K(2P)18.1 translates T cell receptor signals into thymic regulatory T cell development

It remains largely unclear how thymocytes translate relative differences in T cell receptor (TCR) signal strength into distinct developmental programs that drive the cell fate decisions towards conventional (Tconv) or regulatory T cells (Treg). Following TCR activation, intracellular calcium (Ca2+) is the most important second messenger, for which the potassium channel K(2P)18.1 is a relevant regulator. Here, we identify K(2P)18.1 as a central translator of the TCR signal into the thymus-derived Treg (tTreg) selection process. TCR signal was coupled to NF-kappa B-mediated K(2P)18.1 upregulation in tTreg progenitors. K(2P)18.1 provided the driving force for sustained Ca2+ influx that facilitated NF-kappa B- and NFAT-dependent expression of FoxP3, the master transcription factor for Treg development and function. Loss of K(2P)18.1 ion-current function induced a mild lymphoproliferative phenotype in mice, with reduced Treg numbers that led to aggravated experimental autoimmune encephalomyelitis, while a gain-of-function mutation in K(2P)18.1 resulted in increased Treg numbers in mice. Our findings in human thymus, recent thymic emigrants and multiple sclerosis patients with a dominant-negative missense K(2P)18.1 variant that is associated with poor clinical outcomes indicate that K(2P)18.1 also plays a role in human Treg development. Pharmacological modulation of K(2P)18.1 specifically modulated Treg numbers in vitro and in vivo. Finally, we identified nitroxoline as a K(2P)18.1 activator that led to rapid and reversible Treg increase in patients with urinary tract infections. Conclusively, our findings reveal how K(2P)18.1 translates TCR signals into thymic T cell fate decisions and Treg development, and provide a basis for the therapeutic utilization of Treg in several human disorders.Peer reviewe

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Expression tuning in E.coli: discussion of state-of-the-art technologies and multivariate investigation of the pBAD mixed feed system

Background: Tuning of transcription is a powerful process technological tool for efficient recombinant protein production in E. coli. Many challenges such as product toxicity, formation of inclusion bodies, cell death and metabolic burden are associated with non-suitable (too high or too low) levels of recombinant protein expression. Tunable expression systems allow adjusting the recombinant protein expression using process technological means. This enables to exploit the cell's metabolic capacities to a maximum. Aims: In this thesis, tunable recombinant protein expression in E. coli is reviewed thoroughly from a biological and process technological point of view. In a consequent step expression tuning is applied to produce vascular endothelial growth factor-A165 (VEGF-A165), a pharmacologically relevant key player in angiogenesis, in correctly folded and active form in E. coli periplasm. Therefore the well-established system for expression tuning, the E. coli pBAD mixed feed platform, is investigated for the development of an upstream production process. Material and Methods: An E. coli C41 strain with intact L-arabinose metabolism was used in a mixed feed environment with D-glucose as main substrate and L-arabinose as inducing substrate. Following Quality by Design (QbD) principles, the three most promising critical process parameters (CPPs) namely the specific growth rate, specific inducer uptake rate and temperature were investigated in a design of experiments. A 23 factorial design (3 factors at 2 levels) for GIII and 22 factorial design in the case of DsbA signal sequence were conducted, each with a set of 3 center points. Results: So far expression tuning was only addressed in a few studies. For the first time these studies were reviewed with respect to latest findings on induction kinetics and mechanistics. According to the current level of knowledge some promoter system were successfully for expression tuning, in some cases analytical evidence on single cell level is still pending and some attempts did only influence protein expression on population level. For the first time, a promising mixed feed system was applied for tunable protein expression in the periplasm of E. coli. Beside of the observation of quality and quantity dependencies on the investigated CPPs it was demonstrated that the product transcription rate could indirectly be included in an experimental design by the successful use of a tunable promoter system in E. coli. Conclusion: In summary, the use of a tunable expression system was successfully applied in the development of an upstream process for the production of VEGF-A165 in the periplasm of E. coli. We anticipate that expression tuning is able to tackle further issues caused by inappropriate transcription levels and therefore is not only a major benefit for process development, but can pave the way for continuous production of biopharmaceuticals by the issues of constant product quality and culture long term stability14

Tunable recombinant protein expression in E. coli: promoter systems and genetic constraints

Tuning of transcription is a promising strategy to overcome challenges associated with a non-suitable expression rate like outgrowth of segregants, inclusion body formation, metabolic burden and inefficient translocation. By adjusting the expression rate—even on line—to purposeful levels higher product titres and more cost-efficient production processes can be achieved by enabling culture long-term stability and constant product quality. Some tunable systems are registered for patents or already commercially available. Within this contribution, we discuss the induction mechanisms of various Escherichia coli inherent promoter systems with respect to their tunability and review studies using these systems for expression tuning. According to the current level of knowledge, some promoter systems were successfully used for expression tuning, and in some cases, analytical evidence on single-cell level is still pending. However, only a few studies using tunable strains apply a suitable process control strategy. So far, expression tuning has only gathered little attention, but we anticipate that expression tuning harbours great potential for enabling and optimizing the production of a broad spectrum of products in E. coli.5015121

Tunable recombinant protein expression in E. coli: enabler for continuous processing?

The final publication is available at Springer via https://doi.org/10.1007/s00253-016-7550-4.Tuning of transcription is a powerful process technological tool for efficient recombinant protein production in Escherichia coli. Many challenges such as product toxicity, formation of inclusion bodies, cell death, and metabolic burden are associated with non-suitable (too high or too low) levels of recombinant protein expression. Tunable expression systems allow adjusting the recombinant protein expression using process technological means. This enables to exploit the cell’s metabolic capacities to a maximum. Within this article, we review genetic and process technological aspects of tunable expression systems in E. coli, providing a roadmap for the industrial exploitation of the reviewed technologies. We attempt to differentiate the term “expression tuning” from its inflationary use by providing a concise definition and highlight interesting fields of application for this versatile new technology. Dependent on the type of inducer (metabolizable or non-metabolizable), different process strategies are required in order to achieve tuning. To fully profit from the benefits of tunable systems, an independent control of growth rate and expression rate is indispensable. Being able to tackle problems such as long-term culture stability and constant product quality expression tuning is a promising enabler for continuous processing in biopharmaceutical production

On the application of focused ion beam nanotomography in characterizing the 3D pore space geometry of Opalinus clay

The evaluation and optimization of radioactive disposal systems requires a comprehensive understanding of mass transport processes. Among others, mass transport in porous geomaterials depends crucially on the topology and geometry of the pore space. Thus, understanding the mechanism of mass transport processes ultimately requires a 3D characterization of the pore structure. Here, we demonstrate the potential of focused ion beam nanotomography (FIB-nT) in characterizing the 3D geometry of pore space in clay rocks, i.e. Opalinus clay. In order to preserve the microstructure and to reduce sample preparation artefacts we used high pressure freezing and subsequent freeze drying to prepare the samples. Resolution limitations placed the lower limit in pore radii that can be analyzed by FIB-nT to about 10-15 nm. Image analysis and the calculation of pore size distribution revealed that pores with radii larger than 15 nm are related to a porosity of about 3 vol.%. To validate the method, we compared the pores size distribution obtained by FIB-nT with the one obtained by N2 adsorption analysis. The latter yielded a porosity of about 13 vol.%. This means that FIB-nT can describe around 20-30% of the total pore space. For pore radii larger than 15 nm the pore size distribution obtained by FIB-nT and N2 adsorption analysis were in good agreement. This suggests that FIB-nT can provide representative data on the spatial distribution of pores for pore sizes in the range of about 10-100 nm. Based on the spatial analysis of 3D data we extracted information on the spatial distribution of pore space geometrical properties

Mechanical anisotropy of Opalinus Clay shale: a multiscale approach

The mechanical properties of Opalinus Clay shale are strongly affected by the mineralogical composition of the layers and by their spatial arrangement at centimetre to decimetre scales. For this reason, accurate laboratory characterization of the mechanical properties, in many cases, is not representative of the material behaviour at a larger scale. A new approach is proposed to integrate petrophysical information from boreholes with small-scale mechanical laboratory characterization to extrapolate the distribution of mechanical properties to the scale of the engineering problem. The workflow is based on two fundamental concepts: defining a layering setting at different length scales from petrophysical investigations and creating a numerical representation of the mineralogically homogeneous layers. Merging the two concepts, it is possible to define the layered geometry of a finite element analysis of the laboratory tests used to calibrate the mechanical properties of the homogeneous layers. Once the properties of the layers have been assessed, the upscaling process can be performed using the geometric configuration at larger length scales. The layering setting is deduced, at the core scale, from petrophysical measurements and then extended by geostatistical prediction to the borehole scale. The workflow has been applied in a series of high accuracy laboratory tests to obtain the spatial distribution of mechanical properties at the borehole scale on the basis of the layering configuration deduced from a series of X-ray computed tomography tests