A novel MCF-10A line allowing conditional oncogene expression in 3D culture

Introduction Non-transformed mammary epithelial cell lines such as MCF-10A recapitulate epithelial morphogenesis in three-dimensional (3D) tissue culture by forming acinar structures. They represent an important tool to characterize the biological properties of oncogenes and to model early carcinogenic events. So far, however, these approaches were restricted to cells with constitutive oncogene expression prior to the set-up of 3D cultures. Although very informative, this experimental setting has precluded the analysis of effects caused by sudden oncoprotein expression or withdrawal in established epithelial cultures. Here, we report the establishment and use of a stable MCF-10A cell line (MCF-10Atet) fitted with a novel and improved doxycycline (dox)-regulated expression system allowing the conditional expression of any transgene. Methods MCF-10Atet cells were generated by stable transfection with pWHE644, a vector expressing a second generation tetracycline-regulated transactivator and a novel transcriptional silencer. In order to test the properties of this new repressor/activator switch, MCF-10Atet cells were transfected with a second plasmid, pTET-HABRAF-IRES-GFP, which responds to dox treatment with the production of a bi-cistronic transcript encoding hemagglutinin-tagged B-Raf and green fluorescent protein (GFP). This improved conditional expression system was then characterized in detail in terms of its response to various dox concentrations and exposure times. The plasticity of the phenotype provoked by oncogenic B-RafV600E in MCF-10Atet cells was analyzed in 3D cultures by dox exposure and subsequent wash-out. Results MCF-10Atet cells represent a tightly controlled, conditional gene expression system. Using B-RafV600E as a model oncoprotein, we show that its sudden expression in established 3D cultures results in the loss of acinar organization, the induction of an invasive phenotype and hallmarks of epithelial-to-mesenchymal transition (EMT). Importantly, we show for the first time that this severe transformed phenotype can be reversed by dox wash-out and concomitant termination of oncogene expression. Conclusions Taken together, we have generated a stable MCF-10A subline allowing tight dox-controlled and reversible expression of any transgene without the need to modify its product by introducing artificial dimerization or ligand-binding domains. This system will be very valuable to address phenomena such as EMT, oncogene addiction, oncogene-induced senescence and drug resistance

The Hilbert basis method for D-flat directions and the superpotential

We discuss, using the Hilbert basis method, how to efficiently construct a complete basis for D-flat directions in supersymmetric Abelian and non-Abelian gauge theories. We extend the method to discrete (R and non-R) symmetries. This facilitates the construction of a basis of all superpotential terms in a theory with given symmetries.Comment: 11 pages; a related mathematica code can be found at http://einrichtungen.ph.tum.de/T30e/codes/NonAbelianHilbert

Comparing Evapotranspiration from Eddy Covariance Measurements, Water Budgets, Remote Sensing, and Land Surface Models over Canada

This study compares six evapotranspiration ET products for Canada’s landmass, namely, eddy covariance EC measurements; surface water budget ET; remote sensing ET from MODIS; and land surface model (LSM) ET from the Community Land Model (CLM), the Ecological Assimilation of Land and Climate Observations (EALCO) model, and the Variable Infiltration Capacity model (VIC). The ET climatology over the Canadian landmass is characterized and the advantages and limitations of the datasets are discussed. The EC measurements have limited spatial coverage, making it difficult for model validations at the national scale. Water budget ET has the largest uncertainty because of data quality issues with precipitation in mountainous regions and in the north. MODIS ET shows relatively large uncertainty in cold seasons and sparsely vegetated regions. The LSM products cover the entire landmass and exhibit small differences in ET among them. Annual ET from the LSMs ranges from small negative values to over 600 mm across the landmass, with a countrywide average of 256 ± 15 mm. Seasonally, the countrywide average monthly ET varies from a low of about 3 mm in four winter months (November–February) to 67 ± 7 mm in July. The ET uncertainty is scale dependent. Larger regions tend to have smaller uncertainties because of the offset of positive and negative biases within the region. More observation networks and better quality controls are critical to improving ET estimates. Future techniques should also consider a hybrid approach that integrates strengths of the various ET products to help reduce uncertainties in ET estimation

Linking scales and disciplines : an interdisciplinary cross-scale approach to supporting climate-relevant ecosystem management

CITATION: Berger, C. et al. 2019. Linking scales and disciplines : an interdisciplinary cross-scale approach to supporting climate-relevant ecosystem management. Climatic Change, 156:139–150, doi:10.1007/s10584-019-02544-0.The original publication is available at https://www.springer.com/journal/10584Southern Africa is particularly sensitive to climate change, due to both ecological and socioeconomic factors, with rural land users among the most vulnerable groups. The provision of information to support climate-relevant decision-making requires an understanding of the projected impacts of change and complex feedbacks within the local ecosystems, as well as local demands on ecosystem services. In this paper, we address the limitation of current approaches for developing management relevant socio-ecological information on the projected impacts of climate change and human activities.We emphasise the need for linking disciplines and approaches by expounding the methodology followed in our two consecutive projects. These projects combine disciplines and levels of measurements from the leaf level (ecophysiology) to the local landscape level (flux measurements) and from the local household level (socio-economic surveys) to the regional level (remote sensing), feeding into a variety of models at multiple scales. Interdisciplinary, multi-scaled, and integrated socio-ecological approaches, as proposed here, are needed to compliment reductionist and linear, scalespecific approaches. Decision support systems are used to integrate and communicate the data and models to the local decision-makers.https://link.springer.com/article/10.1007/s10584-019-02544-0Publisher's versio

Restricting Glycolysis Preserves T Cell Effector Functions and Augments Checkpoint Therapy

Tumor-derived lactic acid inhibits T and natural killer (NK) cell function and, thereby, tumor immunosurveillance. Here, we report that melanoma patients with high expression of glycolysis-related genes show a worse progression free survival upon anti-PD1 treatment. The non-steroidal anti-inflammatory drug (NSAID) diclofenac lowers lactate secretion of tumor cells and improves anti-PD1-induced T cell killing in vitro. Surprisingly, diclofenac, but not other NSAIDs, turns out to be a potent inhibitor of the lactate transporters monocarboxylate transporter 1 and 4 and diminishes lactate efflux. Notably, T cell activation, viability, and effector functions are preserved under diclofenac treatment and in a low glucose environment in vitro. Diclofenac, but not aspirin, delays tumor growth and improves the efficacy of checkpoint therapy in vivo. Moreover, genetic suppression of glycolysis in tumor cells strongly improves checkpoint therapy. These findings support the rationale for targeting glycolysis in patients with high glycolytic tumors together with checkpoint inhibitors in clinical trials

MiniDAQ-3: Providing concurrent independent subdetector data-taking on CMS production DAQ resources

The data acquisition (DAQ) of the Compact Muon Solenoid (CMS) experiment at CERN, collects data for events accepted by the Level-1 Trigger from the different detector systems and assembles them in an event builder prior to making them available for further selection in the High Level Trigger, and finally storing the selected events for offline analysis. In addition to the central DAQ providing global acquisition functionality, several separate, so-called “MiniDAQ” setups allow operating independent data acquisition runs using an arbitrary subset of the CMS subdetectors. During Run 2 of the LHC, MiniDAQ setups were running their event builder and High Level Trigger applications on dedicated resources, separate from those used for the central DAQ. This cleanly separated MiniDAQ setups from the central DAQ system, but also meant limited throughput and a fixed number of possible MiniDAQ setups. In Run 3, MiniDAQ-3 setups share production resources with the new central DAQ system, allowing each setup to operate at the maximum Level-1 rate thanks to the reuse of the resources and network bandwidth. Configuration management tools had to be significantly extended to support the synchronization of the DAQ configurations needed for the various setups. We report on the new configuration management features and on the first year of operational experience with the new MiniDAQ-3 system

The CMS Orbit Builder for the HL-LHC at CERN

The Compact Muon Solenoid (CMS) experiment at CERN incorporates one of the highest throughput data acquisition systems in the world and is expected to increase its throughput by more than a factor of ten for High-Luminosity phase of Large Hadron Collider (HL-LHC). To achieve this goal, the system will be upgraded in most of its components. Among them, the event builder software, in charge of assembling all the data read out from the different sub-detectors, is planned to be modified from a single event builder to an orbit builder that assembles multiple events at the same time. The throughput of the event builder will be increased from the current 1.6 Tb/s to 51 Tb/s for the HL-LHC orbit builder. This paper presents preliminary network transfer studies in preparation for the upgrade. The key conceptual characteristics are discussed, concerning differences between the CMS event builder in Run 3 and the CMS Orbit Builder for the HL-LHC. For the feasibility studies, a pipestream benchmark, mimicking event-builder-like traffic has been developed. Preliminary performance tests and results are discussed

Towards a container-based architecture for CMS data acquisition

The CMS data acquisition (DAQ) is implemented as a service-oriented architecture where DAQ applications, as well as general applications such as monitoring and error reporting, are run as self-contained services. The task of deployment and operation of services is achieved by using several heterogeneous facilities, custom configuration data and scripts in several languages. In this work, we restructure the existing system into a homogeneous, scalable cloud architecture adopting a uniform paradigm, where all applications are orchestrated in a uniform environment with standardized facilities. In this new paradigm DAQ applications are organized as groups of containers and the required software is packaged into container images. Automation of all aspects of coordinating and managing containers is provided by the Kubernetes environment, where a set of physical and virtual machines is unified in a single pool of compute resources. We demonstrate that a container-based cloud architecture provides an acrossthe-board solution that can be applied for DAQ in CMS. We show strengths and advantages of running DAQ applications in a container infrastructure as compared to a traditional application model

2020 Proceedings of the 3rd International Conference on Trauma Surgery Technology in Giessen

The 3 rd event of the Giessen International Conference on Trauma Surgery Technology on October, the 17th 2020 was hosted on Zoom in accordance with the worldwide corona situation. Dr Mieczakowski, Dr Yu, and Wolfram drafted in 2018 from Jan’s apartment in Bremen the manuscript which was submitted to and approved for funding by the Deutsche Forschungsgemeinschaft (DFG). At that time, we had no idea what substantial changes the conferencing concept would require. This is why we would like to thank again Michele. She first planned this year’s event after the 2019 date and then in the spring of 2020 had to replan for the new situation