Efficiency and environmental indexes to evaluate the sustainability of mineral and organic fertilization in an irrigated melon crop

Melon is traditionally cultivated in fertigated farmlands in the center of Spain with high inputs of water and N fertilizer. Excess N can have a negative impact, from the economic point of view, since it can diminish the production and quality of the fruit, from the environmental point of view, since it is a very mobile element in the soil and can contaminate groundwater. From health point of view, nitrate can be accumulated in fruit pulp, and, in addition, groundwater is the fundamental supply source of human populations. Best management practices are particularly necessary in this region as many zones have been declared vulnerable to NO3- pollution (Directive 91/676/CEE) During successive years, a melon crop (Cucumis melo L.) was grown under field conditions applying mineral and organic fertilizers under drip irrigation. Different doses of ammonium nitrate were used as well as compost derived from the wine-distillery industry which is relevant in this area. The present study reviews the most common N efficiency indexes under the different management options with a view to maximizing yield and minimizing N loss.

Stabilization of embankments on inclined surfaces by micropiles

Embankments constructed on hillsides can have serious problems of stability, generally created by the action of water com-bined with the inclination of the hillside. In order to increase the stability or correct problems of instability already present, there are various methods that can be used: surface and deep drainage, reinforcements with anchored beams, medium and large diameter piles, etc. Standing out among these systems (for its versatility) is the use of micropiles which ?sew? the embankment to a non-unstable area of the hillside. This paper presents research undertaken by means of a finite element code for studying the effect and stress of the micropiles, comparing the results with real measurements taken in the south of Spain.

An integrated modeling approach to assess the impact on road freight transport demand of allowing longer and heavier vehicles (LHVs) in Spain

The assessment on introducing Longer and Heavier Vehicles (LHVs) on the road freight transport demand is performed in this paper by applying an integrated modeling approach composed of a Random Utility-Based Multiregional Input-Output model (RUBMRIO) and a road transport network model. The approach strongly supports the concept that changes in transport costs derived from the LHVs allowance as well as the economic structure of regions have both direct and indirect effects on the road freight transport system. In addition, we estimate the magnitude and extent of demand changes in the road freight transportation system by using the commodity-based structure of the approach to identify the effect on traffic flows and on pollutant emissions over the whole network of Spain by considering a sensitivity analysis of the main parameters which determine the share of Heavy-Goods Vehicles (HGVs) and LHVs. The results show that the introduction of LHVs will strengthen the competitiveness of the road haulage sector by reducing costs, emissions, and the total freight vehicles required.

Native bradyrhizobial symbionts of Lupinus mariae-josephae, a unique endemism thriving in alkaline soils in Eastern Spain

Lupinus mariae-josephae (Lmj) es una especie de lupino endémica de una pequeña y específica área de Comunidad Valenciana (Este de España), donde prospera en suelos alcalinoscalcáreos, un hábitat singular para los altramuces, que crecen preferentemente en suelos ácidos o neutros. Esto hace de Lmj una especie de lupino única. Cuando se inició este trabajo, la extensión conocida de este endemismo abarcaba unos 700 kilómetros cuadrados, confinados en la provincia de Valencia. En esta área, Lmj prospera en pequeñas poblaciones aisladas que contienen un número reducido de plantas por lo que se la consideró una especie en peligro de extinción. Todos los esfuerzos, utilizando estrategias clásicas dirigidas a ampliar el área de crecimiento de Lmj y garantizar su conservación, han tenido un éxito limitado. El trabajo que se presenta está dirigido a mejorar el conocimiento de la ecología de Lmj, en particular la interacción simbiótica que establece con bacterias del suelo denominadas rizobios y se centra en la caracterización fenotípica, filogenética y genómica de esos rizobios. También se investiga la posible contribución de la simbiosis en mejorar la conservación de Lmj. Para este fin, se han estudiado diferentes aspectos que se describen a continuación. El primero objetivo se centró en aislar y estudiar de la diversidad genética de las bacterias endosimbióticas de Lmj. . Se realizó un análisis filogenético de genes esenciales que mostró que las cepas de Lmj pertenecen al género Bradyrhizobium y que presentan una gran diversidad con características fenotípicas y simbióticas diferentes de cepas de Bradyrhizobium que nodulan otras especies de lupinos nativos de España (cepas ISLU). Las cepas estudiadas se dividieron en dos grupos (Clado I y Clado II). El Clado I, incluye a las cepas Lmj, definiendo un nuevo linaje, filogenéticamente relacionado con otras especies de Bradyrhizobium, como B. jicamae y B. elkanii. El Clado II contiene cepas ISLU relacionadas con cepas de B. canariense y B. japonicum que establecen simbiosis con lupinos de suelos ácidos. Otro análisis filogenético basado en genes simbióticos, distribuyó las cepas de Lmj en sólo dos grupos diferentes. La singularidad y gran diversidad de estas cepas en una pequeña área geográfica, hacen de este, un atractivo sistema para el estudio de la evolución y adaptación de las bacterias simbióticas a su respectiva planta huésped. Adicionalmente, se estudio la presencia de bacterias capaces de nodular Lmj en suelos básicos de Chiapas, México. Sorprendentemente, estos suelos contienen bacterias capaces establecer interacciones simbióticas eficientes con Lmj en ensayos de invernadero. A continuación se investigó la taxonomía de los endosimbiontes de Lmj analizando la secuencia de cuatro genes esenciales (16S rRNA, recA, glnII y atpD) y el promedio de identidad de nucleótidos de genomas completos de algunas cepas representativas de la diversidad (ANIm). Se identificaron nuevas especies de Bradyrhizobium dentro del Clado I y se definió una de ellas: 'Bradyrhizobium valentinum' sp. nov (cepa tipo LmjM3T = CECT 8364T, LMG 2761T). También se abordó cómo conservar Lmj en su hábitat natural mediante inoculación con alguna de las cepas aisladas. Se demostró la ausencia de bacterias capaces de nodular Lmj en suelos rojos alcalinos o ‘‘terra rossa’’ de la Península Ibérica y Baleares. Dos cepas, altamente eficientes en cuanto a la fijación de nitrógeno, LmjC y LmjM3T, fueron seleccionadas para ser empleadas como inoculantes. Dos experimentos de campo llevados a cabo en años consecutivos en áreas con características edafoclimáticas similares a las que presentan las poblaciones de Lmj, lograron la reproducción exitosa de la planta. Se concluyó que un ciclo reproductivo exitoso de Lmj es absolutamente dependiente de la inoculación con sus simbiontes naturales y que la simbiosis debe ser considerada un factor esencial en estrategias de conservación de leguminosas en peligro. La obtención de varias secuencias genómicas de cepas aisladas de Lmj y de otras cepas de Bradyrhizobium reveló una alta similitud entre los genomas de las cepas del Clado I, y permitió la identificación de cinco posibles nuevas especies. Además, se estudiaron tres agrupaciones de genes relacionados con la simbiosis (nod, nif y fix) definiendo un nuevo linaje para las cepas de Lmj, diferente del symbiovar “genistearum” de B. canariense y B. japonicum. La baja diversidad encontrada en el análisis filogenético de los genes simbióticos contrasta con la gran diversidad asociada a genes esenciales. La presencia de plásmidos en cepas del género Bradyrhizobium ha sido descrita en muy pocas ocasiones, sin embargo el análisis de la secuencia genómica de la cepa ISLU101, aislada de Lupinus angustifolius, reveló la presencia de un origen de replicación extracromosómico homólogo al operón repABC, presente en el plásmido de Bradyrhizobium sp BTAi1. Gracias a esta secuencia se identificaron genes homólogos en 19 de 72 cepas ISLU. Filogenéticamente, las secuencias de repABC se agruparon en un grupo monofilético con las de pBTAi1 y separadas de los rizobios de crecimiento rápido. Finalmente, se identificaron sistemas de secreción de proteínas de tipo III (T3SS) en nueve genomas de cepas de Lmj. Los T3SS pueden inyectar proteínas efectoras al interior de células vegetales. Su presencia en rizobios se ha relacionado con la gama de hospedador que pueden nodular y puede tener un efecto beneficioso, neutro o perjudicial en la simbiosis. Los T3SS de las cepas de Lmj codifican para una proteína efectora similar a NopE, un efector dependiente de T3SS descrito en B. diazoefficiens USDA 110T. La proteína NopE de la cepa LmjC se ha caracterizado bioquímicamente. ABSTRACT Lupinus mariae-josephae (Lmj) is a lupine species endemic of a unique small area in Valencia region (Eastern Spain) where the lupine plants thrive in alkaline-limed soils, which preferentially grow in acid or neutral soils. This is the type of soils native lupines of Spain. When this work was initiated, the extension of the endemic area of Lmj was of about 700 squared kilometers confined to the Valencia province. In this area, Lmj thrives in small, isolated patches containing a reduced number of plants, and points to an endemism that can easily became endangered or extinct. Consequently, the Valencia Community authorities gave a ‘‘microreserve” status for conservation of the species. All efforts, using classical strategies directed to extend the area of Lmj growth and ensure its conservation have been so far unsuccessful. The work presented here is directed to improve our knowledge of Lmj ecology and it is centered in the characterization of the rhizobial symbiosis by phenotypic, phylogenetic and genomic analysis as well as in investigate the potential contribution of the symbiosis to improve its conservation. To this end, five different topics have been studied, and results are briefly described here. Extensive details can be followed en the attached, published articles. The first topic deals with the indigenous rhizobial symbionts of the Lmj endemism, and its genetic diversity was investigated. The Lmj root symbionts belong to the Bradyrhizobium genus, and phylogenetic analysis based on core genes identified a large diversity of Bradyrhizobium strains with phenotypic and symbiotic characteristics different from rhizobia nodulating other Lupinus spp. native of Spain. The strains were split in two clades. Clade II contained strains close to classical B. canariense and B. japonicum lineages that establish symbioses with lupines in acid soils of the Mediterranean area. Clade I included Lmj strains that define a new lineage, close to other Bradyrhizobium species as B. jicamae and B. elkanii. The phylogenetic analysis based on symbiotic genes identified only two distinct clusters. The singularity and large diversity of these strains in such a small geographical area makes this an attractive system for studying the evolution and adaptation of the rhizobial symbiont to the plant host. Additionally, the presence of bacteria able to nodulate Lmj in basic soils from Chiapas, Mexico was investigated. Surprisingly, these soils contain bacteria able to effectively nodulate and fix nitrogen with Lmj plants in greenhouse assays. In the second topic, the taxonomic status of the endosymbiotic bacteria of Lmj from Valencia endemism and Chiapas was investigated. Results from phylogenetic analysis of core genes and Average Nucleotide Identity (ANIm) using draft genomic sequences identified new Bradyrhizobium species within strains of Clade I of Lmj endosymbiotic bacteria. Only one of these potentially new species has been defined, meanwhile the others are under process of characterization. The name ‘Bradyrhizobium valentinum’ sp. nov. was proposed for the defined species (type strain LmjM3T= CECT 8364T, LMG 2761T). The third topic was directed to conservation of endangered Lmj in its natural habitat. The relevant conclusion of this experimentation is that the symbiosis should be considered as a relevant factor in the conservation strategies for endangered legumes. First, we showed absence of bacteria able to nodulate Lmj in all the inspected ‘‘terra rossa’’ or alkaline red soils of the Iberian Peninsula and Balearic Islands. Then, two efficient nitrogen fixing strains with Lmj plants, LmjC and LmjM3T, were selected as inoculum for seed coating. Two planting experiments were carried out in consecutive years under natural conditions in areas with edapho-climatic characteristics identical to those sustaining natural Lmj populations, and successful reproduction of the plant was achieved. The relevant conclusion from these assays was that the successful reproductive cycle was absolutely dependent on seedling inoculation with effective bradyrhizobia The forth topic deep into the analysis of the genomic of Lmj representative strains. To this end, draft genomic sequences of selected Lmj strains and type strains of Bradyrhizobium spp. were assembled. The comparison analysis of the draft genomic sequences of Lmj strains and related Bradyrhizobium species grouped in Clade I, revealed a high genomic homology among them, and allowed the definition of five potentially new species of Lmj nodulating bacteria. Also, based on the available draft genomic sequences, only three clusters of nod, fix and nif genes from Lmj strains were identified and showed to define a new symbiotic lineage, distant from that of B. canariense and B. japonicum bv. genistearum. The low diversity exhibited by the phylogenetic analysis of symbiotic genes contrast with the large diversity of strains as regards the housekeeping genes analyzed. Besides, the genomic analysis of a Lupinus angustifolius strain ISLU101, revealed the presence of an extrachromosomal replication origin homologous to repABC cluster from plasmid present in Bradyrhizobium spp BTAi1. This repABC cluster gene sequence allowed the identification of extrachromosomic replication origin in 19 out of 72 Bradyrhizobium strains from Lupinus spp., a highly significant result since the absence of plasmids in the Bradyrhizobium genus was traditionally assumed. The repABC gene sequences of these strains grouped them in a unique monophyletic group, related to B. sp. BTAi1 plasmid, but differentiated from the repABC gene cluster of plasmids in fast growing rhizobium strains. The last topic was focused on characterization of type III secreted effectors present in Lmj endosymbiotic bacteria. Type III secretion systems (T3SS) are specialized protein export machineries which can deliver effector proteins into plant cells. The presence of T3SS in rhizobia has frequently been related to the symbiotic nodulation host-range and may have a beneficial or detrimental effect on the symbiosis with legumes. In this context, the presence of T3SS in genomes of nine Lmj strains was investigated, and it was shown the presence of clusters encoding NopE type III-secreted protein similar to the NopE1 and NopE2 of B. diazoefficiens USDA 110T. The putative NopE protein of LmjC strain is at present being characterized regarding its structure and function.

Relationship among slurry characteristics and gaseous emissions at different types of commercial Spanish pig farms

This study aimed to analyse several factors of variation of slurry composition and to establish prediction equations for potential methane (CH4) and ammonia (NH3) emissions. Seventy-nine feed and slurry samples were collected at two seasons (summer and winter) from commercial pig farms sited at two Spanish regions (Centre and Mediterranean). Nursery, growing-fattening, gestating and lactating facilities were sampled. Feed and slurry composition were determined, and potential CH4 and NH3 emissions measured at laboratory. Feed nutrient contents were used as covariates in the analysis. Near infrared reflectance spectroscopy (NIRS) was evaluated as a predicting tool for slurry composition and potential gaseous emissions. A wide variability was found both in feed and slurry composition. Mediterranean farms had a higher pH (p<0.001) and ash (p=0.02) concentration than those located at the Centre of Spain. Also, type of farm affected ether extract content of the slurry (p=0.02), with highest values obtained for the youngest animal facilities. Results suggested a buffer effect of dietary fibre on slurry pH and a direct relationship (p<0.05) with fibre constituents of manure. Dietary protein content did not affect slurry nitrogen content but decreased (p=0.003) total and volatile solids concentration. Prediction models of potential NH3 emissions (R2=0.89) and CH4 yield (R2=0.61) were obtained from slurry composition. Predictions from NIRS showed a high accuracy for most slurry constituents (R2>0.90) and similar accuracy of prediction of potential NH3 and CH4 emissions (R2=0.84 and 0.68, respectively) to models using slurry characteristics, which can be of interest to estimate emissions from commercial farms and establish mitigation strategies or optimize biogas production.

Contribution to Load-Frequency Regulation of a Hydropower Plant with Long Tail-Race Tunnel

In this paper, a hydroelectric power plant with long tail-race tunnel has been modelled for assessing its contribution to secondary regulation reserve. Cavitation problems, caused by the discharge conduit length, are expected downstream the turbine where low pressure appears during regulation manoeuvres. Therefore, governor's gains should be selected taking into account these phenomena. On the other hand, regulation services bidden by the plant operator should fulfil TSO (Transmission System Operator) quality requirements. A methodology for tuning governor PI gains is proposed and applied to a Hydro power plant in pre-design phase in northwest area of Spain. The PI gains adjustment proposed provides a proper plant response, according to some established indexes, while avoiding cavitation phenomena.

Viability of the biochar production from different manure wastes in the Amblés Valley (Ávila, Spain)

In the last years, intensive animal husbandry production has led to a large concentration of animals in small areas. This has resulted in the production of excessive amounts of manures with insufficient nearby land for application. One of this areas is the Amblés Valley located in the centre of Spain, near to Ávila city, with an extension of 167472 ha of which 88.9% is agricultural land. This valley has an important livestock focused on pig, cattle, chicken production which is associated with the generation of more than 200,000 t/year of manure. There are a number of environmental problems associated with these intensive agricultural systems, including N and P pollution of water bodies, methane emissions and odour pollution. These serious environmental threats are called for innovative environmental management approaches. A feasible technology for the management of manures, offering a potential to valorise these wastes, is pyrolysis, which results in the production of biochar. The objective of this work is evaluated the technical and economic feasibility of the production of biochar in Amblés Valley (Spain).

Combine use of Selected Schizosaccharomyces pombe and Lachancea thermotolerans Yeast Strains as an Alternative to the Traditional Malolactic Fermentation in Red Wine Production

Most red wines commercialized in the market use the malolactic fermentationprocess in order to ensure stability from a microbiological point of view. In this secondfermentation, malic acid is converted into L-lactic acid under controlled setups. Howeverthis process is not free from possible collateral effects that on some occasions produceoff-flavors, wine quality loss and human health problems. In warm viticulture regions suchas the south of Spain, the risk of suffering a deviation during the malolactic fermentationprocess increases due to the high must pH. This contributes to produce wines with highvolatile acidity and biogenic amine values. This manuscript develops a new red winemakingmethodology that consists of combining the use of two non-Saccharomyces yeast strains asan alternative to the traditional malolactic fermentation. In this method, malic acid is totallyconsumed by Schizosaccharomyces pombe, thus achieving the microbiological stabilizationobjective, while Lachancea thermotolerans produces lactic acid in order not to reduce andeven increase the acidity of wines produced from low acidity musts. This technique reducesthe risks inherent to the malolactic fermentation process when performed in warm regions.The result is more fruity wines that contain less acetic acid and biogenic amines than thetraditional controls that have undergone the classical malolactic fermentation.

Implications of climate change for rice farming in the Doñana wetland (SW Spain)

Climate change impacts are expected to affect rice farming and wetlands welfare in the Doñana protected Area, due to decreases in quantity and quality water supply and higher temperatures. The largest rice farming area is closely located to the Doñana wetlands in the Guadalquivir river basin estuary (South Western of Spain).

Implementation of road dust resuspension in air quality simulations of particulate matter in Madrid (Spain)

An evaluation of the concentration levels of Particulate Matter (PM) was carried out in Madrid (Spain) by introducing the emissions from road dust resuspension. Road dust resuspension emission factors (EF) for different types of vehicles were calculated from EPA-AP42, a global resuspension factor of 0.097 g veh−1km−1 as described in Amato et al. (2010) and a rain-dependent correction factor. With these resuspension EFs, a simulation at street canyon level was performed with the OSPM model without rainfall. Subsequently, a simulation using the CMAQ model was implemented adding resuspension emissions affected by the rain. These data were compared with monitored data obtained from air quality stations. OSPM model simulations with resuspension EFs but without the effect of rainfall improve the PM estimates in about 20gm−3μ compared to the simulation with default EFs. Total emissions were calculated by adding the emissions estimated with resuspension EFs to the default PM emissions to be used by CMAQ. For the study in the Madrid Area, resuspension emissions are approximately of the same order of magnitude as inventoried emissions. On a monthly scale, rain effects are negligible for resuspension emissions due to the dry weather conditions of Spain. With the exception of April and May, the decrease in resuspension emissions is not >3%. The predicted PM10 concentration increases up to 9μ gm−3 on annual average for each station compared to the same scenario without resuspension. However, in both cases, PM 10 estimates with resuspension are still underestimating observations. It should be noted that although that accounting for resuspension improves the quality of model predictions, other PM sources (e.g., Saharan dust) were not considered in this study.

Yield and sugar accumulating capacities of Airén cultivar. A preliminary study

Airén is the most worldwide spread white grape cultivar, high yielding, well adapted to hot, dry conditions, and not very sensitive to fungal diseases. Its largest growing region is La Mancha, where Airén has been traditionally bush trained, spur pruned and grown with no irrigation. However, grape growing has evolved to meet the need for higher yields and harvest mechanization; and modern cultural practices train grape vines to simple multi-wire trellis systems, cane pruned, and usually irrigated. The aim of the present study was to evaluate the yield and sugar accumulating capacities of Airén cultivar with regard to leaf area, and to assess the influence that different yield components have on yield. In 2014, five commercial irrigated vineyards, located in La Mancha, of different ages, and grafted onto different rootstocks were selected for this study. Canopy surface area (SA) was measured at maturity. Berry weight and sugar concentration were measured during ripening on a weekly basis. Yield and yield components were determined at harvest. Values for shoot density ranged 2.3-5.1 shoots/m2; SA, 0.6-1.1 m2/m2; yield, 20-40 t/ha; fertility, 1.1-1.7 bunches/shoot; bunch weight, 450-650 g; berry weight, 2.5-2.9 g; and sugar concentration, 17-21 ºBrix. The number of bunches per shoot was the yield component that had the greatest influence on yield. The number of berries was the main contributing factor to bunch weight. A lineal relationship between SA/yield and sugar concentration was observed, with values of SA/yield ranging from 0.20 to 0.45 m2/kg. A ratio SA/yield of approximately 0.4 m2/kg was needed to reach a value of 20 ºBrix. Hence it would be necessary a SA of 12000 m2/ha, under the conditions of this study, to achieve a 30 t/ha yield, and a sugar concentration of 20 ºBrix. These results are a step forward in the study of the Airén cultivar, being of help for grape growers in the center area of Spain in order to maximize crop yield and sugar accumulation.

A MULHER ALÉM DO BEM E DO MAL: Malévola e a representação cinematográfica do feminino integrado

Estudo sobre as construções simbólicas e identitárias da mulher presentes na narrativa e na estrutura das personagens femininas do filme Malévola (2014) – produção dos estúdios Disney (EUA). A narrativa é inspirada no conto de fadas “A Bela Adormecida do Bosque” e distingue-se pela perspectiva feminina, modificando as possibilidades de interpretação, além de possibilitar a quebra do paradigma dicotômico relacionado ao Bem e ao Mal. A pesquisa tem por objetivo estudar a evolução das construções imaginárias da mulher no cinema e traçar paralelos entre as características arquetípicas das personagens de Malévola em relação à identidade da mulher na contemporaneidade. Para tal, será tomado como referencial teórico os estudos do imaginário social, com as obras de Gilbert Durand, Edgar Morin e, em especial, Michel Maffesoli; conceitos da psicanálise a partir dos trabalhos de C.G. Jung, Erich Neumann, Marie-Louise Von Franz e Clarissa Pinkola Estés; as teorias de Stuart Hall, Laura Mulvey e Gilles Lipovetsky relacionadas aos estudos culturais com ênfase em gênero; e também o ecofeminismo através dos trabalhos de autoras como Vandana Shiva e Maria Mies. Nosso referencial teórico-metodológico é a Hermenêutica de Profundidade (HP) visando à interpretação da estrutura simbólica de nosso objeto. Resultam desta pesquisa a verificação de um processo de saturação de padrões identitários e simbólicos provindos da modernidade e a evolução de novas dinâmicas nas narrativas presentes nas mídias e na comunicação

Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa

Individuals with hemophilia A require frequent infusion of preparations of coagulation factor VIII. The activity of factor VIII (FVIII) as a cofactor for factor IXa in the coagulation cascade is limited by its instability after activation by thrombin. Activation of FVIII occurs through proteolytic cleavage and generates an unstable FVIII heterotrimer that is subject to rapid dissociation of its subunits. In addition, further proteolytic cleavage by thrombin, factor Xa, factor IXa, and activated protein C can lead to inactivation. We have engineered and characterized a FVIII protein, IR8, that has enhanced in vitro stability of FVIII activity due to resistance to subunit dissociation and proteolytic inactivation. FVIII was genetically engineered by deletion of residues 794-1689 so that the A2 domain is covalently attached to the light chain. Missense mutations at thrombin and activated protein C inactivation cleavage sites provided resistance to proteolysis, resulting in a single-chain protein that has maximal activity after a single cleavage after arginine-372. The specific activity of partially purified protein produced in transfected COS-1 monkey cells was 5-fold higher than wild-type (WT) FVIII. Whereas WT FVIII was inactivated by thrombin after 10 min in vitro, IR8 still retained 38% of peak activity after 4 hr. Whereas binding of IR8 to von Willebrand factor (vWF) was reduced 10-fold compared with WT FVIII, in the presence of an anti-light chain antibody, ESH8, binding of IR8 to vWF increased 5-fold. These results demonstrate that residues 1690–2332 of FVIII are sufficient to support high-affinity vWF binding. Whereas ESH8 inhibited WT factor VIII activity, IR8 retained its activity in the presence of ESH8. We propose that resistance to A2 subunit dissociation abrogates inhibition by the ESH8 antibody. The stable FVIIIa described here provides the opportunity to study the activated form of this critical coagulation factor and demonstrates that proteins can be improved by rationale design through genetic engineering technology.

Mutations in the nebulin gene associated with autosomal recessive nemaline myopathy

The congenital nemaline myopathies are rare hereditary muscle disorders characterized by the presence in the muscle fibers of nemaline bodies consisting of proteins derived from the Z disc and thin filament. In a single large Australian family with an autosomal dominant form of nemaline myopathy, the disease is caused by a mutation in the α-tropomyosin gene TPM3. The typical form of nemaline myopathy is inherited as an autosomal recessive trait, the locus of which we previously assigned to chromosome 2q21.2-q22. We show here that mutations in the nebulin gene located within this region are associated with the disease. The nebulin protein is a giant protein found in the thin filaments of striated muscle. A variety of nebulin isoforms are thought to contribute to the molecular diversity of Z discs. We have studied the 3′ end of the 20.8-kb cDNA encoding the Z disc part of the 800-kDa protein and describe six disease-associated mutations in patients from five families of different ethnic origins. In two families with consanguineous parents, the patients were homozygous for point mutations. In one family with nonconsanguineous parents, the affected siblings were compound heterozygotes for two different mutations, and in two further families with one detected mutation each, haplotypes are compatible with compound heterozygosity. Immunofluorescence studies with antibodies specific to the C-terminal region of nebulin indicate that the mutations may cause protein truncation possibly associated with loss of fiber-type diversity, which may be relevant to disease pathogenesis.

Application of Mathieu functions for the study of non-slanted reflection gratings

In this work we prensent an analysis of non-slanted reflection gratings by using exact solution of the second order differential equation derived from Maxwell equations, in terms of Mathieu functions. The results obtained by using this method will be compared to those obtained by using the well known Kogelnik's Coupled Wave Theory which predicts with great accuracy the response of the efficieny of the zero and first order for volume phase gratings, for both reflection and transmission gratings.