CFD modelling of wind farms in complex terrain

Modelling of entire wind farms in flat and complex terrain using a full 3D Navier–Stokes solver for incompressible flow is presented in this paper. Numerical integration of the governing equations is performed using an implicit pressure correction scheme, where the wind turbines (W/Ts) are modelled as momentum absorbers through their thrust coefficient. The k–ω turbulence model, suitably modified for atmospheric flows, is employed for closure. A correction is introduced to account for the underestimation of the near wake deficit, in which the turbulence time scale is bounded using a general “realizability” constraint for the fluctuating velocities. The second modelling issue that is discussed in this paper is related to the determination of the reference wind speed for the thrust calculation of the machines. Dealing with large wind farms and wind farms in complex terrain, determining the reference wind speed is not obvious when a W/T operates in the wake of another WT and/or in complex terrain. Two alternatives are compared: using the wind speed value at hub height one diameter upstream of the W/T and adopting an induction factor-based concept to overcome the utilization of a wind speed at a certain distance upwind of the rotor. Application is made in two wind farms, a five-machine one located in flat terrain and a 43-machine one located in complex terrain.

Calibration of WAVE in irrigated maize: fallow vs. cover crops.

Nitrate leaching decreases crop available N and increases water contamination. Replacing fallow by cover crops (CC) is an alternative to reduce nitrate contamination, because it reduces overall drainage and soil mineral N accumulation. A study of the soil N and nitrate leaching was conducted during 5 years in a semi-arid irrigated agricultural area of Central Spain. Three treatments were studied during the intercropping period of maize (Zea mays L.): barley (Hordeum vulgare L.), vetch (Vicia villosa L.), and fallow. Cover crops, sown in October, were killed by glyphosate application in March, allowing direct seeding of maize in April. All treatments were irrigated and fertilised following the same procedure. Soil water content was measured using capacity probes. Soil Nmin accumulation was determined along the soil profile before sowing and after harvesting maize. Soil analysis was conducted at six depths every 0.20m in each plot in samples from 0 to 1.2-m depth. The mechanistic water balance model WAVE was applied in order to calculate drainage and plant growth of the different treatments, and apply them to the N balance. We evaluated the water balance of this model using the daily soil water content measurements of this field trial. A new Matlab version of the model was evaluated as well. In this new version improvements were made in the solute transport module and crop module. In addition, this new version is more compatible with external modules for data processing, inverse calibration and uncertainty analysis than the previous Fortran version. The model showed that drainage during the irrigated period was minimized in all treatments, because irrigation water was adjusted to crop needs, leading to nitrate accumulation on the upper layers after maize harvest. Then, during the intercrop period, most of the nitrate leaching occurred. Cover crops usually led to a shorter drainage period, lower drainage water amount and lower nitrate leaching than the treatment with fallow. These effects resulted in larger nitrate accumulation in the upper layers of the soil after CC treatments.

Scaling in Soil and Other Complex Porous Media

Scaling is becoming an increasingly important topic in the earth and environmental sciences as researchers attempt to understand complex natural systems through the lens of an ever-increasing set of methods and scales. The guest editors introduce the papers in this issue’s special section and present an overview of some of the work being done. Scaling remains one of the most challenging topics in earth and environmental sciences, forming a basis for our understanding of process development across the multiple scales that make up the subsurface environment. Tremendous progress has been made in discovery, explanation, and applications of scaling. And yet much more needs to be done and is being done as part of the modern quest to quantify, analyze, and manage the complexity of natural systems. Understanding and succinct representation of scaling properties can unveil underlying relationships between system structure and response functions, improve parameterization of natural variability and heterogeneity, and help us address societal needs by effectively merging knowledge acquired at different scales.

Representing microstates of static granular matter in a stress phase space

A stress phase space is proposed to compare the static packings of a granular system (microstates) that are compatible to a macrostate described by external stresses. The equivalent stress of each particle of a static packing can be obtained from the mechanical interaction forces, and the associated volume is given by the respective Voronoi cell. Therefore, particles can be located at different stress levels and grouped into categories or configurations, which are defined in base of the geometrical features of the local arrangement (in particular, of the number of forces that keep them force-balanced). They can be represented as points in a stress phase space. The nature of this space is analyzed in detail. The integration limits of the stress variables that avoid or limit tensile states and the capability of each configuration to represent specific stress states establish its main features. Furthermore, if some stress variables are used, instead of the usual components of the Cauchy stress tensor, then some symmetries can be found. Results obtained from molecular dynamics simulations are used to check this nature. Finally, some statistical ensembles are written in terms of the coordinates of this phase space. These require some assumptions that are made in base on continuum mechanics principles.

A framework for goal-oriented discovery of resources in the RESTful architecture

One of the challenges facing the current web is the efficient use of all the available information. The Web 2.0 phenomenon has favored the creation of contents by average users, and thus the amount of information that can be found for diverse topics has grown exponentially in the last years. Initiatives such as linked data are helping to build the Semantic Web, in which a set of standards are proposed for the exchange of data among heterogeneous systems. However, these standards are sometimes not used, and there are still plenty of websites that require naive techniques to discover their contents and services. This paper proposes an integrated framework for content and service discovery and extraction. The framework is divided into several layers where the discovery of contents and services is made in a representational stateless transfer system such as the web. It employs several web mining techniques as well as feature-oriented modeling for the discovery of cross-cutting features in web resources. The framework is used in a scenario of electronic newspapers. An intelligent agent crawls the web for related news, and uses services and visits links automatically according to its goal. This scenario illustrates how the discovery is made at different levels and how the use of semantics helps implement an agent that performs high-level tasks.

Fuel poverty as a determinant in energy retrofitting actions

Fuel poverty can be defined as “the inability to afford adequate warmth in the home" and it is the result of the combination of three items: low household income, housing lack of energy efficiency and high energy bills. Although it affects a growing number of households within the European Union only some countries have an official definition for it. In 2013, the European Parliament claimed the Commission and Estate Members to develop different policies in order to fight household energy vulnerability. The importance of tackling fuel poverty is based on the critical consequences it has for human health living below certain temperatures. In Spain some advances have been made in this field but main existing studies remain at the statistical level and do not deepen the understanding of the problem from the perspective of dwelling indoor habitability conditions. What is more, this concept is yet to be officially defined. This paper presents the evaluation of fuel poverty in a building block of social housing located in the centre of Zaragoza and how this issue determined the strategies implemented in the energy retrofitting intervention project. At a first step, fuel poverty was appraised through the exploration of indoor thermal conditions. The adaptive thermal comfort (UNE-EN 15251:2008) method was used to establish the appropriate indoor temperatures and consequently to determine what can be called 'comfort gap'. Results were collated and verified with energy bills collection and a survey work that gathered data from neighbours. All this permitted pointing out those households more in need. Results from the social analysis combined with the evaluation of the building thermal performance determined the intervention. The renovation project was aimed at the implementation of passive strategies that improve households thermal comfort in order to alleviate households fuel poverty situation. This research is part of the project NewSolutions4OldHousing (LIFE10 ENV/ES/439) cofounded by the European Commission under the LIFE+ Programme.

A practical approach to project management in a very small company

This article shows how a very small company has tailored Scrum according to its own needs. The main additions made were the “sprint design” phase and the “sprint test” phase. Before the sprint 0, the requirements elicitation and the functional specification were made in order to meet deadlines and costs agreed with clients. Besides, the introduction of an agile project management tool has supported all the process and it is considered the main success factor for the institutionalization of the Scrum process.

Dust effects on PV array performance: in-field observations with non-uniform patterns

This paper presents the impact of non-homogeneous deposits of dust on the performance of a PV array. The observations have been made in a 2-MW PV park in the southeast region of Spain. The results are that inhomogeneous dust leads to more significant consequences than the mere short-circuit current reduction resulting from transmittance losses. In particular, when the affected PV modules are part of a string together with other cleaned (or less dusty) ones, operation voltage losses arise. These voltage losses can be several times larger than the short-circuit ones, leading to power losses that can be much larger than what measurements suggest when the PV modules are considered separately. Significant hot-spot phenomena can also arise leading to cells exhibiting temperature differences of more than 20 degrees and thus representing a threat to the PV modules' lifetime.

Infrared thermography and shoulder pain in wheelchair users = Termografía infrarroja y dolor de hombro en usuarios de sillas de ruedas

Las personas que usan la silla de ruedas como su forma de movilidad prioritaria presentan una elevada incidencia (73%) de dolor de hombro debido al sobreuso y al movimiento repetitivo de la propulsión. Existen numerosos métodos de diagnóstico para la detección de las patologías del hombro, sin embargo la literatura reclama la necesidad de un test no invasivo y fiable, y sugiere la termografía como una técnica adecuada para evaluar el dolor articular. La termografía infrarroja (IRT) proporciona información acerca de los procesos fisiológicos a través del estudio de las distribuciones de la temperatura de la piel. Debido a la alta correlación entre ambos lados corporales, las asimetrías térmicas entre flancos contralaterales son una buena indicación de patologías o disfunciones físicas subyacentes. La fiabilidad de la IRT ha sido estudiada con anterioridad en sujetos sanos, pero nunca en usuarios de sillas de ruedas. Las características especiales de la población con discapacidad (problemas de sudoración y termorregulación, distribución sanguínea o medicación), hacen necesario estudiar los factores que afectan a la aplicación de la IRT en usuarios de sillas de ruedas. La bibliografía discrepa en cuanto a los beneficios o daños resultantes de la práctica de la actividad física en las lesiones de hombro por sobreuso en usuarios de sillas de ruedas. Recientes resultados apuntan a un aumento del riesgo de rotura del manguito rotador en personas con paraplejia que practican deportes con elevación del brazo por encima de la cabeza. Debido a esta falta de acuerdo en la literatura, surge la necesidad de analizar el perfil termográfico en usuarios de sillas de ruedas sedentarios y deportistas y su relación con el dolor de hombro. Hasta la fecha sólo se han publicado estudios termográficos durante el ejercicio en sujetos sanos. Un mayor entendimiento de la respuesta termográfica al ejercicio en silla de ruedas en relación al dolor de hombro clarificará su aparición y desarrollo y permitirá una apropiada intervención. El primer estudio demuestra que la fiabilidad de la IRT en usuarios de sillas de ruedas varía dependiendo de las zonas analizadas, y corrobora que la IRT es una técnica no invasiva, de no contacto, que permite medir la temperatura de la piel, y con la cual avanzar en la investigación en usuarios de sillas de ruedas. El segundo estudio proporciona un perfil de temperatura para usuarios de sillas de ruedas. Los sujetos no deportistas presentaron mayores asimetrías entre lados corporales que los sedentarios, y ambos obtuvieron superiores asimetrías que los sujetos sin discapacidad reportados en la literatura. Los no deportistas también presentaron resultados más elevados en el cuestionario de dolor de hombro. El área con mayores asimetrías térmicas fue hombro. En deportistas, algunas regiones de interés (ROIs) se relacionaron con el dolor de hombro. Estos resultados ayudan a entender el mapa térmico en usuarios de sillas de ruedas. El último estudio referente a la evaluación de la temperatura de la piel en usuarios de sillas de ruedas en ejercicio, reportó diferencias significativas entre la temperatura de la piel antes del test y 10 minutos después del test de propulsión de silla de ruedas, en 12 ROIs; y entre el post-test y 10 minutos después del test en la mayoría de las ROIs. Estas diferencias se vieron atenuadas cuando se compararon las asimetrías antes y después del test. La temperatura de la piel tendió a disminuir inmediatamente después completar el ejercicio, e incrementar significativamente 10 minutos después. El análisis de las asimetrías vs dolor de hombro reveló relaciones significativas negativas en 5 de las 26 ROIs. No se encontraron correlaciones significativas entre las variables de propulsión y el cuestionario de dolor de hombro. Todas las variables cinemáticas correlacionaron significativamente con las asimetrías en múltiples ROIs. Estos resultados indican que los deportistas en sillas de ruedas exhiben una capacidad similar de producir calor que los deportistas sin discapacidad; no obstante, su patrón térmico es más característico de ejercicios prolongados que de esfuerzos breves. Este trabajo contribuye al conocimiento de la termorregulación en usuarios de sillas de ruedas durante el ejercicio, y aporta información relevante para programas deportivos y de rehabilitación. ABSTRACT Individuals who use wheelchairs as their main means of mobility have a high incidence (73%) of shoulder pain (SP) owing to overuse and repetitive propulsion movement. There are numerous diagnostic methods for the detection of shoulder pathologies, however the literature claims that a noninvasive accurate test to properly assess shoulder pain would be necessary, and suggests thermography as a suitable technique for joint pain evaluation. Infrared thermography (IRT) provides information about physiological processes by studying the skin temperature (Tsk) distributions. Due to the high correlation of skin temperature between both sides of the body, thermal asymmetries between contralateral flanks are an indicator of underlying pathologies or physical dysfunctions. The reliability of infrared thermography has been studied in healthy subjects but there are no studies that have analyzed the reliability of IRT in wheelchair users (WCUs). The special characteristics of people with disabilities (sweating and thermoregulation problems, or blood distribution) make it necessary to study the factors affecting the application of IRT in WCUs. Discrepant reports exist on the benefits of, or damage resulting from, physical exercise and the relationship to shoulder overuse injuries in WCUs. Recent findings have found that overhead sports increase the risk of rotator cuff tears in wheelchair patients with paraplegia. Since there is no agreement in the literature, the thermographic profile of wheelchair athletes and nonathletes and its relation with shoulder pain should also be analysed. Infrared thermographic studies during exercise have been carried out only with able-bodied population at present. The understanding of the thermographic response to wheelchair exercise in relation to shoulder pain will offer an insight into the development of shoulder pain, which is necessary for appropriate interventions. The first study presented in this thesis demonstrates that the reliability of IRT in WCUs varies depending on the areas of the body that are analyzed. Moreover, it corroborates that IRT is a noninvasive and noncontact technique that allows the measurement of Tsk, which will allow for advances to be made in research concerned with WCUs. The second study provides a thermal profile of WCUs. Nonathletic subjects presented higher side-to-side skin temperature differences (ΔTsk) than athletes, and both had greater ΔTsk than the able-bodied results that have been published in the literature. Nonathletes also revealed larger Wheelchair Users Shoulder Pain Index (WUSPI) score than athletes. The shoulder region of interest (ROI) was the area with the highest ΔTsk of the regions measured. The analysis of the athletes’ Tsk showed that some ROIs are related to shoulder pain. These findings help to understand the thermal map in WCUs. Finally, the third study evaluated the thermal response of WCUs in exercise. There were significant differences in Tsk between the pre-test and the post-10 min in 12 ROIs, and between the post-test and the post-10 in most of the ROIs. These differences were attenuated when the ΔTsk was compared before and after exercise. Skin temperature tended to initially decrease immediately after the test, followed by a significant increase at 10 minutes after completing the exercise. The ΔTsk versus shoulder pain analysis yielded significant inverse relationships in 5 of the 26 ROIs. No significant correlations between propulsion variables and the results of the WUSPI questionnaire were found. All kinematic variables were significantly correlated with the temperature asymmetries in multiple ROIs. These results present indications that high performance wheelchair athletes exhibit similar capacity of heat production to able-bodied population; however, they presented a thermal pattern more characteristic of a prolonged exercise rather than brief exercise. This work contributes to improve the understanding about temperature changes in wheelchair athletes during exercise and provides implications to the sports and rehabilitation programs.

Las teorías del gótico y su representación gráfica en España el último tercio del siglo XIX. Un estudio sobre: Some Account of Gothic Architecture in Spain, de George Edmund Street

La tesis se centra en el estudio, descripción y análisis del libro publicado por el arquitecto británico George Edmund Street en 1865, bajo el título Some Account of Gothic Architecture in Spain. El libro dio a conocer una de la colecciones más importantes de dibujos realizada en el siglo XIX sobre la arquitectura gótica española, y por lo tanto fue una primera referencia para su estudio, hasta bien entrado el siglo veinte. El volumen incluyó 107 grabados de diversos tipos de edificios con perspectivas y detalles, y 25 láminas con 45 planos de plantas de iglesias y claustros, muchos de ellos publicados por primera vez. Gracias a varias campañas de verano por la mitad norte del país, la casi inexplorada -desde un punto de vista académico- arquitectura española medieval fue finalmente descubierta. Este hecho conduce a una pregunta intrigante que está en el origen de esta investigación ¿cómo pudo Street en pocos viajes sentar las bases de la historia del gótico español que hasta entonces los estudiosos españoles no habían sido capaces de definir? Esta tesis comienza describiendo la obra de Street en su contexto cultural con un breve repaso a su biografía y a su posición profesional y teórica. También su relación con las personas más representativas que participaron en el estudio de la arquitectura gótica, como Robert Willis, William Whewell, Augustus Pugin, o George G. Scott. Se ha prestado especial atención, en explicar su papel relevante en el Gothic Revival, para entender el significado de su interés en la arquitectura gótica continental. Estos capítulos preliminares son seguidos por una revisión del papel del dibujo como herramienta para la arquitectura de los viajeros en sus rutas en busca de la arquitectura gótica. También se trata la influencia de la Royal Academy y sus académicos, (entre los cuales estuvo Street) y su formación académica. Finalmente la tesis entra en el estudio de los planos arquitectónicos que Street hizo durante sus viajes continentales de arquitectura, seguido por una descripción detallada de sus dibujos de España, analizando su método, su técnica, y las nuevas características aportadas, que fueron una novedad en el contexto español. También se lleva a cabo algunos estudios comparativos de los dibujos de España, gracias a una recopilación exhaustiva de bocetos y dibujos originales de Street, que en su gran mayoría se conservan en los archivos del RIBA, cotejándolos con sus versiones finales, con dibujos de la época de otros autores sobre los mismos edificios, y con fotos recientes. La tesis deja claro por qué y cómo Street, gracias a su soporte teórico y habilidades para el dibujo, pudo realizar algo que había pasado desapercibido para los estudiosos españoles de la época (construcción, historia de los estilos, señalamiento de períodos constructivos), lo que le permitió encontrar el lugar adecuado de la arquitectura gótica española en la historia y en el mapa de la arquitectura gótica europea. ABSTRACT This thesis focuses on the study, description and analysis of the book published by the British architect George Edmund Street in 1865, under the title Some Account of Gothic Architecture in Spain. The book displayed one of the most significant collections of drawings on Spanish Gothic Architecture made in the nineteenth century, and therefore was a first reference for its study, until well into the twentieth century. The book included 107 engravings, the surveying of various types of buildings with perspectives and details, and 25 sheets containing 45 ground plans of churches and cloisters, many of them new and published for the first time. Thanks to several summer campaigns in the north half of the country, the almost unexplored -from a scholar point of view- medieval Spanish architecture was eventually revealed. This fact lead to an intriguing question that is at the origin of this research: how could Street in a few trips lay the foundations of the history of Spanish Gothic that until then Spanish scholars had not been able to define? This thesis begins inscribing this Street's work in his cultural context. A brief review of his biography and professional and theoretical positions has been seen as necessary. Also his debts and relationship with the most representative people involved in the study of Gothic architecture, like Robert Willis, William Whewell, Augustus Pugin, or George G. Scott are discussed. Special attention has been paid, taken into account his relevant role in the Gothic Revival, to understand the significance of his interest in continental Gothic architecture. These preliminary chapters are followed by a review of role of drawing as a tool for Architectural travellers in their Tours in search of the Gothic architecture. The influence of the Royal Academy and its academicians, (among which was Street) and his educational background are here tackled. Eventually this thesis enters into the study of the architectural drawings Street made during his continental architectural journeys, which is followed by a detailed description and analysis of the Spanish ones: his methods, his technique, and the new features which were a novelty in the Spanish context are explored. Also in this thesis is carried out some comparative studies thanks to a previous exhaustive gathering of Street's sketches and original drawings, most of which are preserved in the RIBA archives. Their final versions, drawings of the same buildings from other contemporary draughtsman and pictures of their current state are compared with them. This thesis makes clear why and how Street thanks to his theoretical back-ground and portraying skills could realize what have passed unnoticed by contemporary Spanish scholars (construction, genealogy of forms, dating of periods) allowing him to find the proper place of the Spanish architecture in the history and the map of European Gothic architecture.

Labor risk prevention culture encouragement among students from building degree in the Technical University of Madrid

The influence of training in labor risk prevention and the development of the resulting pre-emptive culture are analyzed within this paper. In order to achieve this, a quantitative analysis of the students of Building Degree in the Technic University of Madrid has been developed. This study has been made in all grades, valuating the previous knowledge acquired during compulsory education. It must be kept in mind that the students in 3rd and 4th grade have received general and specific compulsory training in prevention and safety in the building sector.

Comparing in vitro, in situ, and in vivo experimental data in a three-dimensional model of mammalian cochlear mechanics

Normal mammalian hearing is refined by amplification of the motion of the cochlear partition. This partition, comprising the organ of Corti sandwiched between the basilar and tectorial membranes, contains the outer hair cells that are thought to drive this amplification process. Force generation by outer hair cells has been studied extensively in vitro and in situ, but, to understand cochlear amplification fully, it is necessary to characterize the role played by each of the components of the cochlear partition in vivo. Observations of cochlear partition motion in vivo are severely restricted by its inaccessibility and sensitivity to surgical trauma, so, for the present study, a computer model has been used to simulate the operation of the cochlea under different experimental conditions. In this model, which uniquely retains much of the three-dimensional complexity of the real cochlea, the motions of the basilar and tectorial membranes are fundamentally different during in situ- and in vivo-like conditions. Furthermore, enhanced outer hair cell force generation in vitro leads paradoxically to a decrease in the gain of the cochlear amplifier during sound stimulation to the model in vivo. These results suggest that it is not possible to extrapolate directly from experimental observations made in vitro and in situ to the normal operation of the intact organ in vivo.

The product of ORF O located within the domain of herpes simplex virus 1 genome transcribed during latent infection binds to and inhibits in vitro binding of infected cell protein 4 to its cognate DNA site

The partially overlapping ORF P and ORF O are located within the domains of the herpes simplex virus 1 genome transcribed during latency. Earlier studies have shown that ORF P is repressed by infected cell protein 4 (ICP4), the major viral regulatory protein, binding to its cognate site at the transcription initiation site of ORF P. The ORF P protein binds to p32, a component of the ASF/SF2 alternate splicing factors; in cells infected with a recombinant virus in which ORF P was derepressed there was a significant decrease in the expression of products of key regulatory genes containing introns. We report that (i) the expression of ORF O is repressed during productive infection by the same mechanism as that determining the expression of ORF P; (ii) in cells infected at the nonpermissive temperature for ICP4, ORF O protein is made in significantly lower amounts than the ORF P protein; (iii) the results of insertion of a sequence encoding 20 amino acids between the putative initiator methionine codons of ORF O and ORF P suggest that ORF O initiates at the methionine codon of ORF P and that the synthesis of ORF O results from frameshift or editing of its RNA; and (iv) glutathione S-transferase–ORF O fusion protein bound specifically ICP4 and precluded its binding to its cognate site on DNA in vitro. These and earlier results indicate that ORF P and ORF O together have the capacity to reduce the synthesis or block the expression of regulatory proteins essential for viral replication in productive infection.

Potentially predictive and manipulable blood serum correlates of aging in the healthy human male: Progressive decreases in bioavailable testosterone, dehydroepiandrosterone sulfate, and the ratio of insulin-like growth factor 1 to growth hormone

A cross-sectional survey was made in 56 exceptionally healthy males, ranging in age from 20 to 84 years. Measurements were made of selected steroidal components and peptidic hormones in blood serum, and cognitive and physical tests were performed. Of those blood serum variables that gave highly significant negative correlations with age (r > −0.6), bioavailable testosterone (BT), dehydroepiandrosterone sulfate (DHEAS), and the ratio of insulin-like growth factor 1 (IGF-1) to growth hormone (GH) showed a stepwise pattern of age-related changes most closely resembling those of the age steps themselves. Of these, BT correlated best with significantly age-correlated cognitive and physical measures. Because DHEAS correlated well with BT and considerably less well than BT with the cognitive and physical measures, it seems likely that BT and/or substances to which BT gives rise in tissues play a more direct role in whatever processes are rate-limiting in the functions measured and that DHEAS relates more indirectly to these functions. The high correlation of IGF-1/GH with age, its relatively low correlation with BT, and the patterns of correlations of IGF-1/GH and BT with significantly age-correlated cognitive and physical measures suggest that the GH–IGF-1 axis and BT play independent roles in affecting these functions. Serial determinations made after oral ingestion of pregnenolone and data from the literature suggest there is interdependence of steroid metabolic systems with those operational in control of interrelations in the GH–IGF-1 axis. Longitudinal concurrent measurements of serum levels of BT, DHEAS, and IGF-1/GH together with detailed studies of their correlations with age-correlated functional measures may be useful in detecting early age-related dysregulations and may be helpful in devising ameliorative approaches.

Characterization of the Saccharomyces cerevisiae ERG26 gene encoding the C-3 sterol dehydrogenase (C-4 decarboxylase) involved in sterol biosynthesis

All but two genes involved in the ergosterol biosynthetic pathway in Saccharomyces cerevisiae have been cloned, and their corresponding mutants have been described. The remaining genes encode the C-3 sterol dehydrogenase (C-4 decarboxylase) and the 3-keto sterol reductase and in concert with the C-4 sterol methyloxidase (ERG25) catalyze the sequential removal of the two methyl groups at the sterol C-4 position. The protein sequence of the Nocardia sp NAD(P)-dependent cholesterol dehydrogenase responsible for the conversion of cholesterol to its 3-keto derivative shows 30% similarity to a 329-aa Saccharomyces ORF, YGL001c, suggesting a possible role of YGL001c in sterol decarboxylation. The disruption of the YGL001c ORF was made in a diploid strain, and the segregants were plated onto sterol supplemented media under anaerobic growth conditions. Segregants containing the YGL001c disruption were not viable after transfer to fresh, sterol-supplemented media. However, one segregant was able to grow, and genetic analysis indicated that it contained a hem3 mutation. The YGL001c (ERG26) disruption also was viable in a hem 1Δ strain grown in the presence of ergosterol. Introduction of the erg26 mutation into an erg1 (squalene epoxidase) strain also was viable in ergosterol-supplemented media. We demonstrated that erg26 mutants grown on various sterol and heme-supplemented media accumulate nonesterified carboxylic acid sterols such as 4β,14α-dimethyl-4α-carboxy-cholesta-8,24-dien-3β-ol and 4β-methyl-4α-carboxy-cholesta-8,24-dien-3β-ol, the predicted substrates for the C-3 sterol dehydrogenase. Accumulation of these sterol molecules in a heme-competent erg26 strain results in an accumulation of toxic-oxygenated sterol intermediates that prevent growth, even in the presence of exogenously added sterol.