The role of the yeast cleavage and polyadenylation factor subunit Ydh1p/Cft2p in pre-mRNA 3'-end formation

Cleavage and polyadenylation factor (CPF) is a multi‐protein complex that functions in pre‐mRNA 3′‐end formation and in the RNA polymerase II (RNAP II) transcription cycle. Ydh1p/Cft2p is an essential component of CPF but its precise role in 3′‐end processing remained unclear. We found that mutations in YDH1 inhibited both the cleavage and the polyadenylation steps of the 3′‐end formation reaction in vitro. Recently, we demonstrated that an important function of CPF lies in the recognition of poly(A) site sequences and RNA binding analyses suggesting that Ydh1p/Cft2p interacts with the poly(A) site region. Here we show that mutant ydh1 strains are deficient in the recognition of the ACT1 cleavage site in vivo. The C‐terminal domain (CTD) of RNAP II plays a major role in coupling 3′‐end processing and transcription. We provide evidence that Ydh1p/Cft2p interacts with the CTD of RNAP II, several other subunits of CPF and with Pcf11p, a component of CF IA. We propose that Ydh1p/Cft2p contributes to the formation of important interaction surfaces that mediate the dynamic association of CPF with RNAP II, the recognition of poly(A) site sequences and the assembly of the polyadenylation machinery on the RNA substrate.

Independent functions of yeast Pcf11p in pre-mRNA 3' end processing and in transcription termination

Pcf11p, an essential subunit of the yeast cleavage factor IA, is required for pre‐mRNA 3′ end processing, binds to the C‐terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAP II) and is involved in transcription termination. We show that the conserved CTD interaction domain (CID) of Pcf11p is essential for cell viability. Interestingly, the CTD binding and 3′ end processing activities of Pcf11p can be functionally uncoupled from each other and provided by distinct Pcf11p fragments in trans. Impaired CTD binding did not affect the 3′ end processing activity of Pcf11p and a deficiency of Pcf11p in 3′ end processing did not prevent CTD binding. Transcriptional run‐on analysis with the CYC1 gene revealed that loss of cleavage activity did not correlate with a defect in transcription termination, whereas loss of CTD binding did. We conclude that Pcf11p is a bifunctional protein and that transcript cleavage is not an obligatory step prior to RNAP II termination.

Assessment of cone beam CT registration for prostate radiation therapy : fiducial marker and soft tissue methods

Introduction This investigation aimed to assess the consistency and accuracy of radiation therapists (RTs) performing cone beam computed tomography (CBCT) alignment to fiducial markers (FMs) (CBCTFM) and the soft tissue prostate (CBCTST). Methods Six patients receiving prostate radiation therapy underwent daily CBCTs. Manual alignment of CBCTFM and CBCTST was performed by three RTs. Inter-observer agreement was assessed using a modified Bland–Altman analysis for each alignment method. Clinically acceptable 95% limits of agreement with the mean (LoAmean) were defined as ±2.0 mm for CBCTFM and ±3.0 mm for CBCTST. Differences between CBCTST alignment and the observer-averaged CBCTFM (AvCBCTFM) alignment were analysed. Clinically acceptable 95% LoA were defined as ±3.0 mm for the comparison of CBCTST and AvCBCTFM. Results CBCTFM and CBCTST alignments were performed for 185 images. The CBCTFM 95% LoAmean were within ±2.0 mm in all planes. CBCTST 95% LoAmean were within ±3.0 mm in all planes. Comparison of CBCTST with AvCBCTFM resulted in 95% LoA of −4.9 to 2.6, −1.6 to 2.5 and −4.7 to 1.9 mm in the superior–inferior, left–right and anterior–posterior planes, respectively. Conclusions Significant differences were found between soft tissue alignment and the predicted FM position. FMs are useful in reducing inter-observer variability compared with soft tissue alignment. Consideration needs to be given to margin design when using soft tissue matching due to increased inter-observer variability. This study highlights some of the complexities of soft tissue guidance for prostate radiation therapy.

Sensitivity of an Elekta iView GT a-Si EPID model to delivery errors for pre-treatment verification of IMRT fields

A Monte Carlo model of an Elekta iViewGT amorphous silicon electronic portal imaging device (a-Si EPID) has been validated for pre-treatment verification of clinical IMRT treatment plans. The simulations involved the use of the BEAMnrc and DOSXYZnrc Monte Carlo codes to predict the response of the iViewGT a-Si EPID model. The predicted EPID images were compared to the measured images obtained from the experiment. The measured EPID images were obtained by delivering a photon beam from an Elekta Synergy linac to the Elekta iViewGT a-Si EPID. The a-Si EPID was used with no additional build-up material. Frame averaged EPID images were acquired and processed using in-house software. The agreement between the predicted and measured images was analyzed using the gamma analysis technique with acceptance criteria of 3% / 3 mm. The results show that the predicted EPID images for four clinical IMRT treatment plans have a good agreement with the measured EPID signal. Three prostate IMRT plans were found to have an average gamma pass rate of more than 95.0 % and a spinal IMRT plan has the average gamma pass rate of 94.3 %. During the period of performing this work a routine MLC calibration was performed and one of the IMRT treatments re-measured with the EPID. A change in the gamma pass rate for one field was observed. This was the motivation for a series of experiments to investigate the sensitivity of the method by introducing delivery errors, MLC position and dosimetric overshoot, into the simulated EPID images. The method was found to be sensitive to 1 mm leaf position errors and 10% overshoot errors.

Changes in outdoor workers' sun-related attitudes, beliefs, and behaviors : a pre-post workplace intervention

OBJECTIVE To evaluate changes in outdoor workers' sun-related attitudes, beliefs, and behaviors in response to a health promotion intervention using a participatory action research process. METHODS Fourteen workplaces across four outdoor industry types worked collaboratively with the project team to develop tailored sun protection action plans. Workers were assessed before and after the 18-month intervention. RESULTS Outdoor workers reported increases in workplace support for sun protection (P < 0.01) and personal use of sun protection (P < 0.01). More workers reported seeking natural shade (+20%) and wearing more personal protective equipment, including broad-brimmed hats (+25%), long-sleeved collared shirts (+19%), and long trousers (+16%). The proportion of workers reporting sunburn over the past 12 months was lower at postintervention (-14%) (P = 0.03); however, the intensity of reported sunburn increased. CONCLUSIONS This intervention was successful in increasing workers' sun protective attitudes, beliefs, and behaviors.

Effects of selective oestrogen receptor modulators on proliferation in tissue cultures of pre- and postmenopausal human endometrium

We characterised the effects of selective oestrogen receptor modulators (SERM) in explant cultures of human endometrium tissue. Endometrium tissues were cultured for 24 h in Millicell-CM culture inserts in serum-free medium in the presence of vehicle,17 beta-estradiol (17 beta-E2,1 nM), oestrogen receptor (ER) antagonist ICI 164.384 (40 nM), and 4-OH-tamoxifen (40 nM), raloxifene (4 nM), lasofoxifene (4 nM)and acolbifene (4 nM). Protein expression of ER alpha, ER beta 1 and Ki-67 were evaluated by immunohistochemistry (IHC). The proliferative fraction was assessed by counting the number of Ki-67 positive cells. Nuclear staining of ER( and ER(1 was observed in the glandular epithelium and stroma of pre- and postmenopausal endometrium. ER(1 protein was also localized in the endothelial cells of blood vessels. Treating premenopausal endometrium tissue with 17 beta-E2 increased the fraction of Ki-67 positive cells (p < 0.001) by 55% in glands compared to the control. Raloxifene (4 nM) increased (p < 0.05) the Ki-67 positive fraction. All other SERMS did not affect proliferation in this model. Treating postmenopausal endometrium with 17(-E2 increased (p < 0.001) the fraction of Ki-67 positive cells by 250% in glands compared to the control. A similar effect was also seen for 4-OH-tamoxifen, whereas the rest of SERMs did not stimulate proliferation. We demonstrated that oestradiol increases the fraction of proliferating cells in short term explant cultures of postmenopausal endometrium. In addition, we were able to reveal the agonistic properties of 4-OH-tamoxifen and confirm that raloxifene and next-generation SERMs acolbifene and lasofoxifene were neutral on the human postmenopausal endometrium. (C) 2008 Elsevier Ltd. All rights reserved.

Supine to standing Cobb angle change in idiopathic scoliosis : the effect of endplate pre-selection

Background Supine imaging modalities provide valuable 3D information on scoliotic anatomy, but the altered spine geometry between the supine and standing positions affects the Cobb angle measurement. Previous studies report a mean 7°-10° Cobb angle increase from supine to standing, but none have reported the effect of endplate pre-selection or whether other parameters affect this Cobb angle difference. Methods Cobb angles from existing coronal radiographs were compared to those on existing low-dose CT scans taken within three months of the reference radiograph for a group of females with adolescent idiopathic scoliosis. Reformatted coronal CT images were used to measure supine Cobb angles with and without endplate pre-selection (end-plates selected from the radiographs) by two observers on three separate occasions. Inter and intra-observer measurement variability were assessed. Multi-linear regression was used to investigate whether there was a relationship between supine to standing Cobb angle change and eight variables: patient age, mass, standing Cobb angle, Risser sign, ligament laxity, Lenke type, fulcrum flexibility and time delay between radiograph and CT scan. Results Fifty-two patients with right thoracic Lenke Type 1 curves and mean age 14.6 years (SD 1.8) were included. The mean Cobb angle on standing radiographs was 51.9° (SD 6.7). The mean Cobb angle on supine CT images without pre-selection of endplates was 41.1° (SD 6.4). The mean Cobb angle on supine CT images with endplate pre-selection was 40.5° (SD 6.6). Pre-selecting vertebral endplates increased the mean Cobb change by 0.6° (SD 2.3, range −9° to 6°). When free to do so, observers chose different levels for the end vertebrae in 39% of cases. Multi-linear regression revealed a statistically significant relationship between supine to standing Cobb change and fulcrum flexibility (p = 0.001), age (p = 0.027) and standing Cobb angle (p < 0.001). The 95% confidence intervals for intra-observer and inter-observer measurement variability were 3.1° and 3.6°, respectively. Conclusions Pre-selecting vertebral endplates causes minor changes to the mean supine to standing Cobb change. There is a statistically significant relationship between supine to standing Cobb change and fulcrum flexibility such that this difference can be considered a potential alternative measure of spinal flexibility.

Identification of pre-spike network in patients with mesial temporal lobe epilepsy

Background: Seizures and interictal spikes in mesial temporal lobe epilepsy (MTLE) affect a network of brain regions rather than a single epileptic focus. Simultaneous electroencephalography and functional magnetic resonance imaging (EEG-fMRI) studies have demonstrated a functional network in which hemodynamic changes are time-locked to spikes. However, whether this reflects the propagation of neuronal activity from a focus, or conversely the activation of a network linked to spike generation remains unknown. The functional connectivity (FC) changes prior to spikes may provide information about the connectivity changes that lead to the generation of spikes. We used EEG-fMRI to investigate FC changes immediately prior to the appearance of interictal spikes on EEG in patients with MTLE. Methods/principal findings: Fifteen patients with MTLE underwent continuous EEG-fMRI during rest. Spikes were identified on EEG and three 10 s epochs were defined relative to spike onset: spike (0–10 s), pre-spike (−10 to 0 s), and rest (−20 to −10 s, with no previous spikes in the preceding 45s). Significant spike-related activation in the hippocampus ipsilateral to the seizure focus was found compared to the pre-spike and rest epochs. The peak voxel within the hippocampus ipsilateral to the seizure focus was used as a seed region for FC analysis in the three conditions. A significant change in FC patterns was observed before the appearance of electrographic spikes. Specifically, there was significant loss of coherence between both hippocampi during the pre-spike period compared to spike and rest states. Conclusion/significance: In keeping with previous findings of abnormal inter-hemispheric hippocampal connectivity in MTLE, our findings specifically link reduced connectivity to the period immediately before spikes. This brief decoupling is consistent with a deficit in mutual (inter-hemispheric) hippocampal inhibition that may predispose to spike generation.

Indigenous students transitioning to school : responses to pre-foundational mathematics

Australian Indigenous students' mathematics performance continues to be below that of non-Indigenous students. This occurs from the early years of school, due largely to knowledge and social differences on entry to formal schooling. This paper reports on a mathematics research project conducted in one Aboriginal community school in New South Wales, Australia. The project aimed to identify and explain the ways that young Australian Indigenous students (age 2-4 years) learn number language and processes, specifically attribute language, sorting, 1-1 correspondence and, counting. The project adopted a mixed methods approach. That is, the methodology was decolonising (Smith 1999) in that it collaborated with and gave benefit back to the Indigenous community and school being researched. It was qualitative and interpretative (Burns 2000) and incorporated an action-research teaching-experiment approach where and teachers collaborated with the researchers to try new teaching methods. This paper draws on data pertaining to students' response to diagnostic interview questions, the pre- and post-test results of the interview and photographic evidence as observations during mathematics learning time. Participants referred to in this paper include one female principal (N = 1), and the transition class of students' pre- (N = 6) and post-test (N = 3) results of the pre-foundational processes (also referred to as attributes). The results were encouraging with improvements in colour (34%), patterns (33%); capacity (38%). As a result of this project, our epistemology regarding the importance of finding out about students' pre-foundational knowledge and understandings and providing a culturally appropriate learning environment with resources has been built upon.

Preparing pre-service teachers to teach primary science : an integrated approach using the theme of sustainability

An integrated approach to assessment afforded pre-service teachers the opportunity to learn about a local sustainability issue through three learning areas: science and technology,the arts and studies of society and environment (SOSE). Three sustainability issues chosen by the pre-service teachers are presented in this paper highlighting the science concepts explored. Affordances and constraints of the integrated task are discussed in the conclusion.

The facilitator files : supporting the supporters of nursing students

Nurse education in Australia has undergone a substantial transformation in the course of its professional history. The use of experiential teaching and learning approaches in both pre and post registration courses has escalated dramatically and resulted in the extensive use of hospital based clinical experiences for student learning. The role of the clinical facilitator is used to provide stimulation to the affective and cognitive domains of student professional development and additionally is often used as a vehicle for student assessment. While most tertiary nursing schools utilise hospital clinical experiences widely, few studies have examined whether clinical facilitators have the necessary tools to effectively support and assess students in the clinical environment. Although employed for their clinical expertise and understanding of specific clinical contexts, facilitators are expected to have an understanding of experiential learning, how it informs the undergraduate curriculum and be able to support the development of neophyte nurses as they learn to apply theoretical concepts to the increasingly complex realm of patient care. Additionally, clinical facilitators are often employed on a casual basis and as a consequence of the casualisation of the facilitator workforce, universities are continually recruiting new clinical facilitators to these positions. This paper will explore a trial of facilitator education offered to clinical facilitators at the Queensland University of Technology, School of Nursing campus in Brisbane, Australia. It will report on the use of a series of student/facilitator “vignettes” created specifically for the purpose of facilitator education and how these interactions have been embedded within a wider educational package to support facilitators in their development as facilitators rather than teachers of nursing education. Finally, it will discuss the implications of facilitator preparedness on their ability to support and assess nursing students as they engage in the important transition from neophyte to registered nurse.

Navigating uncertainty in Australia : fostering informed citizenship through teaching controversial issues with pre-service teachers

Australia is a multicultural immigrant society created by public policy and direct state action over a period of two hundred years. It is now one of the world’s most diverse societies. However, like many nations, Australia faces challenges to managing ‘unauthorized arrivals’ who claim to be refugees. The issue of how to deal with unauthorized arrivals is controversial and highly emotive as it challenges public policy and government capacity to manage the multicultural ‘mix’ of Australia’s population. It also raises questions about border security. Given that it is impossible to discern beforehand who is a ‘proper’ refugee and who is not, claims to refugee status by unauthorised arrivals in Australia need to be tested against international convention criteria devised by the United Nations High Commissioner for Refugees (UNHCR). There are no simple solutions to controversial questions such as how and where should unauthorised arrivals, and the children accompanying them, be housed whilst their claims are investigated? Moreover, as this issue continues to prompt division and heated debate in Australian society, teachers new to the profession are often reluctant to explore it in the classroom. However, there are opportunities in national and state curriculum documents for the values dimensions of curriculum inquiries into controversial issues such as this to be addressed. For example, the most recent national statement on the goals for schooling in Australia, the Melbourne Declaration (MCEETYA, 2008), makes clear that Australian students need to be prepared for the challenges of the 21st century and to develop the capacity for innovation and complex problem-solving. The Melbourne Declaration informs the first national curriculum to be implemented in the Australian states and territories, and all other national and state initiatives. Its focus on developing active and informed citizens who can contribute to a socially cohesive society implies a capacity to deal with a range of issues associated with cultural diversity, This chapter explores the ways in which pre-service and early career teachers in one Australian state reflect upon curriculum opportunities to address controversial issues in the social sciences and history classroom. As part of their pre-service education, all the participants in this study completed a final year social science curriculum method unit that embedded a range of controversial issues, including the placement of children in Australian Immigration Detention Centres (IDCs), for investigation. By drawing from interviews and focus groups conducted with different cohorts of pre-service teachers in their final year of university study and beginning years of teaching, this chapter analyses the range of perceptions about how controversial issues can be examined in the secondary classroom as part of fostering informed citizenship. The discussion and analysis of the qualitative data in this study makes no claims for the representativeness of its findings, rather, a range of beginner teacher insights into a complex and important facet of teaching in a period of change and uncertainty is offered.

Pre-processing for approximate Bayesian computation in image analysis

Most of the existing algorithms for approximate Bayesian computation (ABC) assume that it is feasible to simulate pseudo-data from the model at each iteration. However, the computational cost of these simulations can be prohibitive for high dimensional data. An important example is the Potts model, which is commonly used in image analysis. Images encountered in real world applications can have millions of pixels, therefore scalability is a major concern. We apply ABC with a synthetic likelihood to the hidden Potts model with additive Gaussian noise. Using a pre-processing step, we fit a binding function to model the relationship between the model parameters and the synthetic likelihood parameters. Our numerical experiments demonstrate that the precomputed binding function dramatically improves the scalability of ABC, reducing the average runtime required for model fitting from 71 hours to only 7 minutes. We also illustrate the method by estimating the smoothing parameter for remotely sensed satellite imagery. Without precomputation, Bayesian inference is impractical for datasets of that scale.

Multi-modal deformable medical image registration

Non-rigid image registration is an essential tool required for overcoming the inherent local anatomical variations that exist between images acquired from different individuals or atlases. Furthermore, certain applications require this type of registration to operate across images acquired from different imaging modalities. One popular local approach for estimating this registration is a block matching procedure utilising the mutual information criterion. However, previous block matching procedures generate a sparse deformation field containing displacement estimates at uniformly spaced locations. This neglects to make use of the evidence that block matching results are dependent on the amount of local information content. This paper presents a solution to this drawback by proposing the use of a Reversible Jump Markov Chain Monte Carlo statistical procedure to optimally select grid points of interest. Three different methods are then compared to propagate the estimated sparse deformation field to the entire image including a thin-plate spline warp, Gaussian convolution, and a hybrid fluid technique. Results show that non-rigid registration can be improved by using the proposed algorithm to optimally select grid points of interest.