Biomolecular interaction analysis of gestrinone-anti-gestrinone using arrays of high aspect ratio SU-8 nanopillars

In this paper, label-free biosensing for antibody screening by periodic lattices of high-aspect ratio SU-8 nano-pillars (BICELLs) is presented. As a demonstration, the determination of anti-gestrinone antibodies from whole rabbit serum is carried out, and for the first time, the dissociation constant (KD = 6 nM) of antigen-antibody recognition process is calculated using this sensing system. After gestrinone antigen immobilization on the BICELLs, the immunorecognition was performed. The cells were interrogated vertically by using micron spot size Fourier transform visible and IR spectrometry (FT-VIS-IR), and the dip wavenumber shift was monitored. The biosensing assay exhibited good reproducibility and sensitivity (LOD = 0.75 ng/mL).

Light sheet fluorescence microscopy for in situ cell interaction analysis in mouse lymph nodes.

Reactive lymph nodes (LNs) are sites where pMHC-loaded dendritic cells (DCs) interact with rare cognate T cells, leading to their clonal expansion. While DC interactions with T cell subsets critically shape the ensuing immune response, surprisingly little is known on their spatial orchestration at physiologically T cell low precursor frequencies. Light sheet fluorescence microscopy and one of its implementations, selective plane illumination microscopy (SPIM), is a powerful method to obtain precise spatial information of entire organs of 0.5-10mm diameter, the size range of murine LNs. Yet, its usefulness for immunological research has thus far not been comprehensively explored. Here, we have tested and defined protocols that preserve fluorescent protein function during lymphoid tissue clearing required for SPIM. Reconstructions of SPIM-generated 3D data sets revealed that calibrated numbers of adoptively transferred T cells and DCs are successfully detected at a single cell level within optically cleared murine LNs. Finally, we define parameters to quantify specific interactions between antigen-specific T cells and pMHC-bearing DCs in murine LNs. In sum, our studies describe the successful application of light sheet fluorescence microscopy to immunologically relevant tissues.

Helping teachers change their behavior, an inservice training project utilizing sound filmstrips, interaction analysis, self-directed experimentation:

Mode of access: Internet.

A service-oriented user interaction analysis framework supporting adaptive applications

A comprehensive user model, built by monitoring a user's current use of applications, can be an excellent starting point for building adaptive user-centred applications. The BaranC framework monitors all user interaction with a digital device (e.g. smartphone), and also collects all available context data (such as from sensors in the digital device itself, in a smart watch, or in smart appliances) in order to build a full model of user application behaviour. The model built from the collected data, called the UDI (User Digital Imprint), is further augmented by analysis services, for example, a service to produce activity profiles from smartphone sensor data. The enhanced UDI model can then be the basis for building an appropriate adaptive application that is user-centred as it is based on an individual user model. As BaranC supports continuous user monitoring, an application can be dynamically adaptive in real-time to the current context (e.g. time, location or activity). Furthermore, since BaranC is continuously augmenting the user model with more monitored data, over time the user model changes, and the adaptive application can adapt gradually over time to changing user behaviour patterns. BaranC has been implemented as a service-oriented framework where the collection of data for the UDI and all sharing of the UDI data are kept strictly under the user's control. In addition, being service-oriented allows (with the user's permission) its monitoring and analysis services to be easily used by 3rd parties in order to provide 3rd party adaptive assistant services. An example 3rd party service demonstrator, built on top of BaranC, proactively assists a user by dynamic predication, based on the current context, what apps and contacts the user is likely to need. BaranC introduces an innovative user-controlled unified service model of monitoring and use of personal digital activity data in order to provide adaptive user-centred applications. This aims to improve on the current situation where the diversity of adaptive applications results in a proliferation of applications monitoring and using personal data, resulting in a lack of clarity, a dispersal of data, and a diminution of user control.

Layer-by-layer assembly of multilayer films composed of avidin and biotin-labeled antibody for immunosensing

Protein multilayers composed of avidin and biotin-labeled antibody (bio-Ab) were prepared on gold surface by layer-by-layer assembly technology using the high specific binding constant (K-a: approximate to 10(15) M-1) between avidin and biotin. The assembly process of the multilayer films was monitored by using real-time BIA technique based on surface plasmon resonance (SPR). The multilayer films were also characterized by electrochemical impedance spectroscopy (EIS) and reflection absorption Fourier transform infrared spectroscopy (FTIR). The results indicate that the growth of the multilayer is uniform. From response of SPR for each layer, the stoichiometry S for the interaction between avidin and bio-Ab is calculated to be 0.37 in the multilayer whereas 0.82 in the first layer. The protein mass concentration for each layer was also obtained. The schematic figure for the multilayer assembly was proposed according to the layer mass, concentration and S value. The utility of the mutilayer films for immunosensing has been investigated via their subsequent interaction with hIgG. The binding ability of the multilayer increased for one to three layers of antibody, and then reach saturation after the fourth layer. These layer-by-layer constructed antibody multilayers enhance the binding ability than covalently immobilized monolayer antibody. This technology can be also used for construction of other thin films for immunosensing and biosensor.

Real-time immunoassay of ferritin using surface plasmon resonance biosensor

A surface plasmon resonance (SPR) biosensor was used for the first time to determine the concentration of ferritin in both HBS-EP buffer and serum. The monoclonal antibody was immobilized on the carboxymethyl dextran-modified gold surface by an amine coupling method. The interaction of antibody with antigen was monitored in real-time. The signal was enhanced by sandwich amplification strategy to improve the sensitivity and specificity of the immunoassay, especially in serum. The linear range of the assay in serum is over 30-200 ng ml with the detection limit of 28 ng ml(-1). The sensitivity, specificity, and reproducibility of the assay are satisfactory. The analyte and enhancement antibody-binding surface could be regenerated by pH 2.0 glycine-HCl buffer and the same antibody-immobilized surface could be used for more than 50 cycles of ferritin binding and regeneration.

Surface plasmon resonance studies on the electrochemical doping/dedoping processes of anions on polyaniline-modified electrode

The combination of in situ surface plasmon resonance (SPR) with electrochemistry was used to investigate the electrochemical doping/dedoping processes of anions on a polyaniline (PAn)-modified electrode. Electrochemical SPR characteristics of the PAn film before and after doping/dedoping were revealed. The redox transformation between the insulating leucoemeraldine, and the conductive emeraldine, corresponding to the doping/dedoping of anion, can lead to very distinct changes in both the resonance minimum angle and the shape of SPR curve. This is ascribed to the swelling/shrinking effect, and the change of the PAn film in the imaginary part of the dielectric constant resulted from the transition of the film conductivity. In situ recording the time evolution of reflectance change at a fixed angle permits the continuous monitoring of the kinetic processes of doping/dedoping anions. The size and the charge of anions, the film thickness, as well as the concentration of anions are shown to strongly influence the rate of ingress/egress of anions. The time differential of SPR kinetic curves can be well applied in the detecting electroinactive anion by flow injection analysis. The approach has higher sensitivity and reproducibility compared with other kinetic measurements, such as those obtained by amperometry.

Enhanced surface plasmon resonance immunoassay for human complement factor 4

Using an enhanced surface plasmon resonance (SPR) immunosensor, we have determined the concentration of human complement factor 4 (C4). Antibody protein was concentrated into a carboxymethyldextran-modified gold surface by electrostatic attraction force and a simultaneous covalent immobilization of antibody based on amine coupling reaction took place. The sandwich method was applied to enhance the response signal and the specificity of antigen binding assay. The antibody immobilized surface had good response to C4 in the range of 0.02-20 mug/ml by this enhanced immunoassay. The regeneration effect by pH 2 glycine-HC1 buffer was also investigated. The same antibody immobilized surface could be used more than 80 cycles of C4 binding and regeneration. In addition, the ability to determinate C4 directly from serum sample without any purification was investigated. The sensitivity, specificity and reproducibility of the enhanced immunoassay are satisfactory. The results clearly demonstrate the advantages of the enhanced SPR technique for C4 immunoassay.

Real-time immunoassay of antibody activity in serum by surface plasmon resonance biosensor

A surface plasmon resonance biosensor has been used to determine antibody activity in serum. As a model system, the interaction of mouse IgG and sheep anti-mouse IgG polyclonal antibody was investigated in real time. The factors, including pH value, ionic strength, protein concentration, influencing electrostatic adsorption of mouse IgG protein onto carboxylated dextran-coated sensor chip surface, were studied. The procedures of mouse IgG protein immobilization and immune reaction were monitored in real time. The regeneration effect using the different elution reagents was also investigated. The same mouse IgG immobilized surface can be used for 100 cycles of binding and elution with only 0.38% loss per regeneration in reactivity. The results show that the surface plasmon resonance biosensor is a rapid, simple, sensitive, accurate and reliable detection technique for real-time immunoassay of antibody activity. The assay allows antibodies to be detected and studied in their native form without any purification. (C) 2000 Elsevier Science B.V. All rights reserved.

Assembly of alternating polycation and DNA multilayer films by electrostatic layer-by-layer adsorption

The assembly of alternating DNA and positively charged poly(dimethyldiallylammonium chloride) (PDDA) multilayer films by electrostatic layer-by-layer adsorption has been studied. The real-time surface plasmon resonance (BIAcore) technique was used to characterize and monitor the formation of multilayer films in solution in real time continuously. Electrochemical impedance spectroscopy (EIS) and UV-vis absorbance measurements were also used to study the film assembly, and linear film growth was observed. All the results indicate that the uniform multilayer can be obtained on the poly(ethylenimine)- (PEI-) coated substrate surface. The kinetics of the adsorption of DNA on PDDA surface was also studied by the real-time BIAcore technique; the observed rate constant was calculated using a Langmuir model (k(obs) = (1.28 +/- 0.08) x 10(-2) s(-1).

Analysis of the problems associated with dynamic interaction between train, track and structure at transition zones

El gran desarrollo experimentado por la alta velocidad en los principales países de la Unión Europea, en los últimos 30 años, hace que este campo haya sido y aún sea uno de los principales referentes en lo que a investigación se refiere. Por otra parte, la aparición del concepto super − alta velocidad hace que la investigación en el campo de la ingeniería ferroviaria siga adquiriendo importancia en los principales centros de investigación de los países en los que se desea implantar este modo de transporte, o en los que habiendo sido ya implantado, se pretenda mejorar. Las premisas de eficacia, eficiencia, seguridad y confort, que este medio de transporte tiene como razón de ser pueden verse comprometidas por diversos factores. Las zonas de transición, definidas en la ingeniería ferroviaria como aquellas secciones en las que se produce un cambio en las condiciones de soporte de la vía, pueden afectar al normal comportamiento para el que fue diseñada la infraestructura, comprometiendo seriamente los estándares de eficiencia en el tiempo de viaje, confort de los pasajeros y aumentando considerablemente los costes de mantenimiento de la vía, si no se toman las medidas oportunas. En esta tesis se realiza un estudio detallado de la zonas de transición, concretamente de aquellas en las que existe una cambio en la rigidez vertical de la vía debido a la presencia de un marco hidráulico. Para realizar dicho estudio se lleva a cabo un análisis numérico de interacción entre el vehículo y la estructura, con un modelo bidimensional de elemento finitos, calibrado experimentalmente, en estado de tensión plana. En este análisis se tiene en cuenta el efecto de las irregularidades de la vía y el comportamiento mecánico de la interfaz suelo-estructura, con el objetivo de reproducir de la forma más real posible el efecto de interacción entre el vehículo, la vía y la estructura. Otros efectos como la influencia de la velocidad del tren y los asientos diferenciales, debidos a deformaciones por consolidación de los terraplenes a ambos lados el marco hidráulico, son también analizados en este trabajo. En esta tesis, los cálculos de interacción se han llevado a cabo en dos fases diferentes. En la primera, se ha considerado una interacción sencilla debida al paso de un bogie de un tren Eurostar. Los cálculos derivados de esta fase se han denominado cálculos a corto plazo. En la segunda, se ha realizado un análisis considerando múltiples pasos de bogie del tren Eurostar, conformando un análisis de degradación en el que se tiene en cuenta, en cada ciclo, la deformación de la capa de balasto. Los cálculos derivados de esta fase, son denominados en el texto como cálculos a largo plazo. Los resultados analizados muestran que la utilización de los denominados elementos de contacto es fundamental cuando se desea estudiar la influencia de asientos diferenciales, especialmente en transiciones terraplén-estructura en las que la cuña de cimentación no llega hasta la base de cimentación de la estructura. Por otra parte, tener en cuenta los asientos del terraplén, es sumamente importante, cuando se desea realizar un análisis de degradación de la vía ya que su influencia en la interacción entre el vehículo y la vía es muy elevada, especialmente para valores altos de velocidad del tren. En cuanto a la influencia de las irregularidades de la vía, en los cálculos efectuados, se revela que su importancia es muy notable, siendo su influencia muy destacada cuanto mayor sea la velocidad del tren. En este punto cabe destacar la diferencia de resultados derivada de la consideración de perfiles de irregularidades de distinta naturaleza. Los resultados provenientes de considerar perfiles artificiales son en general muy elevados, siendo estos más apropiados para realizar estudios de otra índole, como por ejemplo de seguridad al descarrilamiento. Los resultados provenientes de perfiles reales, dados por diferentes Administradores ferroviarios, presentan resultados menos elevados y más propios del problema analizar. Su influencia en la interacción dinámica entre el vehículo y la vía es muy importante, especialmente para velocidades elevadas del tren. Además el fenómeno de degradación conocido como danza de traviesas, asociado a zonas de transición, es muy susceptible a la consideración de irregularidades de la vía, tal y como se desprende de los cálculos efectuados a largo plazo. The major development experienced by high speed in the main countries of the European Union, in the last 30 years, makes railway research one of the main references in the research field. It should also be mentioned that the emergence of the concept superhigh − speed makes research in the field of Railway Engineering continues to gain importance in major research centers in the countries in which this mode of transportation is already implemented or planned to be implemented. The characteristics that this transport has as rationale such as: effectiveness, efficiency, safety and comfort, may be compromised by several factors. The transition zones are defined in railway engineering as a region in which there is an abrupt change of track stiffness. This stiffness variation can affect the normal behavior for which the infrastructure has been designed, seriously compromising efficiency standards in the travel time, passenger comfort and significantly increasing the costs of track maintenance, if appropriate measures are not taken. In this thesis a detailed study of the transition zones has been performed, particularly of those in which there is a change in vertical stiffness of the track due to the presence of a reinforced concrete culvert. To perform such a study a numerical interaction analysis between the vehicle, the track and the structure has been developed. With this purpose a two-dimensional finite element model, experimentally calibrated, in a state of plane stress, has been used. The implemented numerical models have considered the effects of track irregularities and mechanical behavior of soil-structure interface, with the objective of reproducing as accurately as possible the dynamic interaction between the vehicle the track and the structure. Other effects such as the influence of train speed and differential settlement, due to secondary consolidation of the embankments on both sides of culvert, have also been analyzed. In this work, the interaction analysis has been carried out in two different phases. In the first part a simple interaction due to the passage of a bogie of a Eurostar train has been considered. Calculations derived from this phase have been named short-term analysis. In the second part, a multi-load assessment considering an Eurostar train bogie moving along the transition zone, has been performed. The objective here is to simulate a degradation process in which vertical deformation of the ballast layer was considered. Calculations derived from this phase have been named long-term analysis. The analyzed results show that the use of so-called contact elements is essential when one wants to analyze the influence of differential settlements, especially in embankment-structure transitions in which the wedge-shaped backfill does not reach the foundation base of the structure. Moreover, considering embankment settlement is extremely important when it is desired to perform an analysis of track degradation. In these cases the influence on the interaction behaviour between the vehicle and the track is very high, especially for higher values of speed train. Regarding the influence of the track irregularities, this study has proven that the track’s dynamic response is heavily influenced by the irregularity profile and that this influence is more important for higher train velocities. It should also be noted that the difference in results derived from consideration of irregularities profiles of different nature. The results coming from artificial profiles are generally very high, these might be more appropriate in order to study other effects, such as derailment safety. Results from real profiles, given by the monitoring works of different rail Managers, are softer and they fit better to the context of this thesis. The influence of irregularity profiles on the dynamic interaction between the train and the track is very important, especially for high-speeds of the train. Furthermore, the degradation phenomenon known as hanging sleepers, associated with transition zones, is very susceptible to the consideration of track irregularities, as it can be concluded from the long-term analysis.

How talk and interaction unfold in a digitally enabled preschool classroom

The use of mobile digital devices, such as laptops and tablets, has implications for how teachers interact with young students within the institutional context of educational settings. This article examines language and participation in a digitally enabled preschool classroom as students engage with teachers and peers. Ethnomethodology, conversation analysis and membership categorization analysis are used to explicate video-recorded episodes of students (aged 3-5 years) interacting while using a laptop and a tablet. Attending to the sequential organization (when, how) and the context relevance (where) of talk and interaction, analysis shows how the intersection of interactions involving the teacher, students and digital devices, shape the ways that talk and interactions unfold. Analysis found that the teacher-student interactions were jointly arranged around a participation framework that included: 1) the teacher’s embodied action that mobilizes an accompanying action by a student, 2) allocation of turn-taking and participation while using a digital device and, 3) the affordances of the digital device in relation to the participants’ social organization. In this way, it is possible to understand not just what a digital device is or does, but the affordances of what it makes possible in constituting teachers’ and students’ social and learning relationships.

Semi-analytical finite element analysis of a strip footing on an elastic reinforced soil

A plane strain elastic interaction analysis of a strip footing resting on a reinforced soil bed has been made by using a combined analytical and finite element method (FEM). In this approach the stiffness matrix for the footing has been obtained using the FEM, For the reinforced soil bed (halfplane) the stiffness matrix has been obtained using an analytical solution. For the latter, the reinforced zone has been idealised as (i) an equivalent orthotropic infinite strip (composite approach) and (ii) a multilayered system (discrete approach). In the analysis, the interface between the strip footing and reinforced halfplane has been assumed as (i) frictionless and (ii) fully bonded. The contact pressure distribution and the settlement reduction have been given for different depths of footing and scheme of reinforcement in soil. The load-deformation behaviour of the reinforced soil obtained using the above modelling has been compared with some available analytical and model test results. The equivalent orthotropic approach proposed in this paper is easy to program and is shown to predict the reinforcing effects reasonably well.

Partial Loss Of Support And Frame-Soil Interaction

An interaction analysis has been conducted to study the effects of a local loss of support beneath the beam footing of a two-bay plane frame. The results of the study indicate that the magnitude of increase in the bending moment and axial force in the structure due to the presence of a void are dependent, not only on the extent of support loss, but also on the relative stiffnesses between foundation beam and soil, and between superstructure and soil. The increase in bending moment even for a void span of 1/12 of the foundation beam length can become so significant as to exceed the safety provisions. The study shows that the effect of a void on the superstructure moments can be greatly minimized by a combination of rigid foundation and flexible superstructure.

Frame Soil Interaction And Winkler Model

The Winkler spring model is the most convenient representation of soil support in the domain of linear elasticity for framed structure-soil interaction analyses. The closeness of the analytical results obtained using this model with those corresponding to the elastic half-space continuum has been investigated in the past for foundation beams. The findings, however, are not applicable to framed structures founded on beam or strip footings. Moreover, the past investigations employ the concept of characteristic length which does not adequately account for the stiffness contribution of the superstructure. A framed structure on beam foundation can be described parametrically by the ratios of stiffnesses of superstructure and foundation beams to that of soil. For a practical range of soil allowable pressures, the ranges of these relative stiffness ratios have been established. The present study examines the variation between interactive analyses based on Winkler springs with those using the half-space continuum over these ranges of relative stiffness ratios. The findings enable the analyst to undertake a Winkler spring-based-interaction analysis with knowledge of the likely variation of values with those derived for the more computation-intensive half-space continuum.