Thermodynamic reaction control of nucleoside phosphorolysis

Nucleoside analogs represent a class of important drugs for cancer and antiviral treatments. Nucleoside phosphorylases (NPases) catalyze the phosphorolysis of nucleosides and are widely employed for the synthesis of pentose‐1‐phosphates and nucleoside analogs, which are difficult to access via conventional synthetic methods. However, for the vast majority of nucleosides, it has been observed that either no or incomplete conversion of the starting materials is achieved in NPase‐catalyzed reactions. For some substrates, it has been shown that these reactions are reversible equilibrium reactions that adhere to the law of mass action. In this contribution, we broadly demonstrate that nucleoside phosphorolysis is a thermodynamically controlled endothermic reaction that proceeds to a reaction equilibrium dictated by the substrate‐specific equilibrium constant of phosphorolysis, irrespective of the type or amount of NPase used, as shown by several examples. Furthermore, we explored the temperature‐dependency of nucleoside phosphorolysis equilibrium states and provide the apparent transformed reaction enthalpy and apparent transformed reaction entropy for 24 nucleosides, confirming that these conversions are thermodynamically controlled endothermic reactions. This data allows calculation of the Gibbs free energy and, consequently, the equilibrium constant of phosphorolysis at any given reaction temperature. Overall, our investigations revealed that pyrimidine nucleosides are generally more susceptible to phosphorolysis than purine nucleosides. The data disclosed in this work allow the accurate prediction of phosphorolysis or transglycosylation yields for a range of pyrimidine and purine nucleosides and thus serve to empower further research in the field of nucleoside biocatalysis.DFG, 390540038, EXC 2008: UniSysCatTU Berlin, Open-Access-Mittel - 201

Near Surface Geophysical Archaeological Prospection at the Prehistoric Site of Akrotiri on Santorini/Thera

In February 2014 high-resolution ground penetrating radar and earth resistance tomography measurements have for the first time been used successfully for the distinct mapping of buried archaeological structures in the vicinity of the Bronze Age archaeological site of Akrotiri on Santorini/Thera in Greece

Routes for GMR-Sensor Design in Non-Destructive Testing

GMR sensors are widely used in many industrial segments such as information technology, automotive, automation and production, and safety applications. Each area requires an adaption of the sensor arrangement in terms of size adaption and alignment with respect to the field source involved. This paper deals with an analysis of geometric sensor parameters and the arrangement of GMR sensors providing a design roadmap for non-destructive testing (NDT) applications. For this purpose we use an analytical model simulating the magnetic flux leakage (MFL) distribution of surface breaking defects and investigate the flux leakage signal as a function of various sensor parameters. Our calculations show both the influence of sensor length and height and that when detecting the magnetic flux leakage of µm sized defects a gradiometer base line of 250 µm leads to a signal strength loss of less than 10% in comparison with a magnetometer response. To validate the simulation results we finally performed measurements with a GMR magnetometer sensor on a test plate with artificial µm-range cracks. The differences between simulation and measurement are below 6%. We report on the routes for a GMR gradiometer design as a basis for the fabrication of NDT-adapted sensor arrays. The results are also helpful for the use of GMR in other application when it comes to measure positions, lengths, angles or electrical currents

Documenting Bronze Age Akrotiri on Thera using laser scanning, image-based modelling and geophysical prospection

The excavated architecture of the exceptional prehistoric site of Akrotiri on the Greek island of Thera/Santorini is endangered by gradual decay, damage due to accidents, and seismic shocks, being located on an active volcano in an earthquake-prone area. Therefore, in 2013 and 2014 a digital documentation project has been conducted with support of the National Geographic Society in order to generate a detailed digital model of Akrotiri’s architecture using terrestrial laser scanning and image-based modeling. Additionally, non-invasive geophysical prospection has been tested in order to investigate its potential to explore and map yet buried archaeological remains. This article describes the project and the generated results

Automated Cell Treatment for Competence and Transformation of Escherichia coli in a High-Throughput Quasi-Turbidostat Using Microtiter Plates

Metabolic engineering and genome editing strategies often lead to large strain libraries of a bacterial host. Nevertheless, the generation of competent cells is the basis for transformation and subsequent screening of these strains. While preparation of competent cells is a standard procedure in flask cultivations, parallelization becomes a challenging task when working with larger libraries and liquid handling stations as transformation efficiency depends on a distinct physiological state of the cells. We present a robust method for the preparation of competent cells and their transformation. The strength of the method is that all cells on the plate can be maintained at a high growth rate until all cultures have reached a defined cell density regardless of growth rate and lag phase variabilities. This allows sufficient transformation in automated high throughput facilities and solves important scheduling issues in wet-lab library screenings. We address the problem of different growth rates, lag phases, and initial cell densities inspired by the characteristics of continuous cultures. The method functions on a fully automated liquid handling platform including all steps from the inoculation of the liquid cultures to plating and incubation on agar plates. The key advantage of the developed method is that it enables cell harvest in 96 well plates at a predefined time by keeping fast growing cells in the exponential phase as in turbidostat cultivations. This is done by a periodic monitoring of cell growth and a controlled dilution specific for each well. With the described methodology, we were able to transform different strains in parallel. The transformants produced can be picked and used in further automated screening experiments. This method offers the possibility to transform any combination of strain- and plasmid library in an automated high-throughput system, overcoming an important bottleneck in the high-throughput screening and the overall chain of bioprocess development.BMBF, 031L0018A, ERASysApp2 - Verbundprojekt: LEANPROT - Entwicklung einer Systembiologie-Plattform für die Entwicklung von lean-proteome-Escherichia coli-Stämmen - Deutsches Teilprojekt

From deposit to point cloud: a study of low-cost computer vision approaches for the straightforward documentation of archaeological excavations

Stratigraphic archaeological excavations demand high-resolution documentation techniques for 3D recording. Today, this is typically accomplished using total stations or terrestrial laser scanners. This paper demonstrates the potential of another technique that is low-cost and easy to execute. It takes advantage of software using Structure from Motion (SfM) algorithms, which are known for their ability to reconstruct camera pose and threedimensional scene geometry (rendered as a sparse point cloud) from a series of overlapping photographs captured by a camera moving around the scene. When complemented by stereo matching algorithms, detailed 3D surface models can be built from such relatively oriented photo collections in a fully automated way. The absolute orientation of the model can be derived by the manual measurement of control points. The approach is extremely flexible and appropriate to deal with a wide variety of imagery, because this computer vision approach can also work with imagery resulting from a randomly moving camera (i.e. uncontrolled conditions) and calibrated optics are not a prerequisite. For a few years, these algorithms are embedded in several free and low-cost software packages. This paper will outline how such a program can be applied to map archaeological excavations in a very fast and uncomplicated way, using imagery shot with a standard compact digital camera (even if the images were not taken for this purpose). Archived data from previous excavations of VIAS-University of Vienna has been chosen and the derived digital surface models and orthophotos have been examined for their usefulness for archaeological applications. The absolute georeferencing of the resulting surface models was performed with the manual identification of fourteen control points. In order to express the positional accuracy of the generated 3D surface models, the NSSDA guidelines were applied. Simultaneously acquired terrestrial laser scanning data – which had been processed in our standard workflow – was used to independently check the results. The vertical accuracy of the surface models generated by SfM was found to be within 0.04 m at the 95 % confidence interval, whereas several visual assessments proved a very high horizontal positional accuracy as well

A UV/Vis Spectroscopy-Based Assay for Monitoring of Transformations Between Nucleosides and Nucleobases

Efficient reaction monitoring is crucial for data acquisition in kinetic and mechanistic studies. However, for conversions of nucleosides to their corresponding nucleobases, as observed in enzymatically catalyzed nucleoside phosphorylation reactions, the current analytical arsenal does not meet modern requirements regarding cost, speed of analysis and high throughput. Herein, we present a UV/Vis spectroscopy-based assay employing an algorithm for spectral unmixing in a 96-well plate format. The algorithm relies on fitting of reference spectra of nucleosides and their bases to experimental spectra and allows determination of nucleoside/nucleobase ratios in solution with high precision. The experimental procedure includes appropriate dilution of a sample into aqueous alkaline solution, transfer to a multi-well plate, measurement of a UV/Vis spectrum and subsequent in silico spectral unmixing. This enables data collection in a high-throughput fashion and reduces costs compared to state-of-the-art HPLC analyses by approximately 5-fold while being 20-fold faster and offering comparable precision. Additionally, the method is robust regarding dilution and sample transfer errors as it only considers spectral form and not absolute intensity. It can be applied to all natural nucleosides and nucleobases and even unnatural ones as demonstrated by several examples.DFG, 390540038, EXC 2008: UniSysCa

Production of soluble regulatory hydrogenase from Ralstonia eutropha in Escherichia coli using a fed-batch-based autoinduction system

Background: Autoinduction systems can regulate protein production in Escherichia coli without the need to monitor cell growth or add inducer at the proper time following culture growth. Compared to classical IPTG induction, autoinduction provides a simple and fast way to obtain high protein yields. In the present study, we report on the optimization process for the enhanced heterologous production of the Ralstonia eutropha regulatory hydrogenase (RH) in E. coli using autoinduction. These autoinduction methods were combined with the EnPresso B fed-batch like growth system, which applies slow in situ enzymatic glucose release from a polymer to control cell growth and protein synthesis rate. Results: We were able to produce 125 mg L−1 RH corresponding to a productivity averaged over the whole process time of 3 mg (L h)−1 in shake flasks using classic single-shot IPTG induction. IPTG autoinduction resulted in a comparable volumetric RH yield of 112 mg L−1 and due to the shorter overall process time in a 1.6-fold higher productivity of 5 mg (L h)−1. In contrast, lactose autoinduction increased the volumetric yield more than 2.5-fold and the space time yield fourfold reaching 280 mg L−1 and 11.5 mg (L h)−1, respectively. Furthermore, repeated addition of booster increased RH production to 370 mg L−1, which to our knowledge is the highest RH concentration produced in E. coli to date. Conclusions: The findings of this study confirm the general feasibility of the developed fed-batch based autoinduction system and provide an alternative to conventional induction systems for efficient recombinant protein production. We believe that the fed-batch based autoinduction system developed herein will favor the heterologous production of larger quantities of difficult-to-express complex enzymes to enable economical production of these kinds of proteins.DFG, 390540038, EXC 2008: Unifying Systems in Catalysis "UniSysCat"TU Berlin, Open-Access-Mittel – 202

“BIG DATA” EN PROSPECCIÓN ARQUEOLÓGICA DEL PAISAJE

[EN] While traditionally archaeological research has mainly been focused on individual cultural heritage monuments or distinct archaeological sites, the Austrian based Ludwig Boltzmann Institute for Archaeological Prospection and Virtual Archaeology goes beyond the limitations of discrete sites in order to understand their archaeological context. This is achieved by investigating the space in-between the sites, studying entire archaeological landscapes from the level of individual postholes to the mapping of numerous square kilometres. This large-scale, high-resolution, multi-method prospection approach leads to enormous digital datasets counting many terabytes of data that until recently were technically not manageable. Novel programs and methods of data management had to be developed for data acquisition, processing and archaeological interpretation, in order to permit the extraction of the desired information from the very big amount of data. The analysis of the generated datasets is conducted with the help of semi-automatic algorithms within complex three-, or even four-dimensional geographical information systems. The outcome of landscape archaeological prospection surveys is visually communicated to the scientific community as well as to the general public and stakeholders. In many cases, a visualization of the scientific result and archaeological interpretations can be a powerful and suitable tool to illustrate and communicate even complex contexts to a wide audience. This paper briefly presents the great potential offered by a combination of large-scale non-invasive archaeological prospection methods and standardized workflows for the integration of big data, its interpretation and visualization. The proposed approach provides a context for buried archaeology across entire archaeological landscapes, changing our understanding of known monuments. We address the overcome and remaining challenges with the help of examples taken from outstanding landscape archaeological prospection case studies.[ES] Aunque tradicionalmente la investigación arqueológica ha estado fundamentalmente centrada en monumentos y yacimientos arqueológicos de forma individual, el Ludwig Boltzmann Institute for Archaeological Prospection and Virtual Archaeology (Austria) va más allá de los límites de yacimientos particulares con el objetivo de entender su contexto arqueológico. Esto es conseguido mediante la investigación del espacio entre yacimientos y estudiando paisajes arqueológicos completos yendo desde un hoyo de poste hasta el mapeado de varios kilómetros cuadrados. El enfoque de prospección multi-metodológico a gran escala y de alta resolución conduce hacia un enorme conjunto de datos digital que incluye varios Terabytes de información los cuales no habían podido ser manipulados hasta hace poco debido a limitaciones tecnológicas. Por consiguiente, nuevos programas y métodos de gestión de datos han sido diseñados para la adquisición y procesado de datos así como interpretación arqueológica para así permitir la extracción de la información deseada desde estos enormes bancos de datos. El análisis de estos conjuntos de datos generados es llevado a cabo a través de análisis de sistemas de información geográfica tridimensionales e incluso cuatridimensionales. El resultado de la prospección de paisajes arqueológicos es transferido de forma visual a la comunididad científica así como al gran público e interesados en la materia. En muchos casos una visualización de los resultados científicos e interpretaciones arqueológicas puede ser una herramienta más poderosa y adecuada para ilustrar y comunicar contextos arqueológicos complejos a un público mayor. Este artículo presenta de forma breve el gran potencial ofrecido por la combinación de métodos de prospección arqueológica de gran resolución a gran escala y unos flujos de trabajo estandarizados para integración, interpretación y visualización de datos. La estrategía propuesta proporciona un contexto para restos arqueológicos enmarcados en paisajes arqueológicos que viene a cambiar nuestra forma de entender monumentos ya conocidos. Pretendemos también superar los desafios que quedan con la ayuda de ejemplos sacados de excepcionales paisajes arqueológicos que son nuestros estudios de caso a prospectar.Torrejón Valdelomar, J.; Wallner, M.; Trinks, I.; Kucera, M.; Luznik, N.; Löcker, K.; Neubauer, W. (2016). BIG DATA IN LANDSCAPE ARCHAEOLOGICAL PROSPECTION. En 8th International congress on archaeology, computer graphics, cultural heritage and innovation. Editorial Universitat Politècnica de València. 238-246. https://doi.org/10.4995/arqueologica8.2015.4200OCS23824