High-sensitivity Gd3+-Gd3+ EPR distance measurements that eliminate artefacts seen at short distances.

Gadolinium complexes are attracting increasing attention as spin labels for EPR dipolar distance measurements in biomolecules and particularly for in-cell measurements. It has been shown that flip-flop transitions within the central transition of the high-spin Gd3+ ion can introduce artefacts in dipolar distance measurements, particularly when measuring distances less than 3 nm. Previous work has shown some reduction of these artefacts through increasing the frequency separation between the two frequencies required for the double electron-electron resonance (DEER) experiment. Here we use a high-power (1 kW), wideband, non-resonant system operating at 94 GHz to evaluate DEER measurement protocols using two stiff Gd(III) rulers, consisting of two bis-Gd3+-PyMTA complexes, with separations of 2.1 nm and 6.0 nm, respectively. We show that by avoiding the -12→12 central transition completely, and placing both the pump and the observer pulses on either side of the central transition, we can now observe apparently artefact-free spectra and narrow distance distributions, even for a Gd-Gd distance of 2.1 nm. Importantly we still maintain excellent signal-to-noise ratio and relatively high modulation depths. These results have implications for in-cell EPR measurements at naturally occurring biomolecule concentrations.

Prevention and Recovery Care Services in Australia: Developing a State-Wide Typology of a Subacute Residential Mental Health Service Model.

Aims: Community-based residential alternatives to hospitalization are an emerging service model. Evidence for their acceptability and effectiveness is promising but limited. Prevention and Recovery Care (PARC) services are one such residential model, offering short-term subacute treatment and care (usually between 7 and 28 days). PARC services in Victoria, Australia, are designed to support consumers with severe mental illness to either avoid a psychiatric hospital admission (step-up care) or transition from hospital back into the community (step-down care). As a precursor to a series of studies investigating the appropriateness, effectiveness and efficiency of PARC services, we aimed to investigate whether a typology of PARC services can be developed. Methods: A manager or other appropriately knowledgeable staff member from each of the 19 adult PARC services included in the study completed a tool based on PARC operational guidelines (the Victorian PARC service mapping questionnaire) and a validated instrument measuring the quality of care in residential mental health settings (the Quality Indicator for Rehabilitative Care, QuIRC). Thirty (of 42) stakeholders participated in a modified Delphi study to select 23 from the available 230 variables for entry into a hierarchical cluster analysis. Results: Cluster analysis produced three clusters of equal dissimilarity. At the 90% confidence level, there were four variables which were significantly different between clusters. These were the year the PARC was opened, the QuIRC Living Environment domain score, the proportion of all admissions that were a step-down admission from an inpatient unit, and how often families were invited to care meetings. Sensitivity analyses suggested the findings were robust to the method used to identify clusters. Conclusions: Although PARC services were broadly similar, their identified differences suggest there is variable model implementation across Victoria sufficient to generate a PARC service typology. This typology may prove important for interpreting differences in outcomes experienced by consumers and carers using PARC services, when applied in our analyses of service effectiveness. The value of conducting service mapping and typology studies is underscored. Further research to characterize subacute residential services, including recovery-promoting features of the built environment, is warranted.

The use of composite pulses for improving DEER signal at 94GHz.

The sensitivity of pulsed electron paramagnetic resonance (EPR) measurements on broad-line paramagnetic centers is often limited by the available excitation bandwidth. One way to increase excitation bandwidth is through the use of chirp or composite pulses. However, performance can be limited by cavity or detection bandwidth, which in commercial systems is typically 100-200MHz. Here we demonstrate in a 94GHz spectrometer, with >800MHz system bandwidth, an increase in signal and modulation depth in a 4-pulse DEER experiment through use of composite rather than rectangular π pulses. We show that this leads to an increase in sensitivity by a factor of 3, in line with theoretical predictions, although gains are more limited in nitroxide-nitroxide DEER measurements.

DEER Sensitivity between Iron Centers and Nitroxides in Heme-Containing Proteins Improves Dramatically Using Broadband, High-Field EPR.

This work demonstrates the feasibility of making sensitive nanometer distance measurements between Fe(III) heme centers and nitroxide spin labels in proteins using the double electron-electron resonance (DEER) pulsed EPR technique at 94 GHz. Techniques to measure accurately long distances in many classes of heme proteins using DEER are currently strongly limited by sensitivity. In this paper we demonstrate sensitivity gains of more than 30 times compared with previous lower frequency (X-band) DEER measurements on both human neuroglobin and sperm whale myoglobin. This is achieved by taking advantage of recent instrumental advances, employing wideband excitation techniques based on composite pulses and exploiting more favorable relaxation properties of low-spin Fe(III) in high magnetic fields. This gain in sensitivity potentially allows the DEER technique to be routinely used as a sensitive probe of structure and conformation in the large number of heme and many other metalloproteins.

W-band PELDOR with 1 kW microwave power: molecular geometry, flexibility and exchange coupling.

A technique that is increasingly being used to determine the structure and conformational flexibility of biomacromolecules is Pulsed Electron-Electron Double Resonance (PELDOR or DEER), an Electron Paramagnetic Resonance (EPR) based technique. At X-band frequencies (9.5 GHz), PELDOR is capable of precisely measuring distances in the range of 1.5-8 nm between paramagnetic centres but the orientation selectivity is weak. In contrast, working at higher frequencies increases the orientation selection but usually at the expense of decreased microwave power and PELDOR modulation depth. Here it is shown that a home-built high-power pulsed W-band EPR spectrometer (HiPER) with a large instantaneous bandwidth enables one to achieve PELDOR data with a high degree of orientation selectivity and large modulation depths. We demonstrate a measurement methodology that gives a set of PELDOR time traces that yield highly constrained data sets. Simulating the resulting time traces provides a deeper insight into the conformational flexibility and exchange coupling of three bisnitroxide model systems. These measurements provide strong evidence that W-band PELDOR may prove to be an accurate and quantitative tool in assessing the relative orientations of nitroxide spin labels and to correlate those orientations to the underlying biological structure and dynamics.

High power pulsed dynamic nuclear polarisation at 94 GHz.

In this communication we report initial results using high power pulsed techniques at 94 GHz to perform solid state Dynamic Nuclear Polarisation (DNP) on high volume samples. It is shown that excitation with short pulses, comparable to the pi/2 pulse length, at fast repetition rates can result in higher DNP enhancements relative to continuous wave (cw) excitation for the same average power. Peak enhancements are observed at an average power of only a few hundred mW delivered to the sample.

A kilowatt pulsed 94 GHz electron paramagnetic resonance spectrometer with high concentration sensitivity, high instantaneous bandwidth, and low dead time.

We describe a quasioptical 94 GHz kW pulsed electron paramagnetic resonance spectrometer featuring pi/2 pulses as short as 5 ns and an instantaneous bandwidth of 1 GHz in nonresonant sample holders operating in induction mode and at low temperatures. Low power pulses can be as short as 200 ps and kilowatt pulses as short as 1.5 ns with timing resolution of a few hundred picoseconds. Phase and frequency can be changed on nanosecond time scales and complex high power pulse sequences can be run at repetition rates up to 80 kHz with low dead time. We demonstrate that the combination of high power pulses at high frequencies and nonresonant cavities can offer excellent concentration sensitivity for orientation selective pulsed electron double resonance (double electron-electron resonance), where we demonstrate measurements at 1 microM concentration levels.

Pivotal role of c-Fos in nitric oxide synthase 2 expression in airway epithelial cells.

The regulation of nitric oxide synthase 2 (NOS2) in airway epithelial cells plays a key role in the innate host response to a wide variety of microbial agents and also participates in the generation of pathologic airway inflammation. Among the important signalling cascades that direct NOS2 gene expression are nuclear factor kappaB (NFkappaB) and interferon-gamma (IFNgamma)/signal transducer and activator of transcription 1 (STAT-1). Previous studies suggest activator protein-1 (AP-1), in particular c-Fos component of AP-1, influences NOS2 expression. We investigated the effect of c-Fos modulation using RNA interference siRNA on NOS2 gene expression. A549 cells stably transfected with a plasmid overexpressing a c-Fos siRNA construct (FOSi) resulted in a decrease of NOS2 protein inducibility by IFN gamma. In contrast, classical IFN gamma inducible signal transduction pathways interferon regulated factor-1 (IRF-1) and pSTAT-1 were activated at a similar magnitude in FOSi and control cells. DNA-protein binding assays showed that c-Fos binding was present in wild type cells, but reduced in FOSi clones. FOSi clones had activation of NFkappaB detectable by DNA-protein binding assays, which may have contributed to a decrease of NOS2 expression. Overall, these studies indicate that c-Fos is a requisite and specific component for inducible NOS2 expression.

Force detected electron spin resonance at 94 GHz.

Force detected electron spin resonance (FDESR) detects the presence of unpaired electrons in a sample by measuring the change in force on a mechanical resonator as the magnetization of the sample is modulated under magnetic resonance conditions. The magnetization is coupled to the resonator via a magnetic field gradient. It has been used to both detect and image distributions of electron spins, and it offers both extremely high absolute sensitivity and high spatial imaging resolution. However, compared to conventional induction mode ESR the technique also has a comparatively poor concentration sensitivity and it introduces complications in interpreting and combining both spectroscopy and imaging. One method to improve both sensitivity and spectral resolution is to operate in high magnetic fields in order to increase the sample magnetization and g-factor resolution. In this article we present FDESR measurements on the organic conductor (fluoranthene)(2)PF(6) at 3.2 T, with a corresponding millimeter-wave frequency of 93.5 GHz, which we believe are the highest field results for FDESR reported in the literature to date. A magnet-on-cantilever approach was used, with a high-anisotropy microwave ferrite as the gradient source and employing cyclic saturation to modulate the magnetization at the cantilever fundamental frequency.

Gene expression profile of human airway epithelium induced by hyperoxia in vivo.

Hyperoxia leads to oxidative modification and damage of macromolecules in the respiratory tract with loss of biological functions. Given the lack of antioxidant gene induction with acute exposure to 100% oxygen, we hypothesized that clearance pathways for oxidatively modified proteins may be induced and serve in the immediate cellular response to preserve the epithelial layer. To test this, airway epithelial cells were obtained from individuals under ambient oxygen conditions and after breathing 100% oxygen for 12 h. Gene expression profiling identified induction of genes in the chaperone and proteasome-ubiquitin-conjugation pathways that together comprise an integrated cellular response to manage and degrade damaged proteins. Analyses also revealed gene expression changes associated with oxidoreductase function, cell cycle regulation, and ATP synthesis. Increased HSP70, protein ubiquitination, and intracellular ATP were validated in cells exposed to hyperoxia in vitro. Inhibition of proteasomal degradation revealed the importance of accelerated protein catabolism for energy production of cells exposed to hyperoxia. Thus, the human airway early response to hyperoxia relies predominantly upon induction of cytoprotective chaperones and the ubiquitin-proteasome-dependent protein degradation system to maintain airway homeostatic integrity.