Assessment of a fast generated analytical matrix for rotating slat collimation iterative reconstruction: a possible method to optimize the collimation profile.

In SPECT imaging, improvement or deterioration of performance is mostly due to collimator design. Classical SPECT systems mainly use parallel hole or pinhole collimators. Rotating slat collimators (RSC) can be an interesting alternative to optimize the tradeoff between detection efficiency and spatial resolution. The present study was conducted using a RSC system for small animal imaging called CLiR. The CLiR system was used in planar mode only. In a previous study, planar 2D projections were reconstructed using the well-known filtered backprojection algorithm (FBP). In this paper, we investigated the use of the statistical reconstruction algorithm maximum likelihood expectation maximization (MLEM) to reconstruct 2D images with the CLiR system using a probability matrix calculated using an analytic approach. The primary objective was to propose a method to quickly generate a light system matrix, which facilitates its handling and storage, while providing accurate and reliable performance. Two other matrices were calculated using GATE Monte Carlo simulations to investigate the performance obtained using the matrix calculated analytically. The first matrix calculated using GATE took all the physics processes into account, where the second did not consider for the scattering, as the analytical matrix did not take this physics process into account either. 2D images were reconstructed using FBP and MLEM with the three different probability matrices. Both simulated and experimental data were used. A comparative study of these images was conducted using different metrics: the modulation transfert function, the signal-to-noise ratio and quantification measurement. All the results demonstrated the suitability of using a probability matrix calculated analytically. It provided similar results in terms of spatial resolution (about 0.6 mm with differences <5%), signal-to-noise ratio (differences <10%), or quality of image.

Trends in meat science and technology: the future looks bright, but the journey will be long.

With an increasing world population, an increase in affluence and a substantial growth in the demand for high quality protein, the meat sector faces a fantastic but challenging century. New scientific knowledge, technology and creative minds are the main ingredients in order to reach out for this great opportunity. Efficiency all the way from breeding and farming to processing and dispatch is crucial for success. Technology has brought us far, and there is still a huge potential for increased efficiency by implementing best practices on a global scale. New challenges include: hyper flexible automation, more accurate and faster measurement systems and meeting special consumer demands already at the production line. Systems for optimal animal welfare will be even more important and sustainability is no longer a consumer trend but a license to operate. The scientific meat society must provide knowledge and technology so we together can reach out for a seemingly bright future.

Characterization of a rotating slat collimator system dedicated to small animal imaging.

Some current investigations based on small animal models are dedicated to functional cerebral imaging. They represent a fundamental tool to understand the mechanisms involved in neurodegenerative diseases. In the radiopharmaceutical development approach, the main challenge is to measure the radioactivity distribution in the brain of a subject with good temporal and spatial resolutions. Classical SPECT systems mainly use parallel hole or pinhole collimators. In this paper we investigate the use of a rotating slat collimator system for small animal brain imaging. The proposed prototype consists of a 64-channel multi-anode photomultiplier tube (H8804, Hamamatsu Corp.) coupled to a YAP:Ce crystal highly segmented into 32 strips of 0.575 × 18.4 × 10 mm(3). The parameters of the rotating slat collimator are optimized using GATE Monte Carlo simulations. The performance of the proposed prototype in terms of spatial resolution, detection efficiency and signal-to-noise ratio is compared to that obtained with a gamma camera equipped with a parallel hole collimator. Preliminary experimental results demonstrate that a spatial resolution of 1.54 mm can be achieved with a detection efficiency of 0.012% for a source located at 20 mm, corresponding to the position of the brain in the prototype field of view.

Unfaithfulness and promiscuity of a mutant androgen receptor in a hormone-refractory prostate cancer.

Missense mutations in the androgen receptor (AR) contribute to the failure of hormonal therapy for prostate cancer (PCa), but the underlying molecular bases remain uncharacterized. Here, we describe a new AR variant found in a hormone-refractory metastatic PCa, in which threonine 575 in the DNA binding domain, and threonine 877 in the ligand-binding domain, were both replaced by an alanine. Using gene reporter assays, we demonstrate that the T575A mutation weakened transcriptional activity from promoters containing AR-specific responsive elements, while activity from promoters with AR-non-specific elements was enhanced. Data from gel shift experiments revealed a preferential binding of the T575A mutant to AR-non-specific motifs. We demonstrate that the two mutations T575A and T877A cooperate to confer new functional properties on the AR, and that the mutant AR functions simultaneously as a promiscuous AR due to the T877A mutation, and an unfaithful AR due to the T575A mutation.

DISPLAY-2: a two-dimensional shallow layer model for dense gas dispersion including complex features.

A two-dimensional shallow layer model has been developed to predict dense gas dispersion, under realistic conditions, including complex features such as two-phase releases, obstacles and inclined ground. The model attempts to predict the time and space evolution of the cloud formed after a release of a two-phase pollutant into the atmosphere. The air-pollutant mixture is assumed ideal. The cloud evolution is described mathematically through the Cartesian, two-dimensional, shallow layer conservation equations for mixture mass, mixture momentum in two horizontal directions, total pollutant mass fraction (vapor and liquid) and mixture internal energy. Liquid mass fraction is obtained assuming phase equilibrium. Account is taken in the conservation equations for liquid slip and eventual liquid rainout through the ground. Entrainment of ambient air is modeled via an entrainment velocity model, which takes into account the effects of ground friction, ground heat transfer and relative motion between cloud and surrounding atmosphere. The model additionally accounts for thin obstacles effects in three ways. First a stepwise description of the obstacle is generated, following the grid cell faces, taking into account the corresponding area blockage. Then obstacle drag on the passing cloud is modeled by adding flow resistance terms in the momentum equations. Finally the effect of extra vorticity generation and entrainment enhancement behind obstacles is modeled by adding locally into the entrainment formula without obstacles, a characteristic velocity scale defined from the obstacle pressure drop and the local cloud height.The present model predictions have been compared against theoretical results for constant volume and constant flux gravity currents. It was found that deviations of the predicted cloud footprint area change with time from the theoretical were acceptably small, if one models the frictional forces between cloud and ambient air, neglecting the Richardson dependence.The present model has also been validated in widely different experimental conditions such as the Thorney Island instantaneous isothermal releases 8 (unobstructed) and 21 (with semicircular fence), the EEC-55 two-phase propane experiment (with and without linear fence), the Desert Tortoise 4 two-phase ammonia experiment and the Hamburg DAT-638 instantaneous inclined plate experiment and the model predictions were found in reasonable agreement with the experimental data.

X-ray structure of the orphan nuclear receptor RORbeta ligand-binding domain in the active conformation.

The retinoic acid-related orphan receptor beta (RORbeta) exhibits a highly restricted neuronal-specific expression pattern in brain, retina and pineal gland. So far, neither a natural RORbeta target gene nor a functional ligand have been identified, and the physiological role of the receptor is not well understood. We present the crystal structure of the ligand-binding domain (LBD) of RORbeta containing a bound stearate ligand and complexed with a coactivator peptide. In the crystal, the monomeric LBD adopts the canonical agonist-bound form. The fatty acid ligand-coactivator peptide combined action stabilizes the transcriptionally active conformation. The large ligand-binding pocket is strictly hydrophobic on the AF-2 side and more polar on the beta-sheet side where the carboxylate group of the ligand binds. Site-directed mutagenesis experiments validate the significance of the present structure. Homology modeling of the other isotypes will help to design isotype-selective agonists and antagonists that can be used to characterize the physiological functions of RORs. In addition, our crystallization strategy can be extended to other orphan nuclear receptors, providing a powerful tool to delineate their functions.

Different ligands-different receptor conformations: modeling of the hER alpha LBD in complex with agonists and antagonists.

The aim of this study is to compare crystal structures of nuclear receptor ligand binding domains in complex with different agonists and partial agonists to achieve a better understanding of the three-dimensional structures and their ligand-induced conformational changes. This led to the identification of structurally conserved "rigid" regions and more flexible parts of the proteins. The analysis was found to be of great value in fitting selected non-steroidal compounds into the human estrogen receptor alpha (hER alpha) ligand binding pocket. The experimentally determined binding affinities for a number of 2-aryl indoles and 2-aryl indenones are in good agreement with the subsequently modeled binding interactions. To date, no crystal structure is published for a complex with a pure antagonist. We therefore used the available structural information on complexes with partial agonists and the crystal structure of a mutant protein in complex with estradiol displaying a similar conformation to predict binding interactions for antagonists. The results are discussed in detail.

Crystal structures of the vitamin D receptor complexed to superagonist 20-epi ligands.

The crystal structures of the ligand-binding domain (LBD) of the vitamin D receptor complexed to 1alpha,25(OH)(2)D(3) and the 20-epi analogs, MC1288 and KH1060, show that the protein conformation is identical, conferring a general character to the observation first made for retinoic acid receptor (RAR) that, for a given LBD, the agonist conformation is unique, the ligands adapting to the binding pocket. In all complexes, the A- to D-ring moieties of the ligands adopt the same conformation and form identical contacts with the protein. Differences are observed only for the 17beta-aliphatic chains that adapt their conformation to anchor the 25-hydroxyl group to His-305 and His-397. The inverted geometry of the C20 methyl group induces different paths of the aliphatic chains. The ligands exhibit a low-energy conformation for MC1288 and a more strained conformation for the two others. KH1060 compensates this energy cost by additional contacts. Based on the present data, the explanation of the superagonist effect is to be found in higher stability and longer half-life of the active complex, thereby excluding different conformations of the ligand binding domain.

Crystal structure of a mutant hERalpha ligand-binding domain reveals key structural features for the mechanism of partial agonism.

The crystal structure of a triple cysteine to serine mutant ERalpha ligand-binding domain (LBD), complexed with estradiol, shows that despite the presence of a tightly bound agonist ligand, the protein exhibits an antagonist-like conformation, similar to that observed in raloxifen and 4-hydroxytamoxifen-bound structures. This mutated receptor binds estradiol with wild type affinity and displays transcriptional activity upon estradiol stimulation, but with limited potency (about 50%). This partial activity is efficiently repressed in antagonist competition assays. The comparison with available LBD structures reveals key features governing the positioning of helix H12 and highlights the importance of cysteine residues in promoting an active conformation. Furthermore the present study reveals a hydrogen bond network connecting ligand binding to protein trans conformation. These observations support a dynamic view of H12 positioning, where the control of the equilibrium between two stable locations determines the partial agonist character of a given ligand.

Infection of the glass-eel swimbladder with the nematode Anguillicola crassus.

The ability of the nematode Anguillicola crassus to infect eel larvae (glass-eel stage) was tested. The results show that glass-eels fed on infected copepods, the natural intermediate host of the nematode, can be infected. Light microscopical examination of the infected developing swimbladder tissue revealed that the infection results in a significant thickening of the connective tissue. The basolateral labyrinth of gas gland cells is very much reduced in infected swimbladders, and the distance of gas gland cells to blood capillaries is enlarged. Critical swimming speed, defined as the speed where the larvae were no longer able to swim against the current, was similar in infected and uninfected animals. At intermediate speeds (about 60-80% of critical swimming speed) infected eels showed a slightly higher swimming activity than control animals. Resting oxygen consumption, measured as an index of metabolic activity, within the first 2 months of infection was higher in control animals, which may be due to a reduced rate of activity in infected glass-eels. By 4-5 months after the infection, however, it was significantly higher in infected animals. This may indicate that at this stage a higher activity of the animals is required to compensate for the increase in body density, but swimming performance of infected and non-infected glass-eels was not significantly different. Oxygen consumption during swimming activity, measured in a swim tunnel at 50% of maximal swimming speed, also was not affected. The results thus show that even glass-eels can be infected with A. crassus, and this probably contributes to the rapid spread of the nematode in Europe. While aerobic metabolism during swimming activity is not affected at this stage of infection, the swimbladder tissue shows severe histological changes, which most likely will impair swimbladder function.

Swim bladder gas gland cells produce surfactant: in vivo and in culture.

Electron microscopical examination of gas gland cells of the physostome European eel (Anguilla anguilla) and of the physoclist perch (Perca fluviatilis) revealed the presence of significant numbers of lamellar bodies, which are known to be involved in surfactant secretion. In the perch, in which the gas gland is a compact structure and gas gland cells are connected to the swim bladder lumen via small canals, lamellar bodies were also found in flattened cells forming the swim bladder epithelium. Flat epithelial cells are absent in the eel swim bladder, in which the whole epithelium consists of cuboidal gas gland cells. In both species, Western blot analysis using specific antibodies to human surfactant protein A (SP-A) showed a cross-reaction with swim bladder tissue homogenate proteins of approximately 65 kDa and in the eel occasionally of approximately 120 kDa, probably representing SP-A-like proteins in a dimeric and a tetrameric state. An additional band was observed at approximately 45 kDa. Western blots using antibodies to rat SP-D again resulted in a single band at approximately 45 kDa in both species, suggesting that there might be a cross-reaction of the antibody to human SP-A with an SP-D-like protein of the swim bladder tissue. To localize the surfactant protein, eel gas gland cells were cultured on permeable supports. Under these conditions, the gas gland cells regain their characteristic polarity. Electron microscopy confirmed the presence of lamellar bodies in cultured cells, and occasionally, exocytotic events were observed. Immunohistochemical staining using an antibody to human SP-A demonstrated the presence of surfactant protein only in luminal membranes and in adjacent lateral membranes. Only occasionally, evidence was found for the presence of surfactant protein in lamellar bodies.

A single amino acid mutation of ala-773 in the mineralocorticoid receptor confers agonist properties to 11beta-substituted spirolactones.

Sequence analysis revealed a strong homology between the ligand-binding domain (LBD) of the human mineralocorticoid receptor (hMR) and glucocorticoid receptor (hGR). Nevertheless, steroids with bulky C11-substituents bind to hGR, unlike hMR. In this report, a mutant hMR, in which the residue Ala-773 facing the C11 steroid position was replaced by a glycine (A773G), was assayed for its capacity to bind steroids, to interact with receptor coactivators, and to stimulate transcription. The capacity of A773G to bind aldosterone and C11-substituted spirolactones was the same as that of the wild-type receptor. The agonist properties of aldosterone, as well as the antagonist feature of compounds bearing a 11beta-allenyl group and a C17-ketone function, remain unchanged. In contrast, C11-substituted steroids with a 17gamma-lactonic ring displayed antagonist properties with hMR and acted as potent agonists with A773G. An agonist-dependent hMR interaction with SRC-1 was observed for both the wild-type and the mutant receptors. The hMR activation process is discussed in the light of the hMR-LBD homology model based on the structural data of the human progesterone receptor LBD.

Analysis of data from spilling experiments performed with liquid hydrogen.

This work describes the modelling of liquid hydrogen release experiments using the ADREA-HF 3-D time dependent finite volume code for cloud dispersion, jointly developed by DEMOKRITOS and JRC-Ispra. The experiments were performed by Batelle Ingenieurtechnik for BAM (Bundesanstalt fur Materialforschung und Prufung), Berlin, in the frame of the Euro-Quebec-Hydro-Hydrogen-Pilot-Project and they mainly deal with LH2 near ground releases between buildings. In the present study, the experimental trial #5 was assumed for simulation due to the fact that in this release the largest number of sensor readings were obtained. The simulations illustrated the complex behaviour of LH2 dispersion in presence of buildings, characterized by complicated wind patterns, plume back flow near the source, dense gas behaviour at near range and significant buoyant behaviour at the far range. The simulations showed the strong effect of ground heating in the LH2 dispersion. The model also revealed major features of the dispersion that had to do with the "dense" behaviour of the cold hydrogen and the buoyant behaviour of the "warming-up" gas as well as the interaction of the building and the release wake. Such a behaviour was in qualitative and even quantitative agreement with the experiment. The results are given in terms of concentration time series, scatter plots, contour plots, wind field vector plots and 3-D concentration wireframes. Given all experiment uncertainties, the model gives reasonable results on concentrations levels.

Crucial role of the H11-H12 loop in stabilizing the active conformation of the human mineralocorticoid receptor.

The crystal structures of ligand-free and agonist-associated ligand-binding domain (LBD) of nuclear receptors (NRs) reveal that the amphipathic helix H12 is folded back toward the LBD core in the agonist-associated conformation, allowing the binding of coactivators. We used alanine scanning mutagenesis to explore the role of the residues of the loop connecting H11 and H12 in the activation of the human mineralocorticoid receptor (hMR), a member of the NRs family. H950A retained the ligand binding and transcriptional activities of the wild-type receptor and interacted with coactivators. In contrast F956A had no receptor functions. Aldosterone bound to the mutant hMRs (L952A, K953A, V954A, E955A, P957A) with nearly the same affinity as to the wild-type receptor and caused a receptor conformational change in these mutant hMRs as it does for the wild-type receptor. But the aldosterone-induced transcriptional activity of the mutant hMRs was lower (L952A, E955A, P957A) than that of the wild-type receptor or completely abolished (K953A, V954A) and their interaction with coactivators was impaired (E955A) or suppressed (L952A, K953A, V954A, P957A). In the light of a hMR-LBD model based on the structure of the progesterone-associated receptor-LBD, we propose that the integrity of the H11-H12 loop is crucial for folding the receptor into a ligand-binding competent state and for establishing the network of contacts that stabilize the active receptor conformation.

Residues in the ligand binding domain that confer progestin or glucocorticoid specificity and modulate the receptor transactivation capacity.

To localize regions conferring ligand binding specificity of the human glucocorticoid (hGR) and progesterone (hPR) receptors, we constructed chimeras comprising the DNA-binding domain of the yeast transcription factor GAL4, linked to the ligand binding domain of hGR or hPR. Replacement of a sequence of hGR encompassing helices H6 and H7 with the homologous sequence from hPR creates a chimeric protein GP3, which binds the progestin RU 27987 with high affinity, and results in a concomitant loss of glucocorticoid binding [dexamethasone (DEX), RU 43044]. Moreover, GP3 is not able to mediate RU 27987-induced transactivation. A detailed mutational analysis of this sequence and the study of the recently solved hPR crystal structure revealed five residues that confer progestin responsiveness to GR or modulate ligand binding and transcriptional activation. Notably, the simultaneous presence of residues Ser637 and Phe639 on GP3, lining the ligand binding pocket, is specifically involved in RU 27987 binding. The absence of residues Asp641, Gln642, and Leu647 on GP3 is accountable for the lack of glucocorticoids binding on GP3. Unlike residue 642, residues 641 and 647 are not in direct contact with the ligand and most likely act through steric-mediated interactions. The presence of Gln642 and Leu647 are determinant for transcriptional activation in response to DEX and RU 27987, respectively. DEX-dependent transactivation is further enhanced by the presence of Leu647.

A new model for 20-hydroxyecdysone and dibenzoylhydrazine binding: a homology modeling and docking approach.

The ecdysone receptor (ECR), a nuclear transcription factor controlling insect development, is a novel target for insecticides such as dibenzoylhydrazines with low environmental and toxicological impacts. To understand the high selectivity of such synthetic molecules toward ECR, two homology models of the Chironomus tentans ECR ligand-binding domain (LDB) have been constructed by taking as templates the known LBD crystal structures of the retinoic acid and vitamin D receptors. Docking of 20-hydroxyecdysone (20E) and dibenzoylhydrazines to the receptor suggests a novel superposition of the natural and synthetic molecules; the N-tert-butyl substituent of the dibenzoylhydrazines extends significantly beyond the 20E volume. Our ECR-LBD protein models rationalize how 20E and dibenzoylhydrazines interact with the ligand-binding pocket. The homology model complexes provide new insights that can be exploited in the rational design of new environmentally safe insecticides.

Crystal structure of a heterodimeric complex of RAR and RXR ligand-binding domains.

The crystal structure of a heterodimer between the ligand-binding domains (LBDs) of the human RARalpha bound to a selective antagonist and the constitutively active mouse RXRalphaF318A mutant shows that, pushed by a bulky extension of the ligand, RARalpha helix H12 adopts an antagonist position. The unexpected presence of a fatty acid in the ligand-binding pocket of RXRalpha(F318A is likely to account for its apparent "constitutivity." Specific conformational changes suggest the structural basis of pure and partial antagonism. The RAR-RXR heterodimer interface is similar to that observed in most nuclear receptor (NR) homodimers. A correlative analysis of 3D structures and sequences provides a novel view on dimerization among members of the nuclear receptor superfamily.

Specific recognition of androgens by their nuclear receptor. A structure-function study.

Androgens, like progestins, are 3-ketosteroids with structural differences restricted to the 17beta substituent in the steroid D-ring. To better understand the specific recognition of ligands by the human androgen receptor (hAR), a homology model of the ligand-binding domain (LBD) was constructed based on the progesterone receptor LBD crystal structure. Several mutants of residues potentially involved in the specific recognition of ligands in the hAR were constructed and tested for their ability to bind agonists. Their transactivation capacity in response to agonist (R1881) and antagonists (cyproterone acetate, hydroxyflutamide, and ICI 176344) was also measured. Substitution of His(874) by alanine, only marginally impairs the ligand-binding and transactivation capacity of the hAR receptor. In contrast, mutations of Thr(877) and, to a greater extent, Asn(705) perturb ligand recognition, alter transactivation efficiency, and broaden receptor specificity. Interestingly, the N705A mutant acquires progesterone receptor (PR) properties for agonist ligands but, unlike wild type AR and PR, loses the capacity to repress transactivation with nonsteroidal antagonists. Models of the hAR.LBD complexes with several ligands are presented, which suggests new directions for drug design.

The crystal structure of the nuclear receptor for vitamin D bound to its natural ligand.

The action of 1 alpha, 25-dihydroxyvitamin D3 is mediated by its nuclear receptor (VDR), a ligand-dependent transcription regulator. We report the 1.8 A resolution crystal structure of the complex between a VDR ligand-binding domain (LBD) construct lacking the highly variable VDR-specific insertion domain and vitamin D. The construct exhibits the same binding affinity for vitamin D and transactivation ability as the wild-type protein, showing that the N-terminal part of the LBD is essential for its structural and functional integrity while the large insertion peptide is dispensable. The structure reveals the active conformation of the bound ligand and allows understanding of the different binding properties of some synthetic analogs.