Characterisation of novel cytochrome P450 fusion systems

The biophysical and spectroscopic characterisation of two novel P450 fusion enzymes is reported. The first of these is CYP102A3, which is a fusion of P450 haem and cytochrome P450 reductase (CPR)-like domains and functions as a catalytically self-sufficient fatty acid hydroxylase in its host organism Bacillus subtilis. The elucidation of structural aspects of the isolated haem domain of CYP102A3 (HDCYP102A3) is described. This reveals a strong homology between HDCYP102A3 and the haem domain of the related, well studied enzyme CYP102A1 (known as BM3). Examination of the substrate binding and redox properties of HDCYP102A3 reveals variations in substrate selectivity and the influence of substrate binding over the haem-iron redox potential compared to BM3. Of particular note is the apparent cooperative binding profile displayed for some branched chain fatty acid substrates with CYP102A3. The second system characterised is CYP116B1 from Cupriavidus metallidurans, a P450 fusion with a reductase domain that resembles phthalate dioxygenase reductase (PDOR). The purification of the intact CYP116B1 enzyme, and also of its isolated haem domain (expressed from the relevant gene section), is optimised and biophysical characterisations are reported. The haem iron redox potential is found to be unusually positive (-85 mV) and the influence of thiocarbamate herbicide substrate binding upon this potential is found to be minimal, unlike the case in CYP102A£ with its fatty acid substrates and likely as a consequence of the relatively small degree of shift in haem-iron spin-state towards the high-spin form. From a panel of eight potential substrates for CYP116B1, six were found to stimulate NADPH oxidation, but only two of these were themselves oxidised by the enzyme, with hydroxylated products observable. The genetically dissected reductase domain of CYP116B1 was also expressed and purified, and kinetic studies of the reductase domain revealed a preference for NADPH over NADH coenzyme, and enables comparisons with kinetic features and coenzyme selectivity in other members of the ferredoxin reductase family of enzymes. Collectively, these studies advance our knowledge of the properties of two distinct types of P450-redox partner fusion enzymes, a growing class of enzymes with potential for biotechnological applications.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Making the most of time in quantum metrology: concurrent state preparation and sensing

A quantum metrology protocol for parameter estimation is typically comprised of three stages: probe state preparation, sensing and then readout, where the time required for the first and last stages is usually neglected. In the present work we consider non-negligible state preparation and readout times, and the tradeoffs in sensitivity that come when a limited time resource T must be divided between the three stages. To investigate this, we focus on the problem of magnetic field sensing with spins in one-axis twisted or two-axis twisted states. We find that (accounting for the time necessary to prepare a twisted state) no advantage is gained unless the time T is sufficiently long or the twisting sufficiently strong. However, we also find that the limited time resource is used more effectively if we allow the twisting and the magnetic field to be applied concurrently, which possibly represents a more realistic sensing scenario. We extend this result into an optical setting by utilizing the exact correspondence between a spin system and a bosonic field mode as given by the Holstein-Primakoff transformation

Substrate and Inhibitor-Specific Conformational Changes in the Human Serotonin Transporter Revealed by Voltage-Clamp Fluorometry s

ABSTRACT The serotonin transporter (SERT) regulates neurotransmission by the biogenic monoamine neurotransmitter serotonin (5-HT, 5-hydroxytryptamine) in the central nervous system, and drugs inhibiting SERT are widely used for the treatment of a variety of central nervous system diseases. The conformational dynamics of SERT transport function and inhibition is currently poorly understood. We used voltage-clamp fluorometry to study conformational changes in human SERT (hSERT) during 5-HT transport and inhibitor binding. Cys residues were introduced at 12 positions in hSERT to enable covalent attachment of a rhodamine-based fluorophore. Transport-associated changes in fluorescence from fluorophore-labeled hSERT expressed in Xenopus oocytes could be robustly detected at four positions in hSERT: endogenous Cys109 in the top of transmembrane domain (TM) 1b, Cys substituted for Thr323 in the top of TM6, Ala419 in the interface between TM8 and extracellular loop (EL) 4, and Leu481 in EL5. The reporter positions were used for timeresolved measurement of conformational changes during 5-HT transport and binding of cocaine and the selective serotonin reuptake inhibitors fluoxetine and escitalopram. At all reporter positions, fluorescence changes observed upon substrate application were distinctly different from those observed upon inhibitor application, with respect to relative amplitude or direction. Furthermore, escitalopram, fluoxetine, and cocaine induced a very similar pattern of fluorescent changes overall, which included movements within or around TM1b, EL4, and EL5. Taken together, our data lead us to suggest that competitive inhibitors stabilize hSERT in a state that is different from the apo outward-open conformation as well as inward-facing conformations

Esimulations for blended learning in professional education: capacity building, knowledge transfer and dissemination

A two-year collaborative project by Deakin, RMIT and Charles Sturt universities aims to build in each institution, the academic and professional staff capacities for developing and using a form of online, goal-based, role-play simulation (eSimulation/eSim). While the project embraces 'knowledge transfer', 'capacity building' and 'embedded dissemination', a greater challenge is transforming perceptions of eSims in blended learning contexts to improve flexible learning designs across the higher education sector. The project addresses the need for coordinated research and development in the field of eSimulations in Australian higher education. It aligns the educational, technical, evaluation and research strengths of the three parties to build academic and professional staff capacities for the 'local' development and use of an already successful approach to simulating 'professional workplace experiences' for student learning. This poster presents the ALTC Competitive Grants Program project (2008-2010): 'Building academic staff capacity for using eSimulations in professional education for experience transfer'. The project's website is http://www.deakin.edu.au/itl/insims/altc-project/

Sci-Hub provides access to nearly all scholarly literature

The website Sci-Hub enables users to download PDF versions of scholarly articles, including many articles that are paywalled at their journal\u27s site. Sci-Hub has grown rapidly since its creation in 2011, but the extent of its coverage was unclear. Here we report that, as of March 2017, Sci-Hub\u27s database contains 68.9% of the 81.6 million scholarly articles registered with Crossref and 85.1% of articles published in toll access journals. We find that coverage varies by discipline and publisher, and that Sci-Hub preferentially covers popular, paywalled content. For toll access articles, we find that Sci-Hub provides greater coverage than the University of Pennsylvania, a major research university in the United States. Green open access to toll access articles via licit services, on the other hand, remains quite limited. Our interactive browser at https://greenelab.github.io/scihub allows users to explore these findings in more detail. For the first time, nearly all scholarly literature is available gratis to anyone with an Internet connection, suggesting the toll access business model may become unsustainable

Maternal iron deficiency perturbs embryonic cardiovascular development in mice.

Congenital heart disease (CHD) is the most common class of human birth defects, with a prevalence of 0.9% of births. However, two-thirds of cases have an unknown cause, and many of these are thought to be caused by in utero exposure to environmental teratogens. Here we identify a potential teratogen causing CHD in mice: maternal iron deficiency (ID). We show that maternal ID in mice causes severe cardiovascular defects in the offspring. These defects likely arise from increased retinoic acid signalling in ID embryos. The defects can be prevented by iron administration in early pregnancy. It has also been proposed that teratogen exposure may potentiate the effects of genetic predisposition to CHD through gene-environment interaction. Here we show that maternal ID increases the severity of heart and craniofacial defects in a mouse model of Down syndrome. It will be important to understand if the effects of maternal ID seen here in mice may have clinical implications for women

Testimonial Injustice and Vulnerability: A Qualitative Analysis of Participation in the Court of Protection

This article explores participation in Court of Protection (COP) proceedings by people considered vulnerable. The paper is based on original data obtained from observing COP proceedings and reviewing COP case files. It is argued that the observed absence of the subject of proceedings is a form of testimonial injustice, that is, a failure to value a person in their capacity as a giver of knowledge. The issue of competence to give evidence is considered but it is argued that it is not the formal evidential rules that prohibit a vulnerable adult from giving evidence. Instead, it is the result of a persistent assumption that they are inherently vulnerable and therefore lack credibility as a knowledge giver. This assumption results in the voices of vulnerable adults being routinely absent from legal proceedings. It is argued that having a voice in the courtroom is essential and has a number of intrinsic and instrumental benefits. The paper concludes with a discussion about the implications of the research, including the current trend towards the increased use of special measures, and recommends a presumption in favour of the subject of COP proceedings giving evidence

The Primarily Undergraduate Nanomaterials Cooperative: A New Model for Supporting Collaborative Research at Small Institutions on a National Scale

The Primarily Undergraduate Nanomaterials Cooperative (PUNC) is an organization for research-active faculty studying nanomaterials at Primarily Undergraduate Institutions (PUIs), where undergraduate teaching and research go hand-in-hand. In this perspective, we outline the differences in maintaining an active research group at a PUI compared to an R1 institution. We also discuss the work of PUNC, which focuses on community building, instrument sharing, and facilitating new collaborations. Currently consisting of 37 members from across the United States, PUNC has created an online community consisting of its Web site (nanocooperative.org), a weekly online summer group meeting program for faculty and students, and a Discord server for informal conversations. Additionally, in-person symposia at ACS conferences and PUNC-specific conferences are planned for the future. It is our hope that in the years to come PUNC will be seen as a model organization for community building and research support at primarily undergraduate institutions

Opposing transcriptional programs of KLF5 and AR emerge during therapy for advanced prostate cancer.

Endocrine therapies for prostate cancer inhibit the androgen receptor (AR) transcription factor. In most cases, AR activity resumes during therapy and drives progression to castration-resistant prostate cancer (CRPC). However, therapy can also promote lineage plasticity and select for AR-independent phenotypes that are uniformly lethal. Here, we demonstrate the stem cell transcription factor Krüppel-like factor 5 (KLF5) is low or absent in prostate cancers prior to endocrine therapy, but induced in a subset of CRPC, including CRPC displaying lineage plasticity. KLF5 and AR physically interact on chromatin and drive opposing transcriptional programs, with KLF5 promoting cellular migration, anchorage-independent growth, and basal epithelial cell phenotypes. We identify ERBB2 as a point of transcriptional convergence displaying activation by KLF5 and repression by AR. ERBB2 inhibitors preferentially block KLF5-driven oncogenic phenotypes. These findings implicate KLF5 as an oncogene that can be upregulated in CRPC to oppose AR activities and promote lineage plasticity

Can youth-engaged research facilitate equitable access to contraception in Canada? The qualitative study protocol for the Ask Us project.

INTRODUCTION: There is little to no evidence in Canada on the barriers that youth face when accessing contraception. We seek to identify the contraception access, experiences, beliefs, attitudes, knowledge, and needs of youth in Canada, from the perspectives of youth and youth service providers. METHODS AND ANALYSIS: This prospective, mixed-methods, integrated knowledge mobilisation study, the Ask Us project, will involve a national sample of youth, healthcare and social service providers, and policy makers recruited via a novel relational mapping and outreach approach led by youth. Phase I will centre the voices of youth and their service providers through in-depth one-on-one interviews. We will explore the factors influencing youth access to contraception, theoretically guided by Levesque's Access to Care framework. Phase II will focus on the cocreation and evaluation of knowledge translation products (youth stories) with youth, service providers, and policy makers. ETHICS AND DISSEMINATION: Ethical approval was received from the University of British Columbia's Research Ethics Board (H21-01091). Full open-access publication of the work will be sought in an international peer-reviewed journal. Findings will be disseminated to youth and service providers through social media, newsletters, and communities of practice, and to policy makers through invited evidence briefs and face-to-face presentations