An Engineered Nonsense \u3cem\u3eURA3\u3c/em\u3e Allele Provides a Versatile System to Detect the Presence, Absence and Appearance of the [em\u3ePSI\u3c/em\u3e\u3csup\u3e+\u3c/sup\u3e] Prion in \u3cem\u3eSaccharomyces cerevisiae\u3c/em\u3e

Common methods to identify yeast cells containing the prion form of the Sup35 translation termination factor, [PSI+], involve a nonsense suppressor phenotype. Decreased function of Sup35p in [PSI+] cells leads to readthrough of certain nonsense mutations in a few auxotrophic markers, for example, ade1-14. This readthrough results in growth on adenine deficient media. While this powerful tool has dramatically facilitated the study of [PSI+], it is limited to a narrow range of laboratory strains and cannot easily be used to screen for cells that have lost the [PSI+] prion. Therefore we have engineered a nonsense mutation in the widely used URA3 gene, termed the ura3-14 allele. Introduction of the ura3-14 allele into an array of genetic backgrounds, carrying a loss-of-function URA3 mutation and [PSI+], allows for growth on media lacking uracil, indicative of decreased translational termination efficiency. This ura3-14 allele is able to distinguish various forms of the [PSI+] prion, called variants and is able to detect the de novo appearance of [PSI+] in strains carrying the prion form of Rnq1p, [PIN+]. Furthermore, 5-fluoorotic acid, which kills cells making functional Ura3p, provides a means to select for [psi−] derivatives in a population of [PSI+] cells marked with the ura3-14 allele, making this system much more versatile than previous methods

Two Pathways Determine Yeast Longevity

<p>The longevity of mother cells can be modified by at least two independent interventions: altered ERC levels and CR. In cells lacking Sir2 but containing Fob1, senescence due to ERCs predominates, causing an extremely short life span that cannot be increased by CR. In cells lacking <i>FOB1</i>, ERCs are greatly reduced and the CR pathway predominates. The presence or absence of Sir2 does not impact the longevity benefits of CR under this condition.</p

Regulation of Longevity by CR and Fob1

<div><p>(A) Life span analysis for three genetic models of CR and deletion of <i>FOB1</i>. Strains shown (and mean life spans) are BY4742 (26.7), <i>fob1Δ</i> (37.8), <i>gpa2Δ</i> (34.9), <i>gpr1</i> (34.4), and <i>hxk2Δ</i> (36.7).</p> <p>(B) <i>fob1Δ</i> and <i>hxk2Δ</i> increase life span additively. Strains shown (and mean life spans) are BY4742 (26.6), <i>fob1Δ</i> (37.3), <i>hxk2Δ</i> (36.7), and <i>fob1Δ hxk2Δ</i> (48.3).</p> <p>(C) <i>fob1Δ</i> and <i>gpa2Δ</i> increase life span additively. Strains shown (and mean life spans) are BY4742 (27.2), <i>fob1Δ</i> (37.8), <i>gpa2Δ</i> (36.7), and <i>fob1Δ gpa2Δ</i> (54.5).</p></div

CR Increases the Life Span of Cells Overexpressing <i>SIR2</i>

<div><p>(A) Neither deletion of <i>FOB1</i> nor overexpression of <i>SIR2</i> impact longevity in PSY316. Strains shown (and mean life spans) are PSY316 (21.1), PSY316 <i>fob1Δ</i> (20.7), and PSY316 <i>SIR2</i>-ox (21.7).</p> <p>(B) Overexpression of <i>SIR2</i> and CR increase life span additively in BY4742. Strains shown (and mean life spans) are BY4742 on 2% glucose (26.1), BY4742 on 0.05% glucose (31.8), BY4742 <i>SIR2</i>-ox on 2% glucose (34.6), and BY4742 <i>SIR2</i>-ox on 0.05% glucose (42.2).</p></div

Life Span Extension by CR Does Not Require Sir2

<div><p>(A) CR fails to increase life span of a <i>sir2Δ</i> mutant. Strains shown (and mean life span) are BY4742 (26.7), <i>sir2Δ</i> (14.0), <i>hxk2Δ sir2Δ</i> (12.4), and <i>gpa2Δ sir2Δ</i> (11.7).</p> <p>(B) Deletion of <i>FOB1</i> suppresses the short life span of a <i>sir2Δ</i> strain. Strains shown and mean life spans are: BY4742 (27.5), <i>sir2Δ</i> (14.0), <i>sir2Δ fob1Δ</i> (30.0).</p> <p>(C) Deletion of <i>HXK2</i> increases the life span of a <i>sir2Δ fob1Δ</i> double mutant. Strains shown (and mean life spans) are BY4742 (26.5), <i>sir2Δ fob1Δ</i> (30.0), and <i>sir2Δ fob1Δ hxk2Δ</i> (45.3).</p> <p>(D) Deletion of <i>GPA2</i> increases the life span of a <i>sir2Δ fob1Δ</i> double mutant. Strains shown (and mean life spans) are BY4742 (26.6), <i>sir2Δ fob1Δ</i> (30.0), and <i>sir2Δ fob1Δ gpa2Δ</i> (51.0).</p></div

CR Is More Effective at Enhancing Longevity in a <i>sir2Δ fob1Δ</i> Double Mutant than in Wild-Type Cells

<p>Percent increase in mean life span relative to growth on 2% glucose was determined for 20 mother cells from each strain at 0.5%, 0.1%, and 0.05% glucose.</p

Enhancing screening, brief intervention, and referral to treatment among socioeconomically disadvantaged patients: study protocol for a knowledge exchange intervention involving patients and physicians

Abstract Background Screening, Brief Intervention, and Referral for Treatment (SBIRT) is an effective approach for managing alcohol and other drug misuse in primary care; however, uptake into routine care has been limited. Uptake of SBIRT by healthcare providers may be particularly problematic for disadvantaged populations exhibiting alcohol and other drug problems, and requires creative approaches to enhance patient engagement. This knowledge translation project developed and evaluated a group of patient and health care provider resources designed to enhance the capacity of health care providers to use SBIRT and improve patient engagement with health care. Methods/Design A nonrandomized, two-group, pre-post, quasi-experimental intervention design was used, with baseline, 6-, and 12-month follow-ups. Low income patients using alcohol and other drugs and who sought care in family medicine and emergency medicine settings in Edmonton, Canada, along with physicians providing care in these settings, were recruited. Patients and physicians were allocated to the intervention or control condition by geographic location of care. Intervention patients received a health care navigation booklet developed by inner city community members and also had access to an experienced community member for consultation on health service navigation. Intervention physicians had access to online educational modules, accompanying presentations, point of care resources, addiction medicine champions, and orientations to the inner city. Resource development was informed by a literature review, needs assessment, and iterative consultation with an advisory board and other content experts. Participants completed baseline and follow-up questionnaires (6 months for patients, 6 and 12 months for physicians) and administrative health service data were also retrieved for consenting patients. Control participants were provided access to all resources after follow-up data collection was completed. The primary outcome measure was patient satisfaction with care; secondary outcome measures included alcohol and drug use, health care and addiction treatment use, uptake of SBIRT strategies, and physician attitudes about addiction. Discussion Effective knowledge translation requires careful consideration of the intended knowledge recipient’s context and needs. Knowledge translation in disadvantaged settings may be optimized by using a community-based participatory approach to resource development that takes into account relevant patient engagement issues. Trial registration Northern Alberta Clinical Trials and Research Centre #3009

In Vivo Analysis of Chromosome Condensation in Saccharomyces cerevisiae

Although chromosome condensation in the yeast Saccharomyces cerevisiae has been widely studied, visualization of this process in vivo has not been achieved. Using Lac operator sequences integrated at two loci on the right arm of chromosome IV and a Lac repressor-GFP fusion protein, we were able to visualize linear condensation of this chromosome arm during G2/M phase. As previously determined in fixed cells, condensation in yeast required the condensin complex. Not seen after fixation of cells, we found that topoisomerase II is required for linear condensation. Further analysis of perturbed mitoses unexpectedly revealed that condensation is a transient state that occurs before anaphase in budding yeast. Blocking anaphase progression by activation of the spindle assembly checkpoint caused a loss of condensation that was dependent on Mad2, followed by a delayed loss of cohesion between sister chromatids. Release of cells from spindle checkpoint arrest resulted in recondensation before anaphase onset. The loss of condensation in preanaphase-arrested cells was abrogated by overproduction of the aurora B kinase, Ipl1, whereas in ipl1-321 mutant cells condensation was prematurely lost in anaphase/telophase. In vivo analysis of chromosome condensation has therefore revealed unsuspected relationships between higher order chromatin structure and cell cycle control