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The verification approach of Ultimate Automizer utilizes SMT formulas. This paper presents techniques to keep the size of the formulas small. We focus especially on a normal form, called CommuHash normal form that was easy to implement and had a significant impact on the runtime of our tool.

Ultimate GemCutterverifies concurrent programs using the CEGAR paradigm, by generalizing from spurious counterexample traces to larger sets of correct traces. We integrate classical CEGAR generalization with orthogonal generalization across interleavings. Thereby, we are able to prove correctness of programs otherwise out-of-reach for interpolation-based verification. The competition results show significant advantages over other concurrency approaches in theUltimatefamily.

Given a verification problem for a concurrent program (with a fixed number of threads) over infinite data domains, we can construct a model checking problem for an abstraction of the concurrent program through a Petri net (a problem which can be solved using McMillan’s unfoldings technique). We present a method of abstraction refinement which translates Floyd/Hoare-style proofs for sample traces into additional synchronization constraints for the Petri net.

// Dominik Klumpp 1 , Stephanie C. Frank 2, 3 , Lukas Klumpp 1, 4 , 5 , Efe C. Sezgin 1 , Marita Eckert 1 , Lena Edalat 1, 6 , Martin Bastmeyer 2, 3 , Daniel Zips 1 , Peter Ruth 6 and Stephan M. Huber 1 1 Department of Radiation Oncology, University of Tubingen, Tubingen, Germany 2 Zoological Institute, Department of Cell- and Neurobiology, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany 3 Institute of Functional Interfaces, Karlsruhe Institute of Technology (KIT), Eggenstein-Leopoldshafen, Germany 4 Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart, Germany 5 University of Tubingen, Tubingen, Germany 6 Department of Pharmacology, Toxicology and Clinical Pharmacy, University of Tubingen, Tubingen, Germany Correspondence to: Stephan M. Huber, email: stephan.huber@uni-tuebingen.de Keywords: glioblastoma; radioresistance; TRPM8 channels; Ca 2+ signaling; cell migration Received: July 26, 2017 Accepted: August 25, 2017 Published: September 30, 2017 ABSTRACT TRPM8 is a Ca 2+ -permeable nonselective cation channel belonging to the melastatin sub-group of the transient receptor potential (TRP) family. TRPM8 is aberrantly overexpressed in a variety of tumor entities including glioblastoma multiforme where it reportedly contributes to tumor invasion. The present study aimed to disclose further functions of TRPM8 in glioma biology in particular upon cell injury by ionizing radiation. To this end, TCGA data base was queried to expose the TRPM8 mRNA abundance in human glioblastoma specimens and immunoblotting was performed to analyze the TRPM8 protein abundance in primary cultures of human glioblastoma. Moreover, human glioblastoma cell lines were irradiated with 6 MV photons and TRPM8 channels were targeted pharmacologically or by RNA interference. TRPM8 abundance, Ca 2+ signaling and resulting K + channel activity, chemotaxis, cell migration, clonogenic survival, DNA repair, apoptotic cell death, and cell cycle control were determined by qRT-PCR, fura-2 Ca 2+ imaging, patch-clamp recording, transfilter migration assay, wound healing assay, colony formation assay, immunohistology, flow cytometry, and immunoblotting. As a result, human glioblastoma upregulates TRPM8 channels to variable extent. TRPM8 inhibition or knockdown slowed down cell migration and chemotaxis, attenuated DNA repair and clonogenic survival, triggered apoptotic cell death, impaired cell cycle and radiosensitized glioblastoma cells. Mechanistically, ionizing radiation activated and upregulated TRPM8-mediated Ca 2+ signaling that interfered with cell cycle control probably via CaMKII, cdc25C and cdc2. Combined, our data suggest that TRPM8 channels contribute to spreading, survival and radioresistance of human glioblastoma and, therefore, might represent a promising target in future anti-glioblastoma therapy.

Background Radiotherapy (RT) is used to treat retinoblastoma (Rb), the most frequent ocular tumour in children. Besides eradicating the tumour, RT can cause severe side effects including secondary malignancies. This study aimed to define whether the radioprotector ortho-phospho- L -tyrosine (pTyr) prevents RT-induced side effects and affects local tumour control in a xeno graft and a genetic orthotopic Rb mouse model. Methods B6;129-Rb1tm3Tyj/J (Rb +/− ) and Y79-Rb cell- xeno grafted nude mice were fractionated external beam irradiated (15 fractions of 5 Gy 6 MV photons during 3 weeks) with or without pTyr pre-treatment (100 mg/kg BW, 16 h prior to each irradiation). One, three, six and nine months after RT, tumour control and RT toxicity were evaluated using in vivo imaging and histology. We also analysed pTyr dependant post irradiation cell survival and p53 activity in vitro . Results In vitro pTyr pre-treatment showed no radioprotection on Y79 cells, but led to p53 stabilisation in unirradiated Y79 cells and to a facilitation of radiation-induced p21 up-regulation, confirming a modulation of p53 activity by pTyr. In both mouse models, secondary tumours were undetectable. In Rb +/− mice, pTyr significantly lowered RT-induced greying of the fur, retinal thickness reduction and photoreceptor loss. However, in the xeno grafted Rb model, pTyr considerably decreased RT-mediated tumour control, which was observed in 16 out of 22 control eyes but in none of the 24 pTyr treated eyes. Conclusions In Rb +/− mice pTyr significantly prevents RT-induced greying of the fur as well as retinal degeneration. However, since non-irradiated control mice were not used in our study, a formal possibility exists that the effect shown in the retina of Rb +/− mice may be due to ageing of the animals and/or actions of pTyr alone. Unfortunately, as tested in a xeno graft model, pTyr treatment reduced the control of Rb tumours.

K+ channels crosstalk with biochemical signaling cascades and regulate virtually all cellular processes by adjusting the intracellular K+ concentration, generating the membrane potential, mediating cell volume changes, contributing to Ca2+ signaling, and directly interacting within molecular complexes with membrane receptors and downstream effectors. Tumor cells exhibit aberrant expression and activity patterns of K+ channels. The upregulation of highly "oncogenic" K+ channels such as the Ca2+-activated IK channel may drive the neoplastic transformation, malignant progression, metastasis, or therapy resistance of tumor cells. In particular, ionizing radiation in doses used for fractionated radiotherapy in the clinic has been shown to activate K+ channels. Radiogenic K+ channel activity, in turn, contributes to the DNA damage response and promotes survival of the irradiated tumor cells. Tumor-specific overexpression of certain K+ channel types together with the fact that pharmacological K+ channel modulators are already in clinical use or well tolerated in clinical trials suggests that K+ channel targeting alone or in combination with radiotherapy might become a promising new strategy of anti-cancer therapy. The present article aims to review our current knowledge on K+ channel signaling in irradiated tumor cells. Moreover, it provides new data on molecular mechanisms of radiogenic K+ channel activation and downstream signaling events.

By restructuring and reconfiguring itself at run-time, a collective adaptive system (CAS) is able to fulfill its requirements under uncertain, ever-changing environmental conditions. Indeed, this process of self-organization (SO) is of utmost importance for the ability of the CAS to perform. However, it is hard to design high-performing SO mechanisms, because the environmental conditions are partially unpredictable at design time. Thus, a crucial aid for the development of SO mechanisms is a tool set enabling performance evaluations at design time in order to select the best-fitting mechanism and parametrize it. We present a metric for measuring the performance of an SO mechanism as well as a framework that enables evaluation of this metric. The proposed metric is evaluated for different kinds of SO mechanisms in two case studies: a smart energy management system and a self-organizing production cell.

Although much is known about chemotaxis- induced by gradients of soluble chemical cues – the molecular mechanisms involved in haptotaxis (migration induced by substrate-bound protein gradients) are largely unknown. We used micropatterning to produce discontinuous gradients consisting of μm-sized fibronectin-dots arranged at constant lateral but continuously decreasing axial spacing. Parameters like gradient slope, protein concentration and size or shape of the fibronectin dots were modified to determine optimal conditions for directional cell migration in gradient patterns. We demonstrate that fibroblasts predominantly migrate uphill towards a higher fibronectin density in gradients with a dot size of 2 × 2 μm, a 2% and 6% slope, and a low fibronectin concentration of 1 μg ml−1. Increasing dot size to 3.5 × 3.5 μm resulted in stationary cells, whereas rectangular dots (2 × 3 μm) orientated perpendicular to the gradient axis preferentially induce lateral migration. During haptotaxis, the Golgi apparatus reorients to a posterior position between the nucleus and the trailing edge. Using pharmacological inhibitors, we demonstrate that actomyosin contractility and microtubule dynamics are a prerequisite for gradient recognition indicating that asymmetric intracellular forces are necessary to read the axis of adhesive gradients. In the haptotaxis signalling cascade, RhoA and Cdc42, and the atypical protein kinase C zeta (aPKCζ), but not Rac, are located upstream of actomyosin contractility.

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Neoadjuvant, adjuvant or definitive fractionated radiation therapy are implemented in first line anti-cancer treatment regimens of many tumor entities. Ionizing radiation kills the tumor cells mainly by causing double strand breaks of their DNA through formation of intermediate radicals. Survival of the tumor cells depends on both, their capacity of oxidative defense and their efficacy of DNA repair. By damaging the targeted cells, ionizing radiation triggers a plethora of stress responses. Among those is the modulation of ion channels such as Ca2+-activated K+ channels or Ca2+-permeable nonselective cation channels belonging to the super-family of transient receptor potential channels. Radiogenic activation of these channels may contribute to radiogenic cell death as well as to DNA repair, glucose fueling, radiogenic hypermigration or lowering of the oxidative stress burden. The present review article introduces these channels and summarizes our current knowledge on the mechanisms underlying radiogenic ion channel modulation. This article is part of a Special Issue entitled: Membrane channels and transporters in cancers.

The standard treatment of many tumor entities comprises fractionated radiation therapy which applies ionizing radiation to the tumor-bearing target volume. Ionizing radiation causes double-strand breaks in the DNA backbone that result in cell death if the number of DNA double-strand breaks exceeds the DNA repair capacity of the tumor cell. Ionizing radiation reportedly does not only act on the DNA in the nucleus but also on the plasma membrane. In particular, ionizing radiation-induced modifications of ion channels and transporters have been reported. Importantly, these altered transports seem to contribute to the survival of the irradiated tumor cells. The present review article summarizes our current knowledge on the underlying mechanisms and introduces strategies to radiosensitize tumor cells by targeting plasma membrane ion transports.

Aberrant ion channel expression in the plasma membrane is characteristic for many tumor entities and has been attributed to neoplastic transformation, tumor progression, metastasis, and therapy resistance. The present study aimed to define the function of these "oncogenic" channels for radioresistance of leukemia cells. Chronic myeloid leukemia cells were irradiated (0-6 Gy X ray), ion channel expression and activity, Ca(2+)- and protein signaling, cell cycle progression, and cell survival were assessed by quantitative reverse transcriptase-polymerase chain reaction, patch-clamp recording, fura-2 Ca(2+)-imaging, immunoblotting, flow cytometry, and clonogenic survival assays, respectively. Ionizing radiation-induced G2/M arrest was preceded by activation of Kv3.4-like voltage-gated potassium channels. Channel activation in turn resulted in enhanced Ca(2+) entry and subsequent activation of Ca(2+)/calmodulin-dependent kinase-II, and inactivation of the phosphatase cdc25B and the cyclin-dependent kinase cdc2. Accordingly, channel inhibition by tetraethylammonium and blood-depressing substance-1 and substance-2 or downregulation by RNA interference led to release from radiation-induced G2/M arrest, increased apoptosis, and decreased clonogenic survival. Together, these findings indicate the functional significance of voltage-gated K(+) channels for the radioresistance of myeloid leukemia cells.