"A Sequence in Subdomain 2 of DBL1a of Plasmodium falciparum Erythrocyte Membrane Protein 1 Induces Strain Transcending Antibodies"

Immunity to severe malaria is the first level of immunity acquired to Plasmodium falciparum. Antibodies to the variant antigen PfEMP1 (P. falciparum erythrocyte membrane protein 1) present at the surface of the parasitized red blood cell (pRBC) confer protection by blocking microvascular sequestration. Here we have generated antibodies to peptide sequences of subdomain 2 of PfEMP1-DBL1a previously identified to be associated with severe or mild malaria. A set of sera generated to the amino acid sequence KLQTLTLHQVREYWWALNRKEVWKA, containing the motif ALNRKE, stained the live pRBC. 50% of parasites tested (7/14) were positive both in flow cytometry and immunofluorescence assays with live pRBCs including both laboratory strains and in vitro adapted clinical isolates. Antibodies that reacted selectively with the sequence REYWWALNRKEVWKA in a 15-mer peptide array of DBL1a-domains were also found to react with the pRBC surface. By utilizing a peptide array to map the binding properties of the elicited anti-DBL1a antibodies, the amino acids WxxNRx were found essential for antibody binding. Complementary experiments using 135 degenerate RDSM peptide sequences obtained from 93 Ugandan patient-isolates showed that antibody binding occurred when the amino acids WxLNRKE/D were present in the peptide. The data suggests that the ALNRKE sequence motif, associated with severe malaria, induces strain-transcending antibodies that react with the pRBC surface

A sequence in subdomain 2 of DBL1 alpha of plasmodium falciparum erythrocyte membrane protein 1 induces strain transcending antibodies

Immunity to severe malaria is the first level of immunity acquired to Plasmodium falciparum. Antibodies to the variant antigen PfEMP1 (P. falciparum erythrocyte membrane protein 1) present at the surface of the parasitized red blood cell (pRBC) confer protection by blocking microvascular sequestration. Here we have generated antibodies to peptide sequences of subdomain 2 of PfEMP1-DBL1 alpha previously identified to be associated with severe or mild malaria. A set of sera generated to the amino acid sequence KLQTLTLHQVREYWWALNRKEVWKA, containing the motif ALNRKE, stained the live pRBC. 50% of parasites tested (7/14) were positive both in flow cytometry and immunofluorescence assays with live pRBCs including both laboratory strains and in vitro adapted clinical isolates. Antibodies that reacted selectively with the sequence REYWWALNRKEVWKA in a 15-mer peptide array of DBL1 alpha-domains were also found to react with the pRBC surface. By utilizing a peptide array to map the binding properties of the elicited anti-DBL1 alpha antibodies, the amino acids WxxNRx were found essential for antibody binding. Complementary experiments using 135 degenerate RDSM peptide sequences obtained from 93 Ugandan patient-isolates showed that antibody binding occurred when the amino acids WxLNRKE/D were present in the peptide. The data suggests that the ALNRKE sequence motif, associated with severe malaria, induces strain-transcending antibodies that react with the pRBC surface.

Polimorfismos del gen Butirilcolinesterasa responsables de reacciones adversas en pacientes consumidores de “cocaína”

La butirilcolinesterasa humana (BChE; EC 3.1.1.8) es una enzima polimórfica sintetizada en el hígado y en el tejido adiposo, ampliamente distribuida en el organismo y encargada de hidrolizar algunos ésteres de colina como la procaína, ésteres alifáticos como el ácido acetilsalicílico, fármacos como la metilprednisolona, el mivacurium y la succinilcolina y drogas de uso y/o abuso como la heroína y la cocaína. Es codificada por el gen BCHE (OMIM 177400), habiéndose identificado más de 100 variantes, algunas no estudiadas plenamente, además de la forma más frecuente, llamada usual o silvestre. Diferentes polimorfismos del gen BCHE se han relacionado con la síntesis de enzimas con niveles variados de actividad catalítica. Las bases moleculares de algunas de esas variantes genéticas han sido reportadas, entre las que se encuentra las variantes Atípica (A), fluoruro-resistente del tipo 1 y 2 (F-1 y F-2), silente (S), Kalow (K), James (J) y Hammersmith (H). En este estudio, en un grupo de pacientes se aplicó el instrumento validado Lifetime Severity Index for Cocaine Use Disorder (LSI-C) para evaluar la gravedad del consumo de “cocaína” a lo largo de la vida. Además, se determinaron Polimorfismos de Nucleótido Simple (SNPs) en el gen BCHE conocidos como responsables de reacciones adversas en pacientes consumidores de “cocaína” mediante secuenciación del gen y se predijo el efecto delos SNPs sobre la función y la estructura de la proteína, mediante el uso de herramientas bio-informáticas. El instrumento LSI-C ofreció resultados en cuatro dimensiones: consumo a lo largo de la vida, consumo reciente, dependencia psicológica e intento de abandono del consumo. Los estudios de análisis molecular permitieron observar dos SNPs codificantes (cSNPs) no sinónimos en el 27.3% de la muestra, c.293A>G (p.Asp98Gly) y c.1699G>A (p.Ala567Thr), localizados en los exones 2 y 4, que corresponden, desde el punto de vista funcional, a la variante Atípica (A) [dbSNP: rs1799807] y a la variante Kalow (K) [dbSNP: rs1803274] de la enzima BChE, respectivamente. Los estudios de predicción In silico establecieron para el SNP p.Asp98Gly un carácter patogénico, mientras que para el SNP p.Ala567Thr, mostraron un comportamiento neutro. El análisis de los resultados permite proponer la existencia de una relación entre polimorfismos o variantes genéticas responsables de una baja actividad catalítica y/o baja concentración plasmática de la enzima BChE y algunas de las reacciones adversas ocurridas en pacientes consumidores de cocaína.

Producció i caracterització de variants de la ribonucleasa pancreàtica humana dissenyades per a adquirir propietats citotòxiques

Amb la finalitat d'aprofundir en les bases moleculars de la citotoxicitat de les ribonucleases pancreàtiques, es van construir variants derivades de l'HP-RNasa seguint dues estratègies. En la primera, es van generar variants de l'enzim resistents a l'acció de l'inhibidor proteic de les ribonucleases (hRI), substituint residus implicats en la interfície de contacte entre la ribonucleasa i l'hRI. En la segona, es va addicionar el motiu RGD en regions de superfície de la proteïna implicades en la formació del complex amb l'hRI, a fi de promoure la seva interacció amb la membrana plasmàtica de les cèl·lules i a la vegada disminuir l'afinitat de les variants per l'hRI. Es va comprovar que només les variants portadores de substitucions múltiples adquirien la capacitat de resistència a l'hRI. L'estudi del percentatge d'inhibició de la síntesi proteica en cèl·lules incubades amb cadascuna de les variants va mostrar que només dues de les variants construïdes havien adquirit propietats citotòxiques. La citotoxicitat més elevada la va presentar una variant que no era resistent a l'hRI, amb valors que eren només entre 5 i 15 vegades inferiors als de l'onconasa. Aquest resultat demostrà que la sensibilitat a l'hRI no és necessàriament un paràmetre limitant per a la citotoxicitat de les ribonucleases. Cap de les variants que incorporava un motiu RGD presentà citotoxicitat, evidenciant que aquest motiu no és efectiu a fi de dotar les ribonucleases pancreàtiques de propietats citotòxiques. Es van estudiar les bases moleculars de la citotoxicitat de la variant més citotòxica. En primer lloc, l'anàlisi de la internalització per marcatge radioactiu d'aquesta variant en relació amb l'onconasa i amb altres variants de l'HP-RNasa no citotòxiques, va posar en evidència que només l'onconasa era internalitzada eficientment. Es descartava així la possibilitat que l'acció citotòxica de l'enzim estudiat fos conseqüència d'una major eficiència d'endocitosi. També es va comprovar que l'addició del motiu RGD no era capaç de promoure la internalització de les proteïnes amb més eficàcia. Per microscòpia confocal de fluorescència, les variants humanes només es van començar a detectar a l'interior de la cèl·lula a partir de les 24 h d'incubació. Totes les variants generades van presentar una eficiència catalítica superior al 50 % de l'activitat de la seva proteïna parental, PM5, indicant que probablement l'estructura del centre actiu no havia estat afectada de manera dràstica per les substitucions introduïdes. No obstant, en tots els casos es va produir una disminució en la termoestabilitat respecte a PM5. Aquest resultat indicà que la correlació descrita a la bibliografia entre l'increment de termoestabilitat i l'increment de citotoxicitat per les ribonucleases no sempre es compleix. Per microscòpia confocal es va comprovar que tant la proteïna més citotòxica, com una variant no citotòxica resistent a l'hRI, així com la proteïna parental, seguien la via de degradació lisosomal. Aquesta ruta de trànsit no va ser afectada per l'addició de drogues que alteren les vies de trànsit retrògrad (monensina i brefeldina A), però sí per l'addició de la bafilomicina A1, una droga que neutralitza el pH endosomal i que va actuar alentint el trànsit de les proteïnes als lisosomes. D'acord amb aquests resultats, els valors de citotoxicitat de les variants es van incrementar de manera significativa només en presència de bafilomicina A1, suggerint que les ribonucleases transloquen al citoplasma a partir d'algun punt de la via de trànsit endosomal. Es va comprovar que l'acció de la variant més citotòxica era deguda a que l'addició d'un segon motiu de tres Arg en PE5 dota a aquesta proteïna amb un senyal de transport nuclear. La fracció d'enzim que aconsegueix translocar al citoplasma a partir d'algun punt de la via endosomal previ als lisosomes, és conduït ràpidament al nucli de la cèl·lula per mitjà del mecanisme clàssic de transport actiu. Per la seva afinitat amb l'rRNA, l'enzim es concentra en el nuclèol, on probablement duu a terme la seva activitat catalítica. La interacció d'aquesta variant amb els receptors nucleocitoplasmàtics, les importines, impediria per altra banda el bloqueig de l'enzim per part de l'hRI. Els resultats obtinguts presenten una nova estratègia de disseny de ribonucleases citotòxiques, basada en l'addició de segments NLS a fi de promoure el transport nuclear dels enzims. Aquesta estratègia podria permetre superar limitacions que fins al moment han estat descrites com a limitants de la citotoxicitat de les ribonucleases pancreàtiques, com la sensibilitat a l'hRI o la baixa eficiència d'internalització.

Nitrifying and denitrifying bacterial communities in the sediment and rhizosphere of a free water surface constructed wetland

La contínua descàrrega de nutrients, sobretot fosfats i nitrogen, és la major causa d'eutrofització dels ecosistemes aquàtics. Els sistemes de tractament basats en aiguamolls construïts s'han emprat per reduir ells nivells de nitrogen a l'aigua com a alternativa de baix cost als mètodes de depuració convencionals. L'eliminació del nitrogen a aquests sistemes depèn en bona part de la vegetació, i l'alternança de condicions aeròbiques i anaeròbiques per promoure els processos de nitrificació i desnitrificació. En aquest treball hem volgut investigar les activitats microbianes de nitrificació i desnitrificació en relació a dues espècies de plantes macròfites en un sistema d'aiguamolls de tractament de flux superficial (FS-SAC), dissenyat per minimitzar l'impacte de l'alliberament d'aigua carregada de nutrients a la reserva natural dels Aiguamolls de l'Empordà (Girona, Espanya).

Modeled and observed atmospheric radiation balance during the West African dry season: role of mineral dust, biomass burning aerosol, and surface albedo

The global radiation balance of the atmosphere is still poorly observed, particularly at the surface. We investigate the observed radiation balance at (1) the surface using the ARM Mobile Facility in Niamey, Niger, and (2) the top of the atmosphere (TOA) over West Africa using data from the Geostationary Earth Radiation Budget (GERB) instrument on board Meteosat-8. Observed radiative fluxes are compared with predictions from the global numerical weather prediction (NWP) version of the Met Office Unified Model (MetUM). The evaluation points to major shortcomings in the NWP model's radiative fluxes during the dry season (December 2005 to April 2006) arising from (1) a lack of absorbing aerosol in the model (mineral dust and biomass burning aerosol) and (2) a poor specification of the surface albedo. A case study of the major Saharan dust outbreak of 6–12 March 2006 is used to evaluate a parameterization of mineral dust for use in the NWP models. The model shows good predictability of the large-scale flow out to 4–5 days with the dust parameterization providing reasonable dust uplift, spatial distribution, and temporal evolution for this strongly forced dust event. The direct radiative impact of the dust reduces net downward shortwave (SW) flux at the surface (TOA) by a maximum of 200 W m−2 (150 W m−2), with a SW heating of the atmospheric column. The impacts of dust on terrestrial radiation are smaller. Comparisons of TOA (surface) radiation balance with GERB (ARM) show the “dusty” forecasts reduce biases in the radiative fluxes and improve surface temperatures and vertical thermodynamic structure.

New insights on growth mechanisms of protein clusters at surfaces: an AFM and simulation study

Despite its relevance to a wide range of technological and fundamental areas, a quantitative understanding of protein surface clustering dynamics is often lacking. In inorganic crystal growth, surface clustering of adatoms is well described by diffusion-aggregation models. In such models, the statistical properties of the aggregate arrays often reveal the molecular scale aggregation processes. We investigate the potential of these theories to reveal hitherto hidden facets of protein clustering by carrying out concomitant observations of lysozyme adsorption onto mica surfaces, using atomic force microscopy. and Monte Carlo simulations of cluster nucleation and growth. We find that lysozyme clusters diffuse across the substrate at a rate that varies inversely with size. This result suggests which molecular scale mechanisms are responsible for the mobility of the proteins on the substrate. In addition the surface diffusion coefficient of the monomer can also be extracted from the comparison between experiments and simulations. While concentrating on a model system of lysozyme-on-mica, this 'proof of concept' study successfully demonstrates the potential of our approach to understand and influence more biomedically applicable protein-substrate couples.

Layer-by-layer electrostatic entrapment of protein molecules on superparamagnetic nanoparticle: new strategy to enhance adsorption capacity and maintain biological activity

There is a recent interest to use inorganic-based magnetic nanoparticles as a vehicle to carry biomolecules for various biophysical applications, but direct attachment of the molecules is known to alter their conformation leading to attenuation in activity. In addition, surface immobilization has been limited to monolayer coverage. It is shown that alternate depositions of negatively charged protein molecules, typically bovine serum albumin (BSA) with a positively charged aminocarbohydrate template such as glycol chitosan (GC) on magnetic iron oxide nanoparticle surface as a colloid, are carried out under pH 7.4. Circular dichroism (CD) clearly reveals that the secondary structure of the entrapped BSA sequential depositions in this manner remains totally unaltered which is in sharp contrast to previous attempts. Probing the binding properties of the entrapped BSA using small molecules (Site I and Site II drug compounds) confirms for the first time the full retention of its biological activity as compared with native BSA, which also implies the ready accessibility of the entrapped protein molecules through the porous overlayers. This work clearly suggests a new method to immobilize and store protein molecules beyond monolayer adsorption on a magnetic nanoparticle surface without much structural alteration. This may find applications in magnetic recoverable enzymes or protein delivery.

Ex Vivo Construction of an Artificial Ocular Surface by Combination of Corneal Limbal Epithelial Cells and a Compressed Collagen Scaffold Containing Keratocytes

We have investigated the use of a laminin coated compressed collagen gel containing corneal fibroblasts (keratocytes) as a novel scaffold to support the growth of corneal limbal epithelial stem cells. The growth of limbal epithelial cells was compared between compressed collagen gel and a clinically proven conventional substrate, denuded amniotic membrane. Following compression of the collagen gel, encapsulated keratocytes remained viable and scanning electron microscopy showed that fibres within the compressed gel were dense, homogeneous and similar in structure to those within denuded amniotic membrane. Limbal epithelial cells were successfully expanded upon the compressed collagen resulting in stratified layers of cells containing desmosome and hemidesmosome structures. The resulting corneal constructs of both the groups shared a high degree of transparency, cell morphology and cell stratification. Similar protein expression profiles for cytokeratin 3 and cytokeratin 14 and no significant difference in cytokeratin 12 mRNA expression levels by real time PCR were also observed. This study provides the first line of evidence that a laminin coated compressed collagen gel containing keratocytes can adequately support limbal epithelial cell expansion, stratification and differentiation to a degree that is comparable to the leading conventional scaffold, denuded amniotic membrane.

Land use for England and Wales: evaluation of management options to support 'good ecological status' in surface freshwaters

This paper analyses historic records of agricultural land use and management for England and Wales from 1931 and 1991 and uses export coefficient modelling to hindcast the impact of these practices on the rates of diffuse nitrogen (N) and phosphorus (P) export to water bodies for each of the major geo-climatic regions of England and Wales. Key trends indicate the importance of animal agriculture as a contributor to the total diffuse agricultural nutrient loading on waters, and the need to bring these sources under control if conditions suitable for sustaining 'Good Ecological Status' under the Water Framework Directive are to be generated. The analysis highlights the importance of measuring changes in nutrient loading in relation to the catchment-specific baseline state for different water bodies. The approach is also used to forecast the likely impact of broad regional scale scenarios on nutrient export to waters and highlights the need to take sensitive land out of production, introduce ceilings on fertilizer use and stocking densities, and controls on agricultural practice in higher risk areas where intensive agriculture is combined with a low intrinsic nutrient retention capacity, although the uncertainties associated with the modelling applied at this scale should be taken into account in the interpretation of model output. The paper advocates the need for a two-tiered approach to nutrient management, combining broad regional policies with targeted management in high risk areas at the catchment and farm scale.

Modelling nitrogen fluxes from the land surface to the coastal zone in European systems: the perspective of the INCA project

This paper describes the results and conclusions of the INCA (Integrated Nitrogen Model for European CAtchments) project and sets the findings in the context of the ELOISE (European Land-Ocean Interaction Studies) programme. The INCA project was concerned with the development of a generic model of the major factors and processes controlling nitrogen dynamics in European river systems, thereby providing a tool (a) to aid the scientific understanding of nitrogen transport and retention in catchments and (b) for river-basin management and policy-making. The findings of the study highlight the heterogeneity of the factors and processes controlling nitrogen dynamics in freshwater systems. Nonetheless, the INCA model was able to simulate the in-stream nitrogen concentrations and fluxes observed at annual and seasonal timescales in Arctic, Continental and Maritime-Temperate regimes. This result suggests that the data requirements and structural complexity of the INCA model are appropriate to simulate nitrogen fluxes across a wide range of European freshwater environments. This is a major requirement for the production of coupled fiver-estuary-coastal shelf models for the management of our aquatic environment. With regard to river-basin management, to achieve an efficient reduction in nutrient fluxes from the land to the estuarine and coastal zone, the model simulations suggest that management options must be adaptable to the prevailing environmental and socio-economic factors in individual catchments: 'Blanket approaches' to environmental policy appear too simple. (c) 2004 Elsevier B.V. All rights reserved.

Climate impacts of recent multidecadal changes in Atlantic Ocean Sea Surface Temperature: A multimodel comparison

During the twentieth century sea surface temperatures in the Atlantic Ocean exhibited prominent multidecadal variations. The source of such variations has yet to be rigorously established—but the question of their impact on climate can be investigated. Here we report on a set of multimodel experiments to examine the impact of patterns of warming in the North Atlantic, and cooling in the South Atlantic, derived from observations, that is characteristic of the positive phase of the Atlantic Multidecadal Oscillation (AMO). The experiments were carried out with six atmospheric General Circulation Models (including two versions of one model), and a major goal was to assess the extent to which key climate impacts are consistent between the different models. The major climate impacts are found over North and South America, with the strongest impacts over land found over the United States and northern parts of South America. These responses appear to be driven by a combination of an off-equatorial Gill response to diabatic heating over the Caribbean due to increased rainfall within the region and a Northward shift in the Inter Tropical Convergence Zone (ITCZ) due to the anomalous cross-equatorial SST gradient. The majority of the models show warmer US land temperatures and reduced Mean Sea Level Pressure during summer (JJA) in response to a warmer North Atlantic and a cooler South Atlantic, in line with observations. However the majority of models show no significant impact on US rainfall during summer. Over northern South America, all models show reduced rainfall in southern hemisphere winter (JJA), whilst in Summer (DJF) there is a generally an increase in rainfall. However, there is a large spread amongst the models in the magnitude of the rainfall anomalies over land. Away from the Americas, there are no consistent significant modelled responses. In particular there are no significant changes in the North Atlantic Oscillation (NAO) over the North Atlantic and Europe in Winter (DJF). Additionally, the observed Sahel drying signal in African rainfall is not seen in the modelled responses. Suggesting that, in contrast to some studies, the Atlantic Multidecadal Oscillation was not the primary driver of recent reductions in Sahel rainfall.

Solar modulation in surface atmospheric electricity

The solar wind modulates the flux of galactic cosmic rays impinging on Earth inversely with solar activity. Cosmic ray ionisation is the major source of air’s electrical conductivity over the oceans and well above the continents. Differential solar modulation of the cosmic ray energy spectrum modifies the cosmic ray ionisation at different latitudes,varying the total atmospheric columnar conductance. This redistributes current flow in the global atmospheric electrical circuit, including the local vertical current density and the related surface potential gradient. Surface vertical current density and potential gradient measurements made independently at Lerwick Observatory,Shetland,from 1978 to 1985 are compared with modelled changes in cosmic ray ionisation arising from solar activity changes. Both the lower troposphere atmospheric electricity quantities are significantly increased at cosmic ray maximum(solar minimum),with a proportional change greater than that of the cosmic ray change.

Recent changes in solar outputs and the global mean surface temperature. III. Analysis of contributions to global mean air surface temperature rise

A multivariate fit to the variation in global mean surface air temperature anomaly over the past half century is presented. The fit procedure allows for the effect of response time on the waveform, amplitude and lag of each radiative forcing input, and each is allowed to have its own time constant. It is shown that the contribution of solar variability to the temperature trend since 1987 is small and downward; the best estimate is -1.3% and the 2sigma confidence level sets the uncertainty range of -0.7 to -1.9%. The result is the same if one quantifies the solar variation using galactic cosmic ray fluxes (for which the analysis can be extended back to 1953) or the most accurate total solar irradiance data composite. The rise in the global mean air surface temperatures is predominantly associated with a linear increase that represents the combined effects of changes in anthropogenic well-mixed greenhouse gases and aerosols, although, in recent decades, there is also a considerable contribution by a relative lack of major volcanic eruptions. The best estimate is that the anthropogenic factors contribute 75% of the rise since 1987, with an uncertainty range (set by the 2sigma confidence level using an AR(1) noise model) of 49–160%; thus, the uncertainty is large, but we can state that at least half of the temperature trend comes from the linear term and that this term could explain the entire rise. The results are consistent with the intergovernmental panel on climate change (IPCC) estimates of the changes in radiative forcing (given for 1961–1995) and are here combined with those estimates to find the response times, equilibrium climate sensitivities and pertinent heat capacities (i.e. the depth into the oceans to which a given radiative forcing variation penetrates) of the quasi-periodic (decadal-scale) input forcing variations. As shown by previous studies, the decadal-scale variations do not penetrate as deeply into the oceans as the longer term drifts and have shorter response times. Hence, conclusions about the response to century-scale forcing changes (and hence the associated equilibrium climate sensitivity and the temperature rise commitment) cannot be made from studies of the response to shorter period forcing changes.