High Resolution X-ray and Neutron Crystallographic Studies of \u3cem\u3eEscherichia coli\u3c/em\u3e Dihydrofolate Reductase

Dihydrofolate Reductases (DHFRs) have been identified in nearly every proteome and are essential for most biosynthetic pathways involving one-carbon transfer reactions due to their recycling of tetrahydrofolate (THF). They catalyze the NADPH-dependent reduction of dihydrofolate (DHF), producing THF. Inhibition of DHFR ultimately depletes cellular pools of THF; causing a reduced supply of thymine nucleotides for DNA synthesis, resulting in genomic instability and cell death. Therefore, DHFRs remain important drug targets in antimicrobial and chemotherapeutic treatments. Despite exhaustive investigation of E. coli chromosomal DHFR, controversy persists over the dynamics of regulatory loops (the Met20, the βF-βG, and the βG-βH) and the nature of the interaction between methotrexate (MTX), a tight-binding anti-cancer drug, and Asp 27, the only ionizable residue in the active site. Also of importance is the ionization state of Asp 27 in the apoenzyme and other complexes. Hydrogen atoms (H) likely play a critical role in DHFR ligand binding and catalysis, yet are difficult to directly visualize. High resolution X-ray and neutron crystallography have been utilized in this dissertation to provide accurate positions of H within the DHFR active site and to probe dynamics of the enzyme. The ultrahigh resolution X-ray structures of DHFR/MTX (1.0Å; chapter 4), apo DHFR (1.05Å), and DHFR/MTX/NADPH (1.4Å; both chapter 5) have been solved. Novel features were observed in the electron density maps, including the ability to model the Met20 loop in the apoenzyme as closed (reported disordered previously) and alternate side chain conformations in all the structures. The high data-to-parameter ratio of the apoenzyme and the MTX data sets allowed anisotropic B-factor refinement and full-matrix refinement to calculate carboxylate bond lengths and estimates of their deviations. The apoenzyme has highly different bond lengths for its Asp 27 carboxylate, thus, it is neutral at physiological pH. The carboxylate bond lengths of the Asp 27 in both the monomers of the asymmetric unit of the DHFR/MTX crystal are nearly equal, suggesting it is charged at physiological pH. If H is substituted for deuterium (D), neutrons are especially powerful probes due to D’s strong positive scattering length. To assign protonation states to the MTX and the Asp 27 by the direct identification of D, a neutron structure has been solved to 2.2Å resolution from nearly 80% complete data collected on a 0.3mm3 crystal (chapter 4). Prerequisite to the neutron experiment was the growth and D2O-soaking of large-volume crystals (chapter 3). The DHFR/MTX cocrystal possesses the largest primitive unit cell and is the smallest D2O-soaked crystal used successfully in a neutron diffraction experiment. This is the 11th novel protein ever to be solved by neutron crystallography (the 16th total structure). Nearly 2/3 of the amide backbone has undergone H/D exchange, an indicator of protein dynamics. However, monomer B, where the Met20 loop is closed, is ~10% more exchanged than monomer A, where the Met20 loop is partially occluded. Based on results from D occupancy refinement and analysis of the neutron maps, it is concluded that the MTX N1 is protonated when bound to DHFR. Paired with the X-ray data, this is new strong evidence that the Asp 27·MTX interaction is ionic in nature. To increase the signal-to-noise ratio in future neutron experiments, perdeuterated protein has been produced and its D enrichment measured by mass spectrometry. X-ray data (to 1.2Å) has now been collected on a perdeuterated DHFR/MTX cocrystal and it is isomorphous to the native cocrystals (chapter 3)

Predicting neck pain in Royal Australian Air Force fighter pilots

Objective: Fighter pilots frequently report neck pain and injury, and although risk factors have been suggested, the relationships between risk factors and neck pain have not been quantified. The aim of this study was to identify personal and work behaviors that are significantly associated with neck pain in fighter pilots. Methods: Eighty-two Royal Australian Air Force fighter pilots were surveyed about their flying experience, neck pain prevalence, and prevention. Multinomial logistic regressions were used to fit models between pilots\u27 neck pain during and after flight and a range of personal and work characteristics. Results: In-flight neck pain was very weakly, yet positively associated with flight hours. Duration of postflight pain was positively associated with the weekly desktop work hours and the sum of preventative actions taken in flight. The duration pilots were considered temporarily medically unfit for flying was positively associated with pilots\u27 age and their weekly desktop work hours. Discussion: The risk factors identified by the current study should guide neck pain prevention for fighter pilots. In particular, reducing desktop working hours as well as incorporating specific neck-strengthening exercises and in-flight bracing actions should be considered by agencies to help alleviating neck pain in their pilot

On-Orbit Results from the CanX-7 Drag Sail Deorbit Mission

As a proactive solution to the orbital debris problem, the Space Flight Laboratory (SFL) has developed a passive drag sail deorbit device to remove small satellites from low-Earth orbit (LEO). Upon end-of-mission, the drag sail can be deployed to decrease the ballistic coefficient of the host spacecraft. Without any further operator intervention, the drag sail will interact with Earth’s upper atmosphere to decrease the spacecraft’s orbital energy causing it to eventually deorbit. In order to demonstrate the drag sail technology on-orbit, it has been included as the primary payload on-board the CanX-7 mission, which was launched in September 2016. After successfully completing a seven-month aircraft tracking campaign using the CanX-7 ADS-B payload, the drag sails were deployed in May 2017. This paper provides a first look at the on-orbit results from the CanX-7 mission, focusing on the performance of SFL’s drag sail device

Characterization of Zinc as a Potential Alternative for the Reduction of Nitrate in Water Supplies

Groundwater contamination by nitrate (N03-) is caused mainly by anthropogenic sources including nitrogen fertilizers, animal wastes, septic systems and industrial processes. Due to the potential toxicity of nitrate and nitrite, the United States Environmental Protection Agency (USEPA) has established maximum contaminant levels (MCLs) of 10mg-N/L and 1mg-N/L, respectively, for the two pollutants in drinking water. The significant drawbacks associated with current treatment alternatives for nitrate-contaminated water have resulted in a large research thrust being developed toward the application of reducing nitrate to less toxic forms (i.e. N2(g) or NH4+) with zero-valent iron (Fe°). With the motivation to expand the scope of chemical reductants of nitrate, zinc (Zn°), which is electrochemically similar to iron, brass, a zinc-copper alloy, and copper (Cu°) were tested, using batch reactions, to characterize their effectiveness in reducing nitrate. Since zinc proved to be very effective in reducing nitrate, an empirical rate law was developed to determine the dependency of the reactions between zinc and oxidized nitrogen species on the initial nitrate concentration, zinc dose, and initial pH. The rate law was approximately first order with respect to the initial nitrate concentration and zinc dose, and a fractional order with respect to the initial pH was observed. A direct electron transfer mechanism between the zinc and oxidized nitrogen species is suggested and future work involving potential bench-scale applications with the zinc are proposed.Master of Science in Environmental Engineerin

Informal Release of Information under Section 8 of the Government Information (Public Access) Act 2009 (NSW)

On 30 May 2022, the NSW Information and Privacy Commission (‘IPC’) announced that research would be undertaken to assess the use of informal release pathways by NSW agencies.1 This followed the IPC’s observations in their 2020/21 Report of an ‘unprecedented 30% increase in applications to access government information’ during 2020–21, ‘representing the largest increase in over a decade of reporting’.2 The IPC sought to understand this increase in light of the different information release pathways available to agencies, given that the relevant legislation (Government Information (Public Access) Act 2009 (NSW) (‘GIPA Act’)) is meant to operate as a whole. Due to limited reporting requirements, the informal release pathway was the least understood. The IPC engaged UNSW Sydney (‘UNSW’) to produce this report into the informal release practices of NSW agencies

An electrostatic mechanism for Ca(2+)-mediated regulation of gap junction channels.

Gap junction channels mediate intercellular signalling that is crucial in tissue development, homeostasis and pathologic states such as cardiac arrhythmias, cancer and trauma. To explore the mechanism by which Ca(2+) blocks intercellular communication during tissue injury, we determined the X-ray crystal structures of the human Cx26 gap junction channel with and without bound Ca(2+). The two structures were nearly identical, ruling out both a large-scale structural change and a local steric constriction of the pore. Ca(2+) coordination sites reside at the interfaces between adjacent subunits, near the entrance to the extracellular gap, where local, side chain conformational rearrangements enable Ca(2+)chelation. Computational analysis revealed that Ca(2+)-binding generates a positive electrostatic barrier that substantially inhibits permeation of cations such as K(+) into the pore. Our results provide structural evidence for a unique mechanism of channel regulation: ionic conduction block via an electrostatic barrier rather than steric occlusion of the channel pore

User characteristics and usage of an open access moderated internet support group for depression and other mental disorders: A prospective study

Background: Internet support groups (ISGs) for mental ill-health are common but little is known about the characteristics of users, the usage and predictors of ISG usage and if and how these change over time. Aim: This study evaluated the attributes of a publically accessible ISG for depression and other mental disorders including: (1) the demographic and other characteristics of its users; (2) their patterns of usage; and (3) the factors which predict posts to and retention on the ISG. Method: User characteristics (gender, age, user type, country and location of residence) were collected at the time of registration on the ISG BlueBoard (blueboard.anu.edu.au). All board log data were downloaded for the period October 2008 to May 2014. Predictors of post frequency and retention on the board were examined using logistic regressions. Other data were analysed using descriptive statistics. Results: 2932 users contributed 131,004 posts to the ISG. The majority were female, aged 20 to 34 years, and mental health consumers. Although most users were city dwellers, 19% resided in rural or remote regions. Frequency of posts and retention on the board varied across users, with a moderate association between retention and number of posts. Growth in posts substantially exceeded the growth in new users over the monitoring period. Multivariate analysis demonstrated that consumers posted more often and remained longer than carers or others, and that younger users posted less often; however, the model predicted very little of the variance. Conclusions: A small minority of active users are sufficient to ensure the sustainability and growth of an online mental health ISG. Further research is required to understand why so many support group members limit their contributions to one or a very small number of posts and what factors predict and promote active engagement and long-term retention in virtual mental health communities.At the time of the study Kathleen Griffiths was supported by NHMRC Fellowship 1059620. Bradley Carron-Arthur was supported by an Australian Postgraduate Award. BlueBoard was supported by funding from the Australian Department of Health

Preliminary joint X-ray and neutron protein crystallographic studies of ecDHFR complexed with folate and NADP\u3csup\u3e+\u3c/sup\u3e

A crystal of Escherichia coli dihydrofolate reductase (ecDHFR) complexed with folate and NADP+ of 4 x 1.3 x 0.7 mm (3.6 mm3) in size was obtained by sequential application of microseeding and macroseeding. A neutron diffraction data set was collected to 2.0 A resolution using the IMAGINE diffractometer at the High Flux Isotope Reactor within Oak Ridge National Laboratory. A 1.6 A resolution X-ray data set was also collected from a smaller crystal at room temperature. The neutron and X-ray data were used together for joint refinement of the ecDHFR–folate–NADP+ ternary-complex structure in order to examine the protonation state, protein dynamics and solvent structure of the complex, furthering understanding of the catalytic mechanism