Single-cell functional analysis of parathyroid adenomas reveals distinct classes of calcium sensing behaviour in primary hyperparathyroidism.

Primary hyperparathyroidism (PHPT) is a common endocrine neoplastic disorder caused by a failure of calcium sensing secondary to tumour development in one or more of the parathyroid glands. Parathyroid adenomas are comprised of distinct cellular subpopulations of variable clonal status that exhibit differing degrees of calcium responsiveness. To gain a clearer understanding of the relationship among cellular identity, tumour composition and clinical biochemistry in PHPT, we developed a novel single cell platform for quantitative evaluation of calcium sensing behaviour in freshly resected human parathyroid tumour cells. Live-cell intracellular calcium flux was visualized through Fluo-4-AM epifluorescence, followed by in situ immunofluorescence detection of the calcium sensing receptor (CASR), a central component in the extracellular calcium signalling pathway. The reactivity of individual parathyroid tumour cells to extracellular calcium stimulus was highly variable, with discrete kinetic response patterns observed both between and among parathyroid tumour samples. CASR abundance was not an obligate determinant of calcium responsiveness. Calcium EC50 values from a series of parathyroid adenomas revealed that the tumours segregated into two distinct categories. One group manifested a mean EC50 of 2.40 mM (95% CI: 2.37-2.41), closely aligned to the established normal range. The second group was less responsive to calcium stimulus, with a mean EC50 of 3.61 mM (95% CI: 3.45-3.95). This binary distribution indicates the existence of a previously unappreciated biochemical sub-classification of PHPT tumours, possibly reflecting distinct etiological mechanisms. Recognition of quantitative differences in calcium sensing could have important implications for the clinical management of PHPT.

Structural and Functional Analysis of the Caspase –dependent and –independent Domains of the X-linked Inhibitor of Apoptosis Protein in Inflammatory Breast Cancer Tumor Biology

Inflammatory breast cancer (IBC) is an extremely rare but highly aggressive form of breast cancer characterized by the rapid development of therapeutic resistance leading to particularly poor survival. Our previous work focused on the elucidation of factors that mediate therapeutic resistance in IBC and identified increased expression of the anti-apoptotic protein, X-linked inhibitor of apoptosis protein (XIAP), to correlate with the development of resistance to chemotherapeutics. Although XIAP is classically thought of as an inhibitor of caspase activation, multiple studies have revealed that XIAP can also function as a signaling intermediate in numerous pathways. Based on preliminary evidence revealing high expression of XIAP in pre-treatment IBC cells rather than only subsequent to the development of resistance, we hypothesized that XIAP could play an important signaling role in IBC pathobiology outside of its heavily published apoptotic inhibition function. Further, based on our discovery of inhibition of chemotherapeutic efficacy, we postulated that XIAP overexpression might also play a role in resistance to other forms of therapy, such as immunotherapy. Finally, we posited that targeting of specific redox adaptive mechanisms, which are observed to be a significant barrier to successful treatment of IBC, could overcome therapeutic resistance and enhance the efficacy of chemo-, radio-, and immuno- therapies. To address these hypotheses our objectives were: 1. to determine a role for XIAP in IBC pathobiology and to elucidate the upstream regulators and downstream effectors of XIAP; 2. to evaluate and describe a role for XIAP in the inhibition of immunotherapy; and 3. to develop and characterize novel redox modulatory strategies that target identified mechanisms to prevent or reverse therapeutic resistance.

Using various genomic and proteomic approaches, combined with analysis of cellular viability, proliferation, and growth parameters both in vitro and in vivo, we demonstrate that XIAP plays a central role in both IBC pathobiology in a manner mostly independent of its role as a caspase-binding protein. Modulation of XIAP expression in cells derived from patients prior to any therapeutic intervention significantly altered key aspects IBC biology including, but not limited to: IBC-specific gene signatures; the tumorigenic capacity of tumor cells; and the metastatic phenotype of IBC, all of which are revealed to functionally hinge on XIAP-mediated NFκB activation, a robust molecular determinant of IBC. Identification of the mechanism of XIAP-mediated NFκB activation led to the characterization of novel peptide-based antagonist which was further used to identify that increased NFκB activation was responsible for redox adaptation previously observed in therapy-resistant IBC cells. Lastly, we describe the targeting of this XIAP-NFκB-ROS axis using a novel redox modulatory strategy both in vitro and in vivo. Together, the data presented here characterize a novel and crucial role for XIAP both in therapeutic resistance and the pathobiology of IBC; these results confirm our previous work in acquired therapeutic resistance and establish the feasibility of targeting XIAP-NFκB and the redox adaptive phenotype of IBC as a means to enhance survival of patients.

Functional analysis of the ATM-p53-p21 pathway in the LRF CLL4 trial: blockade at the level of p21 is associated with short response duration.

PURPOSE: This study sought to establish whether functional analysis of the ATM-p53-p21 pathway adds to the information provided by currently available prognostic factors in patients with chronic lymphocytic leukemia (CLL) requiring frontline chemotherapy. EXPERIMENTAL DESIGN: Cryopreserved blood mononuclear cells from 278 patients entering the LRF CLL4 trial comparing chlorambucil, fludarabine, and fludarabine plus cyclophosphamide were analyzed for ATM-p53-p21 pathway defects using an ex vivo functional assay that uses ionizing radiation to activate ATM and flow cytometry to measure upregulation of p53 and p21 proteins. Clinical endpoints were compared between groups of patients defined by their pathway status. RESULTS: ATM-p53-p21 pathway defects of four different types (A, B, C, and D) were identified in 194 of 278 (70%) samples. The type A defect (high constitutive p53 expression combined with impaired p21 upregulation) and the type C defect (impaired p21 upregulation despite an intact p53 response) were each associated with short progression-free survival. The type A defect was associated with chemoresistance, whereas the type C defect was associated with early relapse. As expected, the type A defect was strongly associated with TP53 deletion/mutation. In contrast, the type C defect was not associated with any of the other prognostic factors examined, including TP53/ATM deletion, TP53 mutation, and IGHV mutational status. Detection of the type C defect added to the prognostic information provided by TP53/ATM deletion, TP53 mutation, and IGHV status. CONCLUSION: Our findings implicate blockade of the ATM-p53-p21 pathway at the level of p21 as a hitherto unrecognized determinant of early disease recurrence following successful cytoreduction.

Time-Dependent Density Functional Molecular Orbital and Excited State Calculations on Bis(porphyrinyl)butadiynes in the Monocationic, Neutral, Monoanionic, and Dianionic Oxidation States

We report a theoretical study of the multiple oxidation states (1+, 0, 1−, and 2−) of a meso,meso-linked diporphyrin, namely bis[10,15,20-triphenylporphyrinatozinc(II)-5-yl]butadiyne (4), using Time-Dependent Density Functional Theory (TDDFT). The origin of electronic transitions of singlet excited states is discussed in comparison to experimental spectra for the corresponding oxidation states of the close analogue bis{10,15,20-tris[3‘,5‘-di-tert-butylphenyl]porphyrinatozinc(II)-5-yl}butadiyne (3). The latter were measured in previous work under in situ spectroelectrochemical conditions. Excitation energies and orbital compositions of the excited states were obtained for these large delocalized aromatic radicals, which are unique examples of organic mixed-valence systems. The radical cations and anions of butadiyne-bridged diporphyrins such as 3 display characteristic electronic absorption bands in the near-IR region, which have been successfully predicted with use of these computational methods. The radicals are clearly of the “fully delocalized” or Class III type. The key spectral features of the neutral and dianionic states were also reproduced, although due to the large size of these molecules, quantitative agreement of energies with observations is not as good in the blue end of the visible region. The TDDFT calculations are largely in accord with a previous empirical model for the spectra, which was based simplistically on one-electron transitions among the eight key frontier orbitals of the C4 (1,4-butadiyne) linked diporphyrins.

Investigating the links between muscle strength, sun exposure, dietary vitamin D intake and the vitamin D status of ambulatory older adults in South East Queensland

Vitamin D deficiency and insufficiency are now seen as a contemporary health problem in Australia with possible widespread health effects not limited to bone health1. Despite this, the Vitamin D status (measured as serum 25-hydroxyvitamin D (25(OH)D)) of ambulatory adults has been overlooked in this country. Serum 25(OH)D status is especially important among this group as studies have shown a link between Vitamin D and fall risk in older adults2. Limited data also exists on the contributions of sun exposure via ultraviolet radiation and dietary intake to serum 25(OH)D status in this population. The aims of this project were to assess the serum 25(OH)D status of a group of older ambulatory adults in South East Queensland, to assess the association between their serum 25(OH)D status and functional measures as possible indicators of fall risk, obtain data on the sources of Vitamin D in this population and assess whether this intake was related to serum 25(OH)D status and describe sun protection and exposure behaviors in this group and investigate whether a relationship existed between these and serum 25(OH)D status. The collection of this data assists in addressing key gaps identified in the literature with regard to this population group and their Vitamin D status in Australia. A representative convenience sample of participants (N=47) over 55 years of age was recruited for this cross-sectional, exploratory study which was undertaken in December 2007 in south-east Queensland (Brisbane and Sunshine coast). Participants were required to complete a sun exposure questionnaire in addition to a Calcium and Vitamin D food frequency questionnaire. Timed up and go and handgrip dynamometry tests were used to examine functional capacity. Serum 25(OH)D status and blood measures of Calcium, Phosphorus and Albumin were determined through blood tests. The Mean and Median serum 25-Hydroxyvitamin D (25(OH)D) for all participants in this study was 85.8nmol/L (Standard Deviation 29.7nmol/L) and 81.0nmol/L (Range 22-158nmol/L), respectively. Analysis at the bivariate level revealed a statistically significant relationship between serum 25(OH)D status and location, with participants living on the Sunshine Coast having a mean serum 25(OH)D status 21.3nmol/L higher than participants living in Brisbane (p=0.014). While at the descriptive level there was an apparent trend towards higher outdoor exposure and increasing levels of serum 25(OH)D, no statistically significant associations between the sun measures of outdoor exposure, sun protection behaviors and phenotypic characteristics and serum 25(OH)D status were observed. Intake of both Calcium and Vitamin D was low in this sample with sixty-eight (68%) of participants not meeting the Estimated Average Requirements (EAR) for Calcium (Median=771.0mg; Range=218.0-2616.0mg), while eighty-seven (87%) did not meet the Adequate Intake for Vitamin D (Median=4.46ug; Range=0.13-30.0ug). This raises the question of how realistic meeting the new Adequate Intakes for Vitamin D is, when there is such a low level of Vitamin D fortification in this country. However, participants meeting the Adequate Intake (AI) for Vitamin D were observed to have a significantly higher serum 25(OH)D status compared to those not meeting the AI for Vitamin D (p=0.036), showing that meeting the AI for Vitamin D may play a significant role in determining Vitamin D status in this population. By stratifying our data by categories of outdoor exposure time, a trend was observed between increased importance of Vitamin D dietary intake as a possible determinant of serum 25(OH)D status in participants with lower outdoor exposures. While a trend towards higher Timed Up and Go scores in participants with higher 25(OH) D status was seen, this was only significant for females (p=0.014). Handgrip strength showed statistically significant association with serum 25(OH)D status. The high serum 25(OH)D status in our sample almost certainly explains the limited relationship between functional measures and serum 25(OH)D. However, the observation of an association between slower Time Up and Go speeds, and lower serum 25(OH)D levels, even with a small sample size, is significant as slower Timed Up and Go speeds have been associated with increased fall risk in older adults3. Multivariable regression analysis revealed Location as the only significant determinant of serum 25(OH)D status at p=0.014, with trends (p=>0.1) for higher serum 25(OH)D being shown for participants that met the AI for Vitamin D and rated themselves as having a higher health status. The results of this exploratory study show that 93.6% of participants had adequate 25(OH)D status-possibly due to measurement being taken in the summer season and the convenience nature of the sample. However, many participants do not meet their dietary Calcium and Vitamin D requirements, which may indicate inadequate intake of these nutrients in older Australians and a higher risk of osteoporosis. The relationship between serum 25(OH)D and functional measures in this population also requires further study, especially in older adults displaying Vitamin D insufficiency or deficiency.

Evaluation of functional performance in commercial buildings

This report fully summarises a project designed to enhance commercial real estate performance within both operational and investment contexts through the development of a model aimed at supporting improved decision-making. The model is based on a risk adjusted discounted cash flow, providing a valuable toolkit for building managers, owners, and potential investors for evaluating individual building performance in terms of financial, social and environmental criteria over the complete life-cycle of the asset. The ‘triple bottom line’ approach to the evaluation of commercial property has much significance for the administrators of public property portfolios in particular. It also has applications more generally for the wider real estate industry given that the advent of ‘green’ construction requires new methods for evaluating both new and existing building stocks. The research is unique in that it focuses on the accuracy of the input variables required for the model. These key variables were largely determined by market-based research and an extensive literature review, and have been fine-tuned with extensive testing. In essence, the project has considered probability-based risk analysis techniques that required market-based assessment. The projections listed in the partner engineers’ building audit reports of the four case study buildings were fed into the property evaluation model developed by the research team. The results are strongly consistent with previously existing, less robust evaluation techniques. And importantly, this model pioneers an approach for taking full account of the triple bottom line, establishing a benchmark for related research to follow. The project’s industry partners expressed a high degree of satisfaction with the project outcomes at a recent demonstration seminar. The project in its existing form has not been geared towards commercial applications but it is anticipated that QDPW and other industry partners will benefit greatly by using this tool for the performance evaluation of property assets. The project met the objectives of the original proposal as well as all the specified milestones. The project has been completed within budget and on time. This research project has achieved the objective by establishing research foci on the model structure, the key input variable identification, the drivers of the relevant property markets, the determinants of the key variables (Research Engine no.1), the examination of risk measurement, the incorporation of risk simulation exercises (Research Engine no.2), the importance of both environmental and social factors and, finally the impact of the triple bottom line measures on the asset (Research Engine no. 3).

Poor functional health literacy : the silent disability for older people

In this information age, people are confronted by verbal, visual and written information. This is especially important in the health field, where information is needed to follow directions, understand prescriptions and undertake preventive behaviours. If provided in written form, much of this information may be inaccessible to people who cannot adequately read. Although poor literacy skills affect all groups in the population, older adults with fewer years of education seem to be particularly disadvantaged by an increasing reliance on written communication of health information. With older age comes a higher risk of illness and disability and a greater potential need to access the health system. As a result, poor literacy skills of older individuals may directly impact their health status. This paper explores the link between functional literacy and health, particularly for the older population, provides strategies to practitioners for the management of this problem, and suggests research initiatives in this area.

Estimating total cross sections from neutron-nucleus collisions using a simple functional form

Total cross sections for neutron scattering from nuclei, with energies ranging from 10 to 600 MeV and from many nuclei spanning the mass range 6Li to 238U, have been analyzed using a simple, three-parameter, functional form. The calculated cross sections are compared with results obtained by using microscopic (g-folding) optical potentials as well as with experimental data. The functional form reproduces those total cross sections very well. When allowance is made for Ramsauer-like effects in the scattering, the parameters of the functional form required vary smoothly with energy and target mass. They too can be represented by functions of energy and mass.

Vision, functional and cognitive determinants of motor vehicle incidents in older drivers

Background: The proportion of older individuals in the driving population is predicted to increase in the next 50 years. This has important implications for driving safety as abilities which are important for safe driving, such as vision (which accounts for the majority of the sensory input required for driving), processing ability and cognition have been shown to decline with age. The current methods employed for screening older drivers upon re-licensure are also vision based. This study, which investigated social, behavioural and professional aspects involved with older drivers, aimed to determine: (i) if the current visual standards in place for testing upon re-licensure are effective in reducing the older driver fatality rate in Australia; (ii) if the recommended visual standards are actually implemented as part of the testing procedures by Australian optometrists; and (iii) if there are other non-standardised tests which may be better at predicting the on-road incident-risk (including near misses and minor incidents) in older drivers than those tests recommended in the standards. Methods: For the first phase of the study, state-based age- and gender-stratified numbers of older driver fatalities for 2000-2003 were obtained from the Australian Transportation Safety Bureau database. Poisson regression analyses of fatality rates were considered by renewal frequency and jurisdiction (as separate models), adjusting for possible confounding variables of age, gender and year. For the second phase, all practising optometrists in Australia were surveyed on the vision tests they conduct in consultations relating to driving and their knowledge of vision requirements for older drivers. Finally, for the third phase of the study to investigate determinants of on-road incident risk, a stratified random sample of 600 Brisbane residents aged 60 years and were selected and invited to participate using an introductory letter explaining the project requirements. In order to capture the number and type of road incidents which occurred for each participant over 12 months (including near misses and minor incidents), an important component of the prospective research study was the development and validation of a driving diary. The diary was a tool in which incidents that occurred could be logged at that time (or very close in time to which they occurred) and thus, in comparison with relying on participant memory over time, recall bias of incident occurrence was minimised. Association between all visual tests, cognition and scores obtained for non-standard functional tests with retrospective and prospective incident occurrence was investigated. Results: In the first phase,rivers aged 60-69 years had a 33% lower fatality risk (Rate Ratio [RR] = 0.75, 95% CI 0.32-1.77) in states with vision testing upon re-licensure compared with states with no vision testing upon re-licensure, however, because the CIs are wide, crossing 1.00, this result should be regarded with caution. However, overall fatality rates and fatality rates for those aged 70 years and older (RR=1.17, CI 0.64-2.13) did not differ between states with and without license renewal procedures, indicating no apparent benefit in vision testing legislation. For the second phase of the study, nearly all optometrists measured visual acuity (VA) as part of a vision assessment for re-licensing, however, 20% of optometrists did not perform any visual field (VF) testing and only 20% routinely performed automated VF on older drivers, despite the standards for licensing advocating automated VF as part of the vision standard. This demonstrates the need for more effective communication between the policy makers and those responsible for carrying out the standards. It may also indicate that the overall higher driver fatality rate in jurisdictions with vision testing requirements is resultant as the tests recommended by the standards are only partially being conducted by optometrists. Hence a standardised protocol for the screening of older drivers for re-licensure across the nation must be established. The opinions of Australian optometrists with regard to the responsibility of reporting older drivers who fail to meet the licensing standards highlighted the conflict between maintaining patient confidentiality or upholding public safety. Mandatory reporting requirements of those drivers who fail to reach the standards necessary for driving would minimise potential conflict between the patient and their practitioner, and help maintain patient trust and goodwill. The final phase of the PhD program investigated the efficacy of vision, functional and cognitive tests to discriminate between at-risk and safe older drivers. Nearly 80% of the participants experienced an incident of some form over the prospective 12 months, with the total incident rate being 4.65/10 000 km. Sixty-three percent reported having a near miss and 28% had a minor incident. The results from the prospective diary study indicate that the current vision screening tests (VA and VF) used for re-licensure do not accurately predict older drivers who are at increased odds of having an on-road incident. However, the variation in visual measurements of the cohort was narrow, also affecting the results seen with the visual functon questionnaires. Hence a larger cohort with greater variability should be considered for a future study. A slightly lower cognitive level (as measured with the Mini-Mental State Examination [MMSE]) did show an association with incident involvement as did slower reaction time (RT), however the Useful-Field-of-View (UFOV) provided the most compelling results of the study. Cut-off values of UFOV processing (>23.3ms), divided attention (>113ms), selective attention (>258ms) and overall score (moderate/ high/ very high risk) were effective in determining older drivers at increased odds of having any on-road incident and the occurrence of minor incidents. Discussion: The results have shown that for the 60-69 year age-group, there is a potential benefit in testing vision upon licence renewal. However, overall fatality rates and fatality rates for those aged 70 years and older indicated no benefit in vision testing legislation and suggests a need for inclusion of screening tests which better predict on-road incidents. Although VA is routinely performed by Australian optometrists on older drivers renewing their licence, VF is not. Therefore there is a need for a protocol to be developed and administered which would result in standardised methods conducted throughout the nation for the screening of older drivers upon re-licensure. Communication between the community, policy makers and those conducting the protocol should be maximised. By implementing a standardised screening protocol which incorporates a level of mandatory reporting by the practitioner, the ethical dilemma of breaching patient confidentiality would also be resolved. The tests which should be included in this screening protocol, however, cannot solely be ones which have been implemented in the past. In this investigation, RT, MMSE and UFOV were shown to be better determinants of on-road incidents in older drivers than VA and VF, however, as previously mentioned, there was a lack of variability in visual status within the cohort. Nevertheless, it is the recommendation from this investigation, that subject to appropriate sensitivity and specificity being demonstrated in the future using a cohort with wider variation in vision, functional performance and cognition, these tests of cognition and information processing should be added to the current protocol for the screening of older drivers which may be conducted at licensing centres across the nation.

Functional outcome and usage of the prosthesis of lower limb amputees fitted with osseointegrated fixation

This study established that the core principle underlying categorisation of activities have the potential to provide more comprehensive outcomes than the recognition of activities because it takes into consideration activities other than directional locomotion.

Functional outcome of transfemoral amputees fitted with an osseointegrated fixation : temporal gait characteristics

The purpose of this study was to characterise the functional outcome of 12 transfemoral amputees fitted with osseointegrated fixation using temporal gait characteristics. The objectives were (A) to present the cadence, duration of gait cycle, support and swing phases with an emphasis on the stride-to-stride and participant-to-participant variability, and (B) to compare these temporal variables with normative data extracted from the literature focusing on transfemoral amputees fitted with a socket and able-bodied participants. The temporal variables were extracted from the load applied on the residuum during straight level walking, which was collected at 200 Hz by a transducer. A total of 613 strides were assessed. The cadence (46±4 strides/min), the duration of the gait cycle (1.29±0.11 s), support (0.73±0.07 s, 57±3% of CG) and swing (0.56±0.07 s, 43±3% of GC) phases of the participants were 2% quicker, 3%, 6% shorter and 1% longer than transfemoral amputees using a socket as well as 11% slower, 9%, 6% and 13% longer than able-bodied, respectively. All combined, the results indicated that the fitting of an osseointegrated fixation has enabled this group of amputees to restore their locomotion with a highly functional level. Further longitudinal and cross-sectional studies would be required to confirm these outcomes. Nonetheless, the data presented can be used as benchmark for future comparisons. It can also be used as input in generic algorithms using templates of patterns of loading to recognise activities of daily living and to detect falls.