The GeneOptimizer Algorithm: using a sliding window approach to cope with the vast sequence space in multiparameter DNA sequence optimization

One of the main advantages of de novo gene synthesis is the fact that it frees the researcher from any limitations imposed by the use of natural templates. To make the most out of this opportunity, efficient algorithms are needed to calculate a coding sequence, combining different requirements, such as adapted codon usage or avoidance of restriction sites, in the best possible way. We present an algorithm where a “variation window” covering several amino acid positions slides along the coding sequence. Candidate sequences are built comprising the already optimized part of the complete sequence and all possible combinations of synonymous codons representing the amino acids within the window. The candidate sequences are assessed with a quality function, and the first codon of the best candidates’ variation window is fixed. Subsequently the window is shifted by one codon position. As an example of a freely accessible software implementing the algorithm, we present the Mr. Gene web-application. Additionally two experimental applications of the algorithm are shown

AUC: Accountable Universal Composability

Accountability is a well-established and widely used security concept that allows for obtaining undeniable cryptographic proof of misbehavior, thereby incentivizing honest behavior. There already exist several general purpose accountability frameworks for formal game-based security analyses. Unfortunately, such game-based frameworks do not support modular security analyses, which is an important tool to handle the complexity of modern protocols. Universal composability (UC) models provide native support for modular analyses, including re-use and composition of security results. So far, accountability has mainly been modeled and analyzed in UC models for the special case of MPC protocols, with a general purpose accountability framework for UC still missing. That is, a framework that among others supports arbitrary protocols, a wide range of accountability properties, handling and mixing of accountable and non-accountable security properties, and modular analysis of accountable protocols. To close this gap, we propose AUC, the first general purpose accountability framework for UC models, which supports all of the above, based on several new concepts. We exemplify AUC in three case studies not covered by existing works. In particular, AUC unifies existing UC accountability approaches within a single framework

Regulated overexpression of the survival factor bcl-2 in CHO cells increases viable cell density in batch culture and decreases DNA release in extended fixed-bed cultivation

Using multicistronic expression technology we generated a stable Chinese hamster ovary (CHO) cell line (MG12) expressing a model secreted heterologous glycoprotein, the secreted form of the human placental alkaline phosphatase (SEAP), and bcl-2, best known as an apoptosis inhibitor, in a tetracycline-repressible dicistronic configuration. In batch cultivations in serum-containing medium, MG12 cells reached twice the final viable cell density when Bcl-2 was overexpressed (in the absence oftetracycline) compared to MG12 populations culturedunder tetracycline-containing conditions (bcl-2repressed). However, bcl-2-expressing MG12 cellsshowed no significant retardation of the decline phasecompared to batch cultures in which the dicistronicexpression unit was repressed.Genetic linkage of bcl-2 expression with the reporter protein SEAP in our multicistronic construct allowed online monitoring of Bcl-2 expression over an extended, multistage fixed-bed bioreactor cultivation. The cloned multicistronic expression unit proved to be stable over a 100 day bioreactor run. CHO MG12 cells in the fixed-bed reactor showed a drastic decrease in the release of DNA into the culture supernatant under conditions of reduced tetracycline (and hencederepressed SEAP and bcl-2 overexpression). This observation indicated enhanced robustness associated with bcl-2 overexpression, similar to recent findings for constitutive Bcl-2-overexpressing hybridoma cells under the same bioprocess conditions. These findings indicate, in these serum-containing CHO cell cultures, that overexpression of Bcl-2 results in desirable modifications in culture physiolog

Definition of crop specific areas for planning of distribution of field trials

Auf der Grundlage der Gliederung Deutschlands in Boden-Klima-Räume wurden pflanzenartspezifische Anbaugebiete definiert. Die Anbaugebiete berücksichtigen sowohl die Anbaubedeutung der jeweiligen Pflanzenart als auch deren Präferenzen bezüglich der Boden-Klima-Parameter. Jedes Anbaugebiet stellt gleichzeitig auch ein Beratungsgebiet dar. Das wichtigste Ziel dieser Gebietsgliederung bestand darin, die notwendige Genauigkeit und Repräsentativität der Versuchsergebnisse, die zur Ableitung praxisrelevanter, regionaler Beratungsempfehlungen genutzt werden, auch bei immer stärker eingeschränkten finanziellen und personellen Ressourcen im Versuchswesen zu gewährleisten. Die definierten Anbaugebiete bilden die Grundlage, um die bundesweite pflanzenbauliche Versuchsplanung entsprechend zu optimieren.The segmentation of Germany in soil-climate-spaces allowed to define crop-specific growing areas. The growing areas take into account how significant the growing of a crop is and which soil-climate-parameters a crop prefers. Each growing area represents also a consultation area. The main aim of area segmentation is to guarantee the needed precision and representativeness of experimental results which are used to derive practically oriented, regional advisory recommendations even if financial and personal resources get more and more limited. The defined growing areas form the basis to optimize the nation-wide planning of agronomic experiments

Development of a species-specific RNA polymerase I-based shRNA expression vector

RNA interference (RNAi) can be induced in vitro either by application of synthetic short interfering RNAs (siRNAs), or by intracellular expression of siRNAs or short hairpin RNAs (shRNAs) from transfected vectors. The most widely used promoters for siRNA/shRNA expression are based on polymerase III (Pol III)-dependent transcription. We developed an alternative vector for siRNA/shRNA expression, using a mouse RNA polymerase I (Pol I) promoter. Pol I-dependent transcription serves in cells for production of ribosomal RNA (rRNA), and as such, is ubiquitously and stably active in different cell types. As Pol I-dependent transcription is highly species-specific, Pol I-based system provides an important biosafety advantage with respect to silencing of genes with unknown functions

Aktuelle Trends bei der in vitro-Substanztestung in Deutschland und in der Schweiz

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Study of Field-Induced Magnetic Order in Singlet-Ground-State Magnet CsFeCl3_3

The field-induced magnetic order in the singlet-ground-state system CsFeCl$_3$ has been studied by measuring magnetization and neutron diffraction. The field dependence of intensity for the neutron magnetic reflection has clearly demonstrated that the field-induced ordered phase is described by the order parameter $$. A condensate growth of magnons is investigated through the temperature dependence of $M_z$ and $M_{\perp}$, and this ordering is discussed in the context of a magnon Bose-Einstein condensation. Development of the coherent state and the static correlation length has been observed in the incommensurate phase in the field region of $5 H_{\rm c}$, a satellite peak was found in coexistence with the commensurate peak at the phase boundary around 10 T, which indicates that the tilt of the c-axis would be less than $\sim 0.5^{\circ}$ in the whole experiments.Comment: 5 pages, 5 figure

Multiparameter RNA and Codon Optimization: A Standardized Tool to Assess and Enhance Autologous Mammalian Gene Expression

Autologous expression of recombinant human proteins in human cells for biomedical research and product development is often hampered by low expression yields limiting subsequent structural and functional analyses. Following RNA and codon optimization, 50 candidate genes representing five classes of human proteins – transcription factors, ribosomal and polymerase subunits, protein kinases, membrane proteins and immunomodulators – all showed reliable, and 86% even elevated expression. Analysis of three representative examples showed no detrimental effect on protein solubility while unaltered functionality was demonstrated for JNK1, JNK3 and CDC2 using optimized constructs. Molecular analysis of a sequence-optimized transgene revealed positive effects at transcriptional, translational, and mRNA stability levels. Since improved expression was consistent in HEK293T, CHO and insect cells, it was not restricted to distinct mammalian cell systems. Additionally, optimized genes represent powerful tools in functional genomics, as demonstrated by the successful rescue of an siRNA-mediated knockdown using a sequence-optimized counterpart. This is the first large-scale study addressing the influence of multiparameter optimization on autologous human protein expression

Continuous Adaptive Evolution of a Fast-Growing Corynebacterium glutamicum Strain Independent of Protocatechuate

Graf M, Haas T, Mueller F, et al. Continuous Adaptive Evolution of a Fast-Growing Corynebacterium glutamicum Strain Independent of Protocatechuate. Frontiers in Microbiology . 2019;10: 1648.Corynebacterium glutamicum is a commonly applied host for the industrial production of amino acids. While valued for its robustness, it is somewhat inferior to competing strains such as Escherichia coli because of the relatively low growth rate of 0.40 h(-1) in synthetic, industrial media. Accordingly, adaptive laboratory evolution (ALE) experiments were performed in continuous cultivation mode to select for a growth-improved host. To ensure industrial attractiveness, this ALE study aimed at a reduction of dependency on costly growth-boosting additives such as protocatechuate (RCA) or complex media supplements. Consequently, double selection pressures were installed consisting of a steady increase in growth rate demands and a parallel reduction of complex medium fractions. Selection yielded C. glutamicum EVO5 achieving 0.54 h(-1) and 1.03g(Glc) g(CDW)(-1) h(-1) in minimal medium without above mentioned supplements. Sequencing revealed 10 prominent mutations, three of them in key regulator genes

A Security Framework for Distributed Ledgers

In the past few years blockchains have been a major focus for security research, resulting in significant progress in the design, formalization, and analysis of blockchain protocols. However, the more general class of distributed ledgers, which includes not just blockchains but also prominent non-blockchain protocols, such as Corda and OmniLedger, cannot be covered by the state-of-the-art in the security literature yet. These distributed ledgers often break with traditional blockchain paradigms, such as block structures to store data, system-wide consensus, or global consistency. In this paper, we close this gap by proposing the first framework for defining and analyzing the security of general distributed ledgers, with an ideal distributed ledger functionality, called $\mathcal{F}_\text{ledger}$, at the core of our contribution. This functionality covers not only classical blockchains but also non-blockchain distributed ledgers in a unified way. To illustrate $\mathcal{F}_\text{ledger}$, we first show that the prominent ideal blockchain functionalities $\mathcal{G}_\text{ledger}$ and $\mathcal{G}_\text{PL}$ realize (suitable instantiations of) $\mathcal{F}_\text{ledger}$, which precisely captures their security properties. This immediately implies that their respective implementations, including Bitcoin, Ouroboros Genesis, and Ouroboros Crypsinous, realize $\mathcal{F}_\text{ledger}$ as well. Secondly, we demonstrate that $\mathcal{F}_\text{ledger}$ is capable of precisely modeling also non-blockchain distributed ledgers by performing the first formal security analysis of such a distributed ledger, namely the prominent Corda protocol. Due to the wide spread use of Corda in the industry, in particular the financial sector, this analysis is of independent interest. These results also illustrate that $\mathcal{F}_\text{ledger}$ not just generalizes the modular treatment of blockchains to distributed ledgers, but moreover helps to unify existing results