Search

During the last years CIEMAT has been leading the activities in the European Program to develop an integral breeding blanket with advanced performances to work in a realistic DEMO scenario. This blanket is the Dual Coolant Lithium Lead (DCLL) working at a limited temperature in order to allow the use of conventional materials and technologies. The design of this blanket was finished, including the definition of the tritium extraction system and tritium simulations. Then, determined by the selection of other two concepts as driver blankets for DEMO, the focus was put on developing a novel BB still based on the DCLL concept but working at higher temperatures, thus increasing the plant net efficiency. In this work, a summary of the status of the DCLL is presented, together with some ideas for developing an advanced high temperature DCLL in the near future.

Neurotensin (Nts) is a neuropeptide implicated in the regulation of many facets of physiology, including cardiovascular tone, pain processing, ingestive behaviors, locomotor drive, sleep, addiction and social behaviors. Yet, there is incomplete understanding about how the various populations of Nts neurons distributed throughout the brain mediate such physiology. This knowledge gap largely stemmed from the inability to simultaneously identify Nts cell bodies and manipulate them in vivo. One means of overcoming this obstacle is to study Nts(Cre) mice crossed onto a Cre-inducible green fluorescent reporter line (Nts(Cre);GFP mice), as these mice permit both visualization and in vivo modulation of specific populations of Nts neurons (using Cre-inducible viral and genetic tools) to reveal their function. Here we provide a comprehensive characterization of the distribution and relative densities of the Nts-GFP populations observed throughout the male Nts(Cre);GFP mouse brain, which will pave the way for future work to define their physiologic roles. We also compared the distribution of Nts-GFP neurons with Nts-In situ Hybridization (Nts-ISH) data from the adult mouse brain. By comparing these data sets we can distinguish Nts-GFP populations that may only transiently express Nts during development but not in the mature brain, and hence which populations may not be amenable to Cre-mediated manipulation in adult Nts(Cre);GFP mice. This atlas of Nts-GFP neurons will facilitate future studies using the Nts(Cre);GFP line to describe the physiological functions of individual Nts populations and how modulating them may be useful to treat disease.

Background Pleural fluid homocysteine (HCY) can be useful for diagnosis of malignant pleural effusion (MPE). There are no published studies comparing the diagnostic accuracy of HCY with other tumour markers in pleural fluid for diagnosis of MPE. The aim was to compare the accuracy of HCY with that of carcinoembryonic antigen (CEA), cancer antigen (CA) 15.3, CA19.9 and CA125 in pleural fluid and to develop a probabilistic model using these biomarkers to differentiate benign (BPE) from MPE. Methods Patients with pleural effusion were randomly included. HCY, CEA, CA15.3, CEA19.9 and CA125 were quantified in pleural fluid. Patients were classified into two groups: MPE or BPE. By applying logistic regression analysis, a multivariate probabilistic model was developed using pleural fluid biomarkers. The diagnostic accuracy was determined by receiver operating characteristic (ROC) curves and calculating the area under the curve (AUC). Results Population of study comprised 133 patients (72 males and 61 females) aged between 1 and 96 years (median = 70 years), 81 BPE and 52 MPE. The logistic regression analysis included HCY (p< 0.0001) and CEA (p = 0.0022) in the probabilistic model and excluded the other tumour markers. The probabilistic model was: HCY+CEA = Probability(%) = 100x( 1 +e(-z))(-1), where Z = 0.5471x[ HCY]+0.3846x[ CEA]-8.2671. The AUCs were 0.606, 0.703, 0.778, 0.800, 0.846 and 0.948 for CA125, CA19.9, CEA, CA15.3, HCY and HCY+CEA, respectively. Conclusions Pleural fluid HCY has higher accuracy for diagnosis of MPE than CEA, CA15.3, CA19.9 and CA125. The combination of HCY and CEA concentrations in pleural fluid significantly improves the diagnostic accuracy of the test.

The Liquid Breeder Validation Module (LBVM) will be one of the medium flux irradiation modules of the International Fusion Materials Irradiation Facility (IFMIF) neutron source. The objective of this module – presently under design – is the test of functional materials related to liquid breeders for future nuclear fusion power reactors (DEMO). This paper aims to describe the activation analyses performed to estimate the radioactive inventory and the expected contact dose from the activated materials of the module following a 345 day irradiation period. These calculations supply valuable information for different aspects related to the design of the module, such as the safety evaluation and the waste management and disassembly plan. The neutron transport calculations have been performed using the McDeLicious code. The ACAB nuclear inventory code, with the activation nuclear libraries EAF-2007, has been used for the activation analyses. The main results point out that the contact dose of the LBVM materials is much higher than the hands-on-limits, as expected. Therefore, remote handling operations are requested for disassembling the module. It is important to remark that after 8 h decay time, the contact dose rate of the LBVM decreases 76% for the EUROFER steel components and 46% for the 316 LN components. Regarding the isotopic inventory, although the main activation comes from the module steel structures, the production of tritium and Po-210 in the lithium lead inside the experimental capsules deserved a careful analysis.

The TechnoFusión project involves the construction of a relevant set of scientific-technical facilities in Madrid (Spain), providing new tools to fusion energy community. It will create an infrastructure to support the development of nuclear fusion technology, assuring participation by Spanish research groups and engineering companies.The improvement of liquid lithium technology is one of the milestones of the nuclear fusion pathway. Liquid lithium is a key component in some tritium breeding blankets and more recently, it is considered as a plasma-facing component. The development of this technology requires R&D to many phenomena: Interaction between liquid metal and structural materials as corrosion, ero- sion or embrittlement; chemical behavior of liquid lithium; coatings development; MHD; free surface fluid-dynamics; diagnosis and instrumentation of fluid lithium and any other physical or chemical aspect that will be important for their safe reliable operation and would increase the economic feasibility of commercial fusion energy. Nowadays, there is a significant shortage of experimental facilities committed to the development of the liquid lithium technology in Europe.In the framework of the TechnoFusión Project, an experimental laboratory of liquid lithium technology has been proposed, to back a real technological transfer to regional and national industrial sector. This paper summarizes the current conceptual layout of this facility.

Like their animal counterparts, plant glutamate receptor-like (GLR) homologs are intimately associated with Ca(2+) influx through plasma membrane and participate in various physiological processes. In pathogen-associated molecular patterns (PAMP)-/elicitor-mediated resistance, Ca(2+) fluxes are necessary for activating downstream signaling events related to plant defense. In this study, oligogalacturonides (OGs), which are endogenous elicitors derived from cell wall degradation, were used to investigate the role of Arabidopsis GLRs in defense signaling. Pharmacological investigations indicated that GLRs are partly involved in free cytosolic [Ca(2+)] ([Ca(2+)]cyt) variations, nitric oxide (NO) production, reactive oxygen species (ROS) production and expression of defense-related genes by OGs. In addition, wild-type Col-0 plants treated with the glutamate-receptor antagonist 6,7-dinitriquinoxaline-2,3-dione (DNQX) had a compromised resistance to Botrytis cinerea and Hyaloperonospora arabidopsidis. Moreover, we provide genetic evidence that AtGLR3.3 is a key component of resistance against H. arabidopsidis. In addition, some OGs-triggered immune events such as defense gene expression, NO and ROS production are also to different extents dependent on AtGLR3.3. Taken together, these data provide evidence for the involvement of GLRs in elicitor/pathogen-mediated plant defense signaling pathways in Arabidopsis thaliana.

Due to its applications in the treatment of cancer and autoimmune diseases, the 42 kDa zinc-dependent metalloenzyme carboxypeptidase G2 (CPG2) is of great therapeutic interest. An X-ray crystal structure of unliganded CPG2 reported in 1997 revealed the domain architecture and informed early rational drug design efforts, however further efforts at co-crystallization of CPG2 with ligands, substrates or inhibitors have not been reported. Thus key features of CPG2 such as the location of the active site, the presence of additional ligand-binding sites, stability, oligomeric state, and the molecular basis of activity remain largely unknown, with the current working understanding of CPG2 activity based primarily on computational modelling. To facilitate renewed efforts in CPG2 structural biology, we report the first high-yield (250 mg L(-1)) recombinant expression (and purification) of soluble and active CPG2 using the Escherichia coli expression system. We used this protocol to produce full-length enzyme, as well as protein fragments corresponding to the individual catalytic and dimerization domains, and the activity and stability of each construct was characterised. We adapted our protocol to allow for uniform incorporation of NMR labels ((13)C, (15)N and (2)H) and present preliminary solution-state NMR spectra of high quality. Taken together, our results offer a route for production and solution-state characterization that supports renewed effort in CPG2 structural biology as well as design of significantly truncated CPG2 proteins, which retain activity while yielding (potentially) improved immunogenicity.

Calcium signatures induced by two elicitors of plant defense reactions, namely cryptogein and oligogalacturonides, were monitored at the subcellular level, using apoaequorin-transformed Nicotiana tabacum var Xanthi cells, in which the apoaequorin calcium sensor was targeted either to cytosol, mitochondria or chloroplasts. Our study showed that both elicitors induced specific Ca2+ signatures in each compartment, with the most striking difference relying on duration. Common properties also emerged from the analysis of Ca2+ signatures: both elicitors induced a biphasic cytosolic [Ca2+] elevation together with a single mitochondrial [Ca2+] elevation concomitant with the first cytosolic [Ca2+] peak. In addition, both elicitors induced a chloroplastic [Ca2+] elevation peaking later in comparison to cytosolic [Ca2+] elevation. In cryptogein-treated cells, pharmacological studies indicated that IP3 should play an important role in Ca2+ signaling contrarily to cADPR or nitric oxide, which have limited or no effect on [Ca2+] variations. Our data also showed that, depending on [Ca2+] fluxes at the plasma membrane, cryptogein triggered a mitochondrial respiration increase and affected excess energy dissipation mechanisms in chloroplasts. Altogether the results indicate that cryptogein profoundly impacted cell functions at many levels, including organelles. (C) 2012 Elsevier Ltd. All rights reserved.

343 Background: Abiraterone acetate plus prednisone (AAP) and enzalutamide (ENZ), oral hormonal treatment of metastatic castration resistant prostate cancer (mCRPC), can have neuropsychiatric impact. Methods: The Proportional Reporting Ratio (PRR) was used to analyze evolution of suspected adverse drug reactions (ADRs) reported in real world, in EudraVigilance from March to August 2017. Results: From the total ADRs of AAP and ENZ reported in Eudra from March to August 2017, 1,162 were related to AAP and 5,101 were related to ENZ. ADRs of ENZ reported in Eudra in August were higher than those reported in March 2017, in all the neuropsychiatry ADRs analyzed. In the case of AAP, 44% of the neuropsychiatric suspected ADRs analyzed did not show new cases reported and 56% of the ADRs shown an increase, although it was not higher than 15 new cases reported. Conclusions: In August 2017, the updated ADRs of AAP and ENZ from Eudra were consistent with those observed in March 2017 (Iznaola et al.). It confirms an increasing trend of reporting neuropsychiatric ADRs of ENZ in mCRPC. Suspected ADRs of AAP reported remained steady.[Table: see text]

217 Background: Androgen deprivation is associated with cognitive decline and mood changes. Abiraterone acetate plus prednisone (AAP) and enzalutamide (ENZ) are oral hormonal agents for the treatment of metastatic castration resistant prostate cancer (mCPRC); both target the androgen signaling pathway. Meta-analysis for individual neuropsychiatric adverse effects (NAEs) associated with these drugs has not been available in the literature. Methods: Following the methodology presented by Ruiz et al. a further meta-analysis was performed to estimate the pooled Relative Risk (RR) of NAEs for AAP and ENZ. A complementary analysis of the EudraVigilance database was performed to explore the consistency of the real world adverse drug reactions (ADR) reporting pattern with the meta-analysis. Results: The meta-analysis results indicate that patients treated with ENZ had a statistically significant higher risk of restless leg syndrome (RLS), anxiety, headache and insomnia vs control. Both AAP and ENZ showed significant increased risk for falls vs control. The Proportional Reporting Ratio (PRR) of ADRs reported in Eudra is higher with ENZ than with AAP for all the variables analyzed. Conclusions: NAEs studied are more prevalent with ENZ vs placebo than AAP vs prednisone plus placebo. Reporting trend in Eudra is consistent with this results. [Table: see text]

There are no published studies examining the utility of total homocysteine (HCY) in pleural fluid. The aim was to measure the accuracy of pleural fluid HCY concentration for diagnosis of malignant pleural effusion (MPE). We studied pleural fluids obtained by thoracocentesis in patients with pleural effusion. Pleural fluid HCY concentration was measured by immunonephelometry using N Latex HCY reagent with monoclonal antibody in automated analyzers BNII (Siemens Diagnostics®). Patients were classified into two groups according to the etiology of pleural effusion: benign pleural effusions (BPE) and MPE. Pleural effusion was categorized as MPE if malignant cells were demonstrated in pleural fluid or pleural biopsy. The accuracy of pleural fluid HCY concentration for diagnosis of MPE was determined using receiver operating characteristic (ROC) techniques by analyzing the area under the ROC curve (AUC). We studied 89 patients with ages between 1 and 96 years old (median = 66). Forty-eight patients were BPE and 41 were MPE. Pleural fluid HCY concentration was significantly higher in patients with MPE (median = 13.70 μmol/L) than in those with BPE (median = 8.05 μmol/L). The AUC value was 0.833 (95 % confidence interval (CI) 0.739–0.903). The optimal cutoff value was 13.1 μmol/L exhibiting 56.1 % (95 % CI 39.8–71.5) sensitivity and 85.4 % (95 % CI 72.2–93.9) specificity. Pleural fluid HCY concentration showed high diagnostic accuracy to predict whether a pleural effusion is benign or malignant. Pleural fluid HCY concentration may be measured easily and quickly in automated analyzers and could be a tumor marker commonly used for diagnosis of MPE.

In the fusion plasma experiment TJ-II, two neutral beam injectors provide 2 MW heating power to the plasma. Beam losses occur at different stages due to collisions of beam ions or neutrals with surrounding H 2 molecules. Efficient use of the injected gas is of utmost importance, in order to minimize losses while ensuring optimum neutralization. In the design phase of the vacuum system, extensive use of the computer code OPTIMUS has been made. Calculations show that in order to keep reionization losses at an acceptable level, the maximum allowed pressure during the beam pulse is 10 −4 mbar, which requires an installed pumping speed of 350,000 l/s. Titanium getter pumps have been chosen as the primary vacuum system for these injectors. Base pressures in the range of 5×10 −9 mbar are attainable with titanium pump operation.

OBJECTIVES: To investigate whether the use of low-dose oral cobalamin (Cbl) supplements by older persons, as frequently found in multivitamin preparations, affects their Cbl serum concentrations and function, determined by measurements of the serum Cbl-related metabolites methylmalonic acid (MMA), homocysteine (HCYS), and methylcitric acid (MCTR). DESIGN: Cross-sectional study. SETTING: Community. PARTICIPANTS: Two hundred forty-two independent, active, community-living, older adult volunteers recruited from community events and activities for seniors. MEASUREMENTS: We systematically collected data on vitamin supplement intake, diet, medications, and medical and surgical history. Serum was obtained for Cbl, MMA, HCYS, and MCTR, and creatinine and hematological parameters. RESULTS: Serum levels of Cbl were significantly higher in subjects on oral Cbl supplements (2–37.5 μg/day). Similarly, serum levels of the metabolites MMA, HCYS, and MCTR were also lower in subjects on Cbl supplementation. Intake of low-dose oral supplements of Cbl significantly reduced the odds of low Cbl levels or high MMA. The relationship between Cbl supplement dosage and the biochemical parameters was dose dependent. CONCLUSION: Oral Cbl (2–37.5 μg/day) intake by community-dwelling healthy older adults is associated with higher serum levels of Cbl and improved or normalized Cbl function, as indicated by lower concentrations of the metabolites MMA, HCYS, and MCTR. Use of low-dose oral Cbl replacement therapy might be sufficient to prevent Cbl deficiency in a large proportion of this population.

276 Background: Abiraterone acetate + prednisone (AA) and cabazitaxel (CB) have shown an improvement in overall survival after Docetaxel (DOC) in (mCRPC)1,2. There is little evidence about the optimal sequencing treatment in prospective trials. In this final analysis we now compare the efficacy of AA and CB as second line (2L) treatment in mCRPC patients (pts) in routine clinical practice (RCP) in Spain. Methods: The CAPRO study is a prospective multicenter national observational descriptive study. The primary endpoint was to describe the management in 2L mCRPC pts after DOC in RCP. bPFS (biochemical progression free survival) and PFS (clinical or radiological progression free survival) were assessed by the Kaplan-Meier method. Results: 150 patients were recruited (Jul 2013-Jan 2015). At median follow-up of 7.8 mo (IQR 4.2-12.8) 67% (n = 100) of the pts received AA, 29% (n = 44) CB, and 4% (n = 6) other treatments as 2L. PSA response > 50% was 47.3% and 32.3% for AA group and CB group respectively (p = 0.146). Median bPFS was similar between the AA and the CB group (9.19 mo [95% CI 6.84-11.55] vs 9.92 mo [95% CI 6.08-13.76]; hazard ratio 0.779 [95% CI 0·49–1.24]; p = 0·29), whereas median PFS was significantly longer in the AA group than in the CB group (8.74 mo [95% CI 6.54-10.94] vs 6.41 mo [95% CI 4.99-7.82]; hazard ratio 0·56 [95% CI 0·38–0·85]; p = 0·005). A difference was observed in bPFS and PFS depending on the time of progression to first line (during treatment, < 3 or more than 3 mo) for both AA and CB. The most common toxicities of special interest (all grades/grade 3–4 as % of pts) were fatigue (31/1% in the AA group vs 54/4% in the CB group), oedema (15/0% AA group vs 13/4% CB group), hypertension 7/1% AA group, diarrhea (8/0% AA group vs 31/4% CB group), vomits (9/2% AA group vs 11/0 CB group) and neutropenia 7/4% CB group. Conclusions: In this final analysis AA was the 2L treatment of choice in routine clinical practice in Spain during the period of time in which the study was conducted. PSA response and bPFS were similar between AA and CB, whereas PFS was significantly longer in the AA group. There were no new safety signals observed.

227 Background: Recently treatment options for mCRPC have increased considerably. It remains unclear if there is any predictive factor to recognize which patients (pts) could obtain the most benefit in second line (2L) mCRPC. Recent data have been published addressing long-term predictive factors in a retrospective cohort in 2L1. We aim to reproduce it in this prospective study. Methods: The CAPRO study is a prospective multicenter national observational descriptive study. The primary endpoint was to describe the management in 2L mCRPC pts after DOC in routine clinical practice. To characterize which factors are associated with a better response in 2L mCRPC, we performed a multivariant analysis in the subgroup of pts with a long-term response (long-term responders (LTR): pts receiving abiraterone acetate + prednisone (AA) or Cabazitaxel (CB) for > 12 mo). Results: 150 patients were recruited (Jul 2013-Jan 2015). We identified 36 LTR (29/100 with AA: (29%) and 7/44 (15.9 %) with CB as 2L. No statistical differences in the number of LTR treated with AA vs CB (p = 0.095) were observed. LTR group (before starting 2L): median age 77.2 (51-87) median PSA 56.7 ng/dL (5.3-2627 ng/dL), alkaline phosphatase 92.5 IU/L (44-630 IU/L) and LDH 221.5 IU/L (147-445 IU/L). At median follow-up of 7.8 mo (IQR 4.2- 12.8), median PFS (clinical and/or radiological) was significantly longer in LTR AA group (not yet reached) vs LTR CB group 18.14 [95% CI 12.67-23.6]; HR 0·29 [95% CI 0,092 – 0,918; p = 0.025]. In a multivariate analysis two factors were significantly associated with LTR in 2L mCRPC: Gleason score < 7 (OR: 0.22 95% CI 0.065-0.745, p = 0.015) and normal LDH ( < 250 IU/L) (OR: 0.235, 95% CI 0.093-0.595, p = 0.002). No differences in safety profile between LTR vs non LTR of AA group and CB group were found. Conclusions: In this final subgroup analysis we observed Gleason score < 7 and LDH < 250 IU/L are predictors of long term response in 2L mCRPC. Median PFS in LTR treated with AA was significantly longer than LTR treated with CB. These findings need to be validated in further larger prospective studies.

We report the effect of native cyclodextrins (α, β, and γ) and selected derivatives in modulating the self-assembly of the nonionic surfactant polyoxyethylene cholesteryl ether (ChEO10) and its mixtures with triethylene glycol monododecyl ether (C12EO3), which form wormlike micelles. Cyclodextrins (CDs) generally induce micellar breakup through a host-guest interaction with surfactants; instead, we show that a constructive effect, leading to gel formation, is obtained with specific CDs and that the widely invoked host-guest interaction may not be the only key to the association. When added to wormlike micelles of ChEO10 and C12EO3, native β-CD, 2-hydroxyethyl-β-CD (HEBCD), and a sulfated sodium salt of β-CD (SULFBCD) induce a substantial increase of the viscoelasticity, while methylated CDs rupture the micelles, leading to a loss of the viscosity, and the other CDs studied (native α- and γ- and hydroxypropylated CDs) show a weak interaction. Most remarkably, the addition of HEBCD or SULFBCD to pure ChEO10 solutions (which are low-viscosity, Newtonian fluids of small, ellipsoidal micelles) induces the formation of transparent gels. The combination of small-angle neutron scattering, dynamic light scattering, and cryo-TEM reveals that both CDs drive the elongation of ChEO10 aggregates into an entangled network of wormlike micelles. (1)H NMR and fluorescence spectroscopy demonstrate the formation of inclusion complexes between ChEO10 and methylated CDs, consistent with the demicellization observed. Instead, HEBCD forms a weak complex with ChEO10, while no complex is detected with SULFBCD. This shows that inclusion complex formation is not the determinant event leading to micellar growth. HEBCD:ChEO10 complex, which coexists with the aggregated surfactant, could act as a cosurfactant with a different headgroup area. For SULFBCD, intermolecular interactions via the external surface of the CD may be more relevant.

Heterotrimeric guanosine triphosphate (GTP)-binding proteins (G-proteins) couple many different cell surface receptor types to intracellular effector mechanisms. Uncoupling between receptors and G-proteins and between G-proteins and adenylyl cyclase (AC) and phospholipase C (PLC) has been described for Alzheimer's disease (AD) brain. However, there is little information on whether altered G-protein signaling in AD is just an end-stage phenomenon or is important for the progression of disease pathology. Here we used [(35)S]GTPgammaS autoradiography to study G-protein distribution in sections of entorhinal cortex and hippocampus from 23 cases staged for neurofibrillary changes and amyloid deposits according to Braak and Braak (Acta Neuropathol. [1991] 82:239-259). We also studied the effects of GTP, which has been found to increase [(35)S]GTPgammaS binding in an Mg(2+)-dependent manner. Results show that the ability of GTP (3 microM) to stimulate [(35)S]GTPgammaS binding declined significantly with staging for neurofibrillary changes in the entorhinal cortex (P < 0.05, ANOVA) and CA1 subfield of the hippocampus (P < 0.05, ANOVA). No significant changes were seen for [(35)S]GTPgammaS binding in the absence of GTP. Our results suggest a decrease in G-protein GTP hydrolysis, which correlates with the progression of AD neurofibrillary changes, in the regions most affected by this pathology. These alterations appear to occur prior to stages corresponding to clinical disease and could lead to an impaired regulation of several signaling systems in AD brain.

TJ-II is a four period, low magnetic shear stellarator (R = 1.5 m, a < 0.22 m, B0 ≤ 1.2 T) which was designed to have a high degree of magnetic configuration flexibility. In the most recent experimental campaign, coupling of the full ECRH power (PECRH ≤ 600 kW) to the plasma has been possible using two ECRH transmission lines which have different power densities. Both helium and hydrogen fuelled plasmas have been investigated. The article reviews the latest physics results in particle control, configuration effects, and transport and fluctuation studies.

The binding of [35S]GTPγS was characterised with autoradiography in rat brain. The binding was saturable, but the rate of dissociation was very slow. Analysis of binding isotherms revealed one class of binding sites with a Kd of 0.8 μM. The specific binding was 98%. Different guanine nucleotides were all able to compete with [35S]GTPγS binding. However, no displacement was seen by the ATP-analogue App[NH]p, indicating that [35S]GTPγS does not bind to ATP-sites. Autoradiograms showed a highly homogenous distribution of [35S]GTPγS binding, in grey as well as in white matter. However, the pattern changed dramatically in the presence of GTP, which, unlike the non-hydrolysable GTP-analogues Gpp[NH]p and GTPγS, did not displace [35S]GTPγS binding throughout the brain. In white matter areas the binding was potently displaced, while in many grey matter areas, e.g., the striatum, the binding was seen to increase. This GTP-induced increase in [35S]GTPγS binding was strongly Mg2+-dependent, with an optimum at 10 mM. This, together with the finding that the regional effects of GTP correspond well to previously reported distribution of low Km GTPase, suggest that the levels of binding of [35S]GTPγS in the presence of GTP may reflect functional G-protein activity.

Resveratrol, a natural polyphenol found in grapes, berries and other plants, has been proposed as an ideal chemopreventative agent due to its plethora of health promoting activities. However, despite its lofty promise as a cancer prevention agent its success in human clinical trials has been limited due to its poor bioavailability. Thus, interest in other natural polyphenols is intensifying including the naturally occurring dimethylated analog of resveratrol, pterostilbene. The UDP-glucuronosyltransferase (UGT) family of enzymes plays a vital role in the metabolism of both resveratrol and pterostilbene. The current study sought to elucidate the UGT family members responsible for the metabolism of pterostilbene and to examine gender differences in the glucuronidation of resveratrol and pterostilbene. We demonstrate that UGT1A1 and UGT1A3 are mainly responsible for pterostilbene glucuronidation although UGT1A8, UGT1A9 and UGT1A10 also had detectable activity. Intriguingly, UGT1A1 exhibits the highest activity against both resveratrol and pterostilbene despite altered hydroxyl group specificity. Using pooled human liver microsomes, enzyme kinetics were determined for pterostilbene and resveratrol glucuronides. In all cases females were more efficient than males, indicating potential gender differences in stilbene metabolism. Importantly, the glucuronidation of pterostilbene is much less efficient than that of resveratrol, indicating that pterostilbene will have dramatically decreased metabolism in humans.

Mucus is a ubiquitous feature of mammalian wet epithelial surfaces, where it lubricates and forms a selective barrier that excludes a range of particulates, including pathogens, while hosting a diverse commensal microflora. The major polymeric component of mucus is mucin, a large glycoprotein formed by several MUC gene products, with MUC2 expression dominating intestinal mucus. A satisfactory answer to the question of how these molecules build a dynamic structure capable of playing such a complex role has yet to be found, as recent reports of distinct layers of chemically identical mucin in the colon and anomalously rapid transport of nanoparticles through mucus have emphasized. Here we use atomic force microscopy (AFM) to image a MUC2-rich mucus fraction isolated from pig jejunum. In the freshly isolated mucin fraction, we find direct evidence for trigonally linked structures, and their assembly into lamellar networks with a distribution of pore sizes from 20 to 200 nm. The networks are two-dimensional, with little interaction between lamellae. The existence of persistent cross-links between individual mucin polypeptides is consistent with a non-self-interacting lamellar model for intestinal mucus structure, rather than a physically entangled polymer network. We only observe collapsed entangled structures in purified mucin that has been stored in nonphysiological conditions.

The application of liquid metal technology in fusion devices requires R&D related to many phenomena: interaction between liquid metals and structural material as corrosion, erosion and passivation techniques; magneto-hydrodynamics; free surface fluid-dynamics and any other physical aspect that will be needed for their safe reliable operation. In particular, there is a significant shortage of experimental facilities dedicated to the development of the lithium technology. In the framework of the TECHNOFUSION project, an experimental laboratory devoted to the lithium technology development is proposed, in order to shed some light in the path to IFMIF and the design of chamber's first wall and divertors. The conceptual design foresee a development in two stages, the first one consisting on a material testing loop. The second stage proposes the construction of a mock-up of the IFMIF target that will allow to assess the behaviour of a free-surface lithium target under vacuum conditions. In this paper, such conceptual design is addressed.

G-protein alpha-subunits (Galphao, Galphai, Galphas, Galphaq) and adenylyl cyclase (AC) I and II isoforms were quantified in hippocampus and entorhinal cortex from 22 cases staged for Alzheimer's disease (AD) pathologies according to Braak and Braak. Hippocampal Galphai levels declined significantly with neurofibrillary staging, whereas AC I levels in this region increased. Significant amyloid stage-related reductions of Galphai were seen in both the hippocampus and entorhinal cortex. The hippocampus also showed a significant reduction of Galphao with amyloid staging. It is concluded that levels of inhibitory G-protein subunits Galphao, and in particular Galphai, decrease in parallel to the extent of AD pathology.

Adenylyl cyclase (AC) signal transduction has been shown to be affected in Alzheimer's disease (AD). Deficits have been described in different components of the system, from the receptor to the effector level. [3H]forskolin is a diterpene that binds with high affinity to AC. In the present report, we used autoradiography to study [3H]forskolin binding to sections of entorhinal cortex and hippocampus from 23 cases staged for AD pathology according to Braak and Braak [Acta Neuropathol. 82 (1991) 239-259]. This protocol defines six stages according to neurofibrillary changes, which start in the entorhinal region (stages I-II), spread to the hippocampus (stages III-IV) and finally to the isocortical areas (stages V-VI). The amyloid classification includes three stages in which the basal isocortex is first affected (stage A), followed by other isocortical association areas (stage B) and finally the primary isocortical areas (stage C). We also studied the effects of the GTP-analogue Gpp[NH]p on binding, in order to detect changes in G-protein-AC coupling. We used two different concentrations of Gpp[NH]p, that were previously reported to inhibit and stimulate [3H]forskolin binding via Gi and Gs, respectively. Results showed that [3H]forskolin binding declined significantly with staging for neurofibrillary changes only in the entorhinal region (P0.05, ANOVA). In addition, the decrease in [3H]forskolin binding observed in the presence of 1 microM Gpp[NH]p diminished significantly with staging in the entorhinal region (P0.05, ANOVA). No significant changes were seen with amyloid staging, with the exception of the CA1 subfield of the hippocampus, where [3H]forskolin binding in the absence of Gpp[NH]p was significantly decreased at stage B compared with all other stages (P0.05, ANOVA). In conclusion, our results showed a very limited decrease in [3H]forskolin binding with the progression of AD pathology, suggesting that the AC levels may be largely preserved in the disease. The specific change in the effect of a low concentration of Gpp[NH]p on the binding could indicate the loss of Ca2+/calmodulin-sensitive AC isoforms in AD.

The molecular recognition of specific transfer RNAs by the appropriate aminoacyl-tRNA synthetase is an important step in determining the accuracy of translation of the genetic message from nucleic acids into proteins. Recent studies using variant tRNAs with specific sequence modifications have indicated particular regions that determine their identity. Here we consider whether the base modifications commonly found in tRNAs contribute to their identity. Although unmodified tRNA(Asp) is charged with aspartate as efficiently as the modified native tRNA, it is mischarged with arginine with considerably increased efficiency. Our results indicate that post-transcriptional modification of tRNAs introduces structural 'anti-determinants', restricting the efficiency with which the tRNAs are charged with inappropriate amino acids.

The effect of the GTP-analogue guanylyl 5'-imidodiphosphate (Gpp[NH]p) on [3H]forskolin binding was studied in rat brain using autoradiography. In the striatum, 100 microM Gpp[NH]p produced a 40% increase in binding, whereas a decrease of about 30% was observed with low Gpp[NH]p concentrations (0.1-1 microM). In the molecular layer of the cerebellum all concentrations of Gpp[NH]p decreased [3H]forskolin binding. The decrease in binding disappeared in both striatum and the molecular layer of cerebellum in sections pretreated with 100 microM N-ethylmaleimide (NEM) for 10 min. NEM pretreatment did not significantly affect the stimulation of [3H]forskolin binding by micromolar concentrations of Gpp[NH]p in the striatum, but reversed the decrease observed in the molecular layer of the cerebellum, to an increase. Based on these data we suggest that the effects of the GTP-analogue Gpp[NH]p on [3H]forskolin binding may involve both Gs and Gi, where a stimulation produces an increase and decrease in binding respectively. The regional effects of Gpp[NH]p may reflect differences in the responsiveness of adenylyl cyclase to Gs and Gi-mediated effects.

Nox1 is a member of the NADPH Oxidase family which has not been well characterized in the melanocytic cell lineage. Here we demonstrated that Nox1 and Nox4 were detected in melanocytic lineage, with only Nox1 detected in normal human melanocytes and Nox4 in a subset of metastatic melanoma cell lines. The protein level and enzymatic activity of Nox1 was elevated in all melanoma cells as compared to normal melanocytes. Over-expression of GFP-Nox1 protein in Wm3211 primary melanoma cells increased invasion rate by 4-6 fold as measured by Matrigel invasion assay, while knocking down or inhibiting Nox1 decreased invasion by approximately 40 to 60%. MMP-2 was increased by Nox1 over-expression at the mRNA, protein and activity levels, and decreased by Nox1 knockdown. MMP-2 promoter activity was also regulated by Nox1 knockdown. In addition, stable clones over-expressing Nox1 exhibited an epithelial-mesenchymal transition (EMT) as examined by cell morphology and EMT markers; knockdown or inhibiting Nox1 led to a reversal of EMT. Supplementing MMP-2 to culture media did not induce EMT, suggesting that EMT induction by Nox1 was not through MMP-2 up-regulation. In summary, Nox1 was over-expressed in all melanoma cell lines examined, and enhanced cell invasion by MMP-2 up-regulation and EMT induction. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2418. doi:1538-7445.AM2012-2418

Chemical carcinogens such as the aromatic amide 2-acetylaminofluorene (AAF) are known to induce -1 frameshift mutation hotspots at repetitive sequences. This mutagenesis pathway was suggested to involve slipped intermediates formed during replication. To investigate the stability and structure of such intermediates we have constructed DNA duplexes containing single AAF adducts within a run of three guanine residues. The strand complementary to that bearing the AAF adducts contained either the wild-type sequence (homoduplexes) or lacked one cytosine directly opposite the run of guanines containing the AAF adduct and thus modeled the putative slipped mutagenic intermediates (SMIs). The melting temperature of AAF-modified homoduplexes or the unmodified SMI was reduced by approximately 10 degrees C relative to the unmodified homoduplex. Surprisingly, AAF adducts stabilized the SMIs as evidenced by an increase in melting temperature to a level approaching that of the unmodified homoduplex. The chemical probes hydroxylamine and bromoacetaldehyde were strongly reactive toward cytosine residues opposite the adduct in AAF-modified homoduplexes, indicating adduct-induced denaturation. In contrast, no cytosine reactivities were observed in the AAF-modified SMIs, suggesting that the two cytosines were paired with unmodified guanines. Use of diethyl pyrocarbonate to probe the guanine residues showed that all three guanines in the unmodified SMI adopted a transient single-stranded state which was delocalized along the repetitive sequence. However, when an AAF adduct was present, reduced diethyl pyrocarbonate reactivity at guanines adjacent to the adduct in AAF-modified SMIs reflected localization of the bulge to the adducted base. Our results suggest that AAF exerts a local denaturing and destabilizing effect within the homoduplex which is alleviated by the formation of a bulge. The stabilization by the AAF adduct of the SMIs may contribute to the dramatic increase in -1 frameshift mutation frequency induced by AAF adducts in repetitive sequences.

Chemical footprinting experiments on brewer's yeast tRNA(Asp) complexed to its cognate aspartyl-tRNA synthetase are reported: they demonstrate that bases of the anticodon loop, including the anticodon itself, are in close proximity with the synthetase. Contacts were determined using dimethylsulfate as the probe for testing reactivity of guanine and cytosine residues in free and complexed tRNA. Results correlate with the decrease in aspartylation activity of yeast tRNA(Asp) molecules mutated at these contact positions and will be compared with other structural data arising from solution and crystallographic studies on the aspartic acid complex.

This report presents the conceptual and methodological framework that presently underlies the experiments designed to decipher the structural features in tRNA important for its aminoacylation by aminoacyl-tRNA synthetases. It emphasizes the importance of conformational features in tRNA for an optimized aminoacylation. This is illustrated by selected examples on yeast tRNA(Asp). Using the phage T7 transcriptional system, a series of tRNA(Asp) variants were created in which conformational elements were modified. It is shown that aspartyl-tRNA synthetase tolerates conformational variability in tRNA(Asp) at the level of the D-loop and variable region, of the tertiary Levitt base-pair 15-48 which can be inverted and in the T-arm in which residue 49 can be excised. However, changing the anticodon region completely abolishes the aspartylation capacity of the variants. Transplanting the phenylalanine identity elements into a different tRNA(Asp) variant presenting conformational characteristics of tRNA(Phe) converts this molecule into a phenylalanine acceptor but is less efficient than wild-type tRNA(Phe). This engineered tRNA completely loses its aspartylation capacity, showing that some aspartic acid and phenylalanine identity determinants overlap. The fact that chimeric tRNA(Asp) molecules with altered anticodon regions lose their aspartylation capacity demonstrates that this region is part of the aspartic acid identity of tRNA(Asp).

Yeast tRNA(Ser) is a member of the class II tRNAs, whose characteristic is the presence of an extended variable loop. This additional structural feature raises questions about the recognition of these class II tRNAs by their cognate synthetase and the possibility of the involvement of the extra arm in the recognition process. A footprinting study of yeast tRNA(Ser) complexed with its cognate synthetase, yeast seryl-tRNA synthetase (an alpha 2 dimer), was undertaken. Chemical (ethylnitrosourea) and enzymatic (nucleases S1 and V1) probes were used in the experiments. A map of the contact points between the tRNA and the synthetase was established and results were analyzed with respect to a three-dimensional model of yeast tRNA(Ser). Regions in close vicinity with the synthetase are clustered on one face of tRNA. The extra arm, which is strongly protected from chemical modifications, appears as an essential part of the contact area. The anticodon triplet and a large part of the anticodon arm are, in contrast, still accessible to the probes when the complex is formed. These results are discussed in the context of the recognition of tRNAs in the aminoacylation reaction.

Calcium carbonate production in most freshwater tufa systems is controlled by a combination of physico-chemical precipitation, and biomediation associated with procaryote-microphyte biofilms. Inorganic precipitation is dominant under turbulent flow regimes, whereas biomediation necessitates sluggish flow or static water. We show here that temperature is also likely to affect tufa formation rates and architecture, leading to the development of a clearly definable range of fabrics. Two contrasting models are proposed: Warm semi-arid tufas: well seen in the Ruidera Pools National Park, Albacete Province, Central Spain, where tufa barrages consist of vertical, narrow rimmed arcuate structures. Frequently these are bounded both upstream and downstream by deep lakes. Laterally accreting mammilate stromatolites dominate the upper two thirds of lake margin subaqueous overhangs, whereas conical growth-forms dominate shady basal parts of subaqueous overhangs. Dense reed stands grow in thin peats on top of the vertical lake margin walls. Resistivity survey and percussion augering show up to 20 m of earlier lake fill which is banded into dark and pale beds. The upper 10 m accumulated in 4600 years. There is little or no preservation of organic material within these deposits. Cool temperate tufas: typified by slowly accumulating barrage tufas at Caerwys (N. Wales) and Alport (N. Derbyshire, UK). Transverse barriers across valleys are dominated by arcuate buttress developments built up of downstream-facing tabular carbonate sheets. Pools upstream of the barriers are shallow, dominated by clotted lime mud and organic sapropels in their depocentres, and stromatolite-dominated (laminar to low domes, or oncoidal) facies in broad marginal zones. Marginal stands of semi-aquatic vegetation are rooted in thick peats but frequently are also encrusted around their exposed stems by oncoidal carbonate. Fabric preservation is good for the carbonate material, however, marginal peats rapidly oxidise and stem encrustations collapse to produce cylindrical oncoid beds. Depocentre sapropels are always preserved and contain important pollen records otherwise rare in carbonate regions. Carbonates in both models accumulate relatively rapidly. They have a moderate preservation potential within Late Cenozoic terrestrial successions. The distinctly different characteristics of their preserved fabrics should allow recognition of two contrasting “climatic” models for fluvial barrage tufas in ancient deposits.