Glutamine repeat variants in human RUNX2 associated with decreased femoral neck BMD, broadband ultrasound attenuation and target gene transactivation

RUNX2 is an essential transcription factor required for skeletal development and cartilage formation. Haploinsufficiency of RUNX2 leads to cleidocranial displaysia (CCD) a skeletal disorder characterised by gross dysgenesis of bones particularly those derived from intramembranous bone formation. A notable feature of the RUNX2 protein is the polyglutamine and polyalanine (23Q/17A) domain coded by a repeat sequence. Since none of the known mutations causing CCD characterised to date map in the glutamine repeat region, we hypothesised that Q-repeat mutations may be related to a more subtle bone phenotype. We screened subjects derived from four normal populations for Q-repeat variants. A total of 22 subjects were identified who were heterozygous for a wild type allele and a Q-repeat variant allele: (15Q, 16Q, 18Q and 30Q). Although not every subject had data for all measures, Q-repeat variants had a significant deficit in BMD with an average decrease of 0.7SD measured over 12 BMD-related parameters (p = 0.005). Femoral neck BMD was measured in all subjects (&minus;0.6SD, p = 0.0007). The transactivation function of RUNX2 was determined for 16Q and 30Q alleles using a reporter gene assay. 16Q and 30Q alleles displayed significantly lower transactivation function compared to wild type (23Q). Our analysis has identified novel Q-repeat mutations that occur at a collective frequency of about 0.4%. These mutations significantly alter BMD and display impaired transactivation function, introducing a new class of functionally relevant RUNX2 mutants.<br /

National scale evaluation of the InVEST nutrient retention model in the United Kingdom

A wide variety of tools aim to support decision making by modelling, mapping and quantifying ecosystem services. If decisions are to be properly informed, the accuracy and potential limitations of these tools must be well understood. However, dedicated studies evaluating ecosystem service models against empirical data are rare, especially over large areas. In this paper, we report on the national-scale assessment of a new ecosystem service model for nutrient delivery and retention, the InVEST Nutrient Delivery Ratio model. For 36 river catchments across the UK, we modelled total catchment export of phosphorus (P) and/or nitrogen (N) and compared model outputs to measurements derived from empirical water chemistry data. The model performed well in terms of relative magnitude of nutrient export among catchments (best Spearman’s rank correlation for N and P, respectively: 0.81 and 0.88). However, there was wide variation among catchments in the accuracy of the model, and absolute values of nutrient exports frequently showed high percentage differences between modelled and empirically-derived exports (best median absolute percentage difference for N and P, respectively: ± 64%, ± 44%). The model also showed a high degree of sensitivity to nutrient loads and hydrologic routing input parameters and these sensitivities varied among catchments. These results suggest that the InVEST model can provide valuable information on nutrient fluxes to decision makers, especially in terms of relative differences among catchments. However, caution is needed if using the absolute modelled values for decision-making. Our study also suggests particular attention should be paid to researching input nutrient loadings and retentions, and the selection of appropriate input data resolutions and threshold flow accumulation values. Our results also highlight how availability of empirical data can improve model calibration and performance assessment and reinforce the need to include such data in ecosystem service modelling studies

Re-Evaluation of the Action Potential Upstroke Velocity as a Measure of the Na+ Current in Cardiac Myocytes at Physiological Conditions

Background: The SCN5A encoded sodium current (INa) generates the action potential (AP) upstroke and is a major determinant of AP characteristics and AP propagation in cardiac myocytes. Unfortunately, in cardiac myocytes, investigation of kinetic properties of INa with near-physiological ion concentrations and temperature is technically challenging due to the large amplitude and rapidly activating nature of INa, which may seriously hamper the quality of voltage control over the membrane. We hypothesized that the alternating voltage clamp-current clamp (VC/CC) technique might provide an alternative to traditional voltage clamp (VC) technique for the determination of INa properties under physiological conditions. Principal Findings: We studied INa under close-to-physiological conditions by VC technique in SCN5A cDNA-transfected HEK cells or by alternating VC/CC technique in both SCN5A cDNA-transfected HEK cells and rabbit left ventricular myocytes. In these experiments, peak INa during a depolarizing VC step or maximal upstroke velocity, dV/dtmax, during VC/CC served as an indicator of available INa. In HEK cells, biophysical properties of INa, including current density, voltage dependent (in)activation, development of inactivation, and recovery from inactivation, were highly similar in VC and VC/CC experiments. As an application of the VC/CC technique we studied INa in left ventricular myocytes isolated from control or failing rabbit hearts

The contribution of refractoriness to arrhythmic substrate in hypokalemic Langendorff-perfused murine hearts

The clinical effects of hypokalemia including action potential prolongation and arrhythmogenicity suppressible by lidocaine were reproduced in hypokalemic (3.0 mM K(+)) Langendorff-perfused murine hearts before and after exposure to lidocaine (10 μM). Novel limiting criteria for local and transmural, epicardial, and endocardial re-excitation involving action potential duration (at 90% repolarization, APD(90)), ventricular effective refractory period (VERP), and transmural conduction time (Δlatency), where appropriate, were applied to normokalemic (5.2 mM K(+)) and hypokalemic hearts. Hypokalemia increased epicardial APD(90) from 46.6 ± 1.2 to 53.1 ± 0.7 ms yet decreased epicardial VERP from 41 ± 4 to 29 ± 1 ms, left endocardial APD(90) unchanged (58.2 ± 3.7 to 56.9 ± 4.0 ms) yet decreased endocardial VERP from 48 ± 4 to 29 ± 2 ms, and left Δlatency unchanged (1.6 ± 1.4 to 1.1 ± 1.1 ms; eight normokalemic and five hypokalemic hearts). These findings precisely matched computational predictions based on previous reports of altered ion channel gating and membrane hyperpolarization. Hypokalemia thus shifted all re-excitation criteria in the positive direction. In contrast, hypokalemia spared epicardial APD(90) (54.8 ± 2.7 to 60.6 ± 2.7 ms), epicardial VERP (84 ± 5 to 81 ± 7 ms), endocardial APD(90) (56.6 ± 4.2 to 63.7 ± 6.4 ms), endocardial VERP (80 ± 2 to 84 ± 4 ms), and Δlatency (12.5 ± 6.2 to 7.6 ± 3.4 ms; five hearts in each case) in lidocaine-treated hearts. Exposure to lidocaine thus consistently shifted all re-excitation criteria in the negative direction, again precisely agreeing with the arrhythmogenic findings. In contrast, established analyses invoking transmural dispersion of repolarization failed to account for any of these findings. We thus establish novel, more general, criteria predictive of arrhythmogenicity that may be particularly useful where APD(90) might diverge sharply from VERP

The relation of Vmax to INa, GNa, and h infinity in a model of the cardiac Purkinje fiber.

The inward sodium current in cardiac muscle is difficult to study by voltage clamp methods, so various indirect experimental measures have been used to obtain insight into its characteristics. These methods depend on the relationship between maximal upstroke velocity of the action potential (Vmax) and the sodium current (INa), usually defined in terms of the Hodgkin-Huxley model. These relationships were explored using an adaptation of this model to cardiac Purkinje fibers. In general Vmax corresponded to INa, and it could be used to determine the relationship of membrane potential to GNa, and h infinity. The results, however, depended on the method of stimulation of the action potential, and an optimal stimulation method was determined. A commonly used experimental technique called "membrane responsiveness" was shown to distort seriously the properties of steady-state gating inactivation that is supposed to measure. Estimation of the changes in maximal sodium conductance, such as those produced by tetrodotoxin (TTX), would be accurately measured. Some experimental results have indicated a voltage-dependent effect of TTX. Characteristics of the measures of TTX effect under those conditions were illustrated. In summary, calculations with a model of the cardiac Purkinje fiber action potential provide insight into the accuracy of certain experimental methods using maximal upstroke velocity as a measure of INa, and cast doubt on other experimental methods, such as membrane responsiveness