Building and Use of Infrastructure for Detector Development and Testing at VR-1 Reactor Facility

Activities related to detector development, testing, characterisation and applications belong to key research objectives of the VR-1 reactor facility. The contribution gives a review of related improvements, achievements, used approaches, methods, and trends

Building and Use of Infrastructure for Detector Development and Testing at VR-1 Reactor Facility

Activities related to detector development, testing, characterisation and applications belong to key research objectives of the VR-1 reactor facility. The contribution gives a review of related improvements, achievements, used approaches, methods, and trends

Characterization of the S100A1 protein binding site on TRPC6 C-terminus.

The transient receptor potential (TRP) protein superfamily consists of seven major groups, among them the "canonical TRP" family. The TRPC proteins are calcium-permeable nonselective cation channels activated after the emptying of intracellular calcium stores and appear to be gated by various types of messengers. The TRPC6 channel has been shown to be expressed in various tissues and cells, where it modulates the calcium level in response to external signals. Calcium binding proteins such as Calmodulin or the family of S100A proteins are regulators of TRPC channels. Here we characterized the overlapping integrative binding site for S100A1 at the C-tail of TRPC6, which is also able to accomodate various ligands such as Calmodulin and phosphatidyl-inositol-(4,5)-bisphosphate. Several positively charged amino acid residues (Arg852, Lys856, Lys859, Arg860 and Arg864) were determined by fluorescence anisotropy measurements for their participation in the calcium-dependent binding of S100A1 to the C terminus of TRPC6. The triple mutation Arg852/Lys859/Arg860 exhibited significant disruption of the binding of S100A1 to TRPC6. This indicates a unique involvement of these three basic residues in the integrative overlapping binding site for S100A1 on the C tail of TRPC6

Online training and education from the VR-1 reactor—Lessons learned

Hands-on education and training is a key part of fixing and developing technology knowledge and is an inherent part of many engineering and scientific curricula. However, access to large complex training facilities, such as nuclear reactor, could be limited by various factors, such as unavailability of those facilities in the region, high traveling costs or harmonization of the schedules of hands-on E&T with theoretical lectures and with the operational schedule of the facility. To handle the issue, several success stories have been reached with the introduction of the Internet Reactor Labs (IRL). The Internet Reactor Labs can strongly contribute to accessibility of training at research reactors and can contribute to improvements in their utilization. The paper describes the development of the Internet Reactor Lab at the VR-1 reactor of the Czech Technical University in Prague. Contrary to single-purpose IRLs, it presents various modalities of online teaching and training in experimental reactor physics and reactor operation in general as well as outreach activities that have been developed in recent years

Integrative Binding Sites within Intracellular Termini of TRPV1 Receptor

<div><p>TRPV1 is a nonselective cation channel that integrates wide range of painful stimuli. It has been shown that its activity could be modulated by intracellular ligands PIP2 or calmodulin (CaM). The detailed localization and description of PIP2 interaction sites remain unclear. Here, we used synthesized peptides and purified fusion proteins of intracellular regions of TRPV1 expressed in <em>E.coli</em> in combination with fluorescence anisotropy and surface plasmon resonance measurements to characterize the PIP2 binding to TRPV1. We characterized one PIP2 binding site in TRPV1 N-terminal region, residues F189-V221, and two independent PIP2 binding sites in C–terminus: residues K688-K718 and L777-S820. Moreover we show that two regions, namely F189-V221 and L777-S820, overlap with previously localized CaM binding sites. For all the interactions the equilibrium dissociation constants were estimated. As the structural data regarding C-terminus of TRPV1 are lacking, restraint-based molecular modeling combined with ligand docking was performed providing us with structural insight to the TRPV1/PIP2 binding. Our experimental results are in excellent agreement with our <em>in silico</em> predictions.</p> </div

PIP2 binds to the C-terminal proximal region of TRPV1.

<p>Steady-state fluorescence anisotropy measurement of interaction between fluorescently labeled phosphatidyl inositol-4, 5-bisphosphate (PIP2-Bodipy) and synthetic peptide corresponding to the cytoplasmic tail at the C terminal proximal region K688-K718 of TRPV1 (pTRPV1–CTp) or its Q700A/R701A (pTRPV1–CTp-Q700A/R701A) and K694A/K698A/K710A (pTRPV1–CTp-K694A/K698A/K710A) mutant variant, respectively. PIP2-Bodipy (10 nM) was titrated with with indicated concentrations of the peptides and the bound fraction (F_B) of PIP2 Bodipy was calculated according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048437#pone.0048437.e001" target="_blank">Equation 1</a> as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048437#s4" target="_blank">Material and Methods</a>. The solid lines represent binding isotherms determined by nonlinear least-squares analysis of the isotherm using an <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048437#pone.0048437.e002" target="_blank">Equation 2</a> as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048437#s4" target="_blank">Material and Methods</a>. Values represent the mean ± SD from at least three independent experiments.</p

Surgical Treatment of a Catheter-Induced Iatrogenic Dissection of the Right Coronary Artery following Cardiac Catheterization

Iatrogenic dissections of the ascending aorta are an uncommon and severe complication during cardiac catheterization. A 68-year-old female patient underwent diagnostic cardiac catheterization due to non-ST-elevation myocardial infarction. During the procedure, a catheter-induced 360• Class I dissection of the right coronary artery occurred. The patient developed severe bradycardia, which was treated with a temporary pacemaker. She underwent an emergency operation with ligation and a saphenous vein graft in the right coronary artery. The postoperative course was uneventful; and on postoperative day 6, she was discharged home

PIP2 binds to the C-terminal distal region of TRPV1. A.

<p>Fluorescence anisotropy measurements of interaction between fluorescently labeled phosphatidyl inositol-4, 5-bisphosphate (PIP2-Bodipy) and the distal region of TRPV1 (amino acids 712–838) fusion protein. PIP2-Bodipy (10 nM) was titrated with TRPV1-CT fusion protein WT and the bound fraction (FB) was calculated according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048437#pone.0048437.e001" target="_blank">Equation 1</a> as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048437#s4" target="_blank">Material and Methods</a>. Binding isotherm and the equilibrium dissociation constant KD (3.48+/−0.93 µM) was determined by fitting the data to the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048437#pone.0048437.e002" target="_blank">Equation 2</a> as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048437#s4" target="_blank">Material and Methods</a>. <b>B.</b> Fluorescence anisotropy measurements of interaction between PIP2-Bodipy and thioredoxin. PIP2-Bodipy (10 nM) was titrated with thioredoxin and the bound fraction (FB) of PIP2 Bodipy was calculated as above. <b>C.</b> Steady-state fluorescence anisotropy measurement of interaction between fluorescently labeled phosphatidyl choline (NBD–PC) and TRPV1-CT. NBD-PC (10 nM) was titrated with indicated concentrations of TRPV1-CT fluorescence anisotropy was recorded. Values are expressed as the mean ± standard deviation (SD) measured from at least from six independent experiments.</p

Amino acid senquence of TRPC6 fusion protein.

<p>Native rat TRPC6 801–878 amino acid sequence containing integrative binding site was investigated. Predicted important basic amino acids that were replaced by alanine are in red.</p

The sequence alignment of the C-terminus of TRPV1 A690 - K838 and the fragile histidine triad protein (FHIT) S2-D150.

<p>Identical amino acids are marked with an asterisk. Similar amino acids with the more important groups are indicated with a colon. Dots indicate similar amino acids of the less important groups that are less likely to influence the protein structure.</p