The function of the murine complement *C3a and *C5a anaphylatoxin receptors in endotoxemia, bacteremia and septic shock

The complement system functions as a major effector for both the innate and adaptive immune response. Activation of the complement cascade by either the classical, alternative, or lectin pathway promotes the proteolysis of C3 and C5 thereby generating C3a and C5a. Referred to as anaphylatoxins, the C3a and C5a peptides mediate biological effects upon binding to their respective receptors; C3a binds to the C3a receptor (C3aR) while C5a binds to the C5a receptor (C5aR, CD88). Both C3a and C5a are known for their broad proinflammatory effects. Elevated levels of both peptides have been isolated from patients with a variety of inflammatory diseases such as COPD, asthma, RA, SLE, and sepsis. Recent studies suggest that C5a is a critical component in the acquired neutrophil dysfunction, coagulopathy, and progressive multi-organ dysfunction characteristic of sepsis. The primary hypothesis of this dissertation was that preventing C3a-C3aR and C5a-C5aR mediated pro-inflammatory effects would improve survival in endotoxic, bacteremic and septic shock. To test this hypothesis, the murine C3aR and C5aR genes were disrupted. Following disruption of both the C3aR and C5aR genes, no abnormalities were identified other than the absence of their respective mRNA and protein. In models of both endotoxic and bacteremic shock, C3aR deficient mice suffered increased mortality when compared to their wild type littermates. C3aR deficient mice also had elevated circulating IL-1β levels. Using a model of sepsis, C3aR deficient mice had a higher circulating concentration of IL-6 and decreased peritoneal inflammatory infiltration. While these results were unexpected, they support an emerging role for C3a in immunomodulation. In contrast, following endotoxic or bacteremic shock, C5aR deficient mice experienced increased survival, less hemoconcentration and less thrombocytopenia. It was later determined that C5a mediated histamine release significantly contributes to host morbidity and mortality in bacteremic shock. These studies provide evidence that C5a functions primarily as a proinflammatory molecule in models of endotoxic and bacteremic shock. In the same models, C3a-C3aR interactions suppress the inflammatory response and protect the host. Collectively, these results present in vivo evidence that C3a and C5a have divergent biological functions. ^

Trade Credits under Imperfect Enforcement: A Theory with a Test on Chinese Experience

It is widely recognized that trade credit is an important financial mechanism, particularly in developing economies and transition economies where institutions are weak. This paper documents theoretical analysis and empirical accounts on what facilitates an effective supply of trade credit based on original surveys conducted in P.R. of China. Our theory predicts that trade volume and trade credit are increasing function of cash held by the buyer and enforcement technology of the seller. Furthermore, if the state sector’s enforcement technology is high, it has positive external effect to expand the volumes of trade credit and trades in the whole economy. From the data, we found that government made active commitment in enforcement of trade credit contract and the government owned firms are main supplier and receivers of trade credit, which suggest that enforcement by government and state sector were effective against presumptions in the previous literatures.

An empirical analysis of the money demand function in India

This paper empirically analyzes India’s money demand function during the period of 1980 to 2007 using monthly data and the period of 1976 to 2007 using annual data. Cointegration test results indicated that when money supply is represented by M1 and M2, a cointegrating vector is detected among real money balances, interest rates, and output. In contrast, it was found that when money supply is represented by M3, there is no long-run equilibrium relationship in the　money demand function. Moreover, when the money demand function was estimated using　dynamic OLS, the sign onditions of the coefficients of output and interest rates were found to be　consistent with theoretical rationale, and statistical significance was confirmed when money supply was represented by either M1 or M2. Consequently, though India’s central bank presently uses M3 as an indicator of future price movements, it is thought appropriate to focus on M1 or M2, rather than M3, in managing monetary policy.

Contribution to the Analysis, Design and Assessment of Compact Antenna Test Ranges At Millimeter Wavelengths - Contribución al Análisis, Diseño y Caracterización de Sistemas de Medida de Antenas en Rango Compacto para Frecuencias Milimétricas

This Doctoral Thesis entitled Contribution to the analysis, design and assessment of compact antenna test ranges at millimeter wavelengths aims to deepen the knowledge of a particular antenna measurement system: the compact range, operating in the frequency bands of millimeter wavelengths. The thesis has been developed at Radiation Group (GR), an antenna laboratory which belongs to the Signals, Systems and Radiocommunications department (SSR), from Technical University of Madrid (UPM). The Radiation Group owns an extensive experience on antenna measurements, running at present four facilities which operate in different configurations: Gregorian compact antenna test range, spherical near field, planar near field and semianechoic arch system. The research work performed in line with this thesis contributes the knowledge of the first measurement configuration at higher frequencies, beyond the microwaves region where Radiation Group features customer-level performance. To reach this high level purpose, a set of scientific tasks were sequentially carried out. Those are succinctly described in the subsequent paragraphs. A first step dealed with the State of Art review. The study of scientific literature dealed with the analysis of measurement practices in compact antenna test ranges in addition with the particularities of millimeter wavelength technologies. Joint study of both fields of knowledge converged, when this measurement facilities are of interest, in a series of technological challenges which become serious bottlenecks at different stages: analysis, design and assessment. Thirdly after the overview study, focus was set on Electromagnetic analysis algorithms. These formulations allow to approach certain electromagnetic features of interest, such as field distribution phase or stray signal analysis of particular structures when they interact with electromagnetic waves sources. Properly operated, a CATR facility features electromagnetic waves collimation optics which are large, in terms of wavelengths. Accordingly, the electromagnetic analysis tasks introduce an extense number of mathematic unknowns which grow with frequency, following different polynomic order laws depending on the used algorithmia. In particular, the optics configuration which was of our interest consisted on the reflection type serrated edge collimator. The analysis of these devices requires a flexible handling of almost arbitrary scattering geometries, becoming this flexibility the nucleus of the algorithmia’s ability to perform the subsequent design tasks. This thesis’ contribution to this field of knowledge consisted on reaching a formulation which was powerful at the same time when dealing with various analysis geometries and computationally speaking. Two algorithmia were developed. While based on the same principle of hybridization, they reached different order Physics performance at the cost of the computational efficiency. Inter-comparison of their CATR design capabilities was performed, reaching both qualitative as well as quantitative conclusions on their scope. In third place, interest was shifted from analysis - design tasks towards range assessment. Millimetre wavelengths imply strict mechanical tolerances and fine setup adjustment. In addition, the large number of unknowns issue already faced in the analysis stage appears as well in the on chamber field probing stage. Natural decrease of dynamic range available by semiconductor millimeter waves sources requires in addition larger integration times at each probing point. These peculiarities increase exponentially the difficulty of performing assessment processes in CATR facilities beyond microwaves. The bottleneck becomes so tight that it compromises the range characterization beyond a certain limit frequency which typically lies on the lowest segment of millimeter wavelength frequencies. However the value of range assessment moves, on the contrary, towards the highest segment. This thesis contributes this technological scenario developing quiet zone probing techniques which achieves substantial data reduction ratii. Collaterally, it increases the robustness of the results to noise, which is a virtual rise of the setup’s available dynamic range. In fourth place, the environmental sensitivity of millimeter wavelengths issue was approached. It is well known the drifts of electromagnetic experiments due to the dependance of the re sults with respect to the surrounding environment. This feature relegates many industrial practices of microwave frequencies to the experimental stage, at millimeter wavelengths. In particular, evolution of the atmosphere within acceptable conditioning bounds redounds in drift phenomena which completely mask the experimental results. The contribution of this thesis on this aspect consists on modeling electrically the indoor atmosphere existing in a CATR, as a function of environmental variables which affect the range’s performance. A simple model was developed, being able to handle high level phenomena, such as feed - probe phase drift as a function of low level magnitudes easy to be sampled: relative humidity and temperature. With this model, environmental compensation can be performed and chamber conditioning is automatically extended towards higher frequencies. Therefore, the purpose of this thesis is to go further into the knowledge of millimetre wavelengths involving compact antenna test ranges. This knowledge is dosified through the sequential stages of a CATR conception, form early low level electromagnetic analysis towards the assessment of an operative facility, stages for each one of which nowadays bottleneck phenomena exist and seriously compromise the antenna measurement practices at millimeter wavelengths.

Piezoelectric and electroacoustic properties of Ti-doped AlN thin films as a function of Ti content

In this work we present the assessment of the structural and piezoelectric properties of Al(0.5-x)TixN0.5 compounds (titanium content menor que6% atomic), which are expected to possess improved properties than conventional AlN films, such as larger piezoelectric activity, thermal stability of frequency and temperature resistance. Al:Ti:N films were deposited from a twin concentric target of Al and Ti by reactive AC sputtering, which provided films with a radial gradient of the Ti concentration. The properties of the films were investigated as a function of their composition, which was measured by electron dispersive energy dispersive X-ray spectroscopy and Rutherford backscattering spectrometry. The microstructure and morphology of the films were assessed by X-ray diffraction and infrared reflectance. Their electroacoustic properties and dielectric constant were derived from the frequency response of BAW test resonators. Al:Ti:N films properties appear to be strongly dependent on the Ti content, which modifies the AlN wurtzite crystal structure leading to greater dielectric constant, lower sound velocities, lower electromechanical factor and moderately improved temperature coefficient of the resonant frequency.

Preliminary temperature Accelerated Life Test (ALT) on III-V commercial concentrator triple-junction solar cells

A quantitative temperature accelerated life test on sixty GaInP/GaInAs/Ge triple-junction commercial concentrator solar cells is being carried out. The final objective of this experiment is to evaluate the reliability, warranty period, and failure mechanism of high concentration solar cells in a moderate period of time. The acceleration of the degradation is realized by subjecting the solar cells at temperatures markedly higher than the nominal working temperature under a concentrator Three experiments at three different temperatures are necessary in order to obtain the acceleration factor which relates the time at the stress level with the time at nominal working conditions. . However, up to now only the test at the highest temperature has finished. Therefore, we can not provide complete reliability information but we have analyzed the life data and the failure mode of the solar cells inside the climatic chamber at the highest temperature. The failures have been all of them catastrophic. In fact, the solar cells have turned into short circuits. We have fitted the failure distribution to a two parameters Weibull function. The failures are wear-out type. We have observed that the busbar and the surrounding fingers are completely deteriorate

Determination of the JWL constants for ANFO and emulsion explosives from cylinder test data

The Jones-Wilkins-Lee (JWL) equation of state parameters for ANFO and emulsion-type explosives have been obtained from cylinder test expansion measurements. The calculation method comprises a new radial expansion function, with a non-zero initial velocity at the onset of the expansion in order to comply with a positive Gurney energy at unit relative volume, as the isentropic expansion from the CJ state predicts. The equations reflecting the CJ state conditions and the measured expansion energy were solved for the JWL parameters by a non-linear least squares scheme. The JWL parameters of thirteen ANFO and emulsion type explosives have been determined in this way from their cylinder test expansion data. The results were evaluated through numerical modelling of the tests with the LS-DYNA hydrocode; the expansion histories from the modelling were compared with the measured ones, and excellent agreement was found.

New On-Line Excitation-System Ground Fault Location Method Tested in a 106 MVA Synchronous Generator

In this paper, a novel excitation-system ground-fault location method is described and tested in a 106 MVA synchronous machine. In this unit, numerous rotor ground-fault trips took place always about an hour after the synchronization to the network. However, when the field winding insulation was checked after the trips, there was no failure. The data indicated that the faults in the rotor were caused by centrifugal forces and temperature. Unexpectedly, by applying this new method, the failure was located in a cable between the excitation transformer and the automatic voltage regulator. In addition, several intentional ground faults were performed along the field winding with different fault resistance values, in order to test the accuracy of this method to locate defects in rotor windings of large generators. Therefore, this new on-line rotor ground-fault detection algorithm is tested in high-power synchronous generators with satisfactory results.

Perfil de playas sustentadas : estudio hidrodinámico y experimental : directrices de diseño

Las playas sustentadas por medio de un pie sumergido son una atractiva alternativa de diseño de regeneración de playas especialmente cuando las condiciones física del emplazamiento o las características de la arena nativa y de préstamo producen perfiles de alimentación que no se intersectan. La observación y propuesta de este tipo de solución data de los años 1960’s, así como la experiencia internacional en la construcción de este tipo de playas. Sin embargo, a pesar de su utilización y los estudios en campo y laboratorio, no se dispone de criterios ingenieriles que apoyen el diseño de las mismas. Esta tesis consiste en un análisis experimental del perfil de playas sustentadas en un pie sumergido (o colgadas) que se concreta en una propuesta de directrices de diseño general que permiten estimar la ubicación y características geométricas del pie sumergido frente a un oleaje y material que constituye la playa determinados. En la tesis se describe el experimento bidimensional realizado en el modelo físico de fondo móvil, donde se combinan cinco tipos de oleaje con tres configuraciones del pie sumergido (“Sin estructura”, configuración baja o “Estructura 1” y configuración alta o “Estructura 2”), se presentan los resultados obtenidos y se realiza una discusión detallada de las implicaciones de los resultados desde el punto de vista hidrodinámico utilizando monomios adimensionales. Se ha realizado un análisis detallado del estado del arte sobre playas colgadas, presentando el concepto y las experiencias de realizaciones en distintos países. Se ha realizado una cuidadosa revisión de la literatura publicada sobre estudios experimentales de playas colgadas, modelos teóricos y otras cuestiones auxiliares, necesarias para la formulación de la metodología de la tesis. El estudio realizado se ha estructurado en dos fases. En la primera fase se ha realizado una experimentación en un modelo físico de fondo móvil construido en las instalaciones del Centro de Estudios de Puertos y Costas (CEPYC) del Centro de Estudios y Experimentación de Obras Públicas (CEDEX), consistente en un canal de 36 m de longitud, 3 m de anchura y 1.5 m de altura, provisto de un generador de oleaje de tipo pistón. Se ha diseñado una campaña de 15 ensayos, que se obtienen sometiendo a cinco tipos de oleaje tres configuraciones distintas de playa colgada. En los ensayos se ha medido el perfil de playa en distintos instantes hasta llegar al equilibrio, determinando a partir de esos datos el retroceso de la línea de costa y el volumen de sedimentos perdido. El tiempo total efectivo de ensayo asciende a casi 650 horas, y el número de perfiles de evolución de playa obtenidos totaliza 229. En la segunda fase se ha abordado el análisis de los resultados obtenidos con la finalidad de comprender el fenómeno, identificar las variables de las que depende y proponer unas directrices ingenieriles de diseño. Se ha estudiado el efecto de la altura de ola, del periodo del oleaje, del francobordo adimensional y del parámetro de Dean, constatándose la dificultad de comprensión del funcionamiento de estas obras ya que pueden ser beneficiosas, perjudiciales o inocuas según los casos. También se ha estudiado la respuesta del perfil de playa en función de otros monomios adimensionales, tales como el número de Reynolds o el de Froude. En el análisis se ha elegido el monomio “plunger” como el más significativo, encontrando relaciones de éste con el peralte de oleaje, la anchura de coronación adimensional, la altura del pie de playa adimensional y el parámetro de Dean. Finalmente, se propone un método de diseño de cuatro pasos que permite realizar un primer encaje del diseño funcional de la playa sustentada frente a un oleaje de características determinadas. Las contribuciones más significativas desde el punto de vista científico son: - La obtención del juego de resultados experimentales. - La caracterización del comportamiento de las playas sustentadas. - Las relaciones propuestas entre el monomio plunger y las distintas variables explicativas seleccionadas, que permiten predecir el comportamiento de la obra. - El método de diseño propuesto, en cuatro pasos, para este tipo de esquemas de defensa de costas. Perched beaches are an attractive beach nourishment design alternative especially when either the site conditions or the characteristics of both the native and the borrow sand lead to a non-intersecting profile The observation and suggestion of the use of this type of coastal defence scheme dates back to the 1960’s, as well as the international experience in the construction of this type of beaches. However, in spite of its use and the field and laboratory studies performed to-date, no design engineering guidance is available to support its design. This dissertation is based on the experimental work performed on a movable bed physical model and the use of dimensionless parameters in analyzing the results to provide general functional design guidance that allow the designer, at a particular stretch of coast - to estimate the location and geometric characteristics of the submerged sill as well as to estimate the suitable sand size to be used in the nourishment. This dissertation consists on an experimental analysis of perched beaches by means of a submerged sill, leading to the proposal of general design guidance that allows to estimate the location and geometric characteristics of the submerged sill when facing a wave condition and for a given beach material. The experimental work performed on a bi-dimensional movable bed physical model, where five types of wave conditions are combined with three configurations of the submerged sill (“No structure”, low structure or “Structure 1”, and high structure or “Structure 2”) is described, results are presented, and a detailed discussion of the results - from the hydrodynamic point of view – of the implications of the results by using dimensionless parameters is carried out. A detailed state of the art analysis about perched beaches has been performed, presenting the “perched beach concept” and the case studies of different countries. Besides, a careful revision of the literature about experimental studies on perched beaches, theoretical models, and other topics deemed necessary to formulate the methodology of this work has been completed. The study has been divided into two phases. Within the first phase, experiments on a movable-bed physical model have been developed. The physical model has been built in the Centro de Estudios de Puertos y Costas (CEPYC) facilities, Centro de Estudios y Experimentación de Obras Públicas (CEDEX). The wave flume is 36 m long, 3 m wide and 1.5 m high, and has a piston-type regular wave generator available. The test plan consisted of 15 tests resulting from five wave conditions attacking three different configurations of the perched beach. During the development of the tests, the beach profile has been surveyed at different intervals until equilibrium has been reached according to these measurements. Retreat of the shoreline and relative loss of sediment in volume have been obtained from the measurements. The total effective test time reaches nearly 650 hours, whereas the total number of beach evolution profiles measured amounts to 229. On the second phase, attention is focused on the analysis of results with the aim of understanding the phenomenon, identifying the governing variables and proposing engineering design guidelines. The effect of the wave height, the wave period, the dimensionless freeboard and of the Dean parameter have been analyzed. It has been pointed out the difficulty in understanding the way perched beaches work since they turned out to be beneficial, neutral or harmful according to wave conditions and structure configuration. Besides, the beach profile response as a function of other dimensionless parameters, such as Reynolds number or Froude number, has been studied. In this analysis, the “plunger” parameter has been selected as the most representative, and relationships between the plunger parameter and the wave steepness, the dimensionless crest width, the dimensionless crest height, and the Dean parameter have been identified. Finally, an engineering 4-step design method has been proposed, that allows for the preliminary functional design of the perched beach for a given wave condition. The most relevant contributions from the scientific point of view have been: - The acquisition of a consistent set of experimental results. - The characterization of the behavior of perched beaches. - The proposed relationships between the plunger parameter and the different explanatory variables selected, that allow for the prediction of the beach behavior. - The proposed design method, four-step method, for this type of coastal defense schemes.

Autonomous classification models in ubiquitous environments

Stream-mining approach is defined as a set of cutting-edge techniques designed to process streams of data in real time, in order to extract knowledge. In the particular case of classification, stream-mining has to adapt its behaviour to the volatile underlying data distributions, what has been called concept drift. Moreover, it is important to note that concept drift may lead to situations where predictive models become invalid and have therefore to be updated to represent the actual concepts that data poses. In this context, there is a specific type of concept drift, known as recurrent concept drift, where the concepts represented by data have already appeared in the past. In those cases the learning process could be saved or at least minimized by applying a previously trained model. This could be extremely useful in ubiquitous environments that are characterized by the existence of resource constrained devices. To deal with the aforementioned scenario, meta-models can be used in the process of enhancing the drift detection mechanisms used by data stream algorithms, by representing and predicting when the change will occur. There are some real-world situations where a concept reappears, as in the case of intrusion detection systems (IDS), where the same incidents or an adaptation of them usually reappear over time. In these environments the early prediction of drift by means of a better knowledge of past models can help to anticipate to the change, thus improving efficiency of the model regarding the training instances needed. By means of using meta-models as a recurrent drift detection mechanism, the ability to share concepts representations among different data mining processes is open. That kind of exchanges could improve the accuracy of the resultant local model as such model may benefit from patterns similar to the local concept that were observed in other scenarios, but not yet locally. This would also improve the efficiency of training instances used during the classification process, as long as the exchange of models would aid in the application of already trained recurrent models, that have been previously seen by any of the collaborative devices. Which it is to say that the scope of recurrence detection and representation is broaden. In fact the detection, representation and exchange of concept drift patterns would be extremely useful for the law enforcement activities fighting against cyber crime. Being the information exchange one of the main pillars of cooperation, national units would benefit from the experience and knowledge gained by third parties. Moreover, in the specific scope of critical infrastructures protection it is crucial to count with information exchange mechanisms, both from a strategical and technical scope. The exchange of concept drift detection schemes in cyber security environments would aid in the process of preventing, detecting and effectively responding to threads in cyber space. Furthermore, as a complement of meta-models, a mechanism to assess the similarity between classification models is also needed when dealing with recurrent concepts. In this context, when reusing a previously trained model a rough comparison between concepts is usually made, applying boolean logic. The introduction of fuzzy logic comparisons between models could lead to a better efficient reuse of previously seen concepts, by applying not just equal models, but also similar ones. This work faces the aforementioned open issues by means of: the MMPRec system, that integrates a meta-model mechanism and a fuzzy similarity function; a collaborative environment to share meta-models between different devices; a recurrent drift generator that allows to test the usefulness of recurrent drift systems, as it is the case of MMPRec. Moreover, this thesis presents an experimental validation of the proposed contributions using synthetic and real datasets.

Transient thermal analysis during the ascent phase of a balloon-borne payload. Comparison with SUNRISE test flight measurements

The thermal design of stratospheric balloon payloads usually focuses on the cruise phase of the missions, that is, the floating altitude conditions. The ascent phase usually takes between 2 and 4 h, a very small period compared to the duration of the whole mission, which can last up to 4 weeks. However, during this phase payloads are subjected to very harsh conditions due mainly to the convective cooling that occurs as the balloon passes through the cold atmosphere, with minimum temperatures in the tropopause. The aim of this work is to study the thermal behaviour of a payload carried by a long duration balloon during the ascent phase. Its temperature has been calculated as a function of the altitude from sea level to floating conditions. To perform this analysis it has been assumed that the thermal interactions (convection and radiation) depend on the altitude, on the environmental conditions (which in turn depend also on the altitude) and on the temperature of the system itself. The results have been compared with the measurements taken during the SUNRISE test flight, launched in October 2007 by CSBF from Fort Sumner (New Mexico).

Further examination of the Xist promoter-switch hypothesis in X inactivation: Evidence against the existence and function of a P0 promoter

The onset of X inactivation coincides with accumulation of Xist RNA along the future inactive X chromosome. A recent hypothesis proposed that accumulation is initiated by a promoter switch within Xist. In this hypothesis, an upstream promoter (P0) produces an unstable transcript, while the known downstream promoter (P1) produces a stable RNA. To test this hypothesis, we examined expression and half-life of Xist RNA produced from an Xist transgene lacking P0 but retaining P1. We confirm the previous finding that P0 is dispensable for Xist expression in undifferentiated cells and that P1 can be used in both undifferentiated and differentiated cells. Herein, we show that Xist RNA initiated at P1 is unstable and does not accumulate. Further analysis indicates that the transcriptional boundary at P0 does not represent the 5′ end of a distinct Xist isoform. Instead, P0 is an artifact of cross-amplification caused by a pseudogene of the highly expressed ribosomal protein S12 gene Rps12. Using strand-specific techniques, we find that transcription upstream of P1 originates from the DNA strand opposite Xist and represents the 3′ end of the antisense Tsix RNA. Thus, these data do not support the existence of a P0 promoter and suggest that mechanisms other than switching of functionally distinct promoters control the up-regulation of Xist.

Conserved biological function between Pax-2 and Pax-5 in midbrain and cerebellum development: Evidence from targeted mutations

The development of two major subdivisions of the vertebrate nervous system, the midbrain and the cerebellum, is controlled by signals emanating from a constriction in the neural primordium called the midbrain/hindbrain organizer (Joyner, A. L. (1996) Trends Genet. 12, 15–201). The closely related transcription factors Pax-2 and Pax-5 exhibit an overlapping expression pattern very early in the developing midbrain/hindbrain junction. Experiments carried out in fish (Krauss, S., Maden, M., Holder, N. & Wilson, S. W. (1992) Nature (London) 360, 87–89) with neutralizing antibodies against Pax-b, the orthologue of Pax-2 in mouse, placed this gene family in an regulatory cascade necessary for the development of the midbrain and the cerebellum. The targeted mutation of Pax-5 has been reported to have only slight effects in the development of structures derived from the isthmic constriction, whereas the Pax-2 null mutant mice show a background-dependent phenotype with varying penetrance. To test a possible redundant function between Pax-2 and Pax-5 we analyzed the brain phenotypes of mice expressing different dosages of both genes. Our results demonstrate a conserved biological function of both proteins in midbrain/hindbrain regionalization. Additionally, we show that one allele of Pax-2, but not Pax-5, is necessary and sufficient for midbrain and cerebellum development in C57BL/6 mice.

Recent improvements in prediction of protein structure by global optimization of a potential energy function

Recent improvements of a hierarchical ab initio or de novo approach for predicting both α and β structures of proteins are described. The united-residue energy function used in this procedure includes multibody interactions from a cumulant expansion of the free energy of polypeptide chains, with their relative weights determined by Z-score optimization. The critical initial stage of the hierarchical procedure involves a search of conformational space by the conformational space annealing (CSA) method, followed by optimization of an all-atom model. The procedure was assessed in a recent blind test of protein structure prediction (CASP4). The resulting lowest-energy structures of the target proteins (ranging in size from 70 to 244 residues) agreed with the experimental structures in many respects. The entire experimental structure of a cyclic α-helical protein of 70 residues was predicted to within 4.3 Å α-carbon (Cα) rms deviation (rmsd) whereas, for other α-helical proteins, fragments of roughly 60 residues were predicted to within 6.0 Å Cα rmsd. Whereas β structures can now be predicted with the new procedure, the success rate for α/β- and β-proteins is lower than that for α-proteins at present. For the β portions of α/β structures, the Cα rmsd's are less than 6.0 Å for contiguous fragments of 30–40 residues; for one target, three fragments (of length 10, 23, and 28 residues, respectively) formed a compact part of the tertiary structure with a Cα rmsd less than 6.0 Å. Overall, these results constitute an important step toward the ab initio prediction of protein structure solely from the amino acid sequence.

The copper transporter CTR1 provides an essential function in mammalian embryonic development

Copper serves as an essential cofactor for a variety of proteins in all living organisms. Previously, we described a human gene (CTR1;SLC31A1) that encodes a high-affinity copper-uptake protein and hypothesized that this protein is required for copper delivery to mammalian cells. Here, we test this hypothesis by inactivating the Ctr1 gene in mice by targeted mutagenesis. We observe early embryonic lethality in homozygous mutant embryos and a deficiency in copper uptake in the brains of heterozygous animals. Ctr1−/− embryos can be recovered at E8.5 but are severely developmentally retarded and morphologically abnormal. Histological analysis reveals discontinuities and variable thickness in the basement membrane of the embryonic region and an imperfect Reichert's membrane, features that are likely due to lack of activity in the collagen cross-linking cupro-enzyme lysyl oxidase. A collapsed embryonic cavity, the absence of an allantois, retarded mesodermal migration, and increased cell death are also apparent. In the brains of heterozygous adult mice, which at 16 months are phenotypically normal, copper is reduced to approximately half compared with control littermates, implicating CTR1 as the required port for copper entry into at least this organ. A study of the spatial and temporal expression pattern of Ctr1 during mouse development and adulthood further shows that CTR1 is ubiquitously transcribed with highest expression observed in the specialized epithelia of the choroid plexus and renal tubules and in connective tissues of the eye, ovary, and testes. We conclude that CTR1 is the primary avenue for copper uptake in mammalian cells.