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

Neuropsychological functioning in inpatients with major depression or schizophrenia

Background: Studies that compare neuropsychological functioning in inpatients with mood disorder or schizophrenia come to heterogeneous results. This study aims at investigating the question whether there are different neuropsychological test profiles in stabilised post-acute inpatients with affective disorders or schizophrenia. Method: We were interested in evaluating impairment in specific areas of cognitive functioning in patients with schizophrenia or depression. In clinical reality, patients with depression and schizophrenia are often treated together with little attention to their specific needs. 74 patients with major depression and 38 patients with schizophrenia were assessed in a comprehensive neuropsychological battery. All patients were in a post-acute stage of their illness, i.e. remission of acute symptoms. Results: In spite of a comparable mean score of psychopathological symptoms in the Brief Psychiatric Rating Scale-Expanded (BPRS-E) as well as in the Global Assessment Functioning Scale (GAF), patients with depressive disorder showed significantly better results in verbal and visual short-term memory, verbal fluency, visual-motor coordination, information processing in visual-verbal functioning and selective attention compared to patients with schizophrenia. No significant differences between both samples were found in practical reasoning, general verbal abstraction, spatial-figural functioning, speed of cognitive processing. Conclusions: These results show that there are differences in scores in psychopathology (BPRS-E, GAF) in patients with affective disorders or schizophrenia and different neuropsychological test profiles in the post-acute stage of their illness

Metabolic signature of breast cancer cell line MCF-7: profiling of modified nucleosides via LC-IT MS coupling

<p>Abstract</p> <p>Background</p> <p>Cancer, like other diseases accompanied by strong metabolic disorders, shows characteristic effects on cell turnover rate, activity of modifying enzymes and DNA/RNA modifications, resulting also in elevated amounts of excreted modified nucleosides. For a better understanding of the impaired RNA metabolism in breast cancer cells, we screened these metabolites in the cell culture supernatants of the breast cancer cell line MCF-7 and compared it to the human mammary epithelial cells MCF-10A. The nucleosides were isolated and analyzed via 2D-chromatographic techniques: In the first dimension by cis-diol specific boronate affinity extraction and subsequently by reversed phase chromatography coupled to an ion trap mass spectrometer.</p> <p>Results</p> <p>Besides the determination of ribonucleosides, additional compounds with cis-diol structure, deriving from cross-linked biochemical pathways, like purine-, histidine- and polyamine metabolism were detected. In total, 36 metabolites were identified by comparison of fragmentation patterns and retention time. Relation to the internal standard isoguanosine yielded normalized area ratios for each identified compound and enabled a semi-quantitative metabolic signature of both analyzed cell lines.</p> <p>13 of the identified 26 modified ribonucleosides were elevated in the cell culture supernatants of MCF-7 cells, with 5-methyluridine, <it>N</it>²,<it>N</it>²,7-trimethylguanosine, <it>N</it>⁶-methyl-<it>N</it>⁶-threonylcarbamoyladenosine and 3-(3-aminocarboxypropyl)-uridine showing the most significant differences. 1-ribosylimidazole-4-acetic acid, a histamine metabolite, was solely found in the supernatants of MCF-10A cells, whereas 1-ribosyl-4-carboxamido-5-aminoimidazole and S-adenosylmethionine occurred only in supernatants of MCF-7 cells.</p> <p>Conclusion</p> <p>The obtained results are discussed against the background of pathological changes in cell metabolism, resulting in new perspectives for modified nucleosides and related metabolites as possible biomedical markers for breast carcinoma <it>in vivo</it>.</p

Literatur zum Thema Geschlechterbildung

Rezension zu: 1) Geschlechter bilden - Männer und Frauen in der Katholischen Erwachsenenbildung: eine Arbeitshilfe der Katholischen Erwachsenenbildung Rheinland-Pfalz zur Geschlechtergerechtigkeit. Hg. von der KEB Rheinland-Pfalz, Landesarbeitsgemeinschaft e.V. Mainz 2008. 2) Stefanie Rieger-Goertz: Geschlechterbilder in der Katholischen Erwachsenenbildung. Forum Bildungsethik, Bd. 3. Bielefeld (wbv) 2008. 3) Anne Cottebrune: Der planbare Mensch - Die Deutsche Forschungsgemeinschaft (DFG) und die menschliche Vererbungswissenschaft 1920-1970. Stuttgart: Steiner 2008

A comprehensive evaluation of the activity and selectivity profile of ligands for RGD-binding integrins

Integrins, a diverse class of heterodimeric cell surface receptors, are key regulators of cell structure and behaviour, affecting cell morphology, proliferation, survival and differentiation. Consequently, mutations in specific integrins, or their deregulated expression, are associated with a variety of diseases. In the last decades, many integrin-specific ligands have been developed and used for modulation of integrin function in medical as well as biophysical studies. The IC50-values reported for these ligands strongly vary and are measured using different cell-based and cell-free systems. A systematic comparison of these values is of high importance for selecting the optimal ligands for given applications. In this study, we evaluate a wide range of ligands for their binding affinity towards the RGD-binding integrins avß3, avß5, avß6, avß8, a5ß1, aIIbß3, using homogenous ELISA-like solid phase binding assay.Postprint (published version

Power reduction of a 12-bit 40-MS/s pipeline ADC exploiting partial amplifier sharing

High performance analog-to-digital converters (ADC) are essential elements for the development of high performance image sensors. These circuits need a big number of ADCs to reach the required resolution at a specified speed. Moreover, nowadays power dissipation has become a key performance to be considered in analog designs, specially in those developed for portable devices. Design of such circuits is a challenging task which requires a combination of the most advanced digital circuit, the analog expertise knowledge and an iterative design. Amplifier sharing has been a commonly used technique to reduce power dissipation in pipelined ADCs. In this paper we present a partial amplifier sharing topology of a 12 bit pipeline ADC, developed in 0.35 mum CMOS process. Its performance is compared with a conventional amplifier scaling topology and with a fully amplifier sharing one.This work has been supported by Ministerio de Educación y Ciencia of Spain and the European Regional Development Fund of the European Commission (FEDER) under grant TIN2006-15460-C04-04

Influence of the First Preparation Steps on the Properties of GaN Layers Grown on 6H-SIC by Mbe

AbstractThe Gan heteroepitaxy on 6H-SiC is affected by the bad morphology of the substrate surface. We performed a hydrogen etching at 1550°C on the 6H-SiC(0001) substrates to obtain atomically flat terraces. An improvement of the structural properties of GaN grown by MBE on such substrates after deposition of a LT-AIN buffer layer is observed. A value of less than 220 arcsec of the FWHM of the XRD rocking curve, showing a reduced screw dislocations density, is comparable with the best results reported until now for thick GaN samples. Photoluminescence showed a structured near band edge emission spectrum with evidence of the A, B and C free exciton recombinations

Monitoring Parallel Robotic Cultivations with Online Multivariate Analysis

In conditional microbial screening, a limited number of candidate strains are tested at different conditions searching for the optimal operation strategy in production (e.g., temperature and pH shifts, media composition as well as feeding and induction strategies). To achieve this, cultivation volumes of >10 mL and advanced control schemes are required to allow appropriate sampling and analyses. Operations become even more complex when the analytical methods are integrated into the robot facility. Among other multivariate data analysis methods, principal component analysis (PCA) techniques have especially gained popularity in high throughput screening. However, an important issue specific to high throughput bioprocess development is the lack of so-called golden batches that could be used as a basis for multivariate analysis. In this study, we establish and present a program to monitor dynamic parallel cultivations in a high throughput facility. PCA was used for process monitoring and automated fault detection of 24 parallel running experiments using recombinant E. coli cells expressing three different fluorescence proteins as the model organism. This approach allowed for capturing events like stirrer failures and blockage of the aeration system and provided a good signal to noise ratio. The developed application can be easily integrated in existing data- and device-infrastructures, allowing automated and remote monitoring of parallel bioreactor systems.BMBF, 031L0018A, ERASysApp2 - Verbundprojekt: LEANPROT - Entwicklung einer Systembiologie-Plattform für die Entwicklung von lean-proteome-Escherichia coli-Stämmen - Deutsches Teilprojekt ADFG, 414044773, Open Access Publizieren 2019 - 2020 / Technische Universität Berli

Semi-automated high-throughput substrate screening assay for nucleoside kinases

Nucleoside kinases (NKs) are key enzymes involved in the in vivo phosphorylation of nucleoside analogues used as drugs to treat cancer or viral infections. Having different specificities, the characterization of NKs is essential for drug design and nucleotide analogue production in an in vitro enzymatic process. Therefore, a fast and reliable substrate screening method for NKs is of great importance. Here, we report on the validation of a well-known luciferase-based assay for the detection of NK activity in a 96-well plate format. The assay was semi-automated using a liquid handling robot. Good linearity was demonstrated (r² > 0.98) in the range of 0–500 µM ATP, and it was shown that alternative phosphate donors like dATP or CTP were also accepted by the luciferase. The developed high-throughput assay revealed comparable results to HPLC analysis. The assay was exemplarily used for the comparison of the substrate spectra of four NKs using 20 (8 natural, 12 modified) substrates. The screening results correlated well with literature data, and additionally, previously unknown substrates were identified for three of the NKs studied. Our results demonstrate that the developed semi-automated high-throughput assay is suitable to identify best performing NKs for a wide range of substrates.DFG, 414044773, Open Access Publizieren 2021 - 2022 / Technische Universität BerlinDFG, 392246628, Chemo-enzymatische Synthese von Selen-modifizierten Nukleosiden, Nukleotiden und Oligonukleotide