Generation and characterization of stable, highly concentrated titanium dioxide nanoparticle aerosols for rodent inhalation studies

The intensive use of nano-sized titanium dioxide (TiO2) particles in many different applications necessitates studies on their risk assessment as there are still open questions on their safe handling and utilization. For reliable risk assessment, the interaction of TiO2 nanoparticles (NP) with biological systems ideally needs to be investigated using physico-chemically uniform and well-characterized NP. In this article, we describe the reproducible production of TiO2 NP aerosols using spark ignition technology. Because currently no data are available on inhaled NP in the 10–50 nm diameter range, the emphasis was to generate NP as small as 20 nm for inhalation studies in rodents. For anticipated in vivo dosimetry analyses, TiO2 NP were radiolabeled with 48V by proton irradiation of the titanium electrodes of the spark generator. The dissolution rate of the 48V label was about 1% within the first day. The highly concentrated, polydisperse TiO2 NP aerosol (3–6 × 106 cm−3) proved to be constant over several hours in terms of its count median mobility diameter, its geometric standard deviation, and number concentration. Extensive characterization of NP chemical composition, physical structure, morphology, and specific surface area was performed. The originally generated amorphous TiO2 NP were converted into crystalline anatase TiO2 NP by thermal annealing at 950 °C. Both crystalline and amorphous 20-nm TiO2 NP were chain agglomerated/aggregated, consisting of primary particles in the range of 5 nm. Disintegration of the deposited TiO2 NP in lung tissue was not detectable within 24 h.

The N-terminal domain of Npro of classical swine fever virus determines its stability and regulates type I IFN production

The viral protein Npro is unique to the genus Pestivirus within the family Flaviviridae. After autocatalytic cleavage from the nascent polyprotein, Npro suppresses type I IFN (IFN-α/β) induction by mediating proteasomal degradation of IFN regulatory factor 3 (IRF-3). Previous studies found that the Npro-mediated IRF-3 degradation was dependent of a TRASH domain in the C-terminal half of Npro coordinating zinc by means of the amino acid residues C112, C134, D136 and C138. Interestingly, four classical swine fever virus (CSFV) isolates obtained from diseased pigs in Thailand in 1993 and 1998 did not suppress IFN-α/β induction despite the presence of an intact TRASH domain. Through systematic analyses, it was found that an amino acid mutation at position 40 or mutations at positions 17 and 61 in the N-terminal half of Npro of these four isolates were related to the lack of IRF-3-degrading activity. Restoring a histidine at position 40 or both a proline at position 17 and a lysine at position 61 based on the sequence of a functional Npro contributed to higher stability of the reconstructed Npro compared with the Npro from the Thai isolate. This led to enhanced interaction of Npro with IRF-3 along with its degradation by the proteasome. The results of the present study revealed that amino acid residues in the N-terminal domain of Npro are involved in the stability of Npro, in interaction of Npro with IRF-3 and subsequent degradation of IRF-3, leading to downregulation of IFN-α/β production.

Biogenic and lithogenic silica, silicic acid, production of biogenic silica, and irradiance measurements during the Ross Sea Bloom Project 1994-1996

The development of the seasonal phytoplankton bloom in the Ross Sea was studied during two cruises. The first, conducted in November-December 1994, investigated the initiation and rapid growth of the bloom, whereas the second (December 1995-January 1996) concentrated on the bloom's maximum biomass period and the subsequent decline in biomass. Central to the understanding of the controls of growth and the summer decline of the bloom is a quantitative assessment of the growth rate of phytoplankton. Growth rates were estimated over two time scales with different methods. The first estimated daily growth rates from isotropic incorporation under simulated in situ conditions, including 14C, 15N and 32Si uptake measurements combined with estimates of standing stocks of particulate organic carbon, nitrogen and biogenic silica. The second method used daily to weekly changes in biomass at selected locations, with net growth rates being estimated from changes in standing stocks of phytoplankton. In addition, growth rates were estimated in large-volume experiments under optimal irradiances. Growth rates showed distinct temporal patterns. Early in the growing season, short-term estimates suggested that growth rates of in situ assemblages were less than maximum (relative to the temperature-limited maximum) and were likely reduced due to low irradiance regimes encountered under the ice. Growth rates increased thereafter and appeared to reach their maximum as biomass approached the seasonal peak, but decreased markedly in late December. Differences between the major taxonomic groups present were also noted, especially from the isotopic tracer experiments. The haplophyte Phaeocystic antarctica was dominant in 1994 throughout the growing season, and it exhibited the greatest growth rates (mean 0.41/day) during spring. Diatom standing stocks were low early in the growing season, and growth rates averaged 0.100/day. In summer diatoms were more abundant, but their growth rates remained much lower (mean of 0.08/day) than the potential maximum. Understanding growth rate controls is essential to the development of predictive models of the carbon cycle and food webs in Antarctic waters.

Implications of agricultural production, policy and land use changes on water resource assessment

Una gestión más eficiente y equitativa del agua a escala de cuenca no se puede centrar exclusivamente en el recurso hídrico en sí, sino también en otras políticas y disciplinas científicas. Existe un consenso creciente de que, además de la consideración de las cambiantes condiciones climáticas, es necesaria una integración de ámbitos de investigación tales como la agronomía, planificación del territorio y ciencias políticas y económicas a fin de satisfacer de manera sostenible las demandas de agua por parte de la sociedad y del medio natural. La Política Agrícola Común (PAC) es el principal motor de cambio en las tendencias de paisajes rurales y sistemas agrícolas, pero el deterioro del medio ambiente es ahora una de las principales preocupaciones. Uno de los cambios más relevantes se ha producido con la expansión e intensificación del olivar en España, principalmente con nuevas zonas de regadío o la conversión de olivares de secano a sistemas en regadío. Por otra parte, el cambio de las condiciones climáticas podría ejercer un papel importante en las tendencias negativas de las aportaciones a los ríos, pero no queda claro el papel que podrían estar jugando los cambios de uso de suelo y cobertura vegetal sobre las tendencias negativas de caudal observadas. Esta tesis tiene como objetivo mejorar el conocimiento de los efectos de la producción agrícola, política agraria y cambios de uso de suelo y cobertura vegetal sobre las condiciones de calidad del agua, respuesta hidrológica y apropiación del agua por parte de la sociedad. En primer lugar, el estudio determina las tendencias existentes de nitratos y sólidos en suspensión en las aguas superficiales de la cuenca del río Guadalquivir durante el periodo de 1998 a 2009. Desde una perspectiva de política agraria, la investigación trata de evaluar mediante un análisis de datos de panel las principales variables, incluyendo la reforma de la PAC de 2003, que están teniendo una influencia en ambos indicadores de calidad. En segundo lugar, la apropiación del agua y el nivel de contaminación por nitratos debido a la producción del aceite de oliva en España se determinan con una evaluación de la huella hídrica (HH), teniendo en cuenta una variabilidad espacial y temporal a largo de las provincias españolas y entre 1997 y 2008. Por último, la tesis analiza los efectos de los cambios de uso de suelo y cobertura vegetal sobre las tendencias negativas observadas en la zona alta del Turia, cabecera de la cuenca del río Júcar, durante el periodo 1973-2008 mediante una modelización ecohidrológica. En la cuenca del Guadalquivir cerca del 20% de las estaciones de monitoreo muestran tendencias significativas, lineales o cuadráticas, para cada indicador de calidad de agua. La mayoría de las tendencias significativas en nitratos están aumentando, y la mayoría de tendencias cuadráticas muestran un patrón en forma de U. Los modelos de regresión de datos de panel muestran que las variables más importantes que empeoran ambos indicadores de calidad del agua son la intensificación de biomasa y las exportaciones de ambos indicadores de calidad procedentes de aguas arriba. En regiones en las que el abandono agrícola y/o desintensificación han tenido lugar han mejorado las condiciones de calidad del agua. Para los nitratos, el desacoplamiento de las subvenciones a la agricultura y la reducción de la cuantía de las subvenciones a tierras de regadío subyacen en la reducción observada de la concentración de nitratos. Las medidas de modernización de regadíos y el establecimiento de zonas vulnerables a nitratos reducen la concentración en subcuencas que muestran una tendencia creciente de nitratos. Sin embargo, el efecto de las exportaciones de nitratos procedente de aguas arriba, la intensificación de la biomasa y los precios de los cultivos presentan un mayor peso, explicando la tendencia creciente observada de nitratos. Para los sólidos en suspensión, no queda de forma evidente si el proceso de desacoplamiento ha influido negativa o positivamente. Sin embargo, los mayores valores de las ayudas agrarias aún ligadas a la producción, en particular en zonas de regadío, conllevan un aumento de las tasas de erosión. Aunque la cuenca del Guadalquivir ha aumentado la producción agrícola y la eficiencia del uso del agua, el problema de las altas tasas de erosión aún no ha sido mitigado adecuadamente. El estudio de la huella hídrica (HH) revela que en 1 L de aceite de oliva español más del 99,5% de la HH está relacionado con la producción de la aceituna, mientras que menos del 0,5% se debe a otros componentes, es decir, a la botella, tapón y etiqueta. Durante el período estudiado, la HH verde en secano y en regadío representa alrededor del 72% y 12%, respectivamente, del total de la HH. Las HHs azul y gris representan 6% y 10%, respectivamente. La producción de aceitunas se concentra en regiones con una HH menor por unidad de producto. La producción de aceite de oliva ha aumentado su productividad del agua durante 1997-2008, incentivado por los crecientes precios del aceite, como también lo ha hecho la cantidad de exportaciones de agua virtual. De hecho, las mayores zonas productoras presentan una eficiencia alta del uso y de productividad del agua, así como un menor potencial de contaminación por nitratos. Pero en estas zonas se ve a la vez reflejado un aumento de presión sobre los recursos hídricos locales. El aumento de extracciones de agua subterránea relacionadas con las exportaciones de aceite de oliva podría añadir una mayor presión a la ya estresada cuenca del Guadalquivir, mostrando la necesidad de equilibrar las fuerzas del mercado con los recursos locales disponibles. Los cambios de uso de suelo y cobertura vegetal juegan un papel importante en el balance del agua de la cuenca alta del Turia, pero no son el principal motor que sustenta la reducción observada de caudal. El aumento de la temperatura es el principal factor que explica las mayores tasas de evapotranspiración y la reducción de caudales. Sin embargo, los cambios de uso de suelo y el cambio climático han tenido un efecto compensatorio en la respuesta hidrológica. Por un lado, el caudal se ha visto afectado negativamente por el aumento de la temperatura, mientras que los cambios de uso de suelo y cobertura vegetal han compensado positivamente con una reducción de las tasas de evapotranspiración, gracias a los procesos de disminución de la densidad de matorral y de degradación forestal. El estudio proporciona una visión que fortalece la interdisciplinariedad entre la planificación hidrológica y territorial, destacando la necesidad de incluir las implicaciones de los cambios de uso de suelo y cobertura vegetal en futuros planes hidrológicos. Estos hallazgos son valiosos para la gestión de la cuenca del río Turia, y el enfoque empleado es útil para la determinación del peso de los cambios de uso de suelo y cobertura vegetal en la respuesta hidrológica en otras regiones. ABSTRACT Achieving a more efficient and equitable water management at catchment scale does not only rely on the water resource itself, but also on other policies and scientific knowledge. There is a growing consensus that, in addition to consideration of changing climate conditions, integration with research areas such as agronomy, land use planning and economics and political science is required to meet sustainably the societal and environmental water demands. The Common Agricultural Policy (CAP) is a main driver for trends in rural landscapes and agricultural systems, but environmental deterioration is now a principal concern. One of the most relevant changes has occurred with the expansion and intensification of olive orchards in Spain, taking place mainly with new irrigated areas or with the conversion from rainfed to irrigated systems. Moreover, changing climate conditions might exert a major role on water yield trends, but it remains unclear the role that ongoing land use and land cover changes (LULCC) might have on observed river flow trends. This thesis aims to improve the understanding of the effects of agricultural production, policies and LULCC on water quality conditions, hydrological response and human water appropriation. Firstly, the study determines the existing trends for nitrates and suspended solids in the Guadalquivir river basin’s surface waters (south Spain) during the period from 1998 to 2009. From a policy perspective, the research tries to assess with panel data analysis the main drivers, including the 2003 CAP reform, which are having an influence on both water quality indicators. Secondly, water appropriation and nitrate pollution level originating from the production of olive oil in Spain is determined with a water footprint (WF) assessment, considering a spatial temporal variability across the Spanish provinces and from 1997 to 2008 years. Finally, the thesis analyzes the effects of the LULCC on the observed negative trends over the period 1973-2008 in the Upper Turia basin, headwaters of the Júcar river demarcation (east Spain), with ecohydrological modeling. In the Guadalquivir river basin about 20% of monitoring stations show significant trends, linear or quadratic, for each water quality indicator. Most significant trends of nitrates are augmenting than decreasing, and most significant quadratic terms of both indicators exhibit U-shaped patterns. The panel data models show that the most important drivers that are worsening nitrates and suspended solids in the basin are biomass intensification and exports of both water quality indicators from upland regions. In regions that agricultural abandonment and/or de-intensification have taken place the water quality conditions have improved. For nitrates, the decoupling of agricultural subsidies and the reduction of the amount of subsidies to irrigated land underlie the observed reduction of nitrates concentration. Measures of irrigation modernization and establishment of vulnerable zones to nitrates ameliorate the concentration of nitrates in subbasins showing an increasing trend. However, the effect of nitrates load from upland areas, intensification of biomass and crop prices present a greater weight leading to the final increasing trend in this subbasins group, where annual crops dominate. For suspended solids, there is no clear evidence that decoupling process have influenced negatively or positively. Nevertheless, greater values of subsidies still linked to production, particularly in irrigated regions, lead to increasing erosion rates. Although agricultural production has augmented in the basin and water efficiency in the agricultural sector has improved, the issue of high erosion rates has not yet been properly faced. The water footprint (WF) assessment reveals that for 1 L Spanish olive oil more than 99.5% of the WF is related to the olive fruit production, whereas less than 0.5% is due to other components i.e. bottle, cap and label. Over the studied period, the green WF in rainfed and irrigated systems represents about 72% and 12%, respectively, of the total WF. Blue and grey WFs represent 6% and 10%, respectively. The olive production is concentrated in regions with the smallest WF per unit of product. The olive oil production has increased its apparent water productivity from 1997 to 2008 incentivized by growing trade prices, but also did the amount of virtual water exports. In fact, the largest producing areas present high water use efficiency per product and apparent water productivity as well as less nitrates pollution potential, but this enhances the pressure on the available water resources. Increasing groundwater abstractions related to olive oil exports may add further pressure to the already stressed Guadalquivir basin. This shows the need to balance the market forces with the available local resources. Concerning the effects of LULCC on the Upper Turia basin’s streamflow, LULCC play a significant role on the water balance, but it is not the main driver underpinning the observed reduction on Turia's streamflow. Increasing mean temperature is the main factor supporting larger evapotranspiration rates and streamflow reduction. In fact, LULCC and climate change have had an offsetting effect on the streamflow generation during the study period. While streamflow has been negatively affected by increasing temperature, ongoing LULCC have positively compensated with reduced evapotranspiration rates, thanks to mainly shrubland clearing and forest degradation processes. The research provides insight for strengthening the interdisciplinarity between hydrological and spatial planning, highlighting the need to include the implications of LULCC in future hydrological plans. These findings are valuable for the management of the Turia river basin, as well as a useful approach for the determination of the weight of LULCC on the hydrological response in other regions.

Hybridizing concentrated solar power (CSP) with biogas and biomethane as an alternative to natural gas: Analysis of environmental performance using LCA

Concentrating Solar Power (CSP) plants typically incorporate one or various auxiliary boilers operating in parallel to the solar field to facilitate start up operations, provide system stability, avoid freezing of heat transfer fluid (HTF) and increase generation capacity. The environmental performance of these plants is highly influenced by the energy input and the type of auxiliary fuel, which in most cases is natural gas (NG). Replacing the NG with biogas or biomethane (BM) in commercial CSP installations is being considered as a means to produce electricity that is fully renewable and free from fossil inputs. Despite their renewable nature, the use of these biofuels also generates environmental impacts that need to be adequately identified and quantified. This paper investigates the environmental performance of a commercial wet-cooled parabolic trough 50 MWe CSP plant in Spain operating according to two strategies: solar-only, with minimum technically viable energy non-solar contribution; and hybrid operation, where 12 % of the electricity derives from auxiliary fuels (as permitted by Spanish legislation). The analysis was based on standard Life Cycle Assessment (LCA) methodology (ISO 14040-14040). The technical viability and the environmental profile of operating the CSP plant with different auxiliary fuels was evaluated, including: NG; biogas from an adjacent plant; and BM withdrawn from the gas network. The effect of using different substrates (biowaste, sewage sludge, grass and a mix of biowaste with animal manure) for the production of the biofuels was also investigated. The results showed that NG is responsible for most of the environmental damage associated with the operation of the plant in hybrid mode. Replacing NG with biogas resulted in a significant improvement of the environmental performance of the installation, primarily due to reduced impact in the following categories: natural land transformation, depletion of fossil resources, and climate change. However, despite the renewable nature of the biofuels, other environmental categories like human toxicity, eutrophication, acidification and marine ecotoxicity scored higher when using biogas and BM.

Cloning of a novel T-cell protein FYB that binds FYN and SH2-domain-containing leukocyte protein 76 and modulates interleukin 2 production

T cell receptor ζ (TcRζ)/CD3 ligation initiates a signaling cascade that involves src kinases p56lck and ζ-associated protein 70, leading to the phosphorylation of substrates such as TcRζ, Vav, SH2-domain-containing leukocyte protein 76 (SLP-76), cbl, and p120/130. FYN binding protein (FYB or p120/130) associates with p59fyn, the TcRζ/CD3 complex, and becomes tyrosine-phosphorylated in response to receptor ligation. In this study, we report the cDNA cloning of human and murine FYB and show that it is restricted in expression to T cells and myeloid cells and possesses an overall unique hydrophilic sequence with several tyrosine-based motifs, proline-based type I and type II SH3 domain binding motifs, several putative lysine/glutamic acid-rich nuclear localization motifs, and a SH3-like domain. In addition to binding the src kinase p59fyn, FYB binds specifically to the hematopoietic signaling protein SLP-76, an interaction mediated by the SLP-76 SH2 domain. In keeping with this, expression of FYB augmented interleukin 2 secretion from a T cell hybridoma, DC27.10, in response to TcRζ/CD3 ligation. FYB is therefore a novel hematopoietic protein that acts as a component of the FYN and SLP-76 signaling cascades in T cells.

A stable human-derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes.

We have generated a human 293-derived retroviral packaging cell line (293GPG) capable of producing high titers of recombinant Moloney murine leukemia virus particles that have incorporated the vesicular stomatitis virus G (VSV-G) protein. To achieve expression of the retroviral gag-pol polyprotein, the precise coding sequences for gag-pol were introduced into a vector which utilizes totally nonretroviral signals for gene expression. Because constitutive expression of the VSV-G protein is toxic in 293 cells, we used the tetR/VP 16 transactivator and teto minimal promoter system for inducible, tetracycline-regulatable expression of VSV-G. After stable transfection of the 293GPG packaging cell line with the MFG.SnlsLacZ retroviral vector construct, it was possible to readily isolate stable virus-producing cell lines with titers approaching 10(7) colony-forming units/ml. Transient transfection of 293GPG cells using a modified version of MFG.SnlsLacZ, in which the cytomegalovirus IE promoter was used to drive transcription of the proviral genome, led to titers of approximately 10(6) colony-forming units/ml. The retroviral/VSV-G pseudotypes generated using 293GPG cells were significantly more resistant to human complement than commonly used amphotropic vectors and could be highly concentrated (> 1000-fold). This new packaging cell line may prove to be particularly useful for assessing the potential use of retroviral vectors for direct in vivo gene transfer. The design of the cell line also provides at least theoretical advantages over existing cell lines with regard to the possible release of replication-competent virus.

A defective signal peptide in the maize high-lysine mutant floury 2.

The maize floury 2 (fl2) mutation enhances the lysine content of the grain, but the soft texture of the endosperm makes it unsuitable for commercial production. The mutant phenotype is linked with the appearance of a 24-kDa alpha-zein protein and increased synthesis of binding protein, both of which are associated with irregularly shaped protein bodies. We have cloned the gene encoding the 24-kDa protein and show that it is expressed as a 22-kDa alpha-zein with an uncleaved signal peptide. Comparison of the deduced N-terminal amino acid sequence of the 24-kDa alpha-zein protein with other alpha-zeins revealed an alanine to valine substitution at the C-terminal position of the signal peptide, a histidine insertion within the seventh alpha-helical repeat, and an alanine to threonine substitution with the same alpha-helical repeat of the protein. Structural defects associated with this alpha-zein explain many of the phenotypic effects of the fl2 mutation.

Production of phenol-formaldehyde resin composites

The main objective of this work was to examlne the various stages of the production of industrial laminates based on phenol-formaldehyde resins, with a view of suggesting ways of improving the process economics and/or the physical properties of the final product. Aspects of impregnation, drying, and lamination were investigated. The resins used in all experiments were ammonia-catalysed. Work was concentrated on the lamination stage since this is a labour intensive activity. Paper-phenolic lay-ups were characterised in terms of the temperatures experienced during cure, and a shorter cure-cycle is proposed, utilising the exothermic heat produced during pressing of 25.5 mm thick lay-ups. Significant savings in production costs and improvements in some of the physical properties have been achieved. In particular, water absorption has been reduced by 43-61%. Work on the drying stage has shown that rapid heating of the wet impregnated substrate results in resin solids losses. Drying at lower temperatures by reducing the driving force leads to more resin (up to 6.5%) being retained by the prepregs and therefore more effective use of an expensive raw material. The impregnation work has indicated that residence times above 6 seconds in the varnish bath enhance the insulation resistance of the final product, possibly due to improved resin distribution and reduction in water absorption. In addition, a novel process which involves production of laminates by in situ polymerisation of the phenolic resin on the substrate has been examined. Such a process would eliminate the solvent recovery plant - a necessary stage in current industrial processes. In situ polymerisation has been shown to be chemically feasible.

Aspects of the design and operation of production control systems in manufacturing industry

Computerised production control developments have concentrated on Manufacturing Resources Planning (MRP II) systems. The literature suggests however, that despite the massive investment in hardware, software and management education, successful implementation of such systems in manufacturing industries has proved difficult. This thesis reviews the development of production planning and control systems, in particular, investigates the causes of failures in implementing MRP/MRP II systems in industrial environments and argues that the centralised and top-down planning structure, as well as the routine operational methodology of such systems, is inherently prone to failure. The thesis reviews the control benefits of cellular manufacturing systems but concludes that in more dynamic manufacturing environments, techniques such as Kanban are inappropriate. The basic shortcomings of MRP II systems are highlighted and a new enhanced operational methodology based on distributed planning and control principles is introduced. Distributed Manufacturing Resources Planning (DMRP), was developed as a capacity sensitive production planning and control solution for cellular manufacturing environments. The system utilises cell based, independently operated MRP II systems, integrated into a plant-wide control system through a Local Area Network. The potential benefits of adopting the system in industrial environments is discussed and the results of computer simulation experiments to compare the performance of the DMRP system against the conventional MRP II systems presented. DMRP methodology is shown to offer significant potential advantages which include ease of implementation, cost effectiveness, capacity sensitivity, shorter manufacturing lead times, lower working in progress levels and improved customer service.

Increases in renal ε-(γ-glutamyl)-lysine crosslinks result from compartment-specific changes in tissue transglutaminase in early experimental diabetic nephropathy:Pathologic implications

Diabetic nephropathy (DN) is characterized by an early, progressive expansion and sclerosis of the glomerular mesangium leading to glomerulosclerosis. This is associated with parallel fibrosis of the renal interstitium. In experimental renal scarring, the protein cross-linking enzyme, tissue transglutaminase (tTg), is up-regulated and externalized causing an increase in its crosslink product, e-(γ-glutamyl)-lysine, in the extracellular space. This potentially contributes to the extracellular matrix (ECM) accumulation central to tissue fibrosis by increasing deposition and inhibiting breakdown. We investigated if a similar mechanism may contribute to the ECM expansion characteristic of DN using the rat streptozotocin model over 120 days. Whole kidney e-(γ-glutamyl)-lysine (HPLC analysis) was significantly increased from Day 90 (+337%) and peaked at Day 120 (+650%) (p <0.05). Immunofluorescence showed this increase to be predominantly extracellular in the peritubular interstitial space, but also in individual glomeruli. Total kidney transglutaminase (Tg) was not elevated. However, using a Tg in situ activity assay, increased Tg was detected in both the extracellular interstitial space and glomeruli by Day 60, with a maximal 53% increase at Day 120 (p <0.05). Using a specific anti-tTg antibody, immunohistochemistry showed a similar increase in extracellular enzyme in the interstitium and glomeruli. To biochemically characterize glomerular changes, glomeruli were isolated by selective sieving. In line with whole kidney measurement, there was an increase in glomerular e-(γ-glutamyl) lysine (+ 361%); however, in the glomeruli this was associated with increases in Tg activity (+228%) and tTg antigen by Western blotting (+215%). Importantly, the ratio of glomerular e-(γ-glutamyl) lysine to hydroxyproline increased by 2.2-fold. In DN, changes in the kidney result in increased translocation of tTg to the extracellular environment where high Ca2+ and low GTP levels allow its activation. In the tubulointerstitium this is independent of increased tTg production, but dependent in the glomerulus. This leads to excessive ECM cross-linking, contributing to the renal fibrosis characteristic of progressive DN.

Editorial overview:new protein production tools for structural biology

Full text: The idea of producing proteins from recombinant DNA hatched almost half a century ago. In his PhD thesis, Peter Lobban foresaw the prospect of inserting foreign DNA (from any source, including mammalian cells) into the genome of a λ phage in order to detect and recover protein products from Escherichia coli [ 1 and 2]. Only a few years later, in 1977, Herbert Boyer and his colleagues succeeded in the first ever expression of a peptide-coding gene in E. coli — they produced recombinant somatostatin [ 3] followed shortly after by human insulin. The field has advanced enormously since those early days and today recombinant proteins have become indispensable in advancing research and development in all fields of the life sciences. Structural biology, in particular, has benefitted tremendously from recombinant protein biotechnology, and an overwhelming proportion of the entries in the Protein Data Bank (PDB) are based on heterologously expressed proteins. Nonetheless, synthesizing, purifying and stabilizing recombinant proteins can still be thoroughly challenging. For example, the soluble proteome is organized to a large part into multicomponent complexes (in humans often comprising ten or more subunits), posing critical challenges for recombinant production. A third of all proteins in cells are located in the membrane, and pose special challenges that require a more bespoke approach. Recent advances may now mean that even these most recalcitrant of proteins could become tenable structural biology targets on a more routine basis. In this special issue, we examine progress in key areas that suggests this is indeed the case. Our first contribution examines the importance of understanding quality control in the host cell during recombinant protein production, and pays particular attention to the synthesis of recombinant membrane proteins. A major challenge faced by any host cell factory is the balance it must strike between its own requirements for growth and the fact that its cellular machinery has essentially been hijacked by an expression construct. In this context, Bill and von der Haar examine emerging insights into the role of the dependent pathways of translation and protein folding in defining high-yielding recombinant membrane protein production experiments for the common prokaryotic and eukaryotic expression hosts. Rather than acting as isolated entities, many membrane proteins form complexes to carry out their functions. To understand their biological mechanisms, it is essential to study the molecular structure of the intact membrane protein assemblies. Recombinant production of membrane protein complexes is still a formidable, at times insurmountable, challenge. In these cases, extraction from natural sources is the only option to prepare samples for structural and functional studies. Zorman and co-workers, in our second contribution, provide an overview of recent advances in the production of multi-subunit membrane protein complexes and highlight recent achievements in membrane protein structural research brought about by state-of-the-art near-atomic resolution cryo-electron microscopy techniques. E. coli has been the dominant host cell for recombinant protein production. Nonetheless, eukaryotic expression systems, including yeasts, insect cells and mammalian cells, are increasingly gaining prominence in the field. The yeast species Pichia pastoris, is a well-established recombinant expression system for a number of applications, including the production of a range of different membrane proteins. Byrne reviews high-resolution structures that have been determined using this methylotroph as an expression host. Although it is not yet clear why P. pastoris is suited to producing such a wide range of membrane proteins, its ease of use and the availability of diverse tools that can be readily implemented in standard bioscience laboratories mean that it is likely to become an increasingly popular option in structural biology pipelines. The contribution by Columbus concludes the membrane protein section of this volume. In her overview of post-expression strategies, Columbus surveys the four most common biochemical approaches for the structural investigation of membrane proteins. Limited proteolysis has successfully aided structure determination of membrane proteins in many cases. Deglycosylation of membrane proteins following production and purification analysis has also facilitated membrane protein structure analysis. Moreover, chemical modifications, such as lysine methylation and cysteine alkylation, have proven their worth to facilitate crystallization of membrane proteins, as well as NMR investigations of membrane protein conformational sampling. Together these approaches have greatly facilitated the structure determination of more than 40 membrane proteins to date. It may be an advantage to produce a target protein in mammalian cells, especially if authentic post-translational modifications such as glycosylation are required for proper activity. Chinese Hamster Ovary (CHO) cells and Human Embryonic Kidney (HEK) 293 cell lines have emerged as excellent hosts for heterologous production. The generation of stable cell-lines is often an aspiration for synthesizing proteins expressed in mammalian cells, in particular if high volumetric yields are to be achieved. In his report, Buessow surveys recent structures of proteins produced using stable mammalian cells and summarizes both well-established and novel approaches to facilitate stable cell-line generation for structural biology applications. The ambition of many biologists is to observe a protein's structure in the native environment of the cell itself. Until recently, this seemed to be more of a dream than a reality. Advances in nuclear magnetic resonance (NMR) spectroscopy techniques, however, have now made possible the observation of mechanistic events at the molecular level of protein structure. Smith and colleagues, in an exciting contribution, review emerging ‘in-cell NMR’ techniques that demonstrate the potential to monitor biological activities by NMR in real time in native physiological environments. A current drawback of NMR as a structure determination tool derives from size limitations of the molecule under investigation and the structures of large proteins and their complexes are therefore typically intractable by NMR. A solution to this challenge is the use of selective isotope labeling of the target protein, which results in a marked reduction of the complexity of NMR spectra and allows dynamic processes even in very large proteins and even ribosomes to be investigated. Kerfah and co-workers introduce methyl-specific isotopic labeling as a molecular tool-box, and review its applications to the solution NMR analysis of large proteins. Tyagi and Lemke next examine single-molecule FRET and crosslinking following the co-translational incorporation of non-canonical amino acids (ncAAs); the goal here is to move beyond static snap-shots of proteins and their complexes and to observe them as dynamic entities. The encoding of ncAAs through codon-suppression technology allows biomolecules to be investigated with diverse structural biology methods. In their article, Tyagi and Lemke discuss these approaches and speculate on the design of improved host organisms for ‘integrative structural biology research’. Our volume concludes with two contributions that resolve particular bottlenecks in the protein structure determination pipeline. The contribution by Crepin and co-workers introduces the concept of polyproteins in contemporary structural biology. Polyproteins are widespread in nature. They represent long polypeptide chains in which individual smaller proteins with different biological function are covalently linked together. Highly specific proteases then tailor the polyprotein into its constituent proteins. Many viruses use polyproteins as a means of organizing their proteome. The concept of polyproteins has now been exploited successfully to produce hitherto inaccessible recombinant protein complexes. For instance, by means of a self-processing synthetic polyprotein, the influenza polymerase, a high-value drug target that had remained elusive for decades, has been produced, and its high-resolution structure determined. In the contribution by Desmyter and co-workers, a further, often imposing, bottleneck in high-resolution protein structure determination is addressed: The requirement to form stable three-dimensional crystal lattices that diffract incident X-ray radiation to high resolution. Nanobodies have proven to be uniquely useful as crystallization chaperones, to coax challenging targets into suitable crystal lattices. Desmyter and co-workers review the generation of nanobodies by immunization, and highlight the application of this powerful technology to the crystallography of important protein specimens including G protein-coupled receptors (GPCRs). Recombinant protein production has come a long way since Peter Lobban's hypothesis in the late 1960s, with recombinant proteins now a dominant force in structural biology. The contributions in this volume showcase an impressive array of inventive approaches that are being developed and implemented, ever increasing the scope of recombinant technology to facilitate the determination of elusive protein structures. Powerful new methods from synthetic biology are further accelerating progress. Structure determination is now reaching into the living cell with the ultimate goal of observing functional molecular architectures in action in their native physiological environment. We anticipate that even the most challenging protein assemblies will be tackled by recombinant technology in the near future.

(Table 2) Copepod carbon production in the upper 50 m in Godthabsfjord and Fyllas Banke, West Greenland

This study describes differences in plankton community structure and in chemical and physical gradients between the offshore West Greenland Current system and inland regions close to the Greenland Ice Sheet during the post-bloom in Godthabsfjorden (64° N, 51° W). The offshore region had pronounced vertical mixing, with centric diatoms and Phaeocystis spp. dominating the phytoplankton, chlorophyll (chl) a (0.3 to 3.9 µg/l) was evenly distributed and nutrients were depleted in the upper 50 m. Ciliates and heterotrophic dinoflagellates constituted equal parts of the protozooplankton biomass. Copepod biomass was dominated by Calanus spp. Primary production, copepod production and the vertical flux were high offshore. The water column was stratified in the fjord, causing chl a to be concentrated in a thin sub-surface layer. Nutrients were depleted above the pycnocline, and Thalassiosira spp. dominated the phytoplankton assemblage close to the ice sheet. Dinoflagellates dominated the protozooplankton biomass, whereas copepod biomass was low and was dominated by Pseudocalanus spp. and Metridia longa. Primary production was low in the outer part of the fjord but considerably higher in the inner parts of the fjord. Copepod production was exceeded by protozooplankton production in the fjord. The results of both physical/chemical factors and biological parameters suggest separation of offshore and fjord systems.

Exergetic, energetic, economic and environmental evaluation of concentrated solar power plants in Libya

The PhD project addresses the potential of using concentrating solar power (CSP) plants as a viable alternative energy producing system in Libya. Exergetic, energetic, economic and environmental analyses are carried out for a particular type of CSP plants. The study, although it aims a particular type of CSP plant – 50 MW parabolic trough-CSP plant, it is sufficiently general to be applied to other configurations. The novelty of the study, in addition to modeling and analyzing the selected configuration, lies in the use of a state-of-the-art exergetic analysis combined with the Life Cycle Assessment (LCA). The modeling and simulation of the plant is carried out in chapter three and they are conducted into two parts, namely: power cycle and solar field. The computer model developed for the analysis of the plant is based on algebraic equations describing the power cycle and the solar field. The model was solved using the Engineering Equation Solver (EES) software; and is designed to define the properties at each state point of the plant and then, sequentially, to determine energy, efficiency and irreversibility for each component. The developed model has the potential of using in the preliminary design of CSPs and, in particular, for the configuration of the solar field based on existing commercial plants. Moreover, it has the ability of analyzing the energetic, economic and environmental feasibility of using CSPs in different regions of the world, which is illustrated for the Libyan region in this study. The overall feasibility scenario is completed through an hourly analysis on an annual basis in chapter Four. This analysis allows the comparison of different systems and, eventually, a particular selection, and it includes both the economic and energetic components using the “greenius” software. The analysis also examined the impact of project financing and incentives on the cost of energy. The main technological finding of this analysis is higher performance and lower levelized cost of electricity (LCE) for Libya as compared to Southern Europe (Spain). Therefore, Libya has the potential of becoming attractive for the establishment of CSPs in its territory and, in this way, to facilitate the target of several European initiatives that aim to import electricity generated by renewable sources from North African and Middle East countries. The analysis is presented a brief review of the current cost of energy and the potential of reducing the cost from parabolic trough- CSP plant. Exergetic and environmental life cycle assessment analyses are conducted for the selected plant in chapter Five; the objectives are 1) to assess the environmental impact and cost, in terms of exergy of the life cycle of the plant; 2) to find out the points of weakness in terms of irreversibility of the process; and 3) to verify whether solar power plants can reduce environmental impact and the cost of electricity generation by comparing them with fossil fuel plants, in particular, Natural Gas Combined Cycle (NGCC) plant and oil thermal power plant. The analysis also targets a thermoeconomic analysis using the specific exergy costing (SPECO) method to evaluate the level of the cost caused by exergy destruction. The main technological findings are that the most important contribution impact lies with the solar field, which reports a value of 79%; and the materials with the vi highest impact are: steel (47%), molten salt (25%) and synthetic oil (21%). The “Human Health” damage category presents the highest impact (69%) followed by the “Resource” damage category (24%). In addition, the highest exergy demand is linked to the steel (47%); and there is a considerable exergetic demand related to the molten salt and synthetic oil with values of 25% and 19%, respectively. Finally, in the comparison with fossil fuel power plants (NGCC and Oil), the CSP plant presents the lowest environmental impact, while the worst environmental performance is reported to the oil power plant followed by NGCC plant. The solar field presents the largest value of cost rate, where the boiler is a component with the highest cost rate among the power cycle components. The thermal storage allows the CSP plants to overcome solar irradiation transients, to respond to electricity demand independent of weather conditions, and to extend electricity production beyond the availability of daylight. Numerical analysis of the thermal transient response of a thermocline storage tank is carried out for the charging phase. The system of equations describing the numerical model is solved by using time-implicit and space-backward finite differences and which encoded within the Matlab environment. The analysis presented the following findings: the predictions agree well with the experiments for the time evolution of the thermocline region, particularly for the regions away from the top-inlet. The deviations observed in the near-region of the inlet are most likely due to the high-level of turbulence in this region due to the localized level of mixing resulting; a simple analytical model to take into consideration this increased turbulence level was developed and it leads to some improvement of the predictions; this approach requires practically no additional computational effort and it relates the effective thermal diffusivity to the mean effective velocity of the fluid at each particular height of the system. Altogether the study indicates that the selected parabolic trough-CSP plant has the edge over alternative competing technologies for locations where DNI is high and where land usage is not an issue, such as the shoreline of Libya.

Environmental and genetic effects on production traits of ewes originally from distinct environments.

Wool production and reproductive performance components of similar genotypes, brought from distinct production areas, were evaluated during five years trial at similar environments, such as, joining season and stocking rate on winter improved pasture. The least squares means revealed that the origin (breed) effect concentrated upon the Corriedale ewes wool production, whereas in Romney females it affected the reproductive performance.