Structure-function analysis of RAMP1-RAMP3 chimeras

The role of receptor activity modifying protein 1 (RAMP1) in forming receptors with the calcitonin receptor-like receptor (CLR) and the calcitonin receptor (CTR) was examined by producing chimeras between RAMP1 and RAMP3. RAMPs have three extracellular helices. Exchange of helix 1 of the RAMPs or residues 62-69 in helix 2 greatly reduced CLR trafficking (a marker for CLR association). Modeling suggests that these exchanges alter the CLR recognition site on RAMP1, which is more exposed than on RAMP3. Exchange of residues 86-89 of RAMP1 had no effect on the trafficking of CLR but reduced the potency of human (h) alphaCGRP and adrenomedullin. However, these alterations to RAMP1 had no effect on the potency of hbetaCGRP. These residues of RAMP1 lie at the junction of helix 3 and its connecting loop with helix 2. Modeling suggests that the loop is more exposed in RAMP1 than RAMP3; it may play an important role in peptide binding, either directly or indirectly. Exchange of residues 90-94 of RAMP1 caused a modest reduction in CLR expression and a 15-fold decrease in CGRP potency. It is unlikely that the decrease in expression is enough to explain the reduction in potency, and so these may have dual roles in recognizing CLR and CGRP. For CTR, only 6 out of 26 chimeras covering the extracellular part of RAMP1 did not reduce agonist potency. Thus the association of CTR with RAMP1 seems more sensitive to changes in RAMP1 structure induced by the chimeras than is CLR.

Identification of nonlinear systems by correlation using pseudorandom signals

This thesis is concerned with the measurement of the characteristics of nonlinear systems by crosscorrelation, using pseudorandom input signals based on m sequences. The systems are characterised by Volterra series, and analytical expressions relating the rth order Volterra kernel to r-dimensional crosscorrelation measurements are derived. It is shown that the two-dimensional crosscorrelation measurements are related to the corresponding second order kernel values by a set of equations which may be structured into a number of independent subsets. The m sequence properties determine how the maximum order of the subsets for off-diagonal values is related to the upper bound of the arguments for nonzero kernel values. The upper bound of the arguments is used as a performance index, and the performance of antisymmetric pseudorandom binary, ternary and quinary signals is investigated. The performance indices obtained above are small in relation to the periods of the corresponding signals. To achieve higher performance with ternary signals, a method is proposed for combining the estimates of the second order kernel values so that the effects of some of the undesirable nonzero values in the fourth order autocorrelation function of the input signal are removed. The identification of the dynamics of two-input, single-output systems with multiplicative nonlinearity is investigated. It is shown that the characteristics of such a system may be determined by crosscorrelation experiments using phase-shifted versions of a common signal as inputs. The effects of nonlinearities on the estimates of system weighting functions obtained by crosscorrelation are also investigated. Results obtained by correlation testing of an industrial process are presented, and the differences between theoretical and experimental results discussed for this case;

Cognition, ocular accommodation, and cardiovascular function in emmetropes and late-onset myopes

PURPOSE. To investigate objectively and noninvasively the role of cognitive demand on autonomic control of systemic cardiovascular and ocular accommodative responses in emmetropes and myopes of late-onset. METHODS. Sixteen subjects (10 men, 6 women) aged between 18 and 34 years (mean ± SD: 22.6 ± 4.4 years), eight emmetropes (EMMs; mean spherical equivalent [MSE] refractive error ± SD: 0.05 ± 0.24 D) and eight with late-onset myopia (LOMs; MSE ± SD: -3.66 ± 2.31 D) participated in the study. Subjects viewed stationary numerical digits monocularly within a Badal optical system (at both 0.0 and -3.0 D) while performing a two-alternative, forced-choice paradigm that matched cognitive loading across subjects. Five individually matched cognitive levels of increasing difficulty were used in random order for each subject. Five 20-second, continuous-objective recordings of the accommodative response measured with an open-view infrared autorefractor were obtained for each cognitive level, whereas simultaneous measurement of heart rate was continuously recorded with a finger-mounted piezoelectric pulse transducer for 5 minutes. Fast Fourier transformation of cardiovascular function allowed the relative power of the autonomic components to be assessed in the frequency domain, whereas heart period gave an indication of the time-domain response. RESULTS. Increasing the cognitive demand led to a significant reduction in the accommodative response in all subjects (0.0 D: by -0.35 ± 0.33 D; -3.0 D: by -0.31 ± 0.40 D, P < 0.001). The greater lag of LOMs compared with EMMs was not significant (P = 0.07) at both distance (0.38 ± 0.35 D) and near (0.14 ± 0.42 D). Mean heart period reduced with increasing levels of workload (P < 0.0005). LOMs exhibited a relative elevation in sympathetic system activity compared to EMMs. Within refractive groups, however, accommodative shifts with increasing cognition correlated with parasympathetic activity (r = 0.99, P < 0.001), more than with sympathetic activity (r = 0.62, P > 0.05). CONCLUSIONS. In an equivalent workload paradigm, increasing cognitive demand caused a reduction in accommodative response that was attributable principally to a concurrent reduction in the relative power of the parasympathetic component of the autonomic nervous system (ANS). The disparity in accommodative response between EMMs and LOMs, however, appears to be augmented by changes in the sympathetic nervous component of the systemic ANS. Copyright © Association for Research in Vision and Ophthalmology.

3D reconstruction of complex dielectric function of the glass during the femtosecond micro-fabrication

We measure complex amplitude of scattered wave in the far field, and justify theoretically and numerically solution of the inverse scattering problem. This allows single-shot reconstructing of dielectric function distribution during direct femtosecond laser micro-fabrication.

A randomized controlled trial of the effect of thyroxine replacement on cognitive function in community-living elderly subjects with subclinical hypothyroidism: the Birmingham elderly thyroid study

Context: Subclinical hypothyroidism (SCH) and cognitive dysfunction are both common in the elderly and have been linked. It is important to determine whether T4 replacement therapy in SCH confers cognitive benefit. Objective: Our objective was to determine whether administration of T4 replacement to achieve biochemical euthyroidism in subjects with SCH improves cognitive function. Design and Setting: We conducted a double-blind placebo-controlled randomized controlled trial in the context of United Kingdom primary care. Patients: Ninety-four subjects aged 65 yr and over (57 females, 37 males) with SCH were recruited from a population of 147 identified by screening. Intervention: T4 or placebo was given at an initial dosage of one tablet of either placebo or 25 µg T4 per day for 12 months. Thyroid function tests were performed at 8-weekly intervals with dosage adjusted in one-tablet increments to achieve TSH within the reference range for subjects in treatment arm. Fifty-two subjects received T4 (31 females, 21 males; mean age 73.5 yr, range 65–94 yr); 42 subjects received placebo (26 females, 16 males; mean age 74.2 yr, 66–84 yr). Main Outcome Measures: Mini-Mental State Examination, Middlesex Elderly Assessment of Mental State (covering orientation, learning, memory, numeracy, perception, attention, and language skills), and Trail-Making A and B were administered. Results: Eighty-two percent and 84% in the T4 group achieved euthyroidism at 6- and 12-month intervals, respectively. Cognitive function scores at baseline and 6 and 12 months were as follows: Mini-Mental State Examination T4 group, 28.26, 28.9, and 28.28, and placebo group, 28.17, 27.82, and 28.25 [not significant (NS)]; Middlesex Elderly Assessment of Mental State T4 group, 11.72, 11.67, and 11.78, and placebo group, 11.21, 11.47, and 11.44 (NS); Trail-Making A T4 group, 45.72, 47.65, and 44.52, and placebo group, 50.29, 49.00, and 46.97 (NS); and Trail-Making B T4 group, 110.57, 106.61, and 96.67, and placebo group, 131.46, 119.13, and 108.38 (NS). Linear mixed-model analysis demonstrated no significant changes in any of the measures of cognitive function over time and no between-group difference in cognitive scores at 6 and 12 months. Conclusions: This RCT provides no evidence for treating elderly subjects with SCH with T4 replacement therapy to improve cognitive function.

3D Reconstruction of the complex dielectric function of glass during femtosecond laser micro-fabrication

We report on a new technique to reconstruct the 3D dielectric function change in transparent dielectric materials and the application of the technique for on-line monitoring of refractive index modification in BK7 glass during direct femtosecond laser microfabrication. The complex optical field scattered from the modified region is measured using two-beam, single-shot interferogram and the distribution of the modified refractive index is reconstructed by numerically solving the inverse scattering problem in Born approximation. The optical configuration suggested is further development of digital holographic microscopy (DHM). It takes advantage of high spatial resolution and almost the same optical paths for both interfering beams, and allows ultrafast time resolution.

Contrast summation across eyes and space is revealed along the entire dipper function by a "Swiss cheese" stimulus.

Previous contrast discrimination experiments have shown that luminance contrast is summed across ocular (T. S. Meese, M. A. Georgeson, & D. H. Baker, 2006) and spatial (T. S. Meese & R. J. Summers, 2007) dimensions at threshold and above. However, is this process sufficiently general to operate across the conjunction of eyes and space? Here we used a "Swiss cheese" stimulus where the blurred "holes" in sine-wave carriers were of equal area to the blurred target ("cheese") regions. The locations of the target regions in the monocular image pairs were interdigitated across eyes such that their binocular sum was a uniform grating. When pedestal contrasts were above threshold, the monocular neural images contained strong evidence that the high-contrast regions in the two eyes did not overlap. Nevertheless, sensitivity to dual contrast increments (i.e., to contrast increments in different locations in the two eyes) was a factor of ∼1.7 greater than to single increments (i.e., increments in a single eye), comparable with conventional binocular summation. This provides evidence for a contiguous area summation process that operates at all contrasts and is influenced little, if at all, by eye of origin. A three-stage model of contrast gain control fitted the results and possessed the properties of ocularity invariance and area invariance owing to its cascade of normalization stages. The implications for a population code for pattern size are discussed.

The development of calibration and adaptive filtering procedures for neuromagnetic studies of human visual function

This thesis first considers the calibration and signal processing requirements of a neuromagnetometer for the measurement of human visual function. Gradiometer calibration using straight wire grids is examined and optimal grid configurations determined, given realistic constructional tolerances. Simulations show that for gradiometer balance of 1:104 and wire spacing error of 0.25mm the achievable calibration accuracy of gain is 0.3%, of position is 0.3mm and of orientation is 0.6°. Practical results with a 19-channel 2nd-order gradiometer based system exceed this performance. The real-time application of adaptive reference noise cancellation filtering to running-average evoked response data is examined. In the steady state, the filter can be assumed to be driven by a non-stationary step input arising at epoch boundaries. Based on empirical measures of this driving step an optimal progression for the filter time constant is proposed which improves upon fixed time constant filter performance. The incorporation of the time-derivatives of the reference channels was found to improve the performance of the adaptive filtering algorithm by 15-20% for unaveraged data, falling to 5% with averaging. The thesis concludes with a neuromagnetic investigation of evoked cortical responses to chromatic and luminance grating stimuli. The global magnetic field power of evoked responses to the onset of sinusoidal gratings was shown to have distinct chromatic and luminance sensitive components. Analysis of the results, using a single equivalent current dipole model, shows that these components arise from activity within two distinct cortical locations. Co-registration of the resulting current source localisations with MRI shows a chromatically responsive area lying along the midline within the calcarine fissure, possibly extending onto the lingual and cuneal gyri. It is postulated that this area is the human homologue of the primate cortical area V4.

Abnormal retinal vascular function and lipid levels in a sample of healthy UK South Asians

Background/aims To investigate ethnic differences in retinal vascular function and their relationship to traditional risk indicators for cardiovascular disease (CVD). Methods A total of 90 normoglycaemic subjects (45 South Asian (SA) and 45 age- and gender-matched white Europeans (WEs)) were recruited for the present study. Retinal vessel reactivity to flickering light was assessed by means of the dynamic retinal vessel analyser according to a modified protocol. Fasting plasma glucose, triglycerides (TG), total, LDL and HDL cholesterol were also measured in all individuals. Results SA individuals showed higher fasting triglyceride (p=0.001) and lower HDL levels (p=0.007), leading to a higher TG:HDL-C ratio (p=0.001) than age-matched WE subjects. Additionally, in SAs, the retinal arterial reaction time in response to flicker stimulation was significantly longer in the last flicker cycle than in the WEs (p=0.039), and this change correlated positively with measured plasma TG levels (r=0.60; p=0.01). No such relationship was observed in the WEs (p>0.05). Conclusion Even in the absence of overt vascular disease, in otherwise healthy SAs there are potential signs of retinal vascular function impairment that correlates with established plasma markers for CVD risk.

Structure-function analysis of amino acid 74 of human RAMP1 and RAMP3 and its role in peptide interactions with adrenomedullin and calcitonin gene-related peptide receptors

The receptors for calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) are complexes of the calcitonin receptor-like receptor (CLR) and receptor activity-modifying proteins (RAMP). The CGRP receptor is a CLR/RAMP1 pairing whereas CLR/RAMP2 and CLR/RAMP3 constitute two subtypes of AM receptor: AM(1) and AM(2), respectively. Previous studies identified Glu74 in RAMP3 to be important for AM binding and potency. To further understand the importance of this residue and its equivalent in RAMP1 (Trp74) we substituted the native amino acids with several others. In RAMP3, these were Trp, Phe, Tyr, Ala, Ser, Thr, Arg and Asn; in RAMP1, Glu, Phe, Tyr, Ala and Asn substitutions were made. The mutant RAMPs were co-expressed with CLR in Cos7 cells; receptor function in response to AM, AM(2)/intermedin and CGRP was measured in a cAMP assay and cell surface expression was determined by ELISA. Phe reduced AM potency in RAMP3 but had no effect in RAMP1. In contrast, Tyr had no effect in RAMP3 but enhanced AM potency in RAMP1. Most other substitutions had a small effect on AM potency in both receptors whereas there was little impact on CGRP or AM(2) potency. Overall, these data suggest that the geometry and charge of the residue at position 74 contribute to how AM interacts with the AM(2) and CGRP receptors and confirms the role of this position in dictating differential AM pharmacology at the AM(2) and CGRP receptors.

Extracellular loops 1 and 3 and their associated transmembrane regions of the calcitonin receptor-like receptor are needed for CGRP receptor function

The first and third extracellular loops (ECL) of G protein-coupled receptors (GPCRs) have been implicated in ligand binding and receptor function. This study describes the results of an alanine/leucine scan of ECLs 1 and 3 and loop-associated transmembrane (TM) domains of the secretin-like GPCR calcitonin receptor-like receptor which associates with receptor activity modifying protein 1 to form the CGRP receptor. Leu195Ala, Val198Ala and Ala199Leu at the top of TM2 all reduced aCGRP-mediated cAMP production and internalization; Leu195Ala and Ala199Leu also reduced aCGRP binding. These residues form a hydrophobic cluster within an area defined as the "minor groove" of rhodopsin-like GPCRs. Within ECL1, Ala203Leu and Ala206Leu influenced the ability of aCGRP to stimulate adenylate cyclase. In TM3, His219Ala, Leu220Ala and Leu222Ala have influences on aCGRP binding and cAMP production; they are likely to indirectly influence the binding site for aCGRP as well as having an involvement in signal transduction. On the exofacial surfaces of TMs 6 and 7, a number of residues were identified that reduced cell surface receptor expression, most noticeably Leu351Ala and Glu357Ala in TM6. The residues may contribute to the RAMP1 binding interface. Ile360Ala impaired aCGRP-mediated cAMP production. Ile360 is predicted to be located close to ECL2 and may facilitate receptor activation. Identification of several crucial functional loci gives further insight into the activation mechanism of this complex receptor system and may aid rational drug design.

Ocular and systemic endothelial function in the off spring of diabetics:a pilot study

Purpose To investigate ocular and systemic correlates of endothelial function in the normoglycaemic offspring of Type 2 Diabetics (T2DM). Methods Healthy participants aged between 25-65 with (n=30) and without (n=39) a family history were recruited. Retinal vessel reactivity was assessed by using the Retinal Vessel Analyser (RVA, Imedos GmBH). In addition, systemic endothelial function was assessed by using the flow mediated dilation (FMD) technique. Results Parametric testing showed no significant differences in anthropometric, blood assay or ocular and systemic function between both groups (p>0.05). The average maximum dilation in the measured retinal artery correlated significantly with the maximum dilation of the measured brachial artery (p=0.002 R=0.55) in healthy controls; however, this was not true for subjects with family history of T2DM. Conclusion Subjects with family history of T2DM show possibly early signs of endothelial dysfunction that, in certain conditions, could contribute to the higher risk of this group of developing similar pathology to their parents.

Ascorbate and α-tocopherol differentially modulate reactive oxygen species generation by neutrophils in response to FcγR and TLR agonists

Periodontitis, a ubiquitous chronic inflammatory disease, is associated with reduced antioxidant defences and neutrophil hyperactivity in terms of reactive oxygen species (ROS) generation. Its phenotype is thus characterized by oxidative stress. We have determined the effect of antioxidant micronutrients ascorbate and α-tocopherol on neutrophil ROS generation. Peripheral neutrophils from periodontally-healthy individuals (n = 20) were challenged with phorbol myristate acetate, IgG-opsonised Staphylococcus aureus, Fusobacterium nucleatum or PBS in the presence and absence of micronutrients (50 μM). Total and extracellular ROS were measured by luminol and isoluminol chemiluminescence respectively. Total and extracellular unstimulated, baseline ROS generation was unaffected by α-tocopherol, but inhibited by ascorbate and a combination of both micronutrients. Fcγ-receptor (Fcγ-R)-stimulated total or extracellular ROS generation was not affected by the presence of individual micronutrients. However, the combination significantly reduced extracellular FcγR-stimulated ROS release. Neither micronutrient inhibited TLR-stimulated total ROS, but the combination caused inhibition. Ascorbate and the micronutrient combination, but not α-tocopherol, inhibited extracellular ROS release by TLR-stimulated cells. Such micronutrient effects in vivo could be beneficial in reducing collateral tissue damage in chronic inflammatory diseases, such as periodontitis, while retaining immune-mediated neutrophil function. © The Author(s) 2012 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.