Glycoprotein VI is a major collagen receptor for platelet activation: it recognizes the platelet-activating quaternary structure of collagen, whereas CD36, glycoprotein IIb/IIIa, and von Willebrand factor do not

Simple collagen-related peptides (CRPs) containing a repeat Gly-Pro-Hyp sequence are highly potent platelet agonists. Like collagen, they must exhibit tertiary (triple-helical) and quaternary (polymeric) structure to activate platelets. Platelet signaling events induced by the peptides are the same as most of those induced by collagen. The peptides do not recognize the alpha 2 beta 1 integrin. To identify the signaling receptor involved, we have evaluated the response to the CRP, Gly-Lys-Hyp(Gly-Pro-Hyp)10-Gly-Lys-Hyp-Gly of platelets with defined functional deficiencies. These studies exclude a primary recognition role for CD36, von Willebrand factor (vWF), or glycoprotein (GP) IIb/IIIa. Thus, both CD36 and vWF-deficient platelets exhibited normal aggregation, normal fibrinogen binding, and normal expression of CD62 and CD63, measured by flow cytometry, in response to the peptide, and there was normal expression of CD62 and CD63 on thrombasthenic platelets. In contrast, GPVI-deficient platelets were totally unresponsive to the peptide, indicating that this receptor recognizes the Gly-Pro-Hyp sequence in collagen. GPVI-deficient platelets showed some fibrinogen binding in response to collagen but failed to aggregate and to express CD62 and CD63. Collagen, but not CRP-XL, contains binding sites for alpha 2 beta 1. Therefore, it is possible that collagen still induces some signaling via alpha 2 beta 1, leading to activation of GPIIb/IIIa. Our findings are consistent with a two-site, two-step model of collagen interaction with platelets involving recognition of specific sequences in collagen by an adhesive receptor such as alpha 2 beta 1 to arrest platelets under flow and subsequent recognition of another specific collagen sequence by an activatory receptor, namely GPVI.

Crystal structure of the platelet glycoprotein Ib(alpha) N-terminal domain reveals an unmasking mechanism for receptor activation

Glycoprotein Ib (GPIb) is a platelet receptor with a critical role in mediating the arrest of platelets at sites of vascular damage. GPIb binds to the A1 domain of von Willebrand factor (vWF-A1) at high blood shear, initiating platelet adhesion and contributing to the formation of a thrombus. To investigate the molecular basis of GPIb regulation and ligand binding, we have determined the structure of the N-terminal domain of the GPIb(alpha) chain (residues 1-279). This structure is the first determined from the cell adhesion/signaling class of leucine-rich repeat (LRR) proteins and reveals the topology of the characteristic disulfide-bonded flanking regions. The fold consists of an N-terminal beta-hairpin, eight leucine-rich repeats, a disulfide-bonded loop, and a C-terminal anionic region. The structure also demonstrates a novel LRR motif in the form of an M-shaped arrangement of three tandem beta-turns. Negatively charged binding surfaces on the LRR concave face and anionic region indicate two-step binding kinetics to vWF-A1, which can be regulated by an unmasking mechanism involving conformational change of a key loop. Using molecular docking of the GPIb and vWF-A1 crystal structures, we were also able to model the GPIb.vWF-A1 complex.

A feasibility study on model-based evaluation of kidney perfusion measured by means of FAIR prepared true-FISP arterial spin labeling (ASL) on a 3-T MR scanner

RATIONALE AND OBJECTIVES: A feasibility study on measuring kidney perfusion by a contrast-free magnetic resonance (MR) imaging technique is presented. MATERIALS AND METHODS: A flow-sensitive alternating inversion recovery (FAIR) prepared true fast imaging with steady-state precession (TrueFISP) arterial spin labeling sequence was used on a 3.0-T MR-scanner. The basis for quantification is a two-compartment exchange model proposed by Parkes that corrects for diverse assumptions in single-compartment standard models. RESULTS: Eleven healthy volunteers (mean age, 42.3 years; range 24-55) were examined. The calculated mean renal blood flow values for the exchange model (109 +/- 5 [medulla] and 245 +/- 11 [cortex] ml/min - 100 g) are in good agreement with the literature. Most important, the two-compartment exchange model exhibits a stabilizing effect on the evaluation of perfusion values if the finite permeability of the vessel wall and the venous outflow (fast solution) are considered: the values for the one-compartment standard model were 93 +/- 18 (medulla) and 208 +/- 37 (cortex) ml/min - 100 g. CONCLUSION: This improvement will increase the accuracy of contrast-free imaging of kidney perfusion in treatment renovascular disease.

Two-dimensional assembly and local redox-activity of molecular hybrid structures in an electrochemical environment

The self-assembly and redox-properties of two viologen derivatives, N-hexyl-N-(6-thiohexyl)-4,4-bipyridinium bromide (HS-6V6-H) and N,N-bis(6-thiohexyl)-4,4-bipyridinium bromide (HS-6V6-SH), immobilized on Au(111)-(1x1) macro-electrodes were investigated by cyclic voltammetry, surface enhanced infrared spectroscopy (SEIRAS) and in situ scanning tunneling microscopy (STM). Depending on the assembly conditions one could distinguish three different types of adlayers for both viologens: a low coverage disordered and an ordered striped phase of flat oriented molecules as well as a high coverage monolayer composed of tilted viologen moieties. Both molecules, HS-6V6-H and HS-6V6-SH, were successfully immobilized on Au(poly) nano-electrodes, which gave a well-defined redox-response in the lower pA–current range. An in situ STM configuration was employed to explore electron transport properties of single molecule junctions Au(T)|HS-6V6-SH(HS-6V6-H)|Au(S). The observed sigmoidal potential dependence, measured at variable substrate potential ES and at constant bias voltage (ET–ES), was attributed to electronic structure changes of the viologen moiety during the one-electron reduction/re-oxidation process V2+ V+. Tunneling experiments in asymmetric, STM-based junctions Au(T)-S-6V6-H|Au(S) revealed current (iT)–voltage (ET) curves with a maximum located at the equilibrium potential of the redox-process V2+ V+. The experimental iT–ET characteristics of the HS-6V6-H–modified tunneling junction were tentatively attributed to a sequential two-step electron transfer mechanism.

Quantitative 1H-magnetic resonance spectroscopy of human brain: Influence of composition and parameterization of the basis set in linear combination model-fitting

Localized short-echo-time (1)H-MR spectra of human brain contain contributions of many low-molecular-weight metabolites and baseline contributions of macromolecules. Two approaches to model such spectra are compared and the data acquisition sequence, optimized for reproducibility, is presented. Modeling relies on prior knowledge constraints and linear combination of metabolite spectra. Investigated was what can be gained by basis parameterization, i.e., description of basis spectra as sums of parametric lineshapes. Effects of basis composition and addition of experimentally measured macromolecular baselines were investigated also. Both fitting methods yielded quantitatively similar values, model deviations, error estimates, and reproducibility in the evaluation of 64 spectra of human gray and white matter from 40 subjects. Major advantages of parameterized basis functions are the possibilities to evaluate fitting parameters separately, to treat subgroup spectra as independent moieties, and to incorporate deviations from straightforward metabolite models. It was found that most of the 22 basis metabolites used may provide meaningful data when comparing patient cohorts. In individual spectra, sums of closely related metabolites are often more meaningful. Inclusion of a macromolecular basis component leads to relatively small, but significantly different tissue content for most metabolites. It provides a means to quantitate baseline contributions that may contain crucial clinical information.

Pharmacogenetics-based population pharmacokinetic analysis of etravirine in HIV-1 infected individuals

Objectives: Etravirine (ETV) is metabolized by cytochrome P450 (CYP) 3A, 2C9, and 2C19. Metabolites are glucuronidated by uridine diphosphate glucuronosyltransferases (UGT). To identify the potential impact of genetic and non-genetic factors involved in ETV metabolism, we carried out a two-step pharmacogenetics-based population pharmacokinetic study in HIV-1 infected individuals. Materials and methods: The study population included 144 individuals contributing 289 ETV plasma concentrations and four individuals contributing 23 ETV plasma concentrations collected in a rich sampling design. Genetic variants [n=125 single-nucleotide polymorphisms (SNPs)] in 34 genes with a predicted role in ETV metabolism were selected. A first step population pharmacokinetic model included non-genetic and known genetic factors (seven SNPs in CYP2C, one SNP in CYP3A5) as covariates. Post-hoc individual ETV clearance (CL) was used in a second (discovery) step, in which the effect of the remaining 98 SNPs in CYP3A, P450 cytochrome oxidoreductase (POR), nuclear receptor genes, and UGTs was investigated. Results: A one-compartment model with zero-order absorption best characterized ETV pharmacokinetics. The average ETV CL was 41 (l/h) (CV 51.1%), the volume of distribution was 1325 l, and the mean absorption time was 1.2 h. The administration of darunavir/ritonavir or tenofovir was the only non-genetic covariate influencing ETV CL significantly, resulting in a 40% [95% confidence interval (CI): 13–69%] and a 42% (95% CI: 17–68%) increase in ETV CL, respectively. Carriers of rs4244285 (CYP2C19*2) had 23% (8–38%) lower ETV CL. Co-administered antiretroviral agents and genetic factors explained 16% of the variance in ETV concentrations. None of the SNPs in the discovery step influenced ETV CL. Conclusion: ETV concentrations are highly variable, and co-administered antiretroviral agents and genetic factors explained only a modest part of the interindividual variability in ETV elimination. Opposing effects of interacting drugs effectively abrogate genetic influences on ETV CL, and vice-versa.

The Effect Of Misleading Prior Information On The Subjective Visual Vertical

When tilted sideways participants misperceive the visual vertical assessed by means of a luminous line in otherwise complete dark- ness. A recent modeling approach (De Vrijer et al., 2009) claimed that these typical patterns of errors (known as A- and E-effects) could be explained by as- suming that participants behave in a Bayes optimal manner. In this study, we experimentally manipulate participants’ prior information about body-in-space orientation and measure the effect of this manipulation on the subjective visual vertical (SVV). Specifically, we explore the effects of veridical and misleading instructions about body tilt orientations on the SVV. We used a psychophys- ical 2AFC SVV task at roll tilt angles of 0 degrees, 16 degrees and 4 degrees CW and CCW. Participants were tilted to 4 degrees under different instruction conditions: in one condition, participants received veridical instructions as to their tilt angle, whereas in another condition, participants received the mis- leading instruction that their body position was perfectly upright. Our results indicate systematic differences between the instruction conditions at 4 degrees CW and CCW. Participants did not simply use an ego-centric reference frame in the misleading condition; instead, participants’ estimates of the SVV seem to lie between their head’s Z-axis and the estimate of the SVV as measured in the veridical condition. All participants displayed A-effects at roll tilt an- gles of 16 degrees CW and CCW. We discuss our results in the context of the Bayesian model by De Vrijer et al. (2009), and claim that this pattern of re- sults is consistent with a manipulation of precision of a prior distribution over body-in-space orientations. Furthermore, we introduce a Bayesian Generalized Linear Model for estimating parameters of participants’ psychometric function, which allows us to jointly estimate group level and individual level parameters under all experimental conditions simultaneously, rather than relying on the traditional two-step approach to obtaining group level parameter estimates.

Surface Roughness and Microhardness of Two Recent CAD/CAM-Materials After Storage

Objectives: To investigate surface roughness and microhardness of two recent resin-ceramic materials for computer-aided design/computer-aided manufacturing (CAD/CAM) after polishing with three polishing systems. Surface roughness and microhardness were measured immediately after polishing and after six months storage including monthly artificial toothbrushing. Methods: Sixty specimens of Lava Ultimate (3M ESPE) and 60 specimens of VITA ENAMIC (VITA Zahnfabrik) were roughened in a standardized manner and polished with one of three polishing systems (n=20/group): Sof-Lex XT discs (SOFLEX; three-step (medium-superfine); 3M ESPE), VITA Polishing Set Clinical (VITA; two-step; VITA Zahnfabrik), or KENDA Unicus (KENDA; one-step; KENDA Dental). Surface roughness (Ra; μm) was measured with a profilometer and microhardness (Vickers; VHN) with a surface hardness indentation device. Ra and VHN were measured immediately after polishing and after six months storage (tap water, 37°C) including monthly artificial toothbrushing (500 cycles/month, toothpaste RDA ~70). Ra- and VHN-values were analysed with nonparametric ANOVA followed by Wilcoxon rank sum tests (α=0.05). Results: For Lava Ultimate, Ra (mean [standard deviation] before/after storage) remained the same when polished with SOFLEX (0.18 [0.09]/0.19 [0.10]; p=0.18), increased significantly with VITA (1.10 [0.44]/1.27 [0.39]; p=0.0001), and decreased significantly with KENDA (0.35 [0.07]/0.33 [0.08]; p=0.03). VHN (mean [standard deviation] before/after storage) decreased significantly regardless of polishing system (SOFLEX: 134.1 [5.6]/116.4 [3.6], VITA: 138.2 [10.5]/115.4 [5.9], KENDA: 135.1 [6.2]/116.7 [6.3]; all p<0.0001). For VITA ENAMIC, Ra (mean [standard deviation] before/after storage) increased significantly when polished with SOFLEX (0.37 [0.18]/0.41 [0.14]; p=0.01) and remained the same with VITA (1.32 [0.37]/1.31 [0.40]; p=0.58) and with KENDA (0.81 [0.35]/0.78 [0.32]; p=0.21). VHN (mean [standard deviation] before/after storage) remained the same regardless of polishing system (SOFLEX: 284.9 [24.6]/282.4 [31.8], VITA: 284.6 [28.5]/276.4 [25.8], KENDA: 292.6 [26.9]/282.9 [24.3]; p=0.42-1.00). Conclusion: Surface roughness and microhardness of Lava Ultimate was more affected by storage and artificial toothbrushing than was VITA ENAMIC.

Congenital cervical kyphosis in two young sighthounds

Introduction: Cervical vertebral (C) malformation is rarely reported in large breed dogs. Congenital cervical kyphosis (CCK) may result from defects of vertebral segmentation, failure of formation or both. This report describes two cases of C3-C4 CCK in young sighthounds, treated surgically. Case description: An 18-month-old female Deerhound and a six-week-old female Borzoi dog were presented because of the complaints of reluctance to exercise and signs of of neck pain. Both dogs were neurologically normal. Diagnostic imaging revealed C3-C4 deformity, moderate kyphosis, and spinal canal stenosis associated with chronic spinal cord pressure atrophy. Both dogs underwent surgical treatment. Results: A staged two-step surgery starting with dorsal decompression was elected in the Deerhound. After the first surgical procedure, the dog developed focal myelomalacia and phrenic nerve paralysis and was euthanatized. A ventral distraction-fusion technique with two locking plates was performed in the Borzoi. This patient recovered uneventfully and long-term follow-up computed tomography revealed complete spondylodesis. Clinical significance: Until now, CCK has only been described in sighthounds. Congenital cervical kyphosis might be considered a differential diagnosis in these breeds that are presented with signs of cervical pain. Ventral realignment-fusion and bone grafting may be considered for surgical treatment, although the earliest age at which this procedure can and should be performed remains unclear.

Output processing and automatic reporting with Stata

This talk illustrates how results from various Stata commands can be processed efficiently for inclusion in customized reports. A two-step procedure is proposed in which results are gathered and archived in the first step and then tabulated in the second step. Such an approach disentangles the tasks of computing results (which may take long) and preparing results for inclusion in presentations, papers, and reports (which you may have to do over and over). Examples using results from model estimation commands and various other Stata commands such as tabulate, summarize, or correlate are presented. Users will also be shown how to dynamically link results into word processors or into LaTeX documents.

Recent developments in output processing

This tutorial will show how results from various Stata commands can be processed efficiently for inclusion in customized reports. A two-step procedure is proposed in which results are gathered and archived in the first step and then tabulated in the second step. Such an approach disentangles the tasks of computing results (which may take long) and preparing results for inclusion in presentations, papers, and reports (which you may have to do over and over). Examples using results from model estimation commands and also various other Stata commands such as tabulate, summarize, or correlate are presented. Furthermore, this tutorial shows how to dynamically link results into word processors or into LaTeX documents.

Next-state comfort in learning a vertical stick transportation sequence

Over the last decade, the end-state comfort effect (e.g., Rosenbaum et al., 2006) has received a considerable amount of attention. However, some of the underlying mechanisms are still to be investigated, amongst others, how sequential planning affects end-state comfort and how this effect develops over learning. In a two-step sequencing task, e.g., postural comfort can be planned on the intermediate position (next state) or on the actual end position (final state). It might be hypothesized that, in initial acquisition, next state’s comfort is crucial for action planning but that, in the course of learning, final state’s comfort is taken more and more into account. To test this hypothesis, a variant of Rosenbaum’s vertical stick transportation task was used. Participants (N = 16, right-handed) received extensive practice on a two-step transportation task (10,000 trials over 12 sessions). From the initial position on the middle stair of a staircase in front of the participant, the stick had to be transported either 20 cm upwards and then 40 cm downwards or 20 cm downwards and then 40 cm upwards (N = 8 per subgroup). Participants were supposed to produce fluid movements without changing grasp. In the pre- and posttest, participants were tested on both two-step sequencing tasks as well as on 20 cm single-step upwards and downwards movements (10 trials per condition). For the test trials, grasp height was calculated kinematographically. In the pretest, large end/next/final-state comfort effects for single-step transportation tasks and large next-state comfort effects for sequenced tasks were found. However, no change in grasp height from pre- to posttest could be revealed. Results show that, in vertical stick transportation sequences, the final state is not taken into account when planning grasp height. Instead, action planning seems to be solely based on aspects of the next action goal that is to be reached.

Evaluation of a bolus/infusion protocol for 11C-ABP688, a PET tracer for mGluR5

(11)C-ABP-688 is a selective tracer for the mGluR5 receptor. Its kinetics is fast and thus favourable for an equilibrium approach to determine receptor-related parameters. The purpose of this study was to test the hypothesis that the pattern of the (11)C-ABP688 uptake using a bolus-plus-infusion (B/I) protocol at early time points corresponds to the perfusion and at a later time point to the total distribution volume. METHODS: A bolus and a B/I study (1 h each) was performed in five healthy male volunteers. With the B/I protocol, early and late scans were normalized to gray matter, cerebellum and white matter. The same normalization was done on the maps of the total distribution volume (Vt) and K(1) which were calculated in the study with bolus only injection and the Logan method (Vt) and a two-tissue compartment model (K(1)). RESULTS: There was an excellent correlation close to the identity line between the pattern of the late uptake in the B/I study and Vt of the bolus-only study for all three normalizations. The pattern of the early uptake in the B/I study correlated well with the K(1) maps, but only when normalized to gray matter and cerebellum, not to white matter. CONCLUSION: It is demonstrated that with a B/I protocol the (11)C-ABP688 distribution in late scans reflects the pattern of the total distribution volume and is therefore a measure for the density pattern of mGluR5. The early scans following injection are related to blood flow, although not in a fully quantitative manner. The advantage of the B/I protocol is that no arterial blood sampling is required, which is advantageous in clinical studies.

Retrospective analysis of stored dried blood spots from children with cystic fibrosis and matched controls to assess the performance of a proposed newborn screening protocol in Switzerland

Newborn screening (NBS) for Cystic Fibrosis (CF) has been introduced in many countries, but there is no ideal protocol suitable for all countries. This retrospective study was conducted to evaluate whether the planned two step CF NBS with immunoreactive trypsinogen (IRT) and 7 CFTR mutations would have detected all clinically diagnosed children with CF in Switzerland.

Plasma PCSK9 concentrations during an oral fat load and after short term high-fat, high-fat high-protein and high-fructose diets

Background PCSK9 (Proprotein Convertase Subtilisin Kexin type 9) is a circulating protein that promotes hypercholesterolemia by decreasing hepatic LDL receptor protein. Under non interventional conditions, its expression is driven by sterol response element binding protein 2 (SREBP2) and follows a diurnal rhythm synchronous with cholesterol synthesis. Plasma PCSK9 is associated to LDL-C and to a lesser extent plasma triglycerides and insulin resistance. We aimed to verify the effect on plasma PCSK9 concentrations of dietary interventions that affect these parameters. Methods We performed nutritional interventions in young healthy male volunteers and offspring of type 2 diabetic (OffT2D) patients that are more prone to develop insulin resistance, including: i) acute post-prandial hyperlipidemic challenge (n=10), ii) 4 days of high-fat (HF) or high-fat/high-protein (HFHP) (n=10), iii) 7 (HFruc1, n=16) or 6 (HFruc2, n=9) days of hypercaloric high-fructose diets. An acute oral fat load was also performed in two patients bearing the R104C-V114A loss-of-function (LOF) PCSK9 mutation. Plasma PCSK9 concentrations were measured by ELISA. For the HFruc1 study, intrahepatocellular (IHCL) and intramyocellular lipids were measured by 1H magnetic resonance spectroscopy. Hepatic and whole-body insulin sensitivity was assessed with a two-step hyperinsulinemic-euglycemic clamp (0.3 and 1.0 mU.kg-1.min-1). Findings HF and HFHP short-term diets, as well as an acute hyperlipidemic oral load, did not significantly change PCSK9 concentrations. In addition, post-prandial plasma triglyceride excursion was not altered in two carriers of PCSK9 LOF mutation compared with non carriers. In contrast, hypercaloric 7-day HFruc1 diet increased plasma PCSK9 concentrations by 28% (p=0.05) in healthy volunteers and by 34% (p=0.001) in OffT2D patients. In another independent study, 6-day HFruc2 diet increased plasma PCSK9 levels by 93% (p<0.0001) in young healthy male volunteers. Spearman’s correlations revealed that plasma PCSK9 concentrations upon 7-day HFruc1 diet were positively associated with plasma triglycerides (r=0.54, p=0.01) and IHCL (r=0.56, p=0.001), and inversely correlated with hepatic (r=0.54, p=0.014) and whole-body (r=−0.59, p=0.0065) insulin sensitivity. Conclusions Plasma PCSK9 concentrations vary minimally in response to a short term high-fat diet and they are not accompanied with changes in cholesterolemia upon high-fructose diet. Short-term high-fructose intake increased plasma PCSK9 levels, independent on cholesterol synthesis, suggesting a regulation independent of SREBP-2. Upon this diet, PCSK9 is associated with insulin resistance, hepatic steatosis and plasma triglycerides.