Effect of mutating the regulatory phosphoserine and conserved threonine on the activity of the expressed catalytic domain of Acanthamoeba myosin I heavy chain kinase

Phosphorylation of Ser-627 is both necessary and sufficient for full activity of the expressed 35-kDa catalytic domain of myosin I heavy chain kinase (MIHCK). Ser-627 lies in the variable loop between highly conserved residues DFG and APE at a position at which a phosphorylated Ser/Thr also occurs in many other Ser/Thr protein kinases. The variable loop of MIHCK contains two other hydroxyamino acids: Thr-631, which is conserved in almost all Ser/Thr kinases, and Thr-632, which is not conserved. We determined the effects on the kinase activity of the expressed catalytic domain of mutating Ser-627, Thr-631, and Thr-632 individually to Ala, Asp, and Glu. The S627A mutant was substantially less active than wild type (wt), with a lower kcat and higher Km for both peptide substrate and ATP, but was more active than unphosphorylated wt. The S627D and S627E mutants were also less active than phosphorylated wt, i.e., acidic amino acids cannot substitute for phospho-Ser-627. The activity of the T631A mutant was as low as that of the S627A mutant, whereas the T632A mutant was as active as phosphorylated wt, indicating that highly conserved Thr-631, although not phosphorylated, is essential for catalytic activity. Asp and Glu substitutions for Thr-631 and Thr-632 were inhibitory to various degrees. Molecular modeling indicated that Thr-631 can hydrogen bond with conserved residue Asp-591 in the catalytic loop and that similar interactions are possible for other kinases whose activities also are regulated by phosphorylation in the variable loop. Thus, this conserved Thr residue may be essential for the activities of other Ser/Thr protein kinases as well as for the activity of MIHCK.

Identification by mass spectrometry of the phosphorylated residue responsible for activation of the catalytic domain of myosin I heavy chain kinase, a member of the PAK/STE20 family

Myosin I heavy chain kinase from Acanthamoeba castellanii is activated in vitro by autophosphorylation (8–10 mol of P per mol). The catalytically active C-terminal domain produced by trypsin cleavage of the phosphorylated kinase contains 2–3 mol of P per mol. However, the catalytic domain expressed in a baculovirus–insect cell system is fully active as isolated without autophosphorylation in vitro. We now show that the expressed catalytic domain is inactivated by incubation with acid phosphatase and regains activity upon autophosphorylation. The state of phosphorylation of all of the hydroxyamino acids in the catalytic domain were determined by mass spectrometry of unfractionated protease digests. Ser-627 was phosphorylated in the active, expressed catalytic domain, lost its phosphate when the protein was incubated with phosphatase, and was rephosphorylated when the dephosphorylated protein was incubated with ATP. No other residue was significantly phosphorylated in any of the three samples. Thus, phosphorylation of Ser-627, which is in the same position as the Ser and Thr residues that are phosphorylated in many other kinases, is necessary and sufficient for full activity of the catalytic domain. Ser-627 is also phosphorylated when full-length, native kinase is activated by autophosphorylation.

14–3-3 Inhibits the Dictyostelium Myosin II Heavy-Chain-specific Protein Kinase C Activity by a Direct Interaction: Identification of the 14–3-3 Binding Domain

Myosin II heavy chain (MHC) specific protein kinase C (MHC-PKC), isolated from Dictyostelium discoideum, regulates myosin II assembly and localization in response to the chemoattractant cyclic AMP. Immunoprecipitation of MHC-PKC revealed that it resides as a complex with several proteins. We show herein that one of these proteins is a homologue of the 14–3-3 protein (Dd14–3-3). This protein has recently been implicated in the regulation of intracellular signaling pathways via its interaction with several signaling proteins, such as PKC and Raf-1 kinase. We demonstrate that the mammalian 14–3-3 ζ isoform inhibits the MHC-PKC activity in vitro and that this inhibition is carried out by a direct interaction between the two proteins. Furthermore, we found that the cytosolic MHC-PKC, which is inactive, formed a complex with Dd14–3-3 in the cytosol in a cyclic AMP-dependent manner, whereas the membrane-bound active MHC-PKC was not found in a complex with Dd14–3-3. This suggests that Dd14–3-3 inhibits the MHC-PKC in vivo. We further show that MHC-PKC binds Dd14–3-3 as well as 14–3-3ζ through its C1 domain, and the interaction between these two proteins does not involve a peptide containing phosphoserine as was found for Raf-1 kinase. Our experiments thus show an in vivo function for a member of the 14–3-3 family and demonstrate that MHC-PKC interacts directly with Dd14–3-3 and 14–3-3ζ through its C1 domain both in vitro and in vivo, resulting in the inhibition of the kinase.

Effects of increasing neuromuscular electrical stimulation current intensity on cortical sensorimotor network activation: a time domain fNIRS study

Neuroimaging studies have shown neuromuscular electrical stimulation (NMES)-evoked movements activate regions of the cortical sensorimotor network, including the primary sensorimotor cortex (SMC), premotor cortex (PMC), supplementary motor area (SMA), and secondary somatosensory area (S2), as well as regions of the prefrontal cortex (PFC) known to be involved in pain processing. The aim of this study, on nine healthy subjects, was to compare the cortical network activation profile and pain ratings during NMES of the right forearm wrist extensor muscles at increasing current intensities up to and slightly over the individual maximal tolerated intensity (MTI), and with reference to voluntary (VOL) wrist extension movements. By exploiting the capability of the multi-channel time domain functional near-infrared spectroscopy technique to relate depth information to the photon time-of-flight, the cortical and superficial oxygenated (O2Hb) and deoxygenated (HHb) hemoglobin concentrations were estimated. The O2Hb and HHb maps obtained using the General Linear Model (NIRS-SPM) analysis method, showed that the VOL and NMES-evoked movements significantly increased activation (i.e., increase in O2Hb and corresponding decrease in HHb) in the cortical layer of the contralateral sensorimotor network (SMC, PMC/SMA, and S2). However, the level and area of contralateral sensorimotor network (including PFC) activation was significantly greater for NMES than VOL. Furthermore, there was greater bilateral sensorimotor network activation with the high NMES current intensities which corresponded with increased pain ratings. In conclusion, our findings suggest that greater bilateral sensorimotor network activation profile with high NMES current intensities could be in part attributable to increased attentional/pain processing and to increased bilateral sensorimotor integration in these cortical regions.

Effective Cell-Centred Time-Domain Maxwell's Equations Numerical Solvers

This research work analyses techniques for implementing a cell-centred finite-volume time-domain (ccFV-TD) computational methodology for the purpose of studying microwave heating. Various state-of-the-art spatial and temporal discretisation methods employed to solve Maxwell's equations on multidimensional structured grid networks are investigated, and the dispersive and dissipative errors inherent in those techniques examined. Both staggered and unstaggered grid approaches are considered. Upwind schemes using a Riemann solver and intensity vector splitting are studied and evaluated. Staggered and unstaggered Leapfrog and Runge-Kutta time integration methods are analysed in terms of phase and amplitude error to identify which method is the most accurate and efficient for simulating microwave heating processes. The implementation and migration of typical electromagnetic boundary conditions. from staggered in space to cell-centred approaches also is deliberated. In particular, an existing perfectly matched layer absorbing boundary methodology is adapted to formulate a new cell-centred boundary implementation for the ccFV-TD solvers. Finally for microwave heating purposes, a comparison of analytical and numerical results for standard case studies in rectangular waveguides allows the accuracy of the developed methods to be assessed.

Heavy vehicle suspensions : testing and analysis

Transport regulators consider that, with respect to pavement damage, heavy vehicles (HVs) are the riskiest vehicles on the road network. That HV suspension design contributes to road and bridge damage has been recognised for some decades. This thesis deals with some aspects of HV suspension characteristics, particularly (but not exclusively) air suspensions. This is in the areas of developing low-cost in-service heavy vehicle (HV) suspension testing, the effects of larger-than-industry-standard longitudinal air lines and the characteristics of on-board mass (OBM) systems for HVs. All these areas, whilst seemingly disparate, seek to inform the management of HVs, reduce of their impact on the network asset and/or provide a measurement mechanism for worn HV suspensions. A number of project management groups at the State and National level in Australia have been, and will be, presented with the results of the project that resulted in this thesis. This should serve to inform their activities applicable to this research. A number of HVs were tested for various characteristics. These tests were used to form a number of conclusions about HV suspension behaviours. Wheel forces from road test data were analysed. A “novel roughness” measure was developed and applied to the road test data to determine dynamic load sharing, amongst other research outcomes. Further, it was proposed that this approach could inform future development of pavement models incorporating roughness and peak wheel forces. Left/right variations in wheel forces and wheel force variations for different speeds were also presented. This led on to some conclusions regarding suspension and wheel force frequencies, their transmission to the pavement and repetitive wheel loads in the spatial domain. An improved method of determining dynamic load sharing was developed and presented. It used the correlation coefficient between two elements of a HV to determine dynamic load sharing. This was validated against a mature dynamic loadsharing metric, the dynamic load sharing coefficient (de Pont, 1997). This was the first time that the technique of measuring correlation between elements on a HV has been used for a test case vs. a control case for two different sized air lines. That dynamic load sharing was improved at the air springs was shown for the test case of the large longitudinal air lines. The statistically significant improvement in dynamic load sharing at the air springs from larger longitudinal air lines varied from approximately 30 percent to 80 percent. Dynamic load sharing at the wheels was improved only for low air line flow events for the test case of larger longitudinal air lines. Statistically significant improvements to some suspension metrics across the range of test speeds and “novel roughness” values were evident from the use of larger longitudinal air lines, but these were not uniform. Of note were improvements to suspension metrics involving peak dynamic forces ranging from below the error margin to approximately 24 percent. Abstract models of HV suspensions were developed from the results of some of the tests. Those models were used to propose further development of, and future directions of research into, further gains in HV dynamic load sharing. This was from alterations to currently available damping characteristics combined with implementation of large longitudinal air lines. In-service testing of HV suspensions was found to be possible within a documented range from below the error margin to an error of approximately 16 percent. These results were in comparison with either the manufacturer’s certified data or test results replicating the Australian standard for “road-friendly” HV suspensions, Vehicle Standards Bulletin 11. OBM accuracy testing and development of tamper evidence from OBM data were detailed for over 2000 individual data points across twelve test and control OBM systems from eight suppliers installed on eleven HVs. The results indicated that 95 percent of contemporary OBM systems available in Australia are accurate to +/- 500 kg. The total variation in OBM linearity, after three outliers in the data were removed, was 0.5 percent. A tamper indicator and other OBM metrics that could be used by jurisdictions to determine tamper events were developed and documented. That OBM systems could be used as one vector for in-service testing of HV suspensions was one of a number of synergies between the seemingly disparate streams of this project.

Implementing low intensity CBT in case management of clients with severe mental illness

In a previous chapter (Dean and Kavanagh, Chapter 37), the authors made a case for applying low intensity (LI) cognitive behaviour therapy (CBT) to people with serious mental illness (SMI). As in other populations, LI CBT interventions typically deal with circumscribed problems or behaviours. LI CBT retains an emphasis on self-management, has restricted content and segment length, and does not necessarily require extensive CBT training. In applying these interventions to SMI, adjustments may be needed to address cognitive and symptomatic difficulties often faced by these groups. What may take a single session in a less affected population may require several sessions or a thematic application of the strategy within case management. In some cases, the LI CBT may begin to appear more like a high-intensity (HI) intervention, albeit simple and with many LI CBT characteristics still retained. So, if goal setting were introduced in one or two sessions, it could clearly be seen as an LI intervention. When applied to several different situations and across many sessions, it may be indistinguishable from a simple HI treatment, even if it retains the same format and is effectively applied by a practitioner with limited CBT training. ----- ----- In some ways, LI CBT should be well suited to case management of patients with SMI. treating staff typically have heavy workloads, and find it difficult to apply time-consuming treatments (Singh et al. 2003). LI CBT may allow provision of support to greater numbers of service users, and allow staff to spend more time on those who need intensive and sustained support. However, the introduction of any change in practice has to address significant challenges, and LI CBT is no exception. ----- ----- Many of the issues that we face in applying LI CBT to routine case management in a mnetal health service and their potential solutions are essentially the same as in a range of other problem domains (Turner and Sanders 2006)- and, indeed, are similar to those in any adoption of innovation (Rogers 2003). Over the last 20 years, several commentators have described barriers to implementing evidence-based innovations in mental health services (Corrigan et al. 1992; Deane et al. 2006; Kavanagh et al. 1993). The aim of the current chapter is to present a cognitive behavioural conceptualisation of problems and potential solutions for dissemination of LI CBT.

Mood and interpersonal functioning in heavy smokers

Male and female adult heavy smokers (n = 96) and non-smokers (n = 123) were compared on the Depression Anxiety Stress Scales (DASS), Adult Attachment Scale (AAS), Fear of Intimacy Scale (FIS), Negative Mood Regulation (NMR) Scale and Affect Intensity Measure (AIM). Compared with non-smokers, smokers scored significantly higher on DASS-Stress, DASS-Anxiety, and DASS-Depression, and significantly lower on NMR, AAS-Depend and AAS-Close. Smokers also scored marginally higher on FIS. Results suggest mood and relationship dysfunction in smokers, similar to the findings of a previous investigation of detoxified inpatients undergoing treatment for substance (alcohol, heroin, or methamphetamine) dependence.

Numerical solution of stress intensity factors of multiple cracks in great number with eigen COD boundary integral equations

Based on the eigen crack opening displacement (COD) boundary integral equations, a newly developed computational approach is proposed for the analysis of multiple crack problems. The eigen COD particularly refers to a crack in an infinite domain under fictitious traction acting on the crack surface. With the concept of eigen COD, the multiple cracks in great number can be solved by using the conventional displacement discontinuity boundary integral equations in an iterative fashion with a small size of system matrix. The interactions among cracks are dealt with by two parts according to the distances of cracks to the current crack. The strong effects of cracks in adjacent group are treated with the aid of the local Eshelby matrix derived from the traction BIEs in discrete form. While the relatively week effects of cracks in far-field group are treated in the iteration procedures. Numerical examples are provided for the stress intensity factors of multiple cracks, up to several thousands in number, with the proposed approach. By comparing with the analytical solutions in the literature as well as solutions of the dual boundary integral equations, the effectiveness and the efficiencies of the proposed approach are verified.

Cerebral cortex activation mapping upon electrical muscle stimulation by 32-channel time-domain functional near-infrared spectroscopy

The application of different EMS current thresholds on muscle activates not only the muscle but also peripheral sensory axons that send proprioceptive and pain signals to the cerebral cortex. A 32-channel time-domain fNIRS instrument was employed to map regional cortical activities under varied EMS current intensities applied on the right wrist extensor muscle. Eight healthy volunteers underwent four EMS at different current thresholds based on their individual maximal tolerated intensity (MTI), i.e., 10 % < 50 % < 100 % < over 100 % MTI. Time courses of the absolute oxygenated and deoxygenated hemoglobin concentrations primarily over the bilateral sensorimotor cortical (SMC) regions were extrapolated, and cortical activation maps were determined by general linear model using the NIRS-SPM software. The stimulation-induced wrist extension paradigm significantly increased activation of the contralateral SMC region according to the EMS intensities, while the ipsilateral SMC region showed no significant changes. This could be due in part to a nociceptive response to the higher EMS current intensities and result also from increased sensorimotor integration in these cortical regions.

Comparison between electrically-evoked and voluntary wrist movements on sensorimotor and prefrontal cortical activation : A multi-channel time domain fNIRS study

Neuromuscular electrical stimulation (NMES) has been consistently demonstrated to improve skeletal muscle function in neurological populations with movement disorders, such as poststroke and incomplete spinal cord injury (Vanderthommen and Duchateau, 2007). Recent research has documented that rapid, supraspinal central nervous system reorganisation/neuroplastic mechanisms are also implicated during NMES (Chipchase et al., 2011). Functional neuroimaging studies have shown NMES to activate a network of sub-cortical and cortical brain regions, including the sensorimotor (SMC) and prefrontal (PFC) cortex (Blickenstorfer et al., 2009; Han et al., 2003; Muthalib et al., 2012). A relationship between increase in SMC activation with increasing NMES current intensity up to motor threshold has been previously reported using functional MRI (Smith et al., 2003). However, since clinical neurorehabilitation programmes commonly utilise NMES current intensities above the motor threshold and up to the maximum tolerated current intensity (MTI), limited research has determined the cortical correlates of increasing NMES current intensity at or above MTI (Muthalib et al., 2012). In our previous study (Muthalib et al., 2012), we assessed contralateral PFC activation using 1-channel functional near infrared spectroscopy (fNIRS) during NMES of the elbow flexors by increasing current intensity from motor threshold to greater than MTI and showed a linear relationship between NMES current intensity and the level of PFC activation. However, the relationship between NMES current intensity and activation of the motor cortical network, including SMC and PFC, has not been clarified. Moreover, it is of scientific and clinical relevance to know how NMES affects the central nervous system, especially in comparison to voluntary (VOL) muscle activation. Therefore, the aim of this study was to utilise multi-channel time domain fNIRS to compare SMC and PFC activation between VOL and NMESevoked wrist extension movements.

A general approach to tune the vibration properties of the mounting system in the high-speed and heavy-duty engine

Majority of the current research on the mounting system has emphasised on the low/medium power engine, rare work has been reported for the high-speed and heavy-duty engine, the vibration characteristics of which exhibits significantly increased complexity and uncertainty. In this work, a general dynamics model was firstly established to describe the dynamic properties of a mounting system with various numbers of mounts. Then, this model was employed for the optimization of the mounting system. A modified Powell conjugate direction method was developed to improve the optimization efficiency. Basing on the optimization results obtained from the theoretical model, a mounting system was constructed for a V6 diesel engine. The experimental measurement of the vibration intensity of the mounting systems shows excellent agreement with the theoretical calculations, indicating the validity of the model. This dynamics model opens a new avenue in assessing and designing the mounting system for a high-speed and heavy-duty engine. On the other hand, the delineated dynamics model, and the optimization algorithm should find wide applications for other mounting systems, such as the power transmission system which usually has various uncertain mounts.

Modal parameters identification of heavy-haul railway RC bridges: Experience acquired

Traditionally, it is not easy to carry out tests to identify modal parameters from existing railway bridges because of the testing conditions and complicated nature of civil structures. A six year (2007-2012) research program was conducted to monitor a group of 25 railway bridges. One of the tasks was to devise guidelines for identifying their modal parameters. This paper presents the experience acquired from such identification. The modal analysis of four representative bridges of this group is reported, which include B5, B15, B20 and B58A, crossing the Carajás railway in northern Brazil using three different excitations sources: drop weight, free vibration after train passage, and ambient conditions. To extract the dynamic parameters from the recorded data, Stochastic Subspace Identification and Frequency Domain Decomposition methods were used. Finite-element models were constructed to facilitate the dynamic measurements. The results show good agreement between the measured and computed natural frequencies and mode shapes. The findings provide some guidelines on methods of excitation, record length of time, methods of modal analysis including the use of projected channel and harmonic detection, helping researchers and maintenance teams obtain good dynamic characteristics from measurement data.

Managing cattle grazing intensity: effects on soil organic matter and soil nitrogen

Extensive cattle grazing is the dominant land use in northern Australia. It has been suggested that grazing intensity and rainfall have profound effects on the dynamics of soil nutrients in northern Australia’s semi-arid rangelands. Previous studies have found positive, neutral and negative effects of grazing pressure on soil nutrients. These inconsistencies could be due to short-term experiments that do not capture the slow dynamics of some soil nutrients and the effects of interannual variability in rainfall. In a long-term cattle grazing trial in northern Australia on Brown Sodosol–Yellow Kandosol complex, we analysed soil organic matter and mineral nitrogen in surface soils (0–10 cm depth) 11, 12 and 16 years after trial establishment on experimental plots representing moderate stocking (stocked at the long-term carrying capacity for the region) and heavy stocking (stocked at twice the long-term carrying capacity). Higher soil organic matter was found under heavy stocking, although grazing treatment had little effect on mineral and total soil nitrogen. Interannual variability had a large effect on soil mineral nitrogen, but not on soil organic matter, suggesting that soil nitrogen levels observed in this soil complex may be affected by other indirect pathways, such as climate. The effect of interannual variability in rainfall and the effects of other soil types need to be explored further.

Acoustic emission technique for leak detection in an end shield of a pressurised heavy water reactor

This paper discusses a successful application of the Acoustic Emission Technique (AET) for the detection and location of leak paths present on an inaccessible side of an end shield of a Pressurised Heavy Water Reactor (PHWR). The methodology was based on the fact that air- and water-leak AE signals have different characteristic features. Baseline data was generated from a sound end shield of a PHWR for characterising the background noise. A mock-up end shield system with saw-cut leak paths was used to verify the validity of the methodology. It was found that air-leak signals under pressurisation (as low as 3 psi) could be detected by frequency domain analysis. Signals due to air leaks from various locations of defective end shield were acquired and analysed. It was possible to detect and locate leak paths. The presence of detected leak paths was further confirmed by an alternative test.