A red/far-red light-responsive bi-stable toggle switch to control gene expression in mammalian cells

Growth and differentiation of multicellular systems is orchestrated by spatially restricted gene expression programs in specialized subpopulations. The targeted manipulation of such processes by synthetic tools with high-spatiotemporal resolution could, therefore, enable a deepened understanding of developmental processes and open new opportunities in tissue engineering. Here, we describe the first red/far-red light-triggered gene switch for mammalian cells for achieving gene expression control in time and space. We show that the system can reversibly be toggled between stable on- and off-states using short light pulses at 660 or 740 nm. Red light-induced gene expression was shown to correlate with the applied photon number and was compatible with different mammalian cell lines, including human primary cells. The light-induced expression kinetics were quantitatively analyzed by a mathematical model. We apply the system for the spatially controlled engineering of angiogenesis in chicken embryos. The system's performance combined with cell- and tissue-compatible regulating red light will enable unprecedented spatiotemporally controlled molecular interventions in mammalian cells, tissues and organisms

A comparison of global optimisation algorithms with standard benchmark functions and real-world applications using EnergyPlus

There is an increasing interest in the use of computer algorithms to identify combinations of parameters which optimise the energy performance of buildings. For such problems, the objective function can be multi-modal and needs to be approximated numerically using building energy simulation programs. As these programs contain iterative solution algorithms, they introduce discontinuities in the numerical approximation to the objective function. Metaheuristics often work well for such problems, but their convergence to a global optimum cannot be established formally. Moreover, different algorithms tend to be suited to particular classes of optimization problems. To shed light on this issue we compared the performance of two metaheuristics, the hybrid CMA-ES/HDE and the hybrid PSO/HJ, in minimizing standard benchmark functions and real-world building energy optimization problems of varying complexity. From this we find that the CMA-ES/HDE performs well on more complex objective functions, but that the PSO/HJ more consistently identifies the global minimum for simpler objective functions. Both identified similar values in the objective functions arising from energy simulations, but with different combinations of model parameters. This may suggest that the objective function is multi-modal. The algorithms also correctly identified some non-intuitive parameter combinations that were caused by a simplified control sequence of the building energy system that does not represent actual practice, further reinforcing their utility

A red/far-red light-responsive bi-stable toggle switch to control gene expression in mammalian cells

Growth and differentiation of multicellular systems is orchestrated by spatially restricted gene expression programs in specialized subpopulations. The targeted manipulation of such processes by synthetic tools with high-spatiotemporal resolution could, therefore, enable a deepened understanding of developmental processes and open new opportunities in tissue engineering. Here, we describe the first red/far-red light-triggered gene switch for mammalian cells for achieving gene expression control in time and space. We show that the system can reversibly be toggled between stable on- and off-states using short light pulses at 660 or 740 nm. Red light-induced gene expression was shown to correlate with the applied photon number and was compatible with different mammalian cell lines, including human primary cells. The light-induced expression kinetics were quantitatively analyzed by a mathematical model. We apply the system for the spatially controlled engineering of angiogenesis in chicken embryos. The system's performance combined with cell- and tissue-compatible regulating red light will enable unprecedented spatiotemporally controlled molecular interventions in mammalian cells, tissues and organism

Low Energy Electron Irradiation Is a Potent Alternative to Gamma Irradiation for the Inactivation of (CAR-)NK-92 Cells in ATMP Manufacturing

Background: With increasing clinical use of NK-92 cells and their CAR-modified derivatives in cancer immunotherapy, there is a growing demand for efficient production processes of these “off-the-shelf” therapeutics. In order to ensure safety and prevent the occurrence of secondary tumors, (CAR-)NK-92 cell proliferation has to be inactivated before transfusion. This is commonly achieved by gamma irradiation. Recently, we showed proof of concept that low energy electron irradiation (LEEI) is a new method for NK-92 inactivation. LEEI has several advantages over gamma irradiation, including a faster reaction time, a more reproducible dose rate and much less requirements on radiation shielding. Here, LEEI was further evaluated as a promising alternative to gamma irradiation yielding cells with highly maintained cytotoxic effector function. Methods: Effectiveness and efficiency of LEEI and gamma irradiation were analyzed using NK-92 and CD123-directed CAR-NK-92 cells. LEE-irradiated cells were extensively characterized and compared to gamma-irradiated cells via flow cytometry, cytotoxicity assays, and comet assays, amongst others. Results: Our results show that both irradiation methods caused a progressive decrease in cell viability and are, therefore, suitable for inhibition of cell proliferation. Notably, the NKmediated specific lysis of tumor cells was maintained at stable levels for three days postirradiation, with a trend towards higher activities after LEEI treatment as compared to gamma irradiation. Both gamma irradiation as well as LEEI led to substantial DNA damage and an accumulation of irradiated cells in the G2/M cell cycle phases. In addition, transcriptomic analysis of irradiated cells revealed approximately 12-fold more differentially expressed genes two hours after gamma irradiation, compared to LEEI. Analysis of surface molecules revealed an irradiation-induced decrease in surface expression of CD56, but no changes in the levels of the activating receptors NKp46, NKG2D, or NKp30. Conclusions: The presented data show that LEEI inactivates (CAR-)NK-92 cells as efficiently as gamma irradiation, but with less impact on the overall gene expression. Due to logistic advantages, LEEI might provide a superior alternative for the manufacture of (CAR-)NK-92 cells for clinical application

Probleme der Tourenbildung

Die Tourenbildung beschäftigt sich mit der Konstruktion kostengünstiger Transportrouten zur Belieferung von Verbrauchern. Sie ist eine der weitreichensten Erfolgsgeschichten des Operations Research. Das starke Interesse an diesen Problemen durch Industrie und Forschung liegt zum einen am wirtschaftlichen Potenzial der Tourenbildung und -optimierung, zum anderen macht ihr Reichtum an Struktur sie zu einem faszinierenden Forschungsgebiet. In der vorliegenden Arbeit soll ein Überblick über einige, u. a. auch neuere mathematische Modell- und Lösungsansätze gegeben werden. Auf Grund der hohen Anzahl der Veröffentlichungen auf diesem Gebiet wird nicht zwingend ein Anspruch auf die vollständige Darlegung aller möglichen Problemstellungen im Zusammenhang mit dem TSP sowie dem VRP und deren Lösungsansätze erhoben. An den gegebenen Stellen wird statt dessen auf weiterführende Literatur verwiesen