Towards Physical Hybrid Systems

Some hybrid systems models are unsafe for mathematically correct but physically unrealistic reasons. For example, mathematical models can classify a system as being unsafe on a set that is too small to have physical importance. In particular, differences in measure zero sets in models of cyber-physical systems (CPS) have significant mathematical impact on the mathematical safety of these models even though differences on measure zero sets have no tangible physical effect in a real system. We develop the concept of "physical hybrid systems" (PHS) to help reunite mathematical models with physical reality. We modify a hybrid systems logic (differential temporal dynamic logic) by adding a first-class operator to elide distinctions on measure zero sets of time within CPS models. This approach facilitates modeling since it admits the verification of a wider class of models, including some physically realistic models that would otherwise be classified as mathematically unsafe. We also develop a proof calculus to help with the verification of PHS.Comment: CADE 201

Short- and Long-Term Biomarkers for Bacterial Robustness: A Framework for Quantifying Correlations between Cellular Indicators and Adaptive Behavior

The ability of microorganisms to adapt to changing environments challenges the prediction of their history-dependent behavior. Cellular biomarkers that are quantitatively correlated to stress adaptive behavior will facilitate our ability to predict the impact of these adaptive traits. Here, we present a framework for identifying cellular biomarkers for mild stress induced enhanced microbial robustness towards lethal stresses. Several candidate-biomarkers were selected by comparing the genome-wide transcriptome profiles of our model-organism Bacillus cereus upon exposure to four mild stress conditions (mild heat, acid, salt and oxidative stress). These candidate-biomarkers—a transcriptional regulator (activating general stress responses), enzymes (removing reactive oxygen species), and chaperones and proteases (maintaining protein quality)—were quantitatively determined at transcript, protein and/or activity level upon exposure to mild heat, acid, salt and oxidative stress for various time intervals. Both unstressed and mild stress treated cells were also exposed to lethal stress conditions (severe heat, acid and oxidative stress) to quantify the robustness advantage provided by mild stress pretreatment. To evaluate whether the candidate-biomarkers could predict the robustness enhancement towards lethal stress elicited by mild stress pretreatment, the biomarker responses upon mild stress treatment were correlated to mild stress induced robustness towards lethal stress. Both short- and long-term biomarkers could be identified of which their induction levels were correlated to mild stress induced enhanced robustness towards lethal heat, acid and/or oxidative stress, respectively, and are therefore predictive cellular indicators for mild stress induced enhanced robustness. The identified biomarkers are among the most consistently induced cellular components in stress responses and ubiquitous in biology, supporting extrapolation to other microorganisms than B. cereus. Our quantitative, systematic approach provides a framework to search for these biomarkers and to evaluate their predictive quality in order to select promising biomarkers that can serve to early detect and predict adaptive traits

Heat stress leads to superoxide formation in Bacillus cereus detected using the fluorescent probe MitoSOX

Bacillus cereus is a food-borne human pathogen and food spoilage organism. Spores and vegetative cells of B. cereus can be found almost everywhere and therefore often end up in food processing equipment and food products. To remove spores and vegetative cells from food or equipment, harsh treatments such as high temperatures are applied. The heat stress response of B. cereus and other organisms has been studied and it has been shown that reactive oxygen species may be involved in inactivating the bacterial cells. Using a novel approach with the fluorescent probe MitoSOX, the formation of superoxide in B. cereus cells upon exposure to heat has been confirmed. MitoSOX can be used in combination with other probes, including, SYTOX green, CYTO 9, and CFDA, showing superoxide formation in combination with damaged cell membranes, intact cell membranes, and esterase activity in cells with intact membranes, respectively. MitoSOX in combination with flow cytometry-assisted sorting showed three distinct populations, a low fluorescent population that was still viable, a highly fluorescent population that could not be recovered on agar plates, and a low fluorescent non-viable population that appeared after prolonged exposure to heat. This third population may include dead cells where MitoSOX binds to DNA without reacting with superoxide. Superoxide formation during exposure to lethal temperatures by B. cereus shows that superoxide plays a role in bacterial cell death and its generation may thus contribute to the efficiency of food preservation conditions