Microfabrication and its use in investigating fungal biology

This is the final version. Available on open access from Wiley via the DOI in this recorddata availability statement: Data sharing is not applicable to this article as no new data were created or analyzed in this study.Advances in microfabrication technology, and its increasing accessibility, allow us to explore fungal biology as never before. By coupling molecular genetics with fluorescence live-cell imaging in custom-designed chambers, we can now probe single yeast cell responses to changing conditions over a lifetime, characterise population heterogeneity and investigate its underlying causes. By growing filamentous fungi in complex physical environments, we can identify cross-species commonalities, reveal species-specific growth responses and examine physiological differences relevant to diverse fungal lifestyles. As affordability and expertise broadens, microfluidic platforms will become a standard technique for examining the role of fungi in cross-kingdom interactions, ranging from rhizosphere to microbiome to interconnected human organ systems. This review brings together the perspectives already gained from studying fungal biology in microfabricated systems and outlines their potential in understanding the role of fungi in the environment, health and disease.Wellcome TrustRoyal SocietyMedical Research Council (MRC

Rsr1 Palmitoylation and GTPase Activity Status Differentially Coordinate Nuclear, Septin, and Vacuole Dynamics in Candida albicans

This is the final version. Available on open access from the American Society for Microbiology via the DOI in this recordDirectional growth and tissue invasion by hyphae of the pathogenic fungus, Candida albicans, are disrupted by deletion of the small GTPase, Rsr1, which localizes Cdc42 and its kinase, Cla4, to the site of polarized growth. We investigated additional abnormalities observed in rsr1Δ hyphae, including vacuole development, cytoplasm inheritance, mitochondrial morphology, septin ring organization, nuclear division and migration, and branching frequency, which together demonstrate a fundamental role for Rsr1 in cellular organization. Rsr1 contains a C-terminal CCAAX box, which putatively undergoes both reversible palmitoylation and farnesylation for entry into the secretory pathway. We expressed variants of Rsr1 with mutated C244 or C245, or which lacked GTPase activity (Rsr1K16N and Rsr1G12V), in the rsr1Δ background and compared the resulting phenotypes with those of mutants lacking Bud5 (Rsr1 GEF), Bud2 (Rsr1 GAP), or Cla4. Bud5 was required only for cell size and bud site selection in yeast, suggesting there are alternative activators for Rsr1 in hyphae. Septin ring and vacuole dynamics were restored by expression of unpalmitoylated Rsr1C244S, which localized to endomembranes, but not by cytoplasmic Rsr1C245A or GTP/GDP-locked Rsr1, suggesting Rsr1 functions at intracellular membranes in addition to the plasma membrane. Rsr1K16N or cytoplasmic Rsr1C245A restored normal nuclear division but not septin ring or vacuole dynamics. Rsr1-GDP therefore plays a specific role in suppressing START, which can be signaled from the cytosol. Via differential palmitoylation and activity states, Rsr1 operates at diverse cell sites to orchestrate proper nuclear division and inheritance during constitutive polarized growth. As cla4Δ phenocopied rsr1Δ, it is likely these functions involve Cdc42-Cla4 activity. IMPORTANCE Understanding how single eukaryotic cells self-organize to replicate and migrate is relevant to health and disease. In the fungal pathogen, Candida albicans, the small GTPase, Rsr1, guides the directional growth of hyphae that invade human tissue during life-threatening infections. Rsr1 is a Ras-like GTPase and a homolog of the conserved Rap1 subfamily, which directs migration in mammalian cells. Research into how this single GTPase delivers complex intracellular patterning is challenging established views of GTPase regulation, trafficking, and interaction. Here, we show that Rsr1 directly and indirectly coordinates the spatial and temporal development of key intracellular macrostructures, including septum formation and closure, vacuole dynamics, and nuclear division and segregation, as well as whole-cell morphology by determining branching patterns. Furthermore, we categorize these functions by differential Rsr1 localization and activity state and provide evidence to support the emerging view that the cytosolic pool of Ras-like GTPases is functionally active.Society for Experimental BiologyWellcome TrustRoyal SocietyMedical Research Council (MRC)University of AberdeenUniversity of Exete

Improving fiducial and prostate capsule visualization for radiotherapy planning using MRI.

Background and purpose Intraprostatic fiducial markers (FM) improve the accuracy of radiotherapy (RT) delivery. Here we assess geometric integrity and contouring consistency using a T2*-weighted (T2*W) sequence alone, which allows visualization of the FM.Material and methods Ten patients scanned within the Prostate Advances in Comparative Evidence (PACE) trial (NCT01584258) had prostate images acquired with computed tomography (CT) and Magnetic Resonance (MR) Imaging: T2-weighted (T2W) and T2*W sequences. The prostate was contoured independently on each imaging dataset by three clinicians. Interobserver variability was assessed using comparison indices with Monaco ADMIRE (research version 2.0, Elekta AB) and examined for statistical differences between imaging sets. CT and MR images of two test objects were acquired to assess geometric distortion and accuracy of marker positioning. The first was a linear test object comprising straight tubes in three orthogonal directions, the second was a smaller test object with markers suspended in gel.Results Interobserver variability for prostate contouring was lower for both T2W and T2*W compared to CT, this was statistically significant when comparing CT and T2*W images. All markers are visible in T2*W images with 29/30 correctly identified, only 3/30 are visible in T2W images. Assessment of geometric distortion revealed in-plane displacements were under 0.375 mm in MRI, and through plane displacements could not be detected. The signal loss in the MR images is symmetric in relation to the true marker position shown in CT images.Conclusion Prostate T2*W images are geometrically accurate, and yield consistent prostate contours. This single sequence can be used to identify FM and for prostate delineation in a mixed MR-CT workflow

Gastrointestinal Toxicity Prediction Not Influenced by Rectal Contour or Dose-Volume Histogram Definition.

PURPOSE: Rectal dose delivered during prostate radiation therapy is associated with gastrointestinal toxicity. Treatment plans are commonly optimized using rectal dose-volume constraints, often whole-rectum relative-volumes (%). We investigated whether improved rectal contouring, use of absolute-volumes (cc), or rectal truncation might improve toxicity prediction. METHODS AND MATERIALS: Patients from the CHHiP trial (receiving 74 Gy/37 fractions [Fr] vs 60 Gy/20 Fr vs 57 Gy/19 Fr) were included if radiation therapy plans were available (2350/3216 patients), plus toxicity data for relevant analyses (2170/3216 patients). Whole solid rectum relative-volumes (%) dose-volume-histogram (DVH), as submitted by treating center (original contour), was assumed standard-of-care. Three investigational rectal DVHs were generated: (1) reviewed contour per CHHiP protocol; (2) original contour absolute volumes (cc); and (3) truncated original contour (2 versions; ±0 and ±2 cm from planning target volume [PTV]). Dose levels of interest (V30, 40, 50, 60, 70, 74 Gy) in 74 Gy arm were converted by equivalent-dose-in-2 Gy-Fr (EQD2α/β= 3 Gy) for 60 Gy/57 Gy arms. Bootstrapped logistic models predicting late toxicities (frequency G1+/G2+, bleeding G1+/G2+, proctitis G1+/G2+, sphincter control G1+, stricture/ulcer G1+) were compared by area-undercurve (AUC) between standard of care and the 3 investigational rectal definitions. RESULTS: The alternative dose/volume parameters were compared with the original relative-volume (%) DVH of the whole rectal contour, itself fitted as a weak predictor of toxicity (AUC range, 0.57-0.65 across the 8 toxicity measures). There were no significant differences in toxicity prediction for: (1) original versus reviewed rectal contours (AUCs, 0.57-0.66; P = .21-.98); (2) relative- versus absolute-volumes (AUCs, 0.56-0.63; P = .07-.91); and (3) whole-rectum versus truncation at PTV ± 2 cm (AUCs, 0.57-0.65; P = .05-.99) or PTV ± 0 cm (AUCs, 0.57-0.66; P = .27-.98). CONCLUSIONS: We used whole-rectum relative-volume DVH, submitted by the treating center, as the standard-of-care dosimetric predictor for rectal toxicity. There were no statistically significant differences in prediction performance when using central rectal contour review, with the use of absolute-volume dosimetry, or with rectal truncation relative to PTV. Whole-rectum relative-volumes were not improved upon for toxicity prediction and should remain standard-of-care

Dynamic calcium-mediated stress response and recovery signatures in the fungal pathogen, Candida albicans

Acknowledgements AB conceived the project and wrote the manuscript. CVG conceived the experimental design. SW designed the GCaMP reporter. AM, KL, LV-M, SC and TB constructed strains and optimised imaging. MF developed the image analysis software. CVG and CP carried out the microfluidics experiments and imaging analysis. NG assisted with preparation of the manuscript. PS, SN and DMR developed and undertook the theoretical data analysis and contributed to the interpretation of the results. Funding AB, CG and TB were funded by the Wellcome Trust [Grant number 206412/A/17/Z]. AB and DR were supported by a Wellcome Trust Institutional Strategic Support Award (WT204909/Z/16/Z). CP was funded by a University of Exeter studentship (113516). This work was also supported by a Royal Society URF (UF080611), an MRC NIRG (G0900211/90671) and the MRC-Centre for Medical Mycology at the University of Exeter (MR/N006364/2). DR was funded by the Medical Research Council (MR/P022405/1). SN was supported by the Medical Research Council via the GW4 BioMed2 DTP (MR/W006308/1). MCA was supported by a European Commission ITN ‘FungiBrain’ studentship (607963). LL and SC were funded by a Wellcome Trust Institutional Strategic Support Award to the University of Aberdeen. NG acknowledges support of Wellcome Trust Investigator, Collaborative, Equipment, Strategic and Biomedical Resource awards (101873, 200208, 215599, 224323). NG and AB thank the MRC (MR/M026663/2) for support. This study/research is funded by the National Institute for Health and Care Research (NIHR) Exeter Biomedical Research Centre (BRC). The views expressed are those of the author(s) and not necessarily those of the NIHR or the Department of Health and Social Care. For the purpose of open access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission.Peer reviewedPublisher PD

Physical activity and health related quality of life

Copyright @ 2012 Anokye et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.This article has been made available through the Brunel Open Access Publishing Fund.BACKGROUND: Research on the relationship between Health Related Quality of Life (HRQoL) and physical activity (PA), to date, have rarely investigated how this relationship differ across objective and subjective measures of PA. The aim of this paper is to explore the relationship between HRQoL and PA, and examine how this relationship differs across objective and subjective measures of PA, within the context of a large representative national survey from England. METHODS: Using a sample of 5,537 adults (40–60 years) from a representative national survey in England (Health Survey for England 2008), Tobit regressions with upper censoring was employed to model the association between HRQoL and objective, and subjective measures of PA controlling for potential confounders. We tested the robustness of this relationship across specific types of PA. HRQoL was assessed using the summary measure of health state utility value derived from the EuroQol-5 Dimensions (EQ-5D) whilst PA was assessed via subjective measure (questionnaire) and objective measure (accelerometer- actigraph model GT1M). The actigraph was worn (at the waist) for 7 days (during waking hours) by a randomly selected sub-sample of the HSE 2008 respondents (4,507 adults – 16 plus years), with a valid day constituting 10 hours. Analysis was conducted in 2010. RESULTS: Findings suggest that higher levels of PA are associated with better HRQoL (regression coefficient: 0.026 to 0.072). This relationship is consistent across different measures and types of PA although differences in the magnitude of HRQoL benefit associated with objective and subjective (regression coefficient: 0.047) measures of PA are noticeable, with the former measure being associated with a relatively better HRQoL (regression coefficient: 0.072). CONCLUSION: Higher levels of PA are associated with better HRQoL. Using an objective measure of PA compared with subjective shows a relatively better HRQoL.This project was funded by the NIHR Health Technology Assessment programme (project number 08/72/01)

Evidence for dark matter in the inner Milky Way

The ubiquitous presence of dark matter in the universe is today a central tenet in modern cosmology and astrophysics. Ranging from the smallest galaxies to the observable universe, the evidence for dark matter is compelling in dwarfs, spiral galaxies, galaxy clusters as well as at cosmological scales. However, it has been historically difficult to pin down the dark matter contribution to the total mass density in the Milky Way, particularly in the innermost regions of the Galaxy and in the solar neighbourhood. Here we present an up-to-date compilation of Milky Way rotation curve measurements, and compare it with state-of-the-art baryonic mass distribution models. We show that current data strongly disfavour baryons as the sole contribution to the galactic mass budget, even inside the solar circle. Our findings demonstrate the existence of dark matter in the inner Galaxy while making no assumptions on its distribution. We anticipate that this result will compel new model-independent constraints on the dark matter local density and profile, thus reducing uncertainties on direct and indirect dark matter searches, and will shed new light on the structure and evolution of the Galaxy.Comment: First submitted version of letter published in Nature Physics on Febuary 9, 2015: http://www.nature.com/nphys/journal/vaop/ncurrent/full/nphys3237.htm

No straight lines – young women’s perceptions of their mental health and wellbeing during and after pregnancy: a systematic review and meta-ethnography

Background: Young mothers face mental health challenges during and after pregnancy including increased rates of depression compared to older mothers. While the prevention of teenage pregnancy in countries such as the United States and the United Kingdom has been a focus for policy and research in recent decades, the need to understand young women’s own experiences has been highlighted. The aim of this meta-ethnography was to examine young women’s perceptions of their mental health and wellbeing during and after pregnancy to provide new understandings of those experiences. Methods: A systematic review and meta-ethnographic synthesis of qualitative research was conducted. Seven databases were systematically searched and forward and backward searching conducted. Papers were included if they were from Organisation for Economic Co-operation and Development countries and explored mental health and wellbeing experiences of young mothers (age under 20 in pregnancy; under 25 at time of research) as a primary research question – or where evidence about mental health and wellbeing from participants was foregrounded. Nineteen papers were identified and the Critical Appraisal Skills Programme checklist for qualitative research used to appraise the evidence. Following the seven-step process of meta-ethnography, key constructs were examined within each study and then translated into one another. Results: Seven translated themes were identified forming a new line of argument wherein mental health and wellbeing was analysed as relating to individual bodily experiences; tied into past and present relationships; underpinned by economic insecurity and entangled with feelings of societal surveillance. There were ‘no straight lines’ in young women’s experiences, which were more complex than dominant narratives around overcoming adversity suggest. Conclusions: The synthesis concludes that health and social care professionals need to reflect on the operation of power and stigma in young women’s lives and its impact on wellbeing. It adds to understanding of young women’s mental health and wellbeing during and after pregnancy as located in physical and structural factors rather than individual capacities alone

The Role of Bile in the Regulation of Exocrine Pancreatic Secretion

As early as 1926 Mellanby (1) was able to show that introduction of bile into the duodenum of anesthetized cats produces a copious flow of pancreatic juice. In conscious dogs, Ivy & Lueth (2) reported, bile is only a weak stimulant of pancreatic secretion. Diversion of bile from the duodenum, however, did not influence pancreatic volume secretion stimulated by a meal (3,4). Moreover, Thomas & Crider (5) observed that bile not only failed to stimulate the secretion of pancreatic juice but also abolished the pancreatic response to intraduodenally administered peptone or soap