572 research outputs found

    Simultaneous randomisation of eight key active site residues in E. coli NfsA to generate superior nitroreductases for prodrug activation

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    There is a substantial gap between the levels of enzyme activity Nature can evolve and those that scientists can engineer in the lab. This suggests that conventional directed evolution techniques involving incremental improvements in enzyme activity may frequently fail to ascend even local fitness maxima. This is most likely due to an inability of step-wise evolutionary approaches to effectively retain mutations that are beneficial in combination with one another, but on an individual basis are neutral or even slightly deleterious (i.e., exhibit positive epistasis). To overcome this limitation, we are seeking to “jump” straight to an enzyme with peak activity by conducting simultaneous mass randomisation of eight key active site residues in Escherichia coli NfsA, a nitroreductase enzyme that has several diverse applications in biotechnology. Using degenerate codons, we generated a diverse library containing 425 million unique variants. We then applied a powerful selection system using either or both of two recently identified positive selection compounds, which has enabled us to recover a diverse range of highly active nitroreductase variants. These have been screened against a panel of prodrug substrates to identify variants that are improved with specific prodrug substrates of interest. A primary focus has been developing nitroreductases as tools for targeted cell ablation in zebrafish. The basic system involves co-expression of a nitroreductase and fluorescent reporter under the control of a cell type specific promoter in a transgenic fish. Expression of the nitroreductase selectively sensitises target cells to a prodrug which, following nitroreduction, yields a cytotoxic compound that causes precise targeted cell ablation. We have identified several nil-bystander prodrugs that are able to selectively ablate nitroreductase expressing cells with no harm to nearby cells, and have paired these with highly specialised NfsA variants to improve the efficacy and accuracy of cell ablation. We have also screened our mass-randomisation libraries to recover nitroreductases that have non-overlapping prodrug specificities, to be used in a multiplex cell ablation system. This expands upon the previous system, by using pairs of selective nitroreductases and two different prodrugs to facilitate independent ablation of multiple cell types. For example, we have identified a specialist NfsA variant that has activity for tinidazole and not for metronidazole, achieved by including metronidazole as a simultaneous counter-selection during the initial positive selection process. This elegant positive/negative selection eliminated activity with metronidazole, while still ensuring that some level of nitroreductase activity was retained overall

    Differences in Small Molecule Neurotransmitter Profiles From the Crown-of-Thorns Seastar Radial Nerve Revealed Between Sexes and Following Food-Deprivation

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    Neurotransmitters serve as chemical mediators of cell communication, and are known to have important roles in regulating numerous physiological and metabolic events in eumetazoans. The Crown-of-Thorns Seastar (COTS) is an asteroid echinoderm that has been the focus of numerous ecological studies due to its negative impact on coral reefs when in large numbers. Research devoted to its neural signaling, from basic anatomy to the key small neurotransmitters, would expand our current understanding of neural-driven biological processes, such as growth and reproduction, and offers a new approach to exploring the propensity for COTS population explosions and subsequent collapse. In this study we investigated the metabolomic profiles of small molecule neurotransmitters in the COTS radial nerve cord. Multivariate analysis shows differential abundance of small molecule neurotransmitters in male and female COTS, and in food-deprived individuals with significant differences between sexes in gamma-aminobutyric acid (GABA), histamine and serotonin, and significant differences in histamine and serotonin between satiation states. Annotation established that the majority of biosynthesis enzyme genes are present in the COTS genome. The spatial distribution of GABA, histamine and serotonin in the radial nerve cord was subsequently confirmed by immunolocalization; serotonin is most prominent within the ectoneural regions, including unique neural bulbs, while GABA and histamine localize primarily within neuropil fibers. Glutamic acid, which was also found in high relative abundance and is a precursor of GABA, is known as a spawning inhibitor in seastars, and as such was tested for inhibition of ovulation ex-vivo which resulted in complete inhibition of oocyte maturation and ovulation induced by 1-Methyladenine. These findings not only advance our knowledge of echinoderm neural signaling processes but also identify potential targets for developing novel approaches for COTS biocontrol

    The prevalence of nurse burnout and its association with telomere length pre and during the COVID-19 pandemic

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    Background Burnout is a work-related stress syndrome characterized by emotional exhaustion, depersonalization, and reduced personal accomplishment. Nurse burnout is related to nurses’ deteriorating mental health and poorer patient care quality and thus, is a significant concern in healthcare. The Coronavirus Disease 2019 (COVID-19) pandemic has swept the world and distressed the healthcare systems. Because of the body’s stress mechanism, it is vital to examine the current prevalence of nurse burnout and understand it at a biological level, using an epigenetic biomarker, telomere length. Purpose To determine the prevalence of burnout among nurses in the Peri-Operative and Labor & Delivery settings pre and during the COVID-19 pandemic and to examine the effects of burnout on absolute telomere length. Methods This is a cross-sectional study assessing the prevalence of nurses’ burnout and the relationships between nurses’ burnout and telomere length. Due to the COVID-19 pandemic, we had to stop the study during the mid of data collection. Even though the study was not designed to capture changes before and during the pandemic, we analyzed two groups’ data before and during the pandemic. The study took place in a US hospital. Nurses in the hospital’s Operating Room, Post-Anesthesia Care Unit, and Labor & Delivery Unit participated in the study. Maslach Burnout Inventory survey and nurses’ demographics were administered online. Telomere length was measured via finger-prick blood. Results 146 nurses participated in the study, with 120 participants’ blood samples collected. The high-level burnout rate was 70.5%. Correlation analysis did not reveal a direct correlation between nurse burnout and telomere length. However, in a multiple regression analysis, the final model contained the burnout subscale of emotional exhaustion, years as an RN, and work unit’s nursing care quality. There was a low degree of departure from normality of the mean absolute telomere length in the pre-pandemic group and a substantial degree of departure in the during-pandemic group. Conclusions Nurse burnout is a prevalent phenomenon in healthcare, and this study indicates that nurses currently experience high levels of burnout. Nurses’ cellular biomarker, telomere length, is shorter in the group of nurses during the COVID-19 pandemic than before. Appropriate measures should be implemented to decrease nurses’ burnout symptoms and improve nurses’ psychological and physical health. Nurses, especially those younger than 60, report higher burnout symptoms, particularly emotional exhaustion. This study indicates the need for intervention to promote nurses’ health during the pandemic and beyond. If not appropriately managed, nurse burnout may continue to be a significant issue facing the healthcare system

    Situating the local in global cultural policy

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    David Stevenson - ORCID 0000-0002-8977-1818 https://orcid.org/0000-0002-8977-1818From the growth of city regions to the calls for more localism, engaging with ‘the local’ has become an increasingly important part of cultural policy rhetoric in many countries (UNESCO, 2013; UCLG, 2019). Yet despite apparent recognition that the practices of culture are always situated (and hence local), contemporary cultural policy research tends to privilege the national or international as the primary site at which cultural policy is enacted and thus, can be reformed (Durrer, et al., 2018). For all of its increasing use ‘the local’ remains abstract, seemingly deployed to legitimate activity that is of debatable benefit to the places and practices imagined by its invocation.https://doi.org/10.1080/09548963.2019.164478028pubpub

    The conserved C-terminus of the PcrA/UvrD helicase interacts directly with RNA polymerase

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    Copyright: © 2013 Gwynn et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by a Wellcome Trust project grant to MD (Reference: 077368), an ERC starting grant to MD (Acronym: SM-DNA-REPAIR) and a BBSRC project grant to PM, NS and MD (Reference: BB/I003142/1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

    The neuropeptidome of the Crown-of-Thorns Starfish, Acanthaster planci

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    This study was supported by funds from the Australian federal government Department of the Environment Reef Rescue ‘Caring for Country’ program for funding the ‘The Crown-of-Thorns secretome: Towards a control technology’ project (M. R. H., S. C.). M.R.E. was supported by grants from the BBSRC (BB/M001644/1) and Leverhulme Trust (RPG-2013-351)

    Larger than Life: Humans' Nonverbal Status Cues Alter Perceived Size

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    Social dominance and physical size are closely linked. Nonverbal dominance displays in many non-human species are known to increase the displayer's apparent size. Humans also employ a variety of nonverbal cues that increase apparent status, but it is not yet known whether these cues function via a similar mechanism: by increasing the displayer's apparent size.We generated stimuli in which actors displayed high status, neutral, or low status cues that were drawn from the findings of a recent meta-analysis. We then conducted four studies that indicated that nonverbal cues that increase apparent status do so by increasing the perceived size of the displayer. Experiment 1 demonstrated that nonverbal status cues affect perceivers' judgments of physical size. The results of Experiment 2 showed that altering simple perceptual cues can affect judgments of both size and perceived status. Experiment 3 used objective measurements to demonstrate that status cues change targets' apparent size in the two-dimensional plane visible to a perceiver, and Experiment 4 showed that changes in perceived size mediate changes in perceived status, and that the cue most associated with this phenomenon is postural openness.We conclude that nonverbal cues associated with social dominance also affect the perceived size of the displayer. This suggests that certain nonverbal dominance cues in humans may function as they do in other species: by creating the appearance of changes in physical size

    Airships: A New Horizon for Science

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    The "Airships: A New Horizon for Science" study at the Keck Institute for Space Studies investigated the potential of a variety of airships currently operable or under development to serve as observatories and science instrumentation platforms for a range of space, atmospheric, and Earth science. The participants represent a diverse cross-section of the aerospace sector, NASA, and academia. Over the last two decades, there has been wide interest in developing a high altitude, stratospheric lighter-than-air (LTA) airship that could maneuver and remain in a desired geographic position (i.e., "station-keeping") for weeks, months or even years. Our study found considerable scientific value in both low altitude (< 40 kft) and high altitude (> 60 kft) airships across a wide spectrum of space, atmospheric, and Earth science programs. Over the course of the study period, we identified stratospheric tethered aerostats as a viable alternative to airships where station-keeping was valued over maneuverability. By opening up the sky and Earth's stratospheric horizon in affordable ways with long-term flexibility, airships allow us to push technology and science forward in a project-rich environment that complements existing space observatories as well as aircraft and high-altitude balloon missions.Comment: This low resolution version of the report is 8.6 MB. For the high resolution version see: http://kiss.caltech.edu/study/airship
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