sperm chromatin structure assay (SCSA) and TUNEL assay, can provide improved clinical prognostic values in male fertility status evaluation. Threshold values used to determine an abnormal test are a DNA fragmentation index (DFI) of ≥25% for the SCSA and ≥36% for TUNEL assays. The objective of our study was to evaluate the intra-individual variation of the results of SCSA and TUNEL assays among men presented with a history of infertility.
Methods: Institutional ethics approval was obtained to conduct this study. From January 2012 to June 2016, two semen samples were collected 2-4 months apart from a series of 104 men presented with a clinical history of infertility from a university-based reproductive center. SCSA, TUNEL and Annexin-V binding (for measuring cells demonstrating apoptosis) were performed for all semen samples. The coefficient of variation (CV) of the assays were calculated using the formula (SD/mean) x 100%. We further analyzed the proportion of subjects with variation from the visits that switched their diagnostic category based on their diagnostic thresholds of the assays (25% for SCSA and 36% for TUNEL).
Results: The mean CV was 19.96±15.4% for DFI measured by SCSA, 23.48±18.7% for TUNEL assays and 20.04±18.2% for annexin-V binding. Among the 104 men who had their semen analyzed by SCSA, 15 patients (14.4%) changed their diagnostic category using a cut-off of 25%. In parallel, 18 patients (17.3%) changed category as evaluated by the TUNEL assays using a cut-off of 36%.
Conclusion: In our cohort of infertile men, the CV’s among SCSA, TUNEL assays and Annexin-V binding were similar at about 20%. Interestingly, despite the variations of the assays, over 80% of infertile men remained in the same diagnostic categories from two evaluations. Further studies with a large sample size are required to determine if factors such as age, co-morbidity, conventional semen parameters or etiologies of infertility have an impact on the CV of these assays.
Introduction: The MRN complex (MRE11A, RAD50, and NBS1) has been characterized to play an essential role for genomic stability upstream the homologous recombination (HR) pathway in DNA repair. Germline mutations in these genes, have been previously reported to be in association with the predisposition to hereditary breast and/or ovarian cancer. The recurrence of specific germline mutations in BRCA1, BRCA2, PALB2 and RAD51D, which are cancer predisposing genes involved in the HR pathway, has been reported in the hereditary form of breast and/or ovarian cancer in the French Canadian (FC) population of Quebec due to common ancestors (founder effect).This has had important implications in genetic testing of FCs at risk for these cancers for genetic counselling of risk management. For women of FC descent from Quebec with hereditary ovarian cancer (OC) not due to mutations in BRCA1 and/or BRCA2, the role of germline mutations in MRN genes is still unknown.
Methodology: We used various in silico bioinformatic computational tools that predict potential biologically damaging alleles or identify known pathogenic alleles to analyze a master list of rare genetic variants including MRN complex genes derived by whole-exome sequencing (WES) of the germline of OC cases from 19 FC multi-case OC families and 44 FC women with unusual early-onset OC (88% are FC).
Results: One of 19 women from multi-case OC families harbored a missense mutation in RAD50, while three of 44 women with early onset OC carried two different missense mutations in the same gene, and one in MRE11A. None were found to carry mutations in NBS1. All the missense mutations are present in the heterozygous state, have a reported minor allele frequency < 1% for the general population, and all are classified as variant of uncertain clinical significance (ClinVar). Moreover, the three variants found in cases with early onset OC are predicted to be possibly damaging (PolyPhen, CADD).
Conclusion: We identified four OC cases carrying missense germline variants in genes involved in the MRN complex (RAD50 and MRE11A), but not NBS1 in BRCA1 and BRCA2 mutation-negative women with familial and early-onset OC of FC descent, suggesting these genes might be associated with the hereditary OC warranting further investigation.
Rhizomelic Chondrodysplasia Punctata (RCDP) is a rare autosomal recessive disorder with less than 100 known cases in North America. Children with RCDP suffer from neurological impairments, skeletal dysplasia and severe developmental and growth delays. RCDP is caused by defective biosynthesis of plasmalogens, a unique class of membrane glycerophospholipids representing 20% of total phospholipid mass in the body. Phenotype severity correlates directly with the patients’ level of plasmalogens,
Although RCDP “Primary plasmalogen deficiency “ is a rare disease, secondary plasmalogen deficiency has been documented in patients with more common neurodegenerative disorders, including Alzheimer’s (AD) and Parkinson’s disease (PD). However, research on AD and similar neurodegenerative disorders is inconclusive on the contribution of plasmalogen deficiency to disease pathology. In order to investigate the role of plasmalogen-deficiency in neurodegenerative diseases, we are evaluating the aged RCDP mouse model using behavioral studies and immunohistochemical (IHC) analysis of the brain. We hypothesize that aged, plasmalogen-deficient mice develop evidence of Alzheimer’s more often than wild-type controls. A Y-Maze test was used investigate memory deficit and sagittal sections of brain tissue will be evaluated for amyloid-beta plaques and neurofibrillary tangles of Tau protein, the two primary markers of Alzheimer’s disease and the presence of alpha-synuclein aggregates, a marker of Parkinson’s disease. We also evaluated the number of Purkinje cells, the sole output neuron in the cerebellum which are characteristically degraded in both RCDP and Alzheimer’s patients.
We observed progressive PC loss in aged plasmalogen-deficient mice, with the severity of PC loss directly related to the extent of plasmalogen-deficiency in two strains of severely and moderately plasmalogen-deficient mice. This suggests that plasmalogens may play a critical role in the development and maintenance of PCs and potentially additional neuronal types in other areas of the brain. We will further examine the phenotype of our plasmalogen-deficient mice by investigating number and shape of PCs in the cerebellum, as well as implementing additional IHC methods mentioned previously and continuing behavioral tests using the Y-Maze. Elucidating the relationship between plasmalogen levels and the development of neurodegenerative disease has the potential to improve our understanding of the mechanism of these diseases, and paves the way for the development of drug and gene therapies aiming to rescue plasmalogen levels in patients.
Trafficking of diverse molecules and membrane fusion between vesicle and target membrane are mediated by a set of proteins called SNARE proteins. One such protein is SNAP29, a member of the SNAP (synaptosomal associated proteins) protein family. SNAP29 is encoded by a 5-exon gene located on 22q11.21. Mutations in SNAP29 are responsible for various congenital and infectious diseases. CEDNIK (cerebral dysgenesis, neuropathy, ichthyosis and keratoderma) is a rare congenital syndrome that causes facial dimorphism and developmental failure. CEDNIK is caused by loss of function mutations in SNAP29 which resulted in abnormal epidermal differentiation and ichthyosis. A novel Snap29 mutant mouse line developed in the lab showed skin abnormalities, craniofacial defects and motor defects. Unlike the wild type mice, Snap29 mutant mice were smaller in size with scaly skin. Snap29 mutant mice had reduced grip strength when compared to wild type mice. The goal of this project is to uncover the basis of motor defect in Snap29 mutant mice. Spinal cords will be collected and analyzed by hematoxylin and Eosin. Motor neurons will be stained and counted to determine if they differentiate properly. This study will enable us to uncover the basis of poorly understood motor defects found in human CEDNIK patients and in mouse models with mutations in Snap29.
Tuberous sclerosis complex (TSC) is an inherited tumor syndrome caused by loss of the tumour suppressor genes TSC1 or TSC2 gene, leading in part to constitutive activation of the anabolic kinase ‘mechanistic target of rapamycin’ (mTOR). We and others noted that engineered TSC2-deficient cells, or those obtained from patients, replicate slowly in vitro, perhaps resulting from cellular senescence. Cellular senescence has been described in transformed cells, and is associated with the production of proteins that support the tumour microenvironment (i.e., the senescence-associated secretory phenotype, SASP). We hypothesized that the loss of TSC2 gene leads to cellular senescence and induction of SASP. By Crystal violet assay, we found that cell viability was significantly reduced in TSC2-deficient HEK 293T cells when compared to wild-type controls, and viability could not be restored by the mTOR inhibitor rapamycin. Expression of mRNA for the SASP protein interleukin-6 was increased in TSC2-deficient cells. Therefore, senescence and IL-6 induction appear to result from loss of TSC2, but in mTOR-independent fashion. The molecular mechanisms that drive the SASP in TSC2-deficient cells, as well as the proteins produced during oncogenic senescence, are the subject of current studies, and represent potential therapeutic targets for the control of tumours in patients with tuberous sclerosis complex.
The growth cone of neurons contains an actin cytoskeleton that responds to extracellular cues such as netrin-1 bound to a membrane receptor protein – deleted in colorectal cancer (DCC) - during neuronal development. Complications such as Congenital Mirror Movement, Schizophrenia and Alzheimer’s disease have been associated with mutations of the netrin-1/DCC signaling complex. Compelling evidence has shown that netrin-1/DCC regulates the activity of Rho GTPases to mediate axon outgrowth and guidance. Rho GTPases have been shown to play a critical role in the dynamics of the actin cytoskeleton by operating as molecular switches that alternate between an active GTP-bound and inactive GDP-bound state. The activity of these proteins is regulated by 3 classes of proteins- Guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and GDP-dissociation inhibitors (GDIs). It is well established that netrin-1 stimulation through its receptor DCC induces Rac1 activity through the RacGEF Trio. However, there has yet to be a GAP identified that negatively regulates netrin-1/DCC mediated Rac1 activity in neurons. We intend to investigate chimaerins as candidate Rac1-specific GAPs in netrin-1/DCC-mediated axon outgrowth and guidance. Chimaerins are a unique class of proteins that exclusively display GAP activity for Rac1, and play an essential role in dendritic growth. Previously, a2-chimaerin has been shown to downregulate Rac1 activity downstream of Semaphorin3A/C/PlexinA signaling, however, to date no one has investigated their role in the netrin-1/DCC mediated signaling pathway. We are planning to use a combination of biochemistry and fluorescent microscopy to investigate the possible role of beta2-chimaerin in netrin-1/DCC signaling axis. Using primary rat cortical neurons and HEK293 cells, we will investigate whether beta-2 chimaerin interacts with DCC and if the interaction is netrin-1 inducible. In addition, we will also verify possible changes in the subcellular localization of beta2-chimaerin upon netrin-1 stimulation.
Purpose: Waiting room management software that integrate self-service check-in kiosks have proven to be beneficial for both patients and healthcare providers. At the Cedars Cancer Centre (CCC), the Online Room Management System (ORMS) is used to manage patient check-in and to call patients for their appointments. ORMS frees up reception desks for all but complicated check-ins. The goal of this work was to develop and put in place a software that allows system administrators to visualize both retrospective and current kiosk activity.
Methods: In order to visualize the current and retrospective status of kiosks installed in the CCC, we designed and implemented a web user interface that dynamically interacts with the data logged by the kiosks and displays them using the HighCharts graphing package. During our development we presented mockups and prototypes to the software users. Based on their suggestions, we improved the clarity of kiosk visualization and introduced new features that allow healthcare administrators to observe an hourly trend of kiosk activity within a date range.
Results: Our application allows healthcare administrators to visualize current and retrospective kiosk activity. The application also supports other novel features, such as visualization of frequency of successful and unsuccessful check-ins per hour, as well as abnormal kiosk activity.
Conclusion: The kiosk visualization application is fully operational, and it is currently running on the MUHC clinical network.
Keywords: current source density (CSD), cat, visual cortex, electrophysiology, laminae analysis
The laminar structure is conserved across different areas of cerebral cortex even though the depth of each layer varies across functional areas. There are 6 layers characterized by the distribution of certain types of neurons, and input and output connections. In general, layer 4 and layer 6 act as the sinks of gross transmembrane currents while layer 2, 3 and 5 are extracellular sources. The underlying physiological mechanism is the set of pathways that relay neural activity between different layers by local and long-distance connections. In our experiment, a linear array 32-channel electrode was inserted perpendicularly to the surface of primary visual cortex, in an anesthetized cat. Brief flash stimuli were delivered with both eyes open and we recorded the local field potentials (LFP) generated by the resulting neuronal population activity. Data analysis for current source density (CSD) was done with custom python code using the CSDplotter matlab toolbox by Pettersen et. 2006. The results showed a pattern of sources and sinks consistent with the expected laminar structure in both area 17 and 18. In conclusion, CSD can provide a method to identify cortical laminae for other neurophysiological responses, such as population receptive fields and patterns of LFP covariance.
MammoMC aims to serve as a dose estimation tool for mammography imaging, confirming the accuracy of the approximative methods currently used to estimate Mean Glandular Dose (MGD). MammoMC is a software package built on the Geant4 Monte Carlo simulation toolkit: it is highly flexible and user-friendly, featuring a Qt based Graphical User Interface (GUI) as well as built-in commands for altering the parameters of the simulation, the geometry, and the recording of data. MammoMC simulates the two main parts of a mammography; A) simulation of the x-ray tube with all its components to generate the x-ray spectra, B) sampling from the obtained x-ray spectra to simulate the dose to the patient. The software is accurate and fast, featuring both the ability to load pre-calculated spectra and employ all threads of a multi-core computer or clusters of computers during simulations. In addition to a three dimensional calculation of the dose delivered to patients during imaging, MammoMC also serves as a teaching tool in the fields of imaging and Monte Carlo simulations, and may be useful in the industry for design and development of x-ray tubes. MammoMC was benchmarked against quality assurance measurements taken from mammography machines at the MUHC.
Sleep-disordered breathing (SDB), which includes sleep apnea, is an under-recognized, but highly prevalent sleep disorder in our society. Emerging data suggests that SDB increases in prevalence throughout pregnancy, occurring in 17-45% of women by the third trimester. Maternal SDB has also been found to be associated with adverse maternal-fetal complications, including an increased risk of gestational diabetes (Pamidi et al. Am J Obstet Gynecol 2014). However, it is unknown whether SDB predisposes pregnant women with GDM to worse glucose control. This project investigates the relationship between glucose profiles of pregnant women with GDM and objective SDB measurements using continuous glucose monitoring (CGM), a novel method for measuring continuous 72-hour glucose levels. CGM is emerging as a more representative reflection of glucose control providing useful glucose indices, such as mean nocturnal and daytime glucose levels, measures of glycemic variability and time spent in hyperglycemia.
This cross-sectional analysis of 72-hour glucose profiles and severity of SDB consisted of pregnant women between 24-34 weeks gestational age with diagnosed GDM (WHO 2013 criteria) and assessment of SDB using level 2 at-home sleep recordings. Glucose profiles were obtained from a CGM device (Medtronic). CGM measures interstitial glucose levels every 5 minutes across a consecutive 72-hour period after subcutaneous insertion. The glucose results were blinded from the participant. Unadjusted linear regression modelling was used to determine the impact of the apnea-hypopnea index (AHI) as a measure of SDB severity on mean glucose levels, sleeping glucose levels and glycemic variability over three consecutive days.
A total of 57 participants with GDM were tested for SDB. Thirty-seven (65%) had SDB and 20 (35%) did not have SDB (using AHI cut-off of 10 events/hr). The BMI was significantly higher in the SDB group when compared to the no SDB group (p<0.00). Snoring was significantly greater in the SDB group (p=0.03). The mean AHI in the SDB group was 19.6, indicating moderate severity of SDB. The SDB group was also more likely to be on insulin or metformin for glucose control at baseline (37%; n=14). There was a significant relationship between worsening nocturnal maternal glucose levels and severity of SDB using AHI (p=0.04). No significant relationship was observed between overall mean glucose and glucose variability measurements and AHI.
Untreated SDB is related with increases in nighttime glucose levels in pregnant women with SDB and GDM, a population at an increased risk for cardiometabolic complications and poorer maternal-fetal outcomes.
Figure 1. Univariate linear regression of maternal sleeping glucose levels and AHI in pregnant women with SDB and GDM.
Meta-analysis is a statistical method for combining data from several scientific studies addressing a common research question. We consider the problem of meta-analyzing two-group studies that report the median of the outcome. Often, these studies are excluded from meta-analysis because there are no well-established statistical methods to pool the difference of medians. To include these studies in meta-analysis, several authors have recently proposed methods to estimate the sample mean and standard deviation from the median, sample size, and several commonly reported measures of spread. Researchers frequently apply these methods to estimate the difference of means and its variance for each primary study and pool the difference of means using inverse variance weighting. In this work, we develop several methods to directly meta-analyze the difference of medians. We conduct a simulation study evaluating the performance of the proposed median-based methods and the competing transformation-based methods. The simulation results show that the median-based methods outperform the transformation-based methods when meta-analyzing studies that report the median of the outcome, especially when the outcome is skewed. Moreover, we illustrate the various methods on a real-life data set.
Waiting time is one of the most important factors affecting healthcare quality because it has a significant impact on patients’ overall experience and affects their perceptions of quality and satisfaction. Although healthcare institutions strive to reduce waiting times, in reality doing so is difficult to achieve. One way of possibly reducing the pain of waiting, without actually reducing waiting themselves, is to provide patients with realistic estimates of how long they should expect to wait. This allows patients to better plan around their visits to the hospital and gives them the confidence to move around without the fear of missing their call to be seen by the clinician. In this work, we present an experimental approach to predict minute-by-minute how long patients should expect to wait until the start of the their radiotherapy treatments at the MUHC.
Timestamp data were extracted from the database of the waiting room management software used in the Cedars Cancer Centre (CCC) for each weekday from January 1st to June 30th, for both years 2017 and 2018. Using these historical data, we reconstructed the minute-by-minute state of the radiotherapy clinic, and we calculated remaining waiting times for radiotherapy treatment appointments from every minute of each weekday in the data sample. We then synthesized features from the raw data such as past durations of patients’ treatments, queue occupancy relative to the current minute, and so on. Then, the final data set was split into a test set (n = 29,990 unique clinic snapshots) and a training set (n = 119,960 unique clinic snapshots), and we trained several types of regression models, such as random forest, extremely randomized trees, gradient boosted regression, and extreme gradient boosted regression on the training set.
The extreme gradient boosted regression demonstrated the best performance for minute-by-minute waiting time predictions with a mean absolute deviation of 6.1 minutes and a median absolute deviation of 4.2 minutes. 83% of minute-by-minute waiting times were predicted within 10 minutes, 13% within 20 minutes, and 4% otherwise. When waiting times are less than an hour (n = 17,483 in the test set), the model predicted a mean absolute deviation of 4.6 minutes and a mean deviation of -1.2 minute.
The extreme gradient boosted regression algorithm is able to predict minute-by-minute waiting times with high accuracy. It demonstrates a slightly better performance when waiting times are shorter.
Aim: To conduct an umbrella review of the measurement properties (i.e., content and construct validity, reliability, responsiveness) and the quality of the evidence of mobility measures classified by type of measure, including self-reported outcomes (SRO), patient-reported outcomes (PRO), performance/ clinician-reported outcomes (PerfRO), and technology reported outcomes (TechO) among adults with ABI.
Methods: An Electronic search was performed in MEDLINE, CINAHL, Cochrane and EMBASE followed by hand searches of the reference lists of included reviews. Eligibility criteria were systematic reviews of measurement properties of mobility measures among adults with ABI. Mesh terms included brain injury, mobility, outcome assessment, psychometrics and rehabilitation. The 10 steps Consensus-based Standards for the Selection of Health Measurement Instrument (COSMIN) guideline for systematic reviews was used. The Mobility and ICF frameworks were used to guide evaluation of the conceptual framework and domains assessed. Two independent reviewers screened and retrieved titles, abstracts, and full texts, according to the inclusion criteria and extracted the data. A narrative synthesis of the included studies will be conducted.
Preliminary results: The search resulted in 1290 reviews, and after initial selection based on title and abstract, 80 were assessed for eligibility based on full-text, resulting in 43 systematic reviews. In total 293 mobility outcome measures were identified: 51 PROs/SROs, 198 PerfROs, and 44 TechOs. Results to date indicate reliability estimates of PROs/SROs (ICC: 0.55-0.89); PerfROs (ICC:0.45-0.98); TechOs (ICC: 0.44-0.97)). Estimates for construct validity were PROs/SROs (r: 0.40-0.91); PerfROs (r: 0.43-0.96); TechOs (r: 0.35-0.90)). Responsiveness was assessed in 26 reviews (PROs/SROs: 28%; PerfROs: 62%; TechOs: 10%).
Discussion: This is the first review to examine the properties of different types of mobility measures used among the ABI population. Preliminary results support the reliability and validity of measures. The results of this review will be used to provide clinicians and researchers recommendations for selection of mobility measures to support clinical decision making and individualized treatment protocols for mobility retraining.
Introduction: The Opal patient portal is a mobile app that allows cancer patients to view their medical data and enables clinicians to send them educational material and questionnaires (opalmedapps.com). The current prototype of the Opal app is based at the MUHC. However, cancer patients often receive treatment at multiple hospitals. Our objective is to expand Opal to connect to multiple hospitals simultaneously, allowing patients to access all of their medical data via a single login. Six hospitals in the Montreal area are participating in this project, with funding from the Canadian Partnership Against Cancer.
Methods: The existing versions of the Opal backend system and frontend app were studied to determine which changes needed to be made to implement the system in multiple hospitals and connect each patient's data to a single user account. The data storage system, the process by which the app makes requests for user data and receives responses, and the user registration system were revised after comparing the pros and cons of various design options.
Results: A model for a multi-institutional version of Opal was designed, and a prototype was partially implemented, as an expansion of the current system. The new data storage system involves replicating the existing Opal database and associated backend software identically in each connected hospital, with an additional new shared database and backend software to centrally store user account data. The multi-institutional method for sending requests for data involves adding an index of registered hospitals for every user and targeting app requests to each hospital at which a user is registered. User registration for the multi-institutional system requires users to be registered into the new shared database. A mockup was developed to demonstrate how patients' data can be displayed all together in the frontend app with identifying information indicating the source hospital for each data element. To make this possible, the backend software which sends hospital data to the app must tag the data with its source.
Discussion: This new design for a multi-institutional version of Opal reuses much of the existing system, but involves modifications at each of its levels (database(s), backend software(s), registration system, frontend application). The development of a prototype implementing the new design is partially completed. This proof-of-concept prototype will be used to guide the development of a production version which will be deployed at many institutions in Quebec.
Purpose: Incident reporting and learning improves the quality of radiotherapy by reducing the recurrence of unintended incidents and accidents. Natural language processing (NLP) is a proven technique for extracting information from clinical texts and classifying aviation incident reports, but the utility of NLP for incident learning in radiotherapy is untested. This project applies supervised learning to automate the classification of incident reports by the process step of incident occurrence, and unsupervised learning to cluster similar reports to discern common problem types or contributing factors, using a technique known as topic modelling.
Method: A preliminary analysis that comprised tagging of parts-of-speech and annotation with the Unified Medical Language System was conducted on 3163 incident reports. These reports were then cleaned, filtered by custom functions premised on the results of the preliminary analysis, and vectorized into a matrix of term frequency-inverse document frequency (TF-IDF) features. Multinomial classification by the process step of incident occurrence was transformed into multiple binomial classifications through both the one-vs-all (OVA) and one-vs-one (OVO) strategies. Twenty five regression models as well as a long short-term memory (LSTM) recurrent neural network were cross-validated, and the best performing models were assembled. For topic modelling, the pairwise cosine similarities of the TF-IDF vectors were calculated and then clustered using Ward's method.
Results: The best performing OVA models were Gaussian process regression (GPR) and multilayer-perceptron regression (MPR), and the best performing OVO model was logistic regression, all achieving a correct-classification accuracy of 74%. The accuracy of the LSTM model was 75%, and that of the meta-assembling of GPR, MPR, gradient boosting regression, and extra-trees regression was 80%. For topic modelling, the relationships between all incidents were plotted in a dendrogram, exhibiting promising clusters of problem types and contributing factors upon preliminary inspection.
Uveal melanoma (UM) is the most common ocular tumor in adults and despite effective local treatment, overall outcome has not improved. Moreover, once metastases occur, the outcome is poor. Consequently, detection of novel biomarkers has the potential to aid the development of therapeutic options and is useful to better understand disease progression. Nestin is a neural stem cell marker previously found to be associated with reduced survival in cutaneous melanoma. The purpose of this study is to characterize Nestin expression in UM and its utility as a prognostic marker.
In total, 46 UM enucleated eyes were examined, 24 of which had corresponding survival data. One sample was removed due to complete tumor necrosis. Slides were retrieved from the MUHC-McGill University Ocular Pathology Laboratory. Fully-automated immunohistochemistry using an anti-Nestin polyclonal antibody was performed. Tumors were classified according to cell type (spindle or epithelioid). Slides were graded based on intensity (0=negative, 1=positive, 2=strong positive) and extent of staining (focal=1, diffuse=2). A score was obtained for each tumor by adding extension and intensity grades. Statistical analysis was performed using Mann-Whitney test to assess group differences according to cell type, presence of metastases and metastasis-free survival.
Immunohistochemical staining of ciliary body was used as an internal control. All 45 tumors were positive for Nestin. Staining intensity was not correlated with survival (P=0.523), and staining extent did not correlate with cell type (P=0.224). Within a given tumor, the intensity of Nestin staining was always higher (2) in epithelioid cells compared to the spindle cells (1).
To the best of our knowledge, this is the first study characterizing Nestin expression in UM. Due to the presence of Nestin in all cases, it should be included as part of the immunohistochemical panel for UM diagnosis. Although no correlation between Nestin staining and survival was found in our cohort, the increased positivity in epithelioid cells follows the same pattern as seen in cutaneous melanoma in which increased expression correlated with poor prognosis. Further studies with a larger patient cohort is necessary to confirm our results.
Introduction: Upon being diagnosed with cancer by a clinician, patients move through different parts of Quebec’s public healthcare system without convenient access to their medical data. Our goal is to increase patient involvement and break down the patient-clinician divide through the implementation of a patient portal called Opal (opalmedapps.com). Said portal was created using a participatory stakeholder co-design methodology within the McGill University Health Centre. This design methodology ensured that all stakeholders in the final product were fully and actively engaged in its development. Currently, Opal is in the pilot release phase, which consists of gradually increasing the number of patients using the application to access their appointment schedules and some of their personal health information (PHI) in radiation oncology.
Objective: The goal of this summer research project was to continue the use of stakeholder co-design into the pilot release phase of Opal.
Methods: Patients and staff continue to contribute throughout the pilot stage by providing feedback that the Opal Health Informatics Group will use to improve subsequent Opal updates. In addition, we trained the Cedars Cancer Foundation staff to register patients for Opal with the use of newly-developed documentation, and we observed the registration process as patients signed up. Our observations, as well as the information provided back to us by patients, clinicians, and staff, will allow us to continuously assess and improve the registration workflow.
Results: As a result of patients participating in multiple surveys, we were able to ascertain that 63.8% of patients prefer to have access to all of their PHI immediately once they are available. Of the remaining patients, 23.3% prefer to have access to all their PHI after it had been reviewed with them by their clinician, thus leaving 12.9% of patients who prefer to only receive need-to-know information (ie. their appointment schedule and educational material).
From our initial experience registering patients, we have observed that all patients wish to access all of their medical data as soon as they are available. We also observed that the registration process is too detailed and complex to be used in the full Opal release. As a result, it will need to be fine-tuned and streamlined. However, all patients that have participated in the Opal release to date have reported a positive experience
Discussion: The use of participatory stakeholder co-design throughout the development of Opal allows for the integration of valuable input from both patients and healthcare professionals.
The primary cilium is a fundamental component of vertebrate cells that has been linked to many cellular pathways, one of which is the planar cell polarity (PCP) pathway. It therefore follows that the loss of these microtubule-based projections is correlated with many developmental diseases, one of which is glomerular cystic kidney disease (GCKD). Our preliminary data showed that mutant mice of the PCP effector protein Fuzzy developed GCKD. Functionally, Fuzzy regulates the actin cytoskeleton in Drosophila and seems to be crucial in the development of primary cilia in mammals. However, the functional relationship between Fuzzy and ciliogenesis is not well understood. Previously, it was found that mice who developed GCKD also showed hyperactivity of RhoA. As such, we hypothesize that GCKD in Fuzzy mutant mice may also be attributed to a heightened level of RhoA activity. To test this hypothesis, mouse embryonic fibroblasts (MEF), that were established from wild type (MEF+/+) and Fuzzy mutant mice (MEF-/-), were treated with RhoA inhibitors (ROCK inhibitors). Our data suggest that under normal physiological conditions, MEF-/- cells do not express primary cilia, unlike their wild type counterparts, and their directional cell movement is greatly reduced in comparison to the MEF+/+ variety. Conversely, upon treatment with ROCK inhibitors, Fuzzy mutant MEFs develop cilia and their directional cell movement is partially rescued. Therefore, we conclude that Fuzzy is implicated in both i) ciliogenesis and; ii) directional cell movement, through its control of RhoA activity. Based on these observations, reduction of heightened RhoA activity may represent a therapeutic target for treatment of GCKD.
Background: Defective planar cell polarity (PCP) was postulated to cause cyst formation in polycystic kidney disease (PKD), although the mechanisms that underlie the disease remain unclear. PCP signaling controls the mechanisms of convergent extension (CE, directional cell movements) and oriented cell division (OCD, directional cell divisions during post-natal tubular elongation). In embryonic mice, core PCP gene Van Gogh-like 2 (Vangl2) defects lead to abnormal renal tubules, due to failed CE.
Methods: The focus of the project was to determine whether abnormal PCP in post-natal tissues resulted in tubular dilatation and cyst formation. For this purpose, 1-day postnatal (P1) and 1-month postnatal (P30) mice with a conditional excision of Vangl2 in renal collecting duct tubules were generated, along with the controls. Immunostaining, fluorescence microscopy and image analysis were employed for tissue evaluation.
Results: Our results demonstrate that overall, conditional mutant P1 kidneys had no statistical differences in i) the diameter of the renal tubules; ii) the cross-sectional area; and iii) the number of cells within the collecting duct cross-section. Yet, in a small subset of tubules, visible dilatation was present in the conditional mutant mouse models. However, a normal phenotype, free of cysts and dilatation, was restored by P30, in both conditional mutant and control 1-month kidneys. While our previous results obtained in Vangl2 mutant embryonic kidneys demonstrate significant tubular dilatation in conditional mice compared to the controls, the loss of Vangl2 is not sufficient to cause cyst formation post-natally.
Conclusion: We conclude that while the PCP pathway is required for normal tubular development and control of tubule diameter during embryogenesis, its role post-natally appears to taper off. Therefore, we hypothesize that a switch occurs between the PCP-controlled embryonic renal tubular development and the mechanisms that govern tubular maintenance post-natally. In fact, the disruption of core Vangl2 gene appears to be insufficient for cyst formation, and the mechanism underlying the cystic phenotype may involve defects in additional pathways.
Resting between both neurons and the brain’s microvasculature, astrocytes are non-neural cells of the central nervous system that are essential for brain metabolism and homeostasis. In particular, they have specialized processes called “endfeet” that wrap around cerebral blood vessels, where they actively modulate the interaction between the brain and the microvasculature. Through the regulation water, oxygen and glucose exchange, astrocytic endfeet are vital in ensuring that the brain is well oxygenated and receives enough nutrient supply to meet the high metabolic needs of neurons.
During neuronal firing, local increases in cerebral blood flow are matched to oxygen and glucose consumption by active neurons in a process termed neurovascular coupling (NVC). Astrocytes are not electrically excitable cells, but they respond to both neuronal and vascular activity with transient rises in intracellular calcium (Ca2+). At the endfeet, varying levels of intracellular Ca2+ have been shown to be correlated with dilation and constriction of cerebral blood vessels, yet the source and function of this Ca2+ is unknown. The role of endfoot Ca2+ in NVC is an active area of research given that cerebrovascular tone and flow are potent indicators of health and activity in the brain.
Using a combination of microscopy imaging techniques, we sought to resolve the 3D ultrastructure of the endfeet compartment and its subcellular components, such as mitochondria and endoplasmic reticulum (ER), in order to better understand endfoot Ca2+ signals. The 3D segmentation and reconstruction of astrocytic organelles from serial electron micrographs (EM) indicate that mitochondria are thicker at the endfeet while ER forms cistern-like sheets that are not present in other astrocytic processes. In addition, we quantified mitochondria-ER contact sites and found that they are larger at the endfeet. These contact sites are known to be hosts of a variety of signaling functions, which include lipid synthesis, protein trafficking, and, most importantly, Ca2+ signaling. Accordingly, we performed immunohistochemistry against ER-resident protein GRP78 on tissue sections from mouse brains, in which astrocytes are co-expressing different fluorescent proteins targeted to mitochondria and the cell membrane. Images acquired using super-resolution microscopy show that GRP78 forms a ring-like structure in close apposition with mitochondria in the endfeet, thus supporting our EM observations.
Together, our results suggest a special arrangement of mitochondria and ER in the endfeet that gives insight on the mechanisms underlying endfoot Ca2+ signals. Going forward, Ca2+ imaging and further immunohistochemistry will help us understand the signaling dynamics at the endfeet.
Background: With an increasingly competitive job market, the Desjardins Centre for Advanced Training of the RI-MUHC helps trainees explore their career options. Based on the success of past Career Half-Days (held in 2016 and 2017) and the demand of trainees to maintain communication with professionals in industry and academia, D-CAT wanted to design a mentorship program for the RI-MUHC.
Project objective: Our objective was to create opportunities for trainees to explore their occupational options and to get advice for their career development through the development of a mentorship program. The program would be intended to help trainees recognize and develop skills needed for success and to increase their commitment and network in their area of interest.
Methods: We explored literature on mentorship, mentorship programs existing at other universities and research institutes and consulted with McGill University Career Planning Service.
Project outcome: We have developed the foundations of a mentorship program that will see both mentors and mentees receive informational pamphlets about the RI-MUHC Mentorship Program, the benefits and responsibilities of mentoring and application forms. Professionals who participated to past Career Half-Days will be invited to serve as mentors. Based on the applications and mentors available, trainees will be matched based on compatible interests. The match will be sent into McGill’s Mentorship Program and a four-month unstructured mentorship period will begin. Both mentees and mentors will receive guides for effective mentoring, trainees will participate in a training session and both will complete a mid-way evaluation. Mentoring evaluation forms will be sent to mentors and mentees at the end of the relationship to evaluate the program’s effectiveness and get feedback on which components of the program could be improved.
The Ste20-like protein kinase, SLK, is an ubiquitously expressed kinase in mammals that has well-established roles in mediating apoptosis, the cell cycle and cytoskeletal dynamics, in addition to being essential for embryonic development. In particular, SLK is involved in kidney development, and is prominently expressed in glomerular epithelial cells (GECs). Deletion of SLK in GECs in mice leads to GEC injury and albuminuria. A recent study has shown that SLK may be involved in the maintenance of healthy mitochondria in mouse skeletal muscle. To determine if SLK has a similar role in GECs, we used fluorescence microscopy to study mitochondria in mouse GECs. Wild-type (WT) and SLK knock out (KO) GECs were both transfected with mitochondrially-targeted yellow fluorescent protein (Mito-YFP) and stained with MitoTracker Red CMXRos (a probe that measures mitochondrial membrane potential) to observe and compute any differences in mitochondrial function and distribution patterns. Functional mitochondria were evident diffusely throughout WT GECs, but in the SLK KO GECs, function was preferentially limited to mitochondria in a perinuclear distribution, akin to the effects of the cellular respiration inhibitor, antimycin A in WT GECs. In contrast, mitochondrial structural distribution was diffuse in both WT and KO cells. Additionally, measurements of colocalization between the patterns of mitochondrial function and structure indicated that SLK KO GECs had less functional mitochondria than WT GECs. Future research will examine differences in cellular energy markers between the two cell types, such as the phosphorylation state of AMP-activated protein kinase, to further establish the role of SLK in the maintenance of proper energy levels in GECs.
Hydatidiform mole is a form of gestational trophoblastic disease (GTD) defined as an abnormal human pregnancy characterized by absence of, or less grown, embryonic structures and hyperproliferation of trophoblast. Recurrent hydatidiform mole (RHM) is a rare genetic disorder defined by the occurrence of repeated molar pregnancies in affected women. Two genes, NLRP7 and KHDC3L, play a causal role in RHM and are responsible for 55-80% and 5% of cases, respectively. Our lab performed whole exome sequencing on 65 patients with recurrent hydatidiform moles (≥ 2 hydatidiform moles) and without mutations in two known genes. Bi-allelic mutations in three genes, MEI1, C11orf80 and REC114, were identified in 5 unrelated patients and the three genes have known roles in the formation of meiotic double strand breaks. Interestingly, the brother of one patient was found to be infertile, had been diagnosed with non-obstructive azoospermia, and has bi-allelic MEI1 mutations. Furthermore, some patients and/or their affected sisters with bi-allelic mutations in the identified genes had also miscarriages and infertility, in addition to their RHM. This suggests that MEI1, C11orf80, REC114 are responsible not only for RHM but also for recurrent miscarriages and male and female infertility. The goal of my project is to search for pathogenic variants in these three genes in males with azoospermia. Methodology in the lab involves target sequencing of the three genes in a cohort of 300 infertile azoospermic males, bioinformatics analysis and validation of variants by sanger sequencing. The objective of the study is to determine the frequency of pathogenic variants in these three genes in infertile males.
Background: Glioblastoma Multiforme (GBM) remains a form of brain cancer with poor prognosis. Its hallmarks include vascular abnormalities like florid angiogenesis and systemic venous thromboembolism (VTE), which remains poorly understood. Our laboratory established a link between coagulant phenotype of GBM cells and mutations that drive this disease, including Epidermal Growth Factor Receptor (EGFR) amplification and EGFR variant III (EGFRvIII)1,2. While EGFRvIII modulates different effectors of the haemostatic system, namely tissue factor (TF) which triggers the coagulation cascade, and podoplanin (PDPN) which activates platelets, their role in VTE is confounded by the heterogeneous expression of EGFRvIII, TF and PDPN between and within individual lesions. This gap in knowledge often leads to treating GBM-associated coagulopathy as a uniform and ‘unspecific’ aftermath of tumourigenesis.
Purpose: The objective was to elucidate potential links between genes involved in coagulopathy (termed the "coagulome"), particularly PDPN and TF, and the EGFR oncogene.
Hypothesis: We surmised that cancer cell heterogeneity in expression of molecular drivers may lead to heterogeneity (but not randomness) of their coagulant phenotypes where VTE would result not from the preponderance of one particular molecule but represent a combinatorial input of individual cell subpopulations.
Methods: Single-cell RNA-Seq data is obtained from online databases and gene expression level is assessed using algorithms. The counts were plotted in a heatmap to visualize the expression architecture of the coagulome across tumour cell populations.
Results: Heatmaps indicate that PDPN expression was strongly in contrast with EGFR expression in certain tumours. Notably, within a tumour with generally low levels of both TF and PDPN, there were individual cells with higher expression of these effectors. This is consistent with the notion of GBM subtypes themselves being composits of cells with different phenotypes assembled in different proportions.
Conclusion: The results suggest that GBM can potentially trigger clot formation through a composit mechanism encompassing different effectors (TF and PDPN). This has implications for mechanistic understanding and management of GBM-related coagulopathy given the distinct nature of these pathways (coagulation cascade vs. platelet activation).
1.Magnus N., Garnier D., Rak J. Oncogenic epidermal growth factor receptor up-regulates multiple elements of the tissue factor signaling pathway in human glioma cells. Blood. 2010;116(5):815-18
2.Magnus N., Gerges N., Jabado N., Rak J. Coagulation-related gene expression profile in glioblastoma is defined by molecular disease subtype. J Thromb Haemost. 2013;11(6):1197-200
Introduction: Birth asphyxia and the resulting neonatal encephalopathy still cause significant mortality and long-term morbidities, including cerebral palsy and blindness in term newborns. Therapeutic hypothermia is currently the only available treatment that may prevent brain injuries after birth asphyxia, but its success is limited. Therefore, the development of treatments to repair these brain and retinal injuries and improve long-term outcomes of these newborns is of utmost importance. We have shown in a rat model of term neonatal HIE that sildenafil (Viagra®), when administered after hypoxia-ischemia (HI), improves retinal injuries, with an increase in the number of retinal ganglion cells and bipolar cells in the retina. However, the effects of sildenafil on other retinal cells has not yet been investigated. We are interested in studying amacrine and horizontal cells because they interact with other retinal cells that were affected by HI and sildenafil.
Objective: To investigate the impact of neonatal HI and sildenafil on amacrine cells and horizontal cells in the retina.
Methods: We used a rat model of term neonatal HIE (the Vannucci model), combining a unilateral common carotid artery ligation and a 2-hour exposure to hypoxia (i.e., 8% oxygen) in 10-day-old rat pups since this model is recognized to mimic the brain injuries observed in human term asphyxiated newborns. We randomly administered sildenafil or vehicle 50 mg/kg orally twice a day starting 12 hours after HI and continued the treatment for 7 days. Thus, 3 different experimental groups were used: i.e., sham animals treated with vehicle, HI rat pups treated with vehicle, and HI rat pups treated with sildenafil. The animals were sacrificed at P30, and retinas were extracted and stained with calbindin (a marker of amacrine cells) and calretinin (a marker of horizontal cells).
Results: HI decreased the number of both amacrine and horizontal cells in the retina. Treatment with sildenafil (especially a higher dose of 50mg/kg) reverted the amacrine and horizontal cell number back to sham levels. The amacrine cells were equally damaged throughout the retina while the horizontal cells were less damaged in the periphery compared to the central and mid-peripheral regions.
Conclusion: These results provide us with a more comprehensive view of the impact of HI and sildenafil on retinal cells. Both amacrine and horizontal cells in the retina are affected following neonatal HI, and treatment with sildenafil corrects this impairment.
For several years, bone anchored hearing implant (BAHI) surgeons and researchers have
searched for objective and non-invasive ways to determine implant stability and monitor
the osseointegration process. Although histomorphometric evaluation and mechanical
testing provide valuable information about the implant anchorage in the peri-implant
bone, these tools cannot be applied in the clinical setting. The advent of an implant
stability measure could greatly aid the clinical decision of when to couple the sound
processor to the percutaneous BAHI abutment. In 2004, a consensus was reached, solely
based on surgical experience, stating that a 6-week window should be allocated prior to
coupling the processor to allow sufficient bone-implant osseointegration. However, this
osseointegration is likely dependent on specific implant characteristics, host bone quality,
and bone healing and remodeling abilities. Delaying the sound processor coupling could
have important social and developmental consequences particularly in hearing-impaired
children as it lengthens the period of auditory deprivation. Recently, the use of
Resonance Frequency Analysis (RFA) has been suggested as a noninvasive method to
evaluate the in-vivo stability of osseointegrated BAHIs. We performed a prospective case
series in 29 BAHI recipients to assess if RFA could serve as a predictor for optimal sound
processor coupling time based on peri-operative stability trends over time at our implant
center. The progression of RFA measurements suggest that adults (n = 16) and pediatric
(n = 13) BAHI recipients have different implant stability trends. The results indicate that the
sound processor could be coupled as early as the skin around abutment has healed for
adults, but a 6-week period should be allowed for optimal primary stability in pediatric
patients. Due to its non-invasive nature and clinical value assessing stability, RFA
measurements could be added to BAHI surgery and follow up assessments. Although
initial results are promising, a correlative bio-structural assessment relating to the integrity of the bone-implant interface and the RFA is warranted.
Melanoma is a life-threatening cancer derived from melanocytes with increasing incidence rates. Ocular melanoma (OM) is the leading primary intraocular malignancy in adults and nearly half of patients develop hematogenous dissemination to the liver. Cutaneous melanoma (CM) is the third most common skin cancer, with higher risk of lymphatic involvement if tumor thickness exceeds 0.8mm. The occurrence of both OM and CM in the same individual is rare. In this study we aimed to characterize the epidemiology and clinical features of patients who present with both OM and CM.
A chart review was performed including all patients with histopathologically confirmed OM at the McGill Ocular Pathology Laboratory from 2006 to 2016. Patients who were also diagnosed with CM were included in this study. Demographics, tumor characteristics and disease progression were analyzed.
Among the 61 patients with OM diagnosis, six (9,8%) fulfilled the inclusion criteria. There was a female predominance (n=5/6) and the average age at first diagnosis was 71 years. The interval between diagnoses ranged between 1-15 years. Two patients had mixed type choroidal OM with lymphocytic infiltration and 3-6 mitosis/mm2 as their initial tumor. Both patients developed metastases after enucleation; one had involvement of multiple organs including the skin after 3 years, while the other had skin and liver metastases after 4 years. Four patients first presented with CM; two had rapidly growing epithelioid ocular metastases with concomitant pulmonary lesions after 1-3.5 years. The other 2 patients had no metastases. In both of these patients, a slow growing spindle OM was diagnosed: 7 years after the CM diagnosis in the choroid in one patient, and after 15 years on the lower lid in the other, thus suggestive of new primary tumors. This latter patient had multiple primary CMs, a diffuse large B-cell lymphoma and a renal cell carcinoma in addition to a family history of melanoma. Genetic testing was performed with no evidence of mutations in genes related to familial melanoma.
Patients with OM have an increased risk of developing CM or metastatic disease, especially females. Therefore, multidisciplinary follow-up and systemic workup are recommended for early diagnosis and to confirm the nature of tumor (primary vs. metastatic).
Background: Spontaneous Coronary Artery Dissection (SCAD) is a condition which occurs when a blood vessel leading to the heart forms a tear. This tear can interrupt blood flow to the heart and lead to acute coronary syndrome (ACS). The risk factors associated with SCAD are known to be different from most ACS cases. It most commonly affects women between the ages of 40 and 60. Men are more often affected by atherosclerosis-related SCAD as opposed to the non-atherosclerotic form. Thus, the etiology of SCAD in women may differ from men. Several familial cases have been identified, but more prevalent sporadic cases are thought to originate from de novo genetic variants or combinations of variants. We hypothesize that there is, at least, a partial genetic etiology for SCAD in sporadic cases responsible for the distinction between SCAD and other ACS cases.
Methods: We performed an analysis of risk factor heterogeneity between SCAD patients and other ACS patients in the Premature Acute Coronary Syndrome (PRAXY) cohort (n=863). In addition, 83,665 genetic variants were tested for an association with SCAD in the subset of PRAXY consisting of European-ancestry individuals. Two sets of analyses were performed, one containing all 563 individuals, and one containing only women (n=159).
Results: Risk factor analysis confirmed that those with SCAD had a lower prevalence of traditional risk factors than those with non-SCAD ACS. For the test on all 563 individuals, no variants had a p-value less than 10-5 the threshold for a suggestive association with SCAD. However, 12 variants were associated at p < 10-4.
Conclusions: We confirmed that traditional risk factors are less prevalent in SCAD patients. Twelve variants were also shown to be associated after an exome-wide association study. However, our statistical power was quite low and with an increase in sample size we would expect more variants will be associated with SCAD.
One approach to studying visual perception is to present stimuli to human subjects and measuring how they perform in visual tasks. To perform these experiments, the design and presentation of these stimuli must be carefully-controlled. Typically, this is achieved using a computer that is programmed to present stimuli on a monitor. In recent years, virtual reality (VR) devices have become viable alternatives for presenting stimuli. Head-mounted VR displays like the Oculus Rift envelop the user’s visual field to replace the subject’s visual world with a virtual environment. Although useful for some experiments, the experience can cause discomfort for the user. Additionally, the full visual immersion of VR displays does not allow users to monitor their surroundings for hazards. Many VR devices must outsource their processing to a powerful computer, which means the subject must remain nearby. In contrast to VR, augmented reality (AR) displays project virtual objects (holograms) on top of reality. This has numerous advantages including reducing the amount of work needed to produce a visual environment for the subject, preventing the motion sickness that VR can cause, and affording the user increased awareness of their surroundings.
In our exploration of AR as a tool for studying vision, we are making use of Microsoft’s HoloLens. It provides a head-mounted transparent display with spatial mapping. This means holograms can be anchored to and interact with surrounding surfaces. Built-in voice and gesture commands allow users to interact with the holograms. The processing for the HoloLens is performed in the headset, meaning subjects can move freely while wearing it. Overall this allows subjects to perform more naturally when compared to traditional desktop displays and keyboard input methods. Our lab performs psychophysical studies to investigate visual function in health and disease, with the aim of developing treatments. The portability and unobtrusive nature of the HoloLens lends itself to therapies we have developed that involve prolonged viewing of specific types of stimuli. We have begun piloting a study measuring ocular dominance before and after a monocular eye-cueing task displayed on the HoloLens.
Development for the HoloLens with the Unity game engine allows for a simple and streamlined process for programming visual stimuli. The HoloLens has potential as a future testing device in the field of vision research. Additional uses and features of the HoloLens will be discussed at the poster presentation along with an experimental paradigm demonstration.
Background: Recently, there has been growing attention to young women (≤ 55 years) with acute coronary syndrome (ACS), who experience worse recoveries, poorer quality of life, and higher risk of mortality compared with similar-aged men. While sex is a biological determinant of health, gender is a complex construct with psychosocial components that are largely unrecognized in clinical studies, yet can influence patients care-seeking behavior, access to care, and perception of symptoms. We have previously demonstrated using a gender score developed by our group that feminine characteristics were associated with poorer outcomes in premature ACS. Accessibility is a crucial component of high-quality care that could explain this relationship.
Methods: We merged data from the GENESIS-PRAXY (GENdEr and Sex determInantS of cardiovascular disease: from bench to beyond-Premature Acute Coronary Syndrome) and VIRGO (Variation in Recovery: Role of Gender on Outcomes of Young AMI Patients) studies, two multicenter observational studies containing gender-related characteristics and comprehensive clinical information for 4689 patients (≤ 55 years) hospitalized for acute myocardial infarction (AMI): Data were collected through chart review, patient interviews, and standardized questionnaires.
Gender-related characteristics will include primary earner status, income, employment status, type of work, education level, marital status, stress, social support (ESSI score), and depression (PHQ-9 score). Primary outcomes for access to care will include: having a family doctor or specialist 12 months prior to ACS event (pre-AMI access to care); door-to-needle/balloon time (in-hospital access to care), and number of follow-up visits with a family physician or cardiovascular specialist within 12 months after the event (post-AMI access to care). Differences between sexes in gendered characteristics and access will be examined.
Results: Analysis in progress. Our hypothesis is that both sex and gender-related characteristics listed will be associated with inadequate access to care at different stages of care (pre-AMI, in-hospital, post-AMI).
Significance: The overarching goal is to understand the interactions between gender and access to care in young patients with AMI which will provide direction for changes towards improving ischemic heart disease diagnosis, management, and outcomes in a manner that is equitable for women and men.
Signaling from the epidermal growth factor receptor (EGFR) is a critical step in cell growth and development. Aberrant signaling from this pathway underlies several human diseases, including cancer. Identifying the genes and cellular processes regulating EGFR signaling in vivo is essential in understanding how the pathway can become inappropriately activated. Caenorhabditis elegans vulva development provides an in vivo model to genetically dissect EGFR signaling. Polarized activation of LET-23, the C. elegans homolog of EGFR, is responsible for induction of the vulval cell fate in the vulval precursor cells (VPCs). In order to achieve this polarized activation, LET-23 trafficking is tightly regulated in the VPCs to target the receptor to the basolateral membrane where it can be activated by LIN-3 EGF released by an overlying cell. A previous study identified a role for AGEF-1 and ARF-1.2 GTPases in antagonizing the basolateral localization of LET-23 and characterized them as negative regulators of this pathway. Other than vulva development, these GTPases regulate lysosome size in coelomocytes (phagocytic cells), yolk trafficking in the embryo, and polarized membrane protein localization in the intestinal cells of C. elegans. Loss of the RAB-10 GTPase suppresses the phenotypes associated with loss of AGEF-1 and ARF-1.2, suggesting a fundamental antagonistic relationship between RAB-10 and these GTPases. Although both RAB-10 and AGEF-1/ARF-1.2 colocalize at the Golgi apparatus, interaction between these GTPases is not well established. We hypothesize that RAB-10 regulates polarized protein trafficking in opposition of the AGEF-1/ARF-1.2 pathway in epithelial cells, and will examine this regulation in the context of EGFR localization in the VPCs and transmembrane protein localization in the intestinal cells. Our findings will contribute to the understanding of polarized protein trafficking in epithelial cells and suggest a possible pro-oncogenic role for RAB-10.
Psoriasis is an inflammatory skin disease that affects about 1 million individuals in Canada. The skin condition is characterized by recurring itchy, painful and flaky skin lesions and often described as an autoimmune disease, however, the initial trigger of the disease in unknown. It is clear that the disease has a genetic component: individuals diagnosed with the disorder often have a family member afflicted with psoriasis. However, it has been difficult to definitively pinpoint the mutations that are causing this disorder. Recently, it has been hypothesized that alterations in the microbiota of the skin is activating the immune system and driving psoriatic disease. Preliminary data from our laboratory indicate that genetic deficiency of CD109 in mice results in spontaneous skin inflammation resembling psoriasis. These mice are not born with any visible skin defect, but as the mice age, they begin to lose hair and develop skin inflammation characterized by the thickening of the skin epidermal layer and….. Recently, our lab has shown that treating the CD109 KO mice skin with topical antibiotics decreases inflammation including a reduction in inflammatory cytokines in the skin and a reduction in the thickness of the skin. This finding has led us to hypothesize that loss of CD109 induces alterations in the skin microbiota, which may be in the number of bacteria, the location of the bacteria in the skin or in the diversity of the bacterial population, which is activating the immune system leading to psoriatic disease. My project for the summer has been to examine the abundance and diversity of the skin microbiota at different ages of wildtype and CD109-deficient mice. In addition, we are determining how changes in the microbiota are associated with expression of inflammatory cytokines. The results of this project will provide new insight the role of microbiota in inflammatory skin disease.
Background: The American Journal Experts estimate that in 2016, the world published more than 500,000 scientific articles on medicine. From an abundance of information, how do scientists come to a consensus on what is most pertinent within a specific area of research? One method that can be used is the Delphi method, which involves having experts take a series of questionnaires.
Our research team is interested in gestational diabetes mellitus (GDM), which increases the risk of adverse short- and long-term outcomes, including development of type 2 diabetes. The US Diabetes Prevention Program demonstrates this risk can be halved with an intensive health behaviour change intervention in women with pre-diabetes averaging 12 years since a GDM pregnancy. In recent years, the number of studies looking at changing the behaviours of women with previous GDM closer to the time of delivery has steadily grown, but reported outcomes vary and most studies are not long enough or large enough to examine incident diabetes. Our diabetes research team is using the Delphi method to develop a core outcome set (COS) for interventions seeking to prevent diabetes after pregnancy (DAP) in both women with prior GDM and their families.
Methods/design: The COS-DAP project will use established COS methodology, in four stages: (i) a systematic literature review of DAP prevention intervention studies following GDM, (ii) discussion and cataloguing of outcomes measured and implementation components at an investigator meeting, (iii) a two-round online Delphi survey aimed at prioritising the identified outcomes, and (iv) a consensus meeting with key stakeholders to review, discuss and refine suitable COS measures, using nominal group technique.
Discussion: The COS is expected to enhance opportunities for comparison of future studies and allow for better synthesis of the effects. The inclusion of multiple stakeholder perspectives will increase the final COSs applicability and relevance.
Introduction: According to a study¹, four out of the top five experiences that lead to patient dissatisfaction are related to waiting time, with “time in waiting room” coming in first place. Although it is difficult to decrease waiting times, we can presume that patients will have more control over their schedules by being aware of how much time they can expect to wait for their appointments.
Objective: The goal of this project was to provide waiting time visualizations in the Opal application (opalmedapps.com) for patients’ appointments at the Cedars Cancer Centre, using historical data.
Methods: More than 350,000 medical oncology – mainly chemotherapy – appointments in the Cedars Cancer Centre and their respective timestamped-data were gathered from the centre’s waiting room management system, in order to determine factors related to waiting times, such as the time patients arrive for their appointments or how long they have been waiting after their nominal appointments’ scheduled time. All these data were organized and analysed in data structures specially generated for the purpose, allowing us to keep track of the trajectory of all previous appointments and to provide patients with up-to-date visualizations of their own current appointments’ historical data.
Results: The data structures we have built allow us to easily and efficiently access historical data in real time. In turn, through the Opal application, we can provide patients with historical waiting times of their upcoming appointments, as also visualization of waiting times of their own personal previous appointments.
Discussion: Using our new data structures, we can provide waiting time information to patients regarding their appointments. Our next goal is to deliver visual representations of this information to patients using the Opal application, in order to manage expectations and to raise awareness regarding the effects of arrival time. We believe that these data will help patients to manage their waiting times, and also educate them regarding the scheduled time of their appointments – helping to avoid, for instance, waiting rooms with excessive-early patients.
: Michael, M. , Schaffer, S. D., Egan, P. L., Little, B. B. and Pritchard, P. S. (2013), Improving Wait Times and Patient Satisfaction in Primary Care. Journal for Healthcare Quality, 35: 50-60. doi:10.1111/jhq.12004
Background: 46.X is one of the most common aneuploidy in humans, named Turner syndrome. Although less than 1% of XO embryos survive in the uterus, those that have reached the term can lead normal lives except for metabolic and circulatory problems as well as infertility. Animal models are desirable for understanding the infertility associated with Turner syndrome; however XO female mice are healthy and usually fertile, diminishing their relevance to human cases. Our laboratory has previous found that XO females on the C57BL/6J(B6) genetic background exhibit premature ovarian failure, a major Turner syndrome. However, we rely on the XY non-disjunction during spermatogenesis, caused by the Paf mutation on the X chromosome (XPaf) of C3H.HeSnJ(C3H) origin, to produce XO females, and some XPafY males on advanced backcross generations stop producing XO daughters while their brothers still do so.
Objective: To test the hypothesis that the loss of XO production can be attributed to a low or no incidence of XY non-disjunction in the spermatocytes of XPafY males.
Methods: The rate of XO production by each XPafY male has previously been recorded. In this study, testicular cell suspension was prepared from individual XPafY, C3H, and B6 males, treated with hypotonic solution in a cytospin chamber, and spun down onto histology slides. The slides were immunofluorescence stained for phospho-H3.3, SCP3, and centromeres and mounted with DAPI. Fluorescence signals were captured under a Leica microscope system (DM6000B). At least 30 Second Metaphase (MII) nuclei per male were identified with phospho-H3.3 and no SCP3 staining, and the presence of sex chromosome(s) and the number of autosomal pairs was examined.
Results: We tested four XPafY males on the N8 and four on the N10 generations, which produced XO daughters at 0.0-55.5 and 0.0-22.2%, respectively. The rate of XY non-disjunction ranged between 2.9-19.3 and 2.1-2.5%, respectively. Significant correlation (P<0.01) was found between the rate of XO production and the frequency of XY non-disjunction when all males were combined. However, the rate of XO production decreased after 10 months of age while all N8-males were killed over 12 months. Therefore, the correlation may have been weakened by age differences. We are currently testing XY non-disjunction in young XPafY males. None of two wild-type B6 males showed XY non-disjunction whereas one wild-type C3H male showed 1.9% XY non-disjunction.
Conclusion: The incidence of XY non-disjunction by the PAF mutation can be lost by meiotic recombination between XPaf and XB6 chromosomes.