Maskless projection lithography for the fast and flexible generation of grayscale protein patterns

Protein patterns of different shapes and densities are useful tools for studies of cell behavior and to create biomaterials that induce specific cellular responses. Up to now the dominant techniques for creating protein patterns are mostly based on serial writing processes or require templates such as photomasks or elastomer stamps. Only a few of these techniques permit the creation of grayscale patterns. Herein, the development of a lithography system using a digital mirror device which allows fast patterning of proteins by immobilizing fluorescently labeled molecules via photobleaching is reported. Grayscale patterns of biotin with pixel sizes in the range of 2.5 μm are generated within 10 s of exposure on an area of about 5 mm(2) . This maskless projection lithography method permits the rapid and inexpensive generation of protein patterns definable by any user-defined grayscale digital image on substrate areas in the mm(2) to cm(2) range.     

An automated noncontact deposition interface for liquid chromatography matrix-assisted laser desorption/ionization mass spectrometry

A new multichannel deposition system was developed for off-line liquid chromatography/matrix-assisted laser desorption/ionization mass spectrometry (LC/MALDI-MS). This system employs a pulsed electric field to transfer the eluents from multiple parallel columns directly onto MALDI targets without the column outlets touching the target surface. The deposition device performs well with a wide variety of solvents that have different viscosities, vapor pressures, polarities, and ionic strengths. Surface-modified targets were used to facilitate concentration and precise positioning of samples, allowing for efficient automation of high-throughput MALDI analysis. The operational properties of this system allow the user to prepare samples using MALDI matrixes whose properties range from hydrophilic to hydrophobic. The latter, exemplified by alpha-cyano-4-hydroxycinnamic acid, were typically processed with a multistep deposition method consisting of precoating of individual spots on the target plate, sample deposition, and sample recrystallization steps. Using this method, 50 amol of angiotensin II was detected reproducibly with high signal-to-noise ratio after LC separation. Experimental results show that there is no significant decrease in chromatographic resolution using this device. To assess the behavior of the apparatus for complex mixtures, 5 microg of a tryptic digest of the cytosolic proteins of yeast was analyzed by LC/MALDI-MS and more than 13,500 unique analytes were detected in a single LC/MS analysis.     

Analysis of CheA histidine phosphorylation and its influence on protein stability by high-resolution element and electrospray mass spectrometry

A combination of electrospray mass spectrometry (ESI-MS) and element mass spectrometry (ICPMS) with phosphorus detection was used to characterize histidine phosphorylation (His-48) of the chemotaxis protein CheA quantitatively. The phosphorylation at His-48 was found to be responsible for a stabilization of the protein. For this investigation, the acceptor domain and the kinase domain of the bacterial chemotaxis protein CheA were recombinantly expressed as single proteins. Using in vitro kinase assay conditions, the acceptor domain CheA-H was phosphorylated by the kinase domain CheA-C. The degree of histidine phosphorylation was determined by nanoelectrospray mass spectrometry of intact CheA-H, and was found to be limited to a maximum value of approximately 50%. The site specificity of CheA-H phosphorylation was controlled by nanoESI-MS/MS of the [M + 16H](16+) ion of intact (pHis)-CheA-H and allowed localization of the pHis residue to the region between residues 32 and 86, containing candidates His-48 and His-67, for which His-48 phosphorylation has been described. Analysis of the tryptic digest of in vitro histidine-phosphorylated CheA-H by capillary chromatography coupled to ESI-MS and to ICPMS with phosphorus detection revealed a truncated (pHis)-CheA-H protein as the only phosphorus-containing analyte. Since the truncated (pHis)-CheA-H in the digest was found to exhibit a higher degree of phosphorylation than could be generated by in vitro phosphorylation without trypsin treatment, it is concluded that histidine phosphorylation at His-48 strongly interferes with structural properties of the CheA-H domain in particular with respect to proteolytic degradation by trypsin.     

Flex‐eWare: a flexible model driven solution for designing and implementing embedded distributed systems

The complexity of modern embedded systems increases as they incorporate new concerns such as distribution and mobility. These new features need to be considered as early as possible in the software development life cycle. Model driven engineering promotes an intensive use of models and is now widely seen as a solution to master the development of complex systems such as embedded ones. Component‐based software engineering is another major trend that gains acceptance in the embedded world because of its properties such as reuse, modularity, and flexibility. This article proposes the Flex‐eWare component model (FCM) for designing and implementing modern embedded systems. The FCM unifies model driven engineering and component‐based software engineering and has been evaluated in several application domains with different requirements: wireless sensor networks, distributed client/server applications, and control systems for electrical devices. This approach highlights a new concept: flexibility points that arise at several stages of the development process, that is, in the model (design phase), in the execution platform, and during the execution itself. This flexibility points are captured with model libraries that can extend the FCM. Copyright © 2011 John Wiley & Sons, Ltd.     

Survey on modeling and indexing events in multimedia

Events have gained increasing interest in the area of multimedia in recent years. There have been many approaches published and research conducted on how to extract events from multimedia, represent it using appropriate models, and how to use events in end user applications. In this paper, we conduct an extensive analysis of existing event models along commonly identified aspects of events. In addition, we analyze how the different aspects of events relate to each other and how they can be applied together. Subsequently, we look into different approaches for how to index multimedia data. Finally, we elaborate on how to link the multimedia data with events in order to provide the basis for future event-based multimedia applications.     

Benefiting from users’ gaze: selection of image regions from eye tracking information for provided tags

Providing image annotations is a tedious task. This becomes even more cumbersome when objects shall be annotated in the images. Such region-based annotations can be used in various ways like similarity search or as training set in automatic object detection. We investigate the principle idea of finding objects in images by looking at gaze paths from users, viewing images with an interest in a specific object. We have analyzed 799 gaze paths from 30 subjects viewing image-tag-pairs with the task to decide whether a tag could be found in the image or not. We have compared 13 different fixation measures analyzing the gaze paths. The best performing fixation measure is able to correctly assign a tag to a region for 63 % of the image-tag-pairs and significantly outperforms three baselines. We look into details of the image region characteristics such as the position and size for incorrect and correct assignments. The influence of aggregating multiple gaze paths from several subjects with respect to improving the precision of identifying the correct regions is also investigated. In addition, we look into the possibilities of discriminating different regions in the same image. Here, we are able to correctly identify two regions in the same image from different primings with an accuracy of 38 %.     

Iterative near–far resistant channel estimation by using a linear minimum mean squared error detector

Channel estimation techniques for CDMA system need to combat multiple access interference (MAI) to improve the estimation performance. The linear MMSE detector has certain advantages with respect to the near–far problem and can be used to develop a channel estimation algorithm. In this paper, an efficient iterative method for near–far resistant single-user mobile radio channel estimation in slow fading multi-path direct sequence code division multiple access (DS-CDMA) channels is presented. Computer simulation results demonstrate that a significant performance improvement can be achieved with the proposed method especially under extreme near–far conditions.     

General quadratic performance analysis and synthesis of differential algebraic equation (DAE) systems

In this paper, control of linear differential-algebraic-equation systems, subject to general quadratic constraints, is considered. This setup, especially, includes the H_∞ control problem and the design for strict passivity. Based on linear matrix inequality (LMI) analysis conditions, LMI synthesis conditions for the existence of linear output feedback controllers are derived by means of a linearizing change of variables. This approach is constructive: a procedure for the determination of controller parameterizations is given on the basis of the solution of the LMI synthesis conditions. A discussion of the possible applications of the presented results concludes the paper.     

Detection of Stationary Errors in Multiple Regressions with Integrated Regressors and Cointegration

Abstract

This article studies the problem of detecting sequentially stationary error terms in a multiple regression model with a difference-stationary multivariate I(1)-regressor. The detection of cointegration is covered as a special case. We provide the asymptotic distribution theory for a monitoring procedure that is related to a well-known nonparametric unit root test statistic calculated from sequentially updated least squares residuals. Functional limit theorems for the corresponding sequential processes and central limit theorems for the detectors used to raise an alarm are established under the no-change null hypothesis as well as under change-point models covering a change to I(0)-errors and a change of the regression coefficients as well. We also discuss extensions to the case that continuous time processes are discretely sampled to obtain the data allowing to apply the procedures to high-frequency data as well. Our results show that we can handle the infill asymptotics assuming that nonstationary continuous-time processes such as semimartingales are discretely observed, by virtue of the general assumptions that we impose. The finite sample properties are investigated by a simulation study.