A new role for the tumour suppressor LIN-35 during meiotic recombination in Caenorhabditis elegans

Meiosis is a fundamental biological process used by sexually reproducing species to ensure the faithful transmission of genetic material and to generate genetic diversity. In humans, failure to recombine properly during meiosis causes genetic conditions in the human conceptus such as aneuploidy and spontaneous abortion. An excellent model organism for the investigation of meiotic recombination is the nematode, Caenorhabditis elegans, which has many conserved meiotic processes. In this thesis, I have investigated the role of lin-35 in meiotic crossing over. LIN-35 is the C. elegans ortholog of the retinoblastoma (Rb) protein, well characterized with respect to its role in gene transcription and cell proliferation. My results show that mutation in the lin-35 gene alters recombination frequency differentially for several regions of the chromosome, causing increases in recombinationally suppressed regions and decreases in highly recombinogenic regions. In combination with Rec-1, a mutant known to alter crossover distribution, crossovers across the length of the entire chromosome, were decreased. In addition, other severely detrimental phenotypes were observed. For example, gametic viability was reduced dramatically in the double mutant, compared to either mutant alone. Thus, the Lin-35 and Rec-1 phenotypes were synergistic, indicating non-redundancy. In summary, lin-35 function plays a role in achieving normal levels of meiotic recombination, a role that may be related to its function in chromatin modification and gene transcription.Medicine, Faculty ofMedical Genetics, Department ofGraduat

16S rRNA gene sequence and phylogenetic tree of lactobacillus species from the vagina of healthy Nigerian women

Background: The vaginal microbial community plays a vital role in maintaining women\u27s health. Understanding the precise bacterial composition is challenging because of the diverse and difficult-to-culture nature of many bacterial constituents, necessitating culture-independent methodology. During a natural menstrual cycle, physiological changes could have an impact on bacterial growth, colonization, and community structure. The objective of this study was to assess the stability of the vaginal microbiome of healthy Canadian women throughout a menstrual cycle by using cpn60-based microbiota analysis. Vaginal swabs from 27 naturally cycling reproductive-age women were collected weekly through a single menstrual cycle. Polymerase chain reaction (PCR) was performed to amplify the universal target region of the cpn60 gene and generate amplicons representative of the microbial community. Amplicons were pyrosequenced, assembled into operational taxonomic units, and analyzed. Samples were also assayed for total 16S rRNA gene content and Gardnerella vaginalis by quantitative PCR and screened for the presence of Mollicutes by using family and genus-specific PCR.Results: Overall, the vaginal microbiome of most women remained relatively stable throughout the menstrual cycle, with little variation in diversity and only modest fluctuations in species richness. Microbiomes between women were more different than were those collected consecutively from individual women. Clustering of microbial profiles revealed the expected groupings dominated by Lactobacillus crispatus, Lactobacillus iners, and Lactobacillus jensenii. Interestingly, two additional clusters were dominated by either Bifidobacterium breve or a heterogeneous mixture of nonlactobacilli. Direct G. vaginalis quantification correlated strongly with its pyrosequencing-read abundance, and Mollicutes, including Mycoplasma hominis, Ureaplasma parvum, and Ureaplasma urealyticum, were detected in most samples.Conclusions: Our cpn60-based investigation of the vaginal microbiome demonstrated that in healthy women most vaginal microbiomes remained stable through their menstrual cycle. Of interest in these findings was the presence of Bifidobacteriales beyond just Gardnerella species. Bifidobacteriales are frequently underrepresented in 16S rRNA gene-based studies, and their detection by cpn60-based investigation suggests that their significance in the vaginal community may be underappreciated

Characterization of the vaginal microbiota of healthy Canadian women through the menstrual cycle

BACKGROUND: The vaginal microbial community plays a vital role in maintaining women\u2019s health. Understanding the precise bacterial composition is challenging because of the diverse and difficult-to-culture nature of many bacterial constituents, necessitating culture-independent methodology. During a natural menstrual cycle, physiological changes could have an impact on bacterial growth, colonization, and community structure. The objective of this study was to assess the stability of the vaginal microbiome of healthy Canadian women throughout a menstrual cycle by using cpn60-based microbiota analysis. Vaginal swabs from 27 naturally cycling reproductive-age women were collected weekly through a single menstrual cycle. Polymerase chain reaction (PCR) was performed to amplify the universal target region of the cpn60 gene and generate amplicons representative of the microbial community. Amplicons were pyrosequenced, assembled into operational taxonomic units, and analyzed. Samples were also assayed for total 16S rRNA gene content and Gardnerella vaginalis by quantitative PCR and screened for the presence of Mollicutes by using family and genus-specific PCR. RESULTS: Overall, the vaginal microbiome of most women remained relatively stable throughout the menstrual cycle, with little variation in diversity and only modest fluctuations in species richness. Microbiomes between women were more different than were those collected consecutively from individual women. Clustering of microbial profiles revealed the expected groupings dominated by Lactobacillus crispatus, Lactobacillus iners, and Lactobacillus jensenii. Interestingly, two additional clusters were dominated by either Bifidobacterium breve or a heterogeneous mixture of nonlactobacilli. Direct G. vaginalis quantification correlated strongly with its pyrosequencing-read abundance, and Mollicutes, including Mycoplasma hominis, Ureaplasma parvum, and Ureaplasma urealyticum, were detected in most samples. CONCLUSIONS: Our cpn60-based investigation of the vaginal microbiome demonstrated that in healthy women most vaginal microbiomes remained stable through their menstrual cycle. Of interest in these findings was the presence of Bifidobacteriales beyond just Gardnerella species. Bifidobacteriales are frequently underrepresented in 16S rRNA gene-based studies, and their detection by cpn60-based investigation suggests that their significance in the vaginal community may be underappreciated.Peer reviewed: YesNRC publication: Ye

Oncology Clinic-Based Hereditary Cancer Genetic Testing in a Population-Based Health Care System

New streamlined models for genetic counseling and genetic testing have recently been developed in response to increasing demand for cancer genetic services. To improve access and decrease wait times, we implemented an oncology clinic-based genetic testing model for breast and ovarian cancer patients in a publicly funded population-based health care setting in British Columbia, Canada. This observational study evaluated the oncology clinic-based model as compared to a traditional one-on-one approach with a genetic counsellor using a multi-gene panel testing approach. The primary objectives were to evaluate wait times and patient reported outcome measures between the oncology clinic-based and traditional genetic counselling models. Secondary objectives were to describe oncologist and genetic counsellor acceptability and experience. Wait times from referral to return of genetic testing results were assessed for 400 patients with breast and/or ovarian cancer undergoing genetic testing for hereditary breast and ovarian cancer from June 2015 to August 2017. Patient wait times from referral to return of results were significantly shorter with the oncology clinic-based model as compared to the traditional model (403 vs. 191 days; p < 0.001). A subset of 148 patients (traditional n = 99; oncology clinic-based n = 49) completed study surveys to assess uncertainty, distress, and patient experience. Responses were similar between both models. Healthcare providers survey responses indicated they believed the oncology clinic-based model was acceptable and a positive experience. Oncology clinic-based genetic testing using a multi-gene panel approach and post-test counselling with a genetic counsellor significantly reduced wait times and is acceptable for patients and health care providers.Medicine, Faculty ofNon UBCMedical Genetics, Department ofMedical Oncology, Division ofMedicine, Department ofObstetrics and Gynaecology, Department ofPathology and Laboratory Medicine, Department ofPopulation and Public Health (SPPH), School ofReviewedFacult

Evidence of Subclinical mtDNA Alterations in HIV-Infected Pregnant Women Receiving Combination Antiretroviral Therapy Compared to HIV-Negative Pregnant Women.

Combination antiretroviral therapy (cART) can effectively prevent vertical transmission of HIV but there is potential risk of adverse maternal, foetal or infant effects. Specifically, the effect of cART use during pregnancy on mitochondrial DNA (mtDNA) content in HIV-positive (HIV+) women is unclear. We sought to characterize subclinical alterations in peripheral blood mtDNA levels in cART-treated HIV+ women during pregnancy and the postpartum period.This prospective longitudinal observational cohort study enrolled both HIV+ and HIV-negative (HIV-) pregnant women. Clinical data and blood samples were collected at three time points in pregnancy (13-<23 weeks, 23-<30 weeks, 30-40 weeks), and at delivery and six weeks post-partum in HIV+ women. Peripheral blood mtDNA to nuclear DNA (nDNA) ratio was measured by qPCR.Over a four year period, 63 HIV+ and 42 HIV- women were enrolled. HIV+ women showed significantly lower mtDNA/nDNA ratios compared to HIV- women during pregnancy (p = 0.003), after controlling for platelet count and repeated measurements using a multivariable mixed-effects model. Ethnicity, gestational age (GA) and substance use were also significantly associated with mtDNA/nDNA ratio (p≤0.02). Among HIV+ women, higher CD4 nadir was associated with higher mtDNA/nDNA ratios (p<0.0001), and these ratio were significantly lower during pregnancy compared to the postpartum period (p<0.0001).In the context of this study, it was not possible to distinguish between mtDNA effects related to HIV infection versus cART therapy. Nevertheless, while mtDNA levels were relatively stable over time in both groups during pregnancy, they were significantly lower in HIV+ women compared to HIV- women. Although no immediate clinical impact was observed on maternal or infant health, lower maternal mtDNA levels may exert long-term effects on women and children and remain a concern. Improved knowledge of such subclinical alterations is another step toward optimizing the safety and efficacy of cART regimens during pregnancy

Temporal variation in mtDNA/nDNA ratio during pregnancy, at delivery and postpartum for the HIV+ cohort.

<p>A) mtDNA/nDNA ratio in HIV- women (n = 42), HIV+ women who initiated cART prior to conception (n = 17) and continued throughout pregnancy, and HIV+ women who initiated cART during pregnancy (n = 46) at each sample time point (13–22 weeks, 23–30 weeks, 31–40 weeks, delivery, 6 weeks postpartum); mtDNA/ nDNA ratio among HIV+ women (n = 63) for the significant variables of (B) time of visit (13–22 weeks, 23–30 weeks, 31–40 weeks, delivery, 6 weeks postpartum), where the horizontal line in the boxplots indicates the median value, boxes represent the interquartile range, whiskers indicate 1.5 times the interquartile range, while points indicate outliers; (C) CD4 nadir, where the best-fit line from the mixed-effects model is shown.</p

Means, model estimates, and Chi-squared results for the mixed-effects modelling of mtDNA levels within the 63 HIV+ pregnant women.

<p>^a Means (± SEM) are reported for the raw data without correction for covariates.</p><p>^b Estimated effects after taking covariates into account.</p><p>^d LRT = likelihood-ratio test statistic</p><p>SEM, Standard error on the mean</p><p>df, degrees of freedom</p><p>Means, model estimates, and Chi-squared results for the mixed-effects modelling of mtDNA levels within the 63 HIV+ pregnant women.</p