Current role of enzyme analysis for urea cycle disorders

Urea cycle disorders (UCD) are due to defects of any of its six enzymes or two transporters. The definitive diagnosis of defects of the three mitochondrial enzymes, N-acetylglutamate synthase (NAGS), carbamylphosphate synthetase I (CPS1) and ornithine transcarbamylase (OTC) depends on either molecular mutation analysis or measurement of enzyme activity, whereas the diagnosis of deficiencies of the three cytosolic enzymes argininosuccinate synthetase (ASS), argininosuccinate lyase (ASL) and arginase I (ARG1) is usually straightforward, based on marker metabolites. Enzyme assays for all UCD have been used since their first description, for disease confirmation and in some instances even for prenatal diagnosis. The genetic bases of the UCD have only been unraveled from the 1980s; the last gene cloned being the NAGS gene in 2002. In this review we discuss the enzymatic assays for all urea cycle enzymes from a historical perspective, their potential and drawbacks, and the current role of enzymatic analysis in UCD in general.

Submaximal cardiopulmonary thresholds on a robotics-assisted tilt table, a cycle and a treadmill: a comparative analysis

BACKGROUND: The robotics-assisted tilt table (RATT), including actuators for tilting and cyclical leg movement, is used for rehabilitation of severely disabled neurological patients. Following further engineering development of the system, i.e. the addition of force sensors and visual bio-feedback, patients can actively participate in exercise testing and training on the device. Peak cardiopulmonary performance parameters were previously investigated, but it also important to compare submaximal parameters with standard devices. The aim of this study was to evaluate the feasibility of the RATT for estimation of submaximal exercise thresholds by comparison with a cycle ergometer and a treadmill. METHODS: 17 healthy subjects randomly performed six maximal individualized incremental exercise tests, with two tests on each of the three exercise modalities. The ventilatory anaerobic threshold (VAT) and respiratory compensation point (RCP) were determined from breath-by-breath data. RESULTS: VAT and RCP on the RATT were lower than the cycle ergometer and the treadmill: oxygen uptake (V'O2) at VAT was [mean (SD)] 1.2 (0.3), 1.5 (0.4) and 1.6 (0.5) L/min, respectively (p < 0.001); V'O2 at RCP was 1.7 (0.4), 2.3 (0.8) and 2.6 (0.9) L/min, respectively (p = 0.001). High correlations for VAT and RCP were found between the RATT vs the cycle ergometer and RATT vs the treadmill (R on the range 0.69-0.80). VAT and RCP demonstrated excellent test-retest reliability for all three devices (ICC from 0.81 to 0.98). Mean differences between the test and retest values on each device were close to zero. The ventilatory equivalent for O2 at VAT for the RATT and cycle ergometer were similar and both were higher than the treadmill. The ventilatory equivalent for CO2 at RCP was similar for all devices. Ventilatory equivalent parameters demonstrated fair-to-excellent reliability and repeatability. CONCLUSIONS: It is feasible to use the RATT for estimation of submaximal exercise thresholds: VAT and RCP on the RATT were lower than the cycle ergometer and the treadmill, but there were high correlations between the RATT vs the cycle ergometer and vs the treadmill. Repeatability and test-retest reliability of all submaximal threshold parameters from the RATT were comparable to those of standard devices.

Tp53-dependent repression of cell cycle target genes: Global and mechanistic analysis

Most studies of p53 function have focused on genes transactivated by p53. It is less widely appreciated that p53 can repress target genes to affect a particular cellular response. There is evidence that repression is important for p53-induced apoptosis and cell cycle arrest. It is less clear if repression is important for other p53 functions. A comprehensive knowledge of the genes repressed by p53 and the cellular processes they affect is currently lacking. We used an expression profiling strategy to identify p53-responsive genes following adenoviral p53 gene transfer (Ad-p53) in PC3 prostate cancer cells. A total of 111 genes represented on the Affymetrix U133A microarray were repressed more than two fold (p ≤ 0.05) by p53. An objective assessment of array data quality was carried out using RT-PCR of 20 randomly selected genes. We estimate a confirmation rate of >95.5% for the complete data set. Functional over-representation analysis was used to identify cellular processes potentially affected by p53-mediated repression. Cell cycle regulatory genes exhibited significant enrichment (p ≤ 5E-28) within the repressed targets. Several of these genes are repressed in a p53-dependent manner following DNA damage, but preceding cell cycle arrest. These findings identify novel p53-repressed targets and indicate that p53-induced cell cycle arrest is a function of not only the transactivation of cell cycle inhibitors (e.g., p21), but also the repression of targets that act at each phase of the cell cycle. The mechanism of repression of this set of p53 targets was investigated. Most of the repressed genes identified here do not harbor consensus p53 DNA binding sites but do contain binding sites for E2F transcription factors. We demonstrate a role for E2F/RB repressor complexes in our system. Importantly, p53 is found at the promoter of CDC25A. CDC25A protein is rapidly degraded in response to DNA damage. Our group has demonstrated for the first time that CDC25A is also repressed at the transcript level by p53. This work has important implications for understanding the DNA damage cell cycle checkpoint response and the link between E2F/RB complexes and p53 in the repression of target genes. ^

Modelling Crowd Load for Floor Vibration Analysis

The dynamic floor loads induced by crowds in gymnasium or stadium structures are commonly modelled by superposition of the individual contributions using reduction factors for the different Fourier coefficients. These Fourier coefficients and the reduction factors are calculated using full scale measurements. Generally the testing is performed on platforms or structures that can be considered rigid, such that the natural frequencies are higher than the frequencies of the spectator movement. In this paper we shall present the testing done on a structure that used to be a gymnasium as well as the procedure used to identify its dynamic properties and a first evaluation of the socalled “group effect”.

Load displacement in grape harvesters: Discrete Element Analysis.

Dynamic weighing of the hopper in grape harvesters is affected by a number of factors. One of them is the displacement of the load inside the hopper as a consequence of the terrain topography. In this work, the weight obtained by a load cell in a grape harvester has been analysed and quantified using the discrete element method (DEM). Different models have been developed considering different scenarios for the terrain.

Simulation, Design and Analysis of Air-Breathing Combined-Cycle Engines for High Speed Propulsion

Desde la aparición del turborreactor, el motor aeróbico con turbomaquinaria ha demostrado unas prestaciones excepcionales en los regímenes subsónico y supersónico bajo. No obstante, la operación a velocidades superiores requiere sistemas más complejos y pesados, lo cual ha imposibilitado la ejecución de estos conceptos. Los recientes avances tecnológicos, especialmente en materiales ligeros, han restablecido el interés por los motores de ciclo combinado. La simulación numérica de estos nuevos conceptos es esencial para estimar las prestaciones de la planta propulsiva, así como para abordar las dificultades de integración entre célula y motor durante las primeras etapas de diseño. Al mismo tiempo, la evaluación de estos extraordinarios motores requiere una metodología de análisis distinta. La tesis doctoral versa sobre el diseño y el análisis de los mencionados conceptos propulsivos mediante el modelado numérico y la simulación dinámica con herramientas de vanguardia. Las distintas arquitecturas presentadas por los ciclos combinados basados en sendos turborreactor y motor cohete, así como los diversos sistemas comprendidos en cada uno de ellos, hacen necesario establecer una referencia común para su evaluación. Es más, la tendencia actual hacia aeronaves "más eléctricas" requiere una nueva métrica para juzgar la aptitud de un proceso de generación de empuje en el que coexisten diversas formas de energía. A este respecto, la combinación del Primer y Segundo Principios define, en un marco de referencia absoluto, la calidad de la trasferencia de energía entre los diferentes sistemas. Esta idea, que se ha estado empleando desde hace mucho tiempo en el análisis de plantas de potencia terrestres, ha sido extendida para relacionar la misión de la aeronave con la ineficiencia de cada proceso involucrado en la generación de empuje. La metodología se ilustra mediante el estudio del motor de ciclo combinado variable de una aeronave para el crucero a Mach 5. El diseño de un acelerador de ciclo combinado basado en el turborreactor sirve para subrayar la importancia de la integración del motor y la célula. El diseño está limitado por la trayectoria ascensional y el espacio disponible en la aeronave de crucero supersónico. Posteriormente se calculan las prestaciones instaladas de la planta propulsiva en función de la velocidad y la altitud de vuelo y los parámetros de control del motor: relación de compresión, relación aire/combustible y área de garganta. ABSTRACT Since the advent of the turbojet, the air-breathing engine with rotating machinery has demonstrated exceptional performance in the subsonic and low supersonic regimes. However, the operation at higher speeds requires further system complexity and weight, which so far has impeded the realization of these concepts. Recent technology developments, especially in lightweight materials, have restored the interest towards combined-cycle engines. The numerical simulation of these new concepts is essential at the early design stages to compute a first estimate of the engine performance in addition to addressing airframe-engine integration issues. In parallel, a different analysis methodology is required to evaluate these unconventional engines. The doctoral thesis concerns the design and analysis of the aforementioned engine concepts by means of numerical modeling and dynamic simulation with state-of-the-art tools. A common reference is needed to evaluate the different architectures of the turbine and the rocket-based combined-cycle engines as well as the various systems within each one of them. Furthermore, the actual trend towards more electric aircraft necessitates a common metric to judge the suitability of a thrust generation process where different forms of energy coexist. In line with this, the combination of the First and the Second Laws yields the quality of the energy being transferred between the systems on an absolute reference frame. This idea, which has been since long applied to the analysis of on-ground power plants, was extended here to relate the aircraft mission with the inefficiency of every process related to the thrust generation. The methodology is illustrated with the study of a variable- combined-cycle engine for a Mach 5 cruise aircraft. The design of a turbine-based combined-cycle booster serves to highlight the importance of the engine-airframe integration. The design is constrained by the ascent trajectory and the allocated space in the supersonic cruise aircraft. The installed performance of the propulsive plant is then computed as a function of the flight speed and altitude and the engine control parameters: pressure ratio, air-to-fuel ratio and throat area.

Analysis of advanced European nuclear fuel cycle scenarios including transmutation and economic estimates

Four European fuel cycle scenarios involving transmutation options (in coherence with PATEROS and CPESFR EU projects) have been addressed from a point of view of resources utilization and economic estimates. Scenarios include: (i) the current fleet using Light Water Reactor (LWR) technology and open fuel cycle, (ii) full replacement of the initial fleet with Fast Reactors (FR) burning U?Pu MOX fuel, (iii) closed fuel cycle with Minor Actinide (MA) transmutation in a fraction of the FR fleet, and (iv) closed fuel cycle with MA transmutation in dedicated Accelerator Driven Systems (ADS). All scenarios consider an intermediate period of GEN-III+ LWR deployment and they extend for 200 years, looking for long term equilibrium mass flow achievement. The simulations were made using the TR_EVOL code, capable to assess the management of the nuclear mass streams in the scenario as well as economics for the estimation of the levelized cost of electricity (LCOE) and other costs. Results reveal that all scenarios are feasible according to nuclear resources demand (natural and depleted U, and Pu). Additionally, we have found as expected that the FR scenario reduces considerably the Pu inventory in repositories compared to the reference scenario. The elimination of the LWR MA legacy requires a maximum of 55% fraction (i.e., a peak value of 44 FR units) of the FR fleet dedicated to transmutation (MA in MOX fuel, homogeneous transmutation) or an average of 28 units of ADS plants (i.e., a peak value of 51 ADS units). Regarding the economic analysis, the main usefulness of the provided economic results is for relative comparison of scenarios and breakdown of LCOE contributors rather than provision of absolute values, as technological readiness levels are low for most of the advanced fuel cycle stages. The obtained estimations show an increase of LCOE ? averaged over the whole period ? with respect to the reference open cycle scenario of 20% for Pu management scenario and around 35% for both transmutation scenarios. The main contribution to LCOE is the capital costs of new facilities, quantified between 60% and 69% depending on the scenario. An uncertainty analysis is provided around assumed low and high values of processes and technologies.

Discrete element analysis for the assessment of the accuracy of load cell-based dynamic weighing systems in grape harvesters under different ground conditions

Dynamic weighing systems based on load cells are commonly used to estimate crop yields in the field. There is lack of data, however, regarding the accuracy of such weighing systems mounted on harvesting machinery, especially on that used to collect high value crops such as fruits and vegetables. Certainly, dynamic weighing systems mounted on the bins of grape harvesters are affected by the displacement of the load inside the bin when moving over terrain of changing topography. In this work, the load that would be registered in a grape harvester bin by a dynamic weighing system based on the use of a load cell was inferred by using the discrete element method (DEM). DEM is a numerical technique capable of accurately describing the behaviour of granular materials under dynamic situations and it has been proven to provide successful predictions in many different scenarios. In this work, different DEM models of a grape harvester bin were developed contemplating different influencing factors. Results obtained from these models were used to infer the output given by the load cell of a real bin. The mass detected by the load cell when the bin was inclined depended strongly on the distribution of the load within the bin, but was underestimated in all scenarios. The distribution of the load was found to be dependent on the inclination of the bin caused by the topography of the terrain, but also by the history of inclination (inclination rate, presence of static periods, etc.) since the effect of the inertia of the particles (i.e., representing the grapes) was not negligible. Some recommendations are given to try to improve the accuracy of crop load measurement in the field.

The load dependence of kinesin’s mechanical cycle

Kinesin is a dimeric motor protein that transports organelles in a stepwise manner toward the plus-end of microtubules by converting the energy of ATP hydrolysis into mechanical work. External forces can influence the behavior of kinesin, and force-velocity curves have shown that the motor will slow down and eventually stall under opposing loads of ≈5 pN. Using an in vitro motility assay in conjunction with a high-resolution optical trapping microscope, we have examined the behavior of individual kinesin molecules under two previously unexplored loading regimes: super-stall loads (>5 pN) and forward (plus-end directed) loads. Whereas some theories of kinesin function predict a reversal of directionality under high loads, we found that kinesin does not walk backwards under loads of up to 13 pN, probably because of an irreversible transition in the mechanical cycle. We also found that this cycle can be significantly accelerated by forward loads under a wide range of ATP concentrations. Finally, we noted an increase in kinesin’s rate of dissociation from the microtubule with increasing load, which is consistent with a load dependent partitioning between two recently described kinetic pathways: a coordinated-head pathway (which leads to stepping) and an independent-head pathway (which is static).

A conditional probability analysis of cystic fibrosis transmembrane conductance regulator gating indicates that ATP has multiple effects during the gating cycle

ATP-binding cassette (ABC) transporters bind and hydrolyze ATP. In the cystic fibrosis transmembrane conductance regulator Cl− channel, this interaction with ATP generates a gating cycle between a closed (C) and two open (O1 and O2) conformations. To understand better how ATP controls channel activity, we examined gating transitions from the C to the O1 and O2 states and from these open states to the C conformation. We made three main observations. First, we found that the channel can open into either the O1 or O2 state, that the frequency of transitions to both states was increased by ATP concentration, and that ATP increased the relative proportion of openings into O1 vs. O2. These results indicate that ATP can interact with the closed state to open the channel in at least two ways, which may involve binding to nucleotide-binding domains (NBDs) NBD1 and NBD2. Second, ATP prolonged the burst duration and altered the way in which the channel closed. These data suggest that ATP also interacts with the open channel. Third, the channel showed runs of specific types of open–closed transitions. This finding suggests a mechanism with more than one cycle of gating transitions. These data suggest models to explain how ATP influences conformational transitions in cystic fibrosis transmembrane conductance regulator and perhaps other ABC transporters.

Proteomic analysis of the bacterial cell cycle

A global approach was used to analyze protein synthesis and stability during the cell cycle of the bacterium Caulobacter crescentus. Approximately one-fourth (979) of the estimated C. crescentus gene products were detected by two-dimensional gel electrophoresis, 144 of which showed differential cell cycle expression patterns. Eighty-one of these proteins were identified by mass spectrometry and were assigned to a wide variety of functional groups. Pattern analysis revealed that coexpression groups were functionally clustered. A total of 48 proteins were rapidly degraded in the course of one cell cycle. More than half of these unstable proteins were also found to be synthesized in a cell cycle-dependent manner, establishing a strong correlation between rapid protein turnover and the periodicity of the bacterial cell cycle. This is, to our knowledge, the first evidence for a global role of proteolysis in bacterial cell cycle control.

The beginning of a new political cycle: the results and consequences of a nomination summit. EPC Post-Summit Analysis, 30 June 2014

Despite a broader agenda, the June 2014 European Council was dominated by the decision of EU leaders – taken by qualified majority – to propose to the European Parliament Jean-Claude Juncker as the next Commission President. In this post-summit analysis Janis A. Emmanouilidis argues that recent developments could have four consequences: increasing politicisation at European level; opposition from the side of national governments to what they consider to be an unjustifiable shift of power; further complication, maybe even deterioration of the relationship between London and ‘Brussels; and ‘consolidation’ as the predominant political attitude in the beginning of a new political cycle. Aside from all this, the Summit adopted a Strategic Agenda for the years to come, agreed to new strategic guidelines for the Area of Freedom, Security and Justice, postponed the decision on a new energy and climate framework to October, concluded the fourth European Semester with the adoption of country-specific recommendations, and, last but not least, EU leaders finally signed the Association Agreements with Georgia, Moldova and Ukraine demonstrating that the Union and these countries are ready to deepen political and economic ties.

Life cycle cost analysis. Summary of proceedings: FHWA life cycle cost symposium.

Mode of access: Internet.

Life-cycle cost analysis for utility combinations handbook : departmental staff and program participants.

"December 1985."