Conformational transition of FGFR kinase activation revealed by site-­specific unnatural amino acid reporter and single molecule FRET

Protein kinases share significant structural similarity; however, structural features alone are insufficient to explain their diverse functions. Thus, bridging the gap between static structure and function requires a more detailed understanding of their dynamic properties. For example, kinase activation may occur via a switch-like mechanism or by shifting a dynamic equilibrium between inactive and active states. Here, we utilize a combination of FRET and molecular dynamics (MD) simulations to probe the activation mechanism of the kinase domain of Fibroblast Growth Factor Receptor (FGFR). Using genetically-encoded, site-specific incorporation of unnatural amino acids in regions essential for activation, followed by specific labeling with fluorescent moieties, we generated a novel class of FRET-based reporter to monitor conformational differences corresponding to states sampled by non phosphorylated/inactive and phosphorylated/active forms of the kinase. Single molecule FRET analysis in vitro, combined with MD simulations, shows that for FGFR kinase, there are populations of inactive and active states separated by a high free energy barrier resulting in switch-like activation. Compared to recent studies, these findings support diversity in features of kinases that impact on their activation mechanisms. The properties of these FRET-based constructs will also allow further studies of kinase dynamics as well as applications in vivo

Relativistic effects in the chaotic Sitnikov problem

We investigate the phase space structure of the relativistic Sitnikov problem in the first post-Newtonian approximation. The phase space portraits show a strong dependence on the gravitational radius which describes the strength of the relativistic pericentre advance. Bifurcations appearing at increasing the gravitational radius are presented. Transient chaotic behavior related to escapes from the primaries are also studied. Finally, the numerically determined chaotic saddle is investigated in the context of hyperbolic and non-hyperbolic dynamics as a function of the gravitational radius.Comment: 8 pages, 11 figure

Conformational transition of FGFR kinase activation revealed by site-specific unnatural amino acid reporter and single molecule FRET

Protein kinases share significant structural similarity; however, structural features alone are insufficient to explain their diverse functions. Thus, bridging the gap between static structure and function requires a more detailed understanding of their dynamic properties. For example, kinase activation may occur via a switch-like mechanism or by shifting a dynamic equilibrium between inactive and active states. Here, we utilize a combination of FRET and molecular dynamics (MD) simulations to probe the activation mechanism of the kinase domain of Fibroblast Growth Factor Receptor (FGFR). Using genetically-encoded, site-specific incorporation of unnatural amino acids in regions essential for activation, followed by specific labeling with fluorescent moieties, we generated a novel class of FRET-based reporter to monitor conformational differences corresponding to states sampled by non phosphorylated/inactive and phosphorylated/active forms of the kinase. Single molecule FRET analysis in vitro, combined with MD simulations, shows that for FGFR kinase, there are populations of inactive and active states separated by a high free energy barrier resulting in switch-like activation. Compared to recent studies, these findings support diversity in features of kinases that impact on their activation mechanisms. The properties of these FRET-based constructs will also allow further studies of kinase dynamics as well as applications in vivo

The impact of altered haemodynamics on the development of the epicardium

During embryo development the heart is the first functioning organ. Although quiescent in the adult heart, the epicardium is essential during development to form a normal functioning heart. Epicardial derived cells contribute to the heart as it develops, including fibroblasts and vascular smooth muscle cells. Previous studies have shown that a heartbeat is required for epicardium formation. Further, preliminary studies from our laboratory have shown that the development of the epicardium is aberrant when the haemodynamics are altered. This study aims to investigate how the epicardium and some of its derived cell lineages respond to altered haemodynamics in the developing embryo. Since the aetiology of many congenital heart diseases (CHDs) is unknown, we suggest that an alteration in the heart’s haemodynamics might provide an explanatory basis for some of them. In order to change the heart’s haemodynamics, outflow tract (OFT) banding using a double overhang knot was performed on Hamburger and Hamilton (HH) stage 21 chick embryos, with harvesting at different developmental stages. Upon alteration of haemodynamics, the epicardium exhibited abnormal morphology and minor bleeding at HH29 using morphological analysis. This phenotype was exacerbated at HH35 with severe changes in the structure of the extracellular matrix (ECM) and the coronary vasculature. A number of genes tied to ECM production were also differentially expressed in HH29 and HH35 OTB hearts including, collagen I and collagen XII. At HH35, there was also downregulation in a number of vascular genes. It is the first time that the importance of the epicardium is shown regarding CHDs that are caused by altered haemodynamics. In this study, the epicardium was found to be severely impacted by OFT banding. The altered phenotype also showed signs of becoming embryonically lethal as development ensued. More studies should be conducted regarding the effects of haemodynamics on the epicardium with respect to ECM and coronary vessel maturation

Characterisation of the developing heart in a pressure overloaded model utilising RNA sequencing to direct functional analysis

Cardiogenesis is influenced by both environmental and genetic factors, with blood flow playing a critical role in cardiac remodelling. Perturbation of any of these factors could lead to abnormal heart development and hence the formation of congenital heart defects. Although abnormal blood flow has been associated with a number of heart defects, the effects of abnormal pressure load on the developing heart gene expression profile have to date not clearly been defined. To determine the heart transcriptional response to haemodynamic alteration during development, outflow tract (OFT) banding was employed in the chick embryo at Hamburger and Hamilton stage (HH) 21. Stereological and expression studies, including the use of global expression analysis by RNA sequencing with an optimised procedure for effective globin depletion, were subsequently performed on HH29 OFT‐banded hearts and compared with sham control hearts, with further targeted expression investigations at HH35. The OFT‐banded hearts were found to have an abnormal morphology with a rounded appearance and left‐sided dilation in comparison with controls. Internal analysis showed they typically had a ventricular septal defect and reductions in the myocardial wall and trabeculae, with an increase in the lumen on the left side of the heart. There was also a significant reduction in apoptosis. The differentially expressed genes were found to be predominately involved in contraction, metabolism, apoptosis and neural development, suggesting a cardioprotective mechanism had been induced. Therefore, altered haemodynamics during development leads to left‐sided dilation and differential expression of genes that may be associated with stress and maintaining cardiac output

Regional differences in AIDS and non-AIDS related mortality in HIV-positive individuals across Europe and Argentina: the EuroSIDA study

BACKGROUND Differences in access to care and treatment have been reported in Eastern Europe, a region with one of the fastest growing HIV epidemics, compared to the rest of Europe. This analysis aimed to establish whether there are regional differences in the mortality rate of HIV-positive individuals across Europe, and Argentina. METHODS 13,310 individuals under follow-up were included in the analysis. Poisson regression investigated factors associated with the risk of death. FINDINGS During 82,212 person years of follow-up (PYFU) 1,147 individuals died (mortality rate 14.0 per 1,000 PYFU (95% confidence interval [CI] 13.1-14.8). Significant differences between regions were seen in the rate of all-cause, AIDS and non-AIDS related mortality (global p<0.0001 for all three endpoints). Compared to South Europe, after adjusting for baseline demographics, laboratory measurements and treatment, a higher rate of AIDS related mortality was observed in East Europe (IRR 2.90, 95%CI 1.97-4.28, p<.0001), and a higher rate of non-AIDS related mortality in North Europe (IRR 1.51, 95%CI 1.24-1.82, p<.0001). The differences observed in North Europe decreased over calendar-time, in 2009-2011, the higher rate of non-AIDS related mortality was no longer significantly different to South Europe (IRR 1.07, 95%CI 0.66-1.75, p = 0.77). However, in 2009-2011, there remained a higher rate of AIDS-related mortality (IRR 2.41, 95%CI 1.11-5.25, p = 0.02) in East Europe compared to South Europe in adjusted analysis. INTERPRETATIONS There are significant differences in the rate of all-cause mortality among HIV-positive individuals across different regions of Europe and Argentina. Individuals in Eastern Europe had an increased risk of mortality from AIDS related causes and individuals in North Europe had the highest rate of non-AIDS related mortality. These findings are important for understanding and reviewing HIV treatment strategies and policies across the European region

A compressed-sensing approach for ultrasound imaging

Ultrasonography uses multiple piezo-electric element probes to image tissues. Current time-domain beamforming techniques require the signal at each transducer-element to be sampled at a rate higher than the Nyquist criterion, resulting in an extensive amount of data to be received, stored and processed. In this work, we propose to exploit sparsity of the signal received at each transducer-element. The proposed approach uses multiple compressive multiplexers for signal encoding and solves an l1-minimization in the decoding step, resulting in the reduction of 75 % of the amount of data, the number of cables and the number of analog-to-digital converters required to perform high quality reconstruction

Joint Sparsity with Partially Known Support and Application to Ultrasound Imaging

We investigate the benefits of known partial support for the recovery of joint-sparse signals and demonstrate that it is advantageous in terms of recovery performance for both rank-blind and rank-aware algorithms. We suggest extensions of several joint-sparse recovery algorithms, e.g. simultaneous normalized iterative hard thresholding, subspace greedy methods and subspace-augmented multiple signal classification (MUSIC) techniques. We describe a direct application of the proposed methods for compressive multiplexing of ultrasound (US) signals. The technique exploits the compressive multiplexer architecture for signal compression and relies on joint-sparsity of US signals in the frequency domain for signal reconstruction. We validate the proposed algorithms on numerical experiments and show their superiority against state-of-the-art approaches in rank-defective cases. We also demonstrate that the techniques lead to a significant increase of the image quality on in vivo carotid images compared to reconstruction without partially known support. The supporting code is available on https://github.com/AdriBesson/ spl2018_joint_sparse