High-thoughput Screen to Identify Small Molecule Inhibitors of the Canonical Wnt Signaling Pathway

Wnt signaling is important in human development and disease, thus dysregulated beta-catenin constitutes an attractive target for drug intervention. The few functional inhibitors currently available target transcriptional activation, therefore, identifying novel upstream modulators would be of tremendous importance to elucidating the mechanisms involved in regulatingbeta-catenin activity. To achieve this, I developed a high-throughput screen to assess beta-catenin stability in mammalian cells using a luciferase tagged beta-catenin molecule. This assay was used to screen three chemical libraries to identify small molecule modulators of the pathway. Identified inhibitors/activators of the pathway were investigated via secondary assays. The most promising inhibitor, 21H7, significantly attenuated activated beta-catenin signaling in colon cancer cells, decreasing beta-catenin stability. The inhibitory effects of 21H7 and a structurally similar compound were shown to not only inhibit Wnt target gene expression in colon cancer cells, but also prostate cancer lines. Thus, 21H7 represents an attractive lead compound for further study.MAS

Molecular Analysis of Antimicrobial Resistance Mechanisms in Neisseria gonorrhoeae Isolates from Ontario, Canada▿

Surveillance of gonococcal antimicrobial resistance and the molecular characterization of the mechanisms underlying these resistance phenotypes are essential in order to establish correct empirical therapies, as well as to describe the emergence of new mechanisms in local bacterial populations. To address these goals, 149 isolates were collected over a 1-month period (October-November 2008) at the Ontario Public Health Laboratory, Toronto, Canada, and susceptibility profiles (8 antibiotics) were examined. Mutations in previously identified targets or the presence of some enzymes related to resistance (r), nonsusceptibility (ns) (resistant plus intermediate categories), or reduced susceptibility (rs) to the antibiotics tested were also studied. A significant proportion of nonsusceptibility to penicillin (PEN) (89.2%), tetracycline (TET) (72.3%), ciprofloxacin (CIP) (29%), and macrolides (erythromycin [ERY] and azithromycin; 22.3%) was found in these strains. Multidrug resistance was observed in 18.8% of the collection. Although all the strains were susceptible to spectinomycin and extended-spectrum cephalosporins (ESC) (ceftriaxone and cefixime), 9.4% of them displayed reduced susceptibility to extended-spectrum cephalosporins. PBP 2 mosaic structures were found in all of these ESCrs isolates. Alterations in the mtrR promoter, MtrR repressor (TETr, PENns, ESCrs, and ERYns), porin PIB (TETr and PENns), and ribosomal protein S10 (TETr) and double mutations in gyrA and parC quinolone resistance-determining regions (QRDRs) (CIPr) were associated with and presumably responsible for the resistance phenotypes observed. This is the first description of ESCrs in Canada. The detection of this phenotype indicates a change in the epidemiology of this resistance and highlights the importance of continued surveillance to preserve the last antimicrobial options available

Use of Genome Sequencing to Define Institutional Influenza Outbreaks, Toronto, Ontario, Canada, 2014–15

Adequacy of the current clinical definition of institutional influenza outbreaks is unclear. We performed a retrospective genome sequencing and epidemiologic analysis of institutional influenza outbreaks that occurred during the 2014–15 influenza season in Toronto, Canada. We sequenced the 2 earliest submitted samples positive for influenza A(H3N2) from each of 38 reported institutional outbreaks in long-term care facilities. Genome sequencing showed most outbreak pairs identified by using the current clinical definition were highly related. Inclusion of surveillance samples demonstrated that outbreak sources were likely introductions from broader circulating lineages. Pairwise distance analysis using majority genome and hemagglutinin-specific genes enabled identification of thresholds for discrimination of within and between outbreak pairs; the area under the curve ranged 0.93–0.95. Routine genome sequencing for defining influenza outbreaks in long-term care facilities is unlikely to add significantly to the current clinical definition. Sequencing may prove most useful for investigating sources of outbreak introductions

The epidemiology of sexually transmitted co-infections in HIV-positive and HIV-negative African-Caribbean women in Toronto

Abstract Background HIV disproportionately affects African-Caribbean women in Canada but the frequency and distribution of sexually transmitted infections in this community have not been previously studied. Methods We recruited women based on HIV status through a Toronto community health centre. Participants completed a socio-behavioural questionnaire using Audio Computer Assisted Self-Interview (ACASI) and provided blood for syphilis, HIV, hepatitis B and C, herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), and human cytomegalovirus (CMV) serology, urine for chlamydia and gonorrhea molecular testing and vaginal secretions for bacterial vaginosis (BV) and human papillomavirus (HPV). Differences in prevalence were assessed for statistical significance using chi-square. Results We recruited 126 HIV-positive and 291 HIV-negative women, with a median age of 40 and 31 years, respectively (p < 0.001). Active HBV infection and lifetime exposure to HBV infection were more common in HIV-positive women (4.8% vs. 0.34%, p = 0.004; and 47.6% vs. 21.2%, p < 0.0001), as was a self-reported history of HBV vaccination (66.1% vs. 44.0%, p = 0.0001). Classical STIs were rare in both groups; BV prevalence was low and did not vary by HIV status. HSV-2 infection was markedly more frequent in HIV-positive (86.3%) than HIV-negative (46.6%) women (p < 0.0001). Vaginal HPV infection was also more common in HIV-positive than in HIV-negative women (50.8% vs. 22.6%, p < 0.0001) as was infection with high-risk oncogenic HPV types (48.4% vs. 17.3%, p < 0.0001). Conclusions Classical STIs were infrequent in this clinic-based population of African-Caribbean women in Toronto. However, HSV-2 prevalence was higher than that reported in previous studies in the general Canadian population and was strongly associated with HIV infection, as was infection with hepatitis B and HPV

Epidemiology of Enterovirus D68 in Ontario.

In August 2014, children's hospitals in Kansas City, Missouri and Chicago, Illinois notified the Centers for Disease Control and Prevention (CDC) about increased numbers of pediatric patients hospitalized with severe respiratory illness (SRI). In response to CDC reports, Public Health Ontario Laboratories (PHOL) launched an investigation of patients being tested for enterovirus D-68 (EV-D68) in Ontario, Canada. The purpose of this investigation was to enhance our understanding of EV-D68 epidemiology and clinical features. Data for this study included specimens submitted for EV-D68 testing at PHOL from September 1, 2014 to October 31, 2014. Comparisons were made between patients who tested positive for the virus (cases) and those testing negative (controls). EV-D68 was identified in 153/907 (16.8%) of patients tested. In the logistic regression model adjusting for age, sex, setting and time to specimen collection, individuals younger than 20 years of age were more likely to be diagnosed with EV-D68 compared to those 20 and over, with peak positivity at ages 5-9 years. Cases were not more likely to be hospitalized than controls. Cases were more likely to be identified in September than October (OR 8.07; 95% CI 5.15 to 12.64). Routine viral culture and multiplex PCR were inadequate methods to identify EV-D68 due to poor sensitivity and inability to differentiate EV-D68 from other enterovirus serotypes or rhinovirus. Testing for EV-D68 in Ontario from July to December, 2014 detected the presence of EV-D68 virus among young children during September-October, 2014, with most cases detected in September. There was no difference in hospitalization status between cases and controls. In order to better understand the epidemiology of this virus, surveillance for EV-D68 should include testing of symptomatic individuals from all treatment settings and patient age groups, with collection and analysis of comprehensive clinical and epidemiological data

Number of positive patients and percent positivity for EV-D68, PHOL/NML testing, September 1 to October 31, 2014.

<p>*Date when specimen was collected was used for this analysis and date when specimen was received in laboratory was used in the event the date of specimen collection was missing.</p

Geographical distribution of EV-D68 cases in Ontario, PHOL/NML, September 1 to October 31, 2014.

<p>A) Health unit reflects the jurisdiction in which the patient resides. In the event this was not available, health unit of the specimen submitter was used.</p