DNA replication stress restricts ribosomal DNA copy number

Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100–200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how “normal” copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a “normal” rDNA copy number

The iPrevent Online Breast Cancer Risk Assessment and Risk Management Tool: Usability and Acceptability Testing

Background: iPrevent estimates breast cancer (BC) risk and provides tailored risk management information. Objective: The objective of this study was to assess the usability and acceptability of the iPrevent prototype. Methods: Clinicians were eligible for participation in the study if they worked in primary care, breast surgery, or genetics clinics. Female patients aged 18-70 years with no personal cancer history were eligible. Clinicians were first familiarized with iPrevent using hypothetical paper-based cases and then actor scenarios; subsequently, they used iPrevent with their patients. Clinicians and patients completed the System Usability Scale (SUS) and an Acceptability questionnaire 2 weeks after using iPrevent; patients also completed measures of BC worry, anxiety, risk perception, and knowledge pre- and 2 weeks post-iPrevent. Data were summarized using descriptive statistics. Results: The SUS and Acceptability questionnaires were completed by 19 of 20 clinicians and 37 of 43 patients. Usability was above average (SUS score >68) for 68% (13/19) clinicians and 76% (28/37) patients. The amount of information provided by iPrevent was reported as “about right” by 89% (17/19) clinicians and 89% (33/37) patients and 95% (18/19) and 97% (36/37), respectively, would recommend iPrevent to others, although 53% (10/19) clinicians and 27% (10/37) patients found it too long. Exploratory analyses suggested that iPrevent could improve risk perception, decrease frequency of BC worry, and enhance BC prevention knowledge without changing state anxiety. Conclusions: The iPrevent prototype demonstrated good usability and acceptability. Because concerns about length could be an implementation barrier, data entry has been abbreviated in the publicly available version of iPrevent

A double blind, randomised placebo controlled trial of topical 2% viscous lidocaine in improving oral intake in children with painful infectious mouth conditions

<p>Abstract</p> <p>Background</p> <p>Painful infectious mouth conditions are a common presentation to emergency departments. Although self limiting, painful ulcerative lesions and inflamed mucosa can decrease oral intake and can lead to dehydration. Oral analgesia is of limited efficacy and is often refused by the patient. Despite widespread use of oral 2% viscous lidocaine for many years, there is little evidence for its efficacy as an analgesic and in aiding oral intake in children with painful infectious mouth conditions. This study aims to establish the effectiveness of 2% viscous lidocaine in increasing oral intake in these children by comparing it with placebo.</p> <p>Methods/Design</p> <p>This study is a randomised double-blind placebo controlled trial of children between 6 months and 8 years of age with painful infectious mouth conditions defined as gingivostomatitis (herpetic or non herpetic), ulcerative pharyngitis, herpangina and hand foot and mouth disease as assessed by the treating clinician in association with a history of poor oral fluid intake. It will be conducted at a single tertiary paediatric emergency department in Melbourne Australia.</p> <p>20 patients have already been randomised to receive 2% lidocaine or placebo in a pilot study to determine the sample size in a preplanned adaptive design. A further 80 patients will be randomised to receive either 2% lidocaine or placebo. The placebo agent is identical to lidocaine in terms of appearance, flavour and smell. All clinical and research staff involved, patients and their parents will be blinded to treatment allocation.</p> <p>The primary endpoint is the amount of fluid ingested by each child, expressed in ml/kg, within 60 minutes from the time of administration of the study mixture. Secondary endpoints are the proportion of patients ingesting 5 ml/kg and 10 ml/kg at 30 and 60 minutes after drug administration and the incidence of adverse events. Longer term outcomes will include the proportion of patients requiring hospital admission and length of emergency department stay.</p> <p>Discussion</p> <p>This trial will define the role of 2% lidocaine in the treatment of painful infectious mouth conditions</p> <p>Trial registration</p> <p>The trial is registered with the Australian and New Zealand Clinical Trials Registry - <a href="http://www.anzctr.org.au/ACTRN12609000566235.aspx">ACTRN12609000566235</a>.</p

Genetic Factors Influence the Clustering of Depression among Individuals with Lower Socioeconomic Status

Objective: To investigate the extent to which shared genetic factors can explain the clustering of depression among individuals with lower socioeconomic status, and to examine if neuroticism or intelligence are involved in these pathways. Methods: In total 2,383 participants (1,028 men and 1,355 women) of the Erasmus Rucphen Family Study were assessed with the Center for Epidemiologic Studies Depression Scale (CES-D) and the Hospital Anxiety and Depression Scale (HADSD). Socioeconomic status was assessed as the highest level of education obtained. The role of shared genetic factors was quantified by estimating genetic correlations (rG) between symptoms of depression and education level, with and without adjustment for premorbid intelligence and neuroticism scores. Results: Higher level of education was associated with lower depression scores (partial correlation coefficient 20.09 for CESD and 20.17 for HADS-D). Significant genetic correlations were found between education and bo

CYP17 genetic polymorphism, breast cancer, and breast cancer risk factors: Australian Breast Cancer Family Study

INTRODUCTION: Because CYP17 can influence the degree of exposure of breast tissues to oestrogen, the interaction between polymorphisms in this gene and hormonal risk factors is of particular interest. We attempted to replicate the findings of studies assessing such interactions with the -34T→C polymorphism. METHODS: Risk factor and CYP17 genotyping data were derived from a large Australian population-based case-control-family study of 1,284 breast cancer cases and 679 controls. Crude and adjusted odds ratio (OR) estimates and 95% confidence intervals (CIs) were calculated by unconditional logistic regression analyses. RESULTS: We found no associations between the CYP17 genotype and breast cancer overall. Premenopausal controls with A(2)/A(2 )genotype had a later age at menarche (P < 0.01). The only associations near statistical significance were that postmenopausal women with A(1)/A(1 )(wild-type) genotype had an increased risk of breast cancer if they had ever used hormone replacement therapy (OR 2.40, 95% CI 1.0 to 5.7; P = 0.05) and if they had menopause after age 47 years (OR 2.59, 95% CI 1.0 to 7.0; P = 0.06). We found no associations in common with any other studies, and no evidence for interactions. CONCLUSION: We observed no evidence of effect modification of reproductive risk factors by CYP17 genotype, although the experiment did not have sufficient statistical power to detect small main effects and modest effects in subgroups. Associations found only in subgroup analyses based on relatively small numbers require cautious interpretation without confirmation by other studies. This emphasizes the need for replication in multiple and large population-based studies to provide convincing evidence for gene–environment interactions

A discrete choice experiment of preferences for genetic counselling among Jewish women seeking cancer genetics services

To determine which aspects of breast cancer genetic counselling are important to Ashkenazi Jewish women, a discrete choice experiment was conducted. Participants consisted of 339 Australian Ashkenazi Jewish women who provided a blood sample for research used to test for Ashkenazi Jewish ancestral mutations in the genes BRCA1 and BRCA2, and were offered their genetic test result through a cancer genetics service. Main outcome measures were women's preferences for, and trade-offs between, the genetic counselling aspects of providing cancer, gene, and risk information (information); giving advice about cancer surveillance (surveillance); preparing for genetic testing (preparation); and, assistance with decision-making (direction). Respondents most valued information, about twice as much as advice about surveillance, four times as much as preparation for testing, and nine times as much as assistance with decision-making, which was least valued. Women's preferences were consistent with the major goals of genetic counselling, which include providing information and surveillance advice, and avoiding direction by facilitating autonomous decision-making. There were differences between the women in which aspects they most favoured, suggesting that counselling that elicits and responds to clients' preferences is more likely to meet clients' needs

Reproductive profiles and risk of breast cancer subtypes : a multi-center case-only study

Background: Previous studies have shown that reproductive factors are differentially associated with breast cancer (BC) risk by subtypes. The aim of this study was to investigate associations between reproductive factors and BC subtypes, and whether these vary by age at diagnosis. Methods: We used pooled data on tumor markers (estrogen and progesterone receptor, human epidermal growth factor receptor-2 (HER2)) and reproductive risk factors (parity, age at first full-time pregnancy (FFTP) and age at menarche) from 28,095 patients with invasive BC from 34 studies participating in the Breast Cancer Association Consortium (BCAC). In a case-only analysis, we used logistic regression to assess associations between reproductive factors and BC subtype compared to luminal A tumors as a reference. The interaction between age and parity in BC subtype risk was also tested, across all ages and, because age was modeled non-linearly, specifically at ages 35, 55 and 75 years. Results: Parous women were more likely to be diagnosed with triple negative BC (TNBC) than with luminal A BC, irrespective of age (OR for parity = 1.38, 95% CI 1.16-1.65, p = 0.0004; p for interaction with age = 0.076). Parous women were also more likely to be diagnosed with luminal and non-luminal HER2-like BCs and this effect was slightly more pronounced at an early age (p for interaction with age = 0.037 and 0. 030, respectively). For instance, women diagnosed at age 35 were 1.48 (CI 1.01-2.16) more likely to have luminal HER2-like BC than luminal A BC, while this association was not significant at age 75 (OR = 0.72, CI 0.45-1.14). While age at menarche was not significantly associated with BC subtype, increasing age at FFTP was non-linearly associated with TNBC relative to luminal A BC. An age at FFTP of 25 versus 20 years lowered the risk for TNBC (OR = 0.78, CI 0.70-0.88, p <0.0001), but this effect was not apparent at a later FFTP. Conclusions: Our main findings suggest that parity is associated with TNBC across all ages at BC diagnosis, whereas the association with luminal HER2-like BC was present only for early onset BC.Peer reviewe

VapC Toxins from Mycobacterium tuberculosis Are Ribonucleases that Differentially Inhibit Growth and Are Neutralized by Cognate VapB Antitoxins

The chromosome of Mycobacterium tuberculosis (Mtb) encodes forty seven toxin-antitoxin modules belonging to the VapBC family. The role of these modules in the physiology of Mtb and the function(s) served by their expansion are unknown. We investigated ten vapBC modules from Mtb and the single vapBC from M. smegmatis. Of the Mtb vapCs assessed, only Rv0549c, Rv0595c, Rv2549c and Rv2829c were toxic when expressed from a tetracycline-regulated promoter in M. smegmatis. The same genes displayed toxicity when conditionally expressed in Mtb. Toxicity of Rv2549c in M. smegmatis correlated with the level of protein expressed, suggesting that the VapC level must exceed a threshold for toxicity to be observed. In addition, the level of Rv2456 protein induced in M. smegmatis was markedly lower than Rv2549c, which may account for the lack of toxicity of this and other VapCs scored as ‘non-toxic’. The growth inhibitory effects of toxic VapCs were neutralized by expression of the cognate VapB as part of a vapBC operon or from a different chromosomal locus, while that of non-cognate antitoxins did not. These results demonstrated a specificity of interaction between VapCs and their cognate VapBs, a finding corroborated by yeast two-hybrid analyses. Deletion of selected vapC or vapBC genes did not affect mycobacterial growth in vitro, but rendered the organisms more susceptible to growth inhibition following toxic VapC expression. However, toxicity of ‘non-toxic’ VapCs was not unveiled in deletion mutant strains, even when the mutation eliminated the corresponding cognate VapB, presumably due to insufficient levels of VapC protein. Together with the ribonuclease (RNase) activity demonstrated for Rv0065 and Rv0617 – VapC proteins with similarity to Rv0549c and Rv3320c, respectively – these results suggest that the VapBC family potentially provides an abundant source of RNase activity in Mtb, which may profoundly impact the physiology of the organism