The Biotechnology Facility for International Space Station

The primary mission of the Cellular Biotechnology Program is to advance microgravity as a tool in basic and applied cell biology. The microgravity environment can be used to study fundamental principles of cell biology and to achieve specific applications such as tissue engineering. The Biotechnology Facility (BTF) will provide a state-of-the-art facility to perform cellular biotechnology research onboard the International Space Station (ISS). The BTF will support continuous operation, which will allow performance of long-duration experiments and will significantly increase the on-orbit science throughput. With the BTF, dedicated ground support, and a community of investigators, the goals of the Cellular Biotechnology Program at Johnson Space Center are to: Support approximately 400 typical investigator experiments during the nominal design life of BTF (10 years). Support a steady increase in investigations per year, starting with stationary bioreactor experiments and adding rotating bioreactor experiments at a later date. Support at least 80% of all new cellular biotechnology investigations selected through the NASA Research Announcement (NRA) process. Modular components - to allow sequential and continuous experiment operations without cross-contamination Increased cold storage capability (+4 C, -80 C, -180 C). Storage of frozen cell culture inoculum - to allow sequential investigations. Storage of post-experiment samples - for return of high quality samples. Increased number of cell cultures per investigation, with replicates - to provide sufficient number of samples for data analysis and publication of results in peer-reviewed scientific journals

Destabilisation of dimeric 14-3-3 proteins as a novel approach to anti-cancer therapeutics

14-3-3 proteins play a pivotal role in controlling cell proliferation and survival, two commonly dysregulated hallmarks of cancers. 14-3-3 protein expression is enhanced in many human cancers and correlates with more aggressive tumors and poor prognosis, suggesting a role for 14-3-3 proteins in tumorigenesis and/or progression. We showed previously that the dimeric state of 14-3-3 proteins is regulated by the lipid sphingosine, a physiological inducer of apoptosis. As the functions of 14-3-3 proteins are dependent on their dimeric state, this sphingosine-mediated 14-3-3 regulation provides a possible means to target dimeric 14-3-3 for therapeutic effect. However, sphingosine mimics are needed that are not susceptible to sphingolipid metabolism. We show here the identification and optimization of sphingosine mimetics that render dimeric 14-3-3 susceptible to phosphorylation at a site buried in the dimer interface and induce mitochondrial-mediated apoptosis. Two such compounds, RB-011 and RB-012, disrupt 14-3-3 dimers at low micromolar concentrations and induce rapid down-regulation of Raf-MAPK and PI3K-Akt signaling in Jurkat cells. Importantly, both RB-011 and RB-012 induce apoptosis of human A549 lung cancer cells and RB-012, through disruption of MAPK signaling, reduces xenograft growth in mice. Thus, these compounds provide proof-of-principle for this novel 14-3-3-targeting approach for anti-cancer drug discovery

Destabilisation of dimeric 14-3-3 proteins as a novel approach to anti-cancer therapeutics

14-3-3 proteins play a pivotal role in controlling cell proliferation and survival, two commonly dysregulated hallmarks of cancers. 14-3-3 protein expression is enhanced in many human cancers and correlates with more aggressive tumors and poor prognosis, suggesting a role for 14-3-3 proteins in tumorigenesis and/or progression. We showed previously that the dimeric state of 14-3-3 proteins is regulated by the lipid sphingosine, a physiological inducer of apoptosis. As the functions of 14-3-3 proteins are dependent on their dimeric state, this sphingosine-mediated 14-3-3 regulation provides a possible means to target dimeric 14-3-3 for therapeutic effect. However, sphingosine mimics are needed that are not susceptible to sphingolipid metabolism. We show here the identification and optimization of sphingosine mimetics that render dimeric 14-3-3 susceptible to phosphorylation at a site buried in the dimer interface and induce mitochondrial-mediated apoptosis. Two such compounds, RB-011 and RB-012, disrupt 14-3-3 dimers at low micromolar concentrations and induce rapid down-regulation of Raf-MAPK and PI3K-Akt signaling in Jurkat cells. Importantly, both RB-011 and RB-012 induce apoptosis of human A549 lung cancer cells and RB-012, through disruption of MAPK signaling, reduces xenograft growth in mice. Thus, these compounds provide proof-of-principle for this novel 14-3-3-targeting approach for anti-cancer drug discovery

Role of salt bridges in the dimer interface of 14-3-3ζ in dimer dynamics, N-terminal α-helical order and molecular chaperone activity

The 14-3-3 family of intracellular proteins are dimeric, multifunctional adaptor proteins that bind to and regulate the activities of many important signaling proteins. The subunits within 14-3-3 dimers are predicted to be stabilized by salt bridges that are largely conserved across the 14-3-3 protein family and allow the different isoforms to form heterodimers. Here, we have examined the contributions of conserved salt-bridging residues in stabilizing the dimeric state of 14-3-3ζ. Using analytical ultracentrifugation, our results revealed that Asp21 and Glu89 both play key roles in dimer dynamics and contribute to dimer stability. Furthermore, hydrogen-deuterium exchange coupled with mass spectrometry showed that mutation of Asp21 promoted disorder in the N-terminal helices of 14-3-3ζ, suggesting that this residue plays an important role in maintaining structure across the dimer interface. Intriguingly, a D21N 14-3-3ζ mutant exhibited enhanced molecular chaperone ability that prevented amorphous protein aggregation, suggesting a potential role for N-terminal disorder in 14-3-3ζ's poorly understood chaperone action. Taken together, these results imply that disorder in the N-terminal helices of 14-3-3ζ is a consequence of the dimer–monomer dynamics and may play a role in conferring chaperone function to 14-3-3ζ protein.This work was supported in part by Australian National Health and Medical Research Council Project Grant 1068087 (to J. A. C.), National Health and Medical Research Council Program Grant 1071897 (to A. F. L.), and the Fay Fuller Foundation

Discovering Argumentative Patterns in Energy Polylogues: A Macroscope for Argument Mining

A macroscope is proposed and tested here for the discovery of the unique argumentative footprint that characterizes how a collective (e.g., group, online community) manages differences and pursues disagreement through argument in a polylogue. The macroscope addresses broader analytic problems posed by various conceptualizations of large-scale argument, such as fields, spheres, communities, and institutions. The design incorporates a two-tier methodology for detecting argument patterns of the arguments performed in arguing by an interactive collective that produces views, or topographies, of the ways that issues are generated in the making and defending of standpoints. The design premises for the macroscope build on insights about argument patterns from pragma-dialectical theory by incorporating research and theory on disagreement management and the Argumentum Model of Topics. The design reconceptualizes prototypical and stereotypical argument patterns for characterizing large-scale argumentation. A prototype of the macroscope is tested on data drawn from six threads about oil-drilling and fracking from the subreddit Changemyview. The implementation suggests the efficacy of the macroscope’s design and potential for identifying what communities make controversial and how the disagreement space in a polylogue is managed through stereotypical argument patterns in terms of claims/premises, inferential relations, and presentational devices

Adjunctive rifampicin for Staphylococcus aureus bacteraemia (ARREST): a multicentre, randomised, double-blind, placebo-controlled trial.

BACKGROUND: Staphylococcus aureus bacteraemia is a common cause of severe community-acquired and hospital-acquired infection worldwide. We tested the hypothesis that adjunctive rifampicin would reduce bacteriologically confirmed treatment failure or disease recurrence, or death, by enhancing early S aureus killing, sterilising infected foci and blood faster, and reducing risks of dissemination and metastatic infection. METHODS: In this multicentre, randomised, double-blind, placebo-controlled trial, adults (≥18 years) with S aureus bacteraemia who had received ≤96 h of active antibiotic therapy were recruited from 29 UK hospitals. Patients were randomly assigned (1:1) via a computer-generated sequential randomisation list to receive 2 weeks of adjunctive rifampicin (600 mg or 900 mg per day according to weight, oral or intravenous) versus identical placebo, together with standard antibiotic therapy. Randomisation was stratified by centre. Patients, investigators, and those caring for the patients were masked to group allocation. The primary outcome was time to bacteriologically confirmed treatment failure or disease recurrence, or death (all-cause), from randomisation to 12 weeks, adjudicated by an independent review committee masked to the treatment. Analysis was intention to treat. This trial was registered, number ISRCTN37666216, and is closed to new participants. FINDINGS: Between Dec 10, 2012, and Oct 25, 2016, 758 eligible participants were randomly assigned: 370 to rifampicin and 388 to placebo. 485 (64%) participants had community-acquired S aureus infections, and 132 (17%) had nosocomial S aureus infections. 47 (6%) had meticillin-resistant infections. 301 (40%) participants had an initial deep infection focus. Standard antibiotics were given for 29 (IQR 18-45) days; 619 (82%) participants received flucloxacillin. By week 12, 62 (17%) of participants who received rifampicin versus 71 (18%) who received placebo experienced treatment failure or disease recurrence, or died (absolute risk difference -1·4%, 95% CI -7·0 to 4·3; hazard ratio 0·96, 0·68-1·35, p=0·81). From randomisation to 12 weeks, no evidence of differences in serious (p=0·17) or grade 3-4 (p=0·36) adverse events were observed; however, 63 (17%) participants in the rifampicin group versus 39 (10%) in the placebo group had antibiotic or trial drug-modifying adverse events (p=0·004), and 24 (6%) versus six (2%) had drug interactions (p=0·0005). INTERPRETATION: Adjunctive rifampicin provided no overall benefit over standard antibiotic therapy in adults with S aureus bacteraemia. FUNDING: UK National Institute for Health Research Health Technology Assessment

Bat Population Monitoring in National Parks of The Great Lakes Region and Evaluation of Bat Acoustic Analysis Software

North American bats face multiple threats, prompting an increase in bat research and conservation efforts in recent decades. Researchers often use acoustic monitoring, which entails recording bats’ echolocation calls and subsequently identifying them to species, typically using automated software. Chapter 1 describes an acoustic monitoring program at eight U.S. national parks that aims to assess changes in bat populations over time. Data collected in 2016-2017 showed that activity levels of the little brown bat (Myotis lucifigus) decreased significantly while other species remained stable. Little brown bats have undergone similar population declines elsewhere due to the disease white-nose syndrome. Chapter 2 investigates whether different versions of bat call identification software are comparable to each other and how accurate they are. For the two software programs tested, agreement among versions was variable and species-dependent. Furthermore, newer versions were more conservative in assigning identifications, though not, on average, more accurate

Localisation of the molecular chaperone site of 14-3-3ζ: an intracellular protein associated with toxic neurological protein aggregates

14-3-3 proteins are a family of acidic, dimeric, phospho-serine binding proteins, which are ubiquitously expressed in all mammals. There are 7 known isoforms in mammals (β, γ, ε, ζ, η, τ, σ), which have similar structures and roles. 14-3-3 proteins interact with over 200 target proteins and regulate many roles including apoptosis, protein transportation, mitosis and signal transduction. Due to these diverse roles, 14-3-3 proteins are associated with many diseases, e.g. cancer and neurodegenerative diseases. 14-3-3 is co-located with many neurological protein aggregates; however the role of 14-3-3 in these diseases is unknown. Recently the molecular chaperone action of 14-3-3ζ was described whereby 14-3-3ζ is able to interact with and stabilise aggregating target proteins. Previous investigations into the regions responsible for chaperone action showed that the C-terminal extension and the polar face of the amphipathic binding groove of 14-3-3ζ are unlikely to be involved in chaperone action. Here, the investigation into the site and mechanism of the molecular chaperone action is extended to target two major hydrophobic regions of 14-3-3ζ: the hydrophobic face of the amphipathic binding groove and the dimer interface. The hydrophobic face of the amphipathic binding groove is not a critical region for the chaperone action of 14-3-3ζ. This was determined by the mutations of exposed hydrophobic residues, V176, L216, L220 and L227 and assessing the chaperone ability of these proteins compared with WT 14-3-3ζ proteins against the amorphous aggregation of alcohol dehydrogenase and reduced insulin. The dimer interface was determined to be involved in the chaperone activity of 14-3-3ζ. This region was investigated by targeting hypothesised salt bridging sites in the dimer interface (D21 and E89). Disruption of this region can also be achieved via phosphorylation of S58. The 14-3-3ζ protein, S58D, is a phospho-mimic which exhibits a similar dimer disruption. The disruption caused by these mutations was assessed and the chaperone ability was tested against amorphous aggregation of alcohol dehydrogenase and reduced insulin and compared to WT 14-3-3ζ. These dimer disrupted proteins exhibited enhanced chaperone ability, implying exposure of the dimer interface is important for the chaperone action of 14-3-3ζ. In addition these 14-3-3ζ mutants also exhibited a shift in the monomer-dimer equilibrium which results in the increased production of monomeric 14-3-3ζ. This shift in the monomer-dimer equilibrium correlates with the enhanced chaperone ability of 14-3-3ζ, suggestive of the 14-3-3ζ monomer being an important chaperone active unit. To further the investigation of the role of the dimer interface in the chaperone action of 14-3-3ζ the interaction with a physiological lipid mimic was undertaken. The physiological lipid, sphingosine, is known to interact with 14-3-3 and cause disruption to the dimer interface in order to allow phosphorylation of S58. The interaction with a sphingosine mimic caused disruption to the dimer, and the chaperone ability in the presence of this mimic was assessed. There no observed effect on the chaperone activity of 14-3-3ζ as a result of this interaction. However the disruption caused by this interaction is minor and may not be sufficient to cause enough disruption to expose significant region of the dimer interface required for enhanced chaperone ability. Small angle scattering studies confirmed that the dimer interface is involved in the chaperone action of 14-3-3ζ. Modelling the interaction between aggregating ADH and 14-3-3ζ revealed that ADH interacts with dimeric 14-3-3ζ via a region of the dimer interface. This allows the formation of the dimeric structure which maintains the stability of the ADH-14-3-3 complex. The independent movement of the two interaction regions of the dimer interface makes it possible for one side of the dimer interface to dissociate allowing the interaction with the aggregating target protein. The interactions on the other side of the dimer interface remain intact allowing the dimer to be maintained. This maintains the stability that comes with the dimeric form of 14-3-3ζ whilst still allowing the interaction with aggregating protein via the hydrophobic dimer interface. This investigation of the chaperone activity of 14-3-3ζ has revealed that 14-3-3 acts as a molecular chaperone via the dimer interface. This interaction occurs when half of the dimer interface dissociates to allow access to amorphously aggregating target proteins. This allows the other side of the dimer interface to maintain salt bridging interactions and retain the dimeric state of the 14-3-3ζ protein with the associated stability. This investigation is the first instance in which a chaperone protein has been modelled interacting with an aggregating target protein. It also opens up the role of the dimer interface in 14-3-3 function, and clarifies that the monomeric 14-3-3 unit is unlikely to have a role in 14-3-3 pathology due to its inherent reduced stability. This thesis has provided more in depth knowledge about the chaperone capability of 14-3-3ζ and the potential role of 14-3-3ζ in neurodegenerative disease. The precise function of 14-3-3 proteins in these diseases is not well understood, with 14-3-3 acting as both positive and negative regulator of protein aggregation. The determination of the mechanism of chaperone function of 14-3-3ζ provides important information which can be utilised to further investigate the role of 14-3-3 in these diseases, leading to the development of new therapeutic techniques to overcome these debilitating diseases.Thesis (Ph.D.) -- University of Adelaide, School of Physical Sciences, 2015