MAVEN Deliverable 7.2: Impact Assessment - Technical Report

This deliverable focuses on an important topic within the MAVEN project - evaluation of the project impact. This is an important step that will allow us to say what the results and impact of the different technologies, functionalities as well as assumptions are. It covers different dimensions of the impact assessment as stated in the Deliverable D7.1 - Impact assessment plan [10]. The field tests proved that the technology in the vehicle works together with the infrastructure and the solution is technically feasible. This was demonstrated also during particular events and is reported in the attached test protocols. At the same time, the emulation and simulation in Dominion software proved the functionality, for example with respect to the cooperative perception or safety indicators. The tests also proved that the key performance indicator "minimum time to the collision" decreases when applying the cooperative sensing. Also, the number of human interventions needed was zero in all the tests. This deliverable also discussed selected results of a detailed user survey aiming at understanding the expected impacts and transition of automated vehicles. The overall number of respondents reached 209. The responses have revealed some interesting facts. For example, over 80% of the respondents believe that CAVs will decrease the number of traffic accidents. Similarly, about 70% of the respondents expect improvements in traffic congestions. Over 82% of respondents declared that they would accept some detour when driving if it helps the overall traffic situation. The literature review, however, indicated that autonomous vehicles will have either a positive or a negative effect on the environment, depending on the policies. For example, opening cars as a mode of transport to new user groups (seniors, children etc.) together with improvements of the traffic, flow parameters can increase the traffic volume on roads. Policy makers shall focus on the integration of the CAVs into a broader policy concept including car or ride-sharing, electromobility and others. In order to evaluate the transition, for example, the influence of different penetration rates of CAVs on the performance, a microscopic traffic simulation was performed. Here the particular MAVEN use cases, as well as their combination, was addressed. The results of the simulation are rather promising. The potential for improvements in traffic performance is clearly there. It was demonstrated that a proper integration of CAVs into city traffic management can, for example, help with respect to the environmental goals (Climate Action of the European Commission) and reduce CO2 emissions by up to 12 % (a combination of GLOSA and signal optimization). On corridors with a green wave, a capacity increase of up to 34% was achieved. The conclusions from this project can be used not only by other researchers but mainly by traffic managers and decision-makers in cities. The findings can get a better idea about the real impacts of particular use cases (such as green wave, GLOSA and others) in the cities. An important added value is also the focus on the transition phase. It was demonstrated that already for lower penetration rates (even 20% penetration of automated vehicles), there are significant improvements in traffic performance. For example, the platooning leads to a decrease of CO2 emissions of 2,6% or the impact indicator by 17,7%

YAP regulates cell mechanics by controlling focal adhesion assembly

Hippo effectors YAP/TAZ act as on-off mechanosensing switches by sensing modifications in extracellular matrix (ECM) composition and mechanics. The regulation of their activity has been described by a hierarchical model in which elements of Hippo pathway are under the control of focal adhesions (FAs). Here we unveil the molecular mechanism by which cell spreading and RhoA GTPase activity control FA formation through YAP to stabilize the anchorage of the actin cytoskeleton to the cell membrane. This mechanism requires YAP co-transcriptional function and involves the activation of genes encoding for integrins and FA docking proteins. Tuning YAP transcriptional activity leads to the modification of cell mechanics, force development and adhesion strength, and determines cell shape, migration and differentiation. These results provide new insights into the mechanism of YAP mechanosensing activity and qualify this Hippo effector as the key determinant of cell mechanics in response to ECM cues.Peer reviewe

AFM Monitoring the Influence of Selected Cryoprotectants on Regeneration of Cryopreserved Cells Mechanical Properties

Cryopreservation of cells (mouse embryonic fibroblasts) is a fundamental task for wide range of applications. In practice, cells are protected against damage during freezing by applications of specific cryoprotectants and freezing/melting protocols. In this study by using AFM and fluorescence microscopy we showed how selected cryoprotectants (dimethyl sulfoxide and polyethylene glycol) affected the cryopreserved cells mechanical properties (stiffness) and how these parameters are correlated with cytoskeleton damage and reconstruction. We showed how cryopreserved (frozen and thawed) cells' stiffness change according to type of applied cryoprotectant and its functionality in extracellular or intracellular space. We showed that AFM can be used as technique for investigation of cryopreserved cells surfaces state and development ex vivo. Our results offer a new perspective on the monitoring and characterization of frozen cells recovery by measuring changes in elastic properties by nanoindentation technique. This may lead to a new and detailed way of investigating the post-thaw development of cryopreserved cells which allows to distinguish between different cell parts

Advanced and Rationalized Atomic Force Microscopy Analysis Unveils Specific Properties of Controlled Cell Mechanics

The cell biomechanical properties play a key role in the determination of the changes during the essential cellular functions, such as contraction, growth, and migration. Recent advances in nano-technologies have enabled the development of new experimental and modeling approaches to study cell biomechanics, with a level of insights and reliability that were not possible in the past. The use of atomic force microscopy (AFM) for force spectroscopy allows nanoscale mapping of the cell topography and mechanical properties under, nearly physiological conditions. A proper evaluation process of such data is an essential factor to obtain accurate values of the cell elastic properties (primarily Young's modulus). Several numerical models were published in the literature, describing the depth sensing indentation as interaction process between the elastic surface and indenting probe. However, many studies are still relying on the nowadays outdated Hertzian model from the nineteenth century, or its modification by Sneddon. The lack of comparison between the Hertz/Sneddon model with their modern modifications blocks the development of advanced analysis software and further progress of AFM promising technology into biological sciences. In this work, we applied a rationalized use of mechanical models for advanced postprocessing and interpretation of AFM data. We investigated the effect of the mechanical model choice on the final evaluation of cellular elasticity. We then selected samples subjected to different physicochemical modulators, to show how a critical use of AFM data handling can provide more information than simple elastic modulus estimation. Our contribution is intended as a methodological discussion of the limitations and benefits of AFM-based advanced mechanical analysis, to refine the quantification of cellular elastic properties and its correlation to undergoing cellular processes in vitro

YAP regulates cell mechanics by controlling focal adhesion assembly

Hippo effectors YAP/TAZ act as on–off mechanosensing switches by sensing modifications inextracellular matrix (ECM) composition and mechanics. The regulation of their activity hasbeen described by a hierarchical model in which elements of Hippo pathway are under thecontrol of focal adhesions (FAs). Here we unveil the molecular mechanism by which cellspreading and RhoA GTPase activity control FA formation through YAP to stabilize theanchorage of the actin cytoskeleton to the cell membrane. This mechanism requires YAPco-transcriptional function and involves the activation of genes encoding for integrins and FAdocking proteins. Tuning YAP transcriptional activity leads to the modification of cellmechanics, force development and adhesion strength, and determines cell shape, migrationand differentiation. These results provide new insights into the mechanism of YAPmechanosensing activity and qualify this Hippo effector as the key determinant of cellmechanics in response to ECM cues.</p

Economically Optimal Road Subnetwork

The paper deals with the following situation: An area is served by a transportation network, usually the road one. The current quality of the network is not satisfactory for the owner (e.g. a company or public administration), but the reconstruction or recovery of the whole network is not feasible for the economic reasons. Managers, who are responsible for the network, make decisions how to reduce the network and then to reconstruct or recover it meeting certain conditions and minimizing costs. The condition is formulated by means of the set W of important pairs of sources and sinks of transport flows and by the number q ≥ 1 representing the maximal acceptable elongation rate of routes between nodes (vertices) from the set W. Such a problem can be met e.g. in rural road network reduction for winter maintenance, choice of tram or trolleybus network as a subnetwork of the bus one etc. The paper describes the mathematical support for that decision making. The mathematical model of the problem is presented. Then a depth-first search type exact method is proposed and verified. Afterwards, a heuristics is described and verified as well. Finally, linear programming version of the problem is added. The results were applied to urban bus network of Slovak town Piestany

Cisplatin enhances cell stiffness and decreases invasiveness rate in prostate cancer cells by actin accumulation: dataset of confocal and atomic force microscopy

Summary Dataset of imaging data of the experiment "Cisplatin enhances cell stiffness: Biomechanical profiling of prostate cancer cells". This dataset includes image data of atomic force microcopy (Young modulus) and confocal microscopy(staining of F-actin and β-tubulin) of prostate cell lines PNT1A, 22Rv1, and PC-3. Materials and Methods Cells, cell culture conditions Cells confluent up to 50–60% were washed with a FBS-free medium and treated with a fresh medium with FBS and required antineoplastic drug concentration (IC50 concentration for the particular cell line). The cells were treated with 93 µM (PC-3), 38 µM (PNT1A), and 24 µM (22Rv1) of cisplatin (Sigma-Aldrich, St. Louis, Missouri), respectively. IC50 concentrations used for treatment with docetaxel (Sigma-Aldrich, St. Louis, Missouri) were 200nM for PC-3, 70nM for PNT1A, and 150nM for 22Rv1. Long-term zinc (II) treatment of cell cultures Cells were cultivated in the constant presence of zinc(II) ions. Concentrations of zinc(II) sulphate in the medium were increased gradually by small changes of 25 or 50 µM. The cells were cultivated at each concentration no less than one week before harvesting and their viability was checked before adding more zinc. This process was used to select zinc resistant cells naturally and to ensure better accumulation of zinc within the cells (accumulation of zinc is usually poor during the short-term treatment of prostate cancer cells). Total time of the cultivation of cell lines in the zinc(II)-containing media exceeded one year. Resulting concentrations of zinc(II) in the media (IC50 for the particular cell line) were 50 µM for the PC-3 cell line, 150 µM for the PNT1A cell line, and 400 µM for the 22Rv1 cell line. The concentrations of zinc(II) in the media and FBS were taken into account. Actin and tubulin staining β-tubulin was labeled with anti- β tubulin antibody [EPR1330] (ab108342) at a working dilution of 1/300. The secondary antibody used was Alexa Fluor® 555 donkey anti-rabbit (ab150074) at a dilution of 1/1000. Actin was labeled with Alexa Fluor™ 488 Phalloidin (A12379, Invitrogen); 1 unit per slide. For mounting Duolink® In Situ Mounting Medium with DAPI (DUO82040) was used. The cells were fixed in 3.7% paraformaldehyde and permeabilized using 0.1% Triton X-100. Confocal microscopy The microscopy of samples was performed at the Institute of Biophysics, Czech Academy of Sciences, Brno, Czech Republic. Leica DM RXA microscope (equipped with DMSTC motorized stage, Piezzo z-movement, MicroMax CCD camera, CSU-10 confocal unit and 488, 562, and 714 nm laser diodes with AOTF) was used for acquiring detailed cell images (100× oil immersion Plan Fluotar lens, NA 1.3). Total 50 Z slices was captured with Z step size 0.3 μm. Atomic force microscopy We used the bioAFM microscope JPK NanoWizard 3 (JPK, Berlin, Germany) placed on the inverted optical microscope Olympus IX‑81 (Olympus, Tokyo, Japan) equipped with the fluorescence and confocal module, thus allowing a combined experiment (AFM‑optical combined images). The maximal scanning range of the AFM microscope in X‑Y‑Z range was 100‑100‑15 µm. The typical approach/retract settings were identical with a 15 μm extend/retract length, Setpoint value of 1 nN, a pixel rate of 2048 Hz and a speed of 30 µm/s. The system operated under closed-loop control. After reaching the selected contact force, the cantilever was retracted. The retraction length of 15 μm was sufficient to overcome any adhesion between the tip and the sample and to make sure that the cantilever had been completely retracted from the sample surface. Force‑distance (FD) curve was recorded at each point of the cantilever approach/retract movement. AFM measurements were obtained at 37°C (Petri dish heater, JPK) with force measurements recorded at a pulling speed of 30 µm/s (extension time 0.5 sec). The Young's modulus (E) was calculated by fitting the Hertzian‑Sneddon model on the FD curves measured as force maps (64x64 points) of the region containing either a single cell or multiple cells. JPK data evaluation software was used for the batch processing of measured data. The adjustment of the cantilever position above the sample was carried out under the microscope by controlling the position of the AFM‑head by motorized stage equipped with Petri dish heater (JPK) allowing precise positioning of the sample together with a constant elevated temperature of the sample for the whole period of the experiment. Soft uncoated AFM probes HYDRA-2R-100N (Applied NanoStructures, Mountain View, CA, USA), i.e. silicon nitride cantilevers with silicon tips are used for stiffness studies because they are maximally gentle to living cells (not causing mechanical stimulation). Moreover, as compared with coated cantilevers, these probes are very stable under elevated temperatures in liquids – thus allowing long-time measurements without nonspecific changes in the measured signal. Identification of files Files are separated into individual zip files. The dataset of confocal microscopy is separated based on treatments: untreated control, docetaxel-treated cells, cisplatin-treated cells, zinc-treated cells. Filenames actin_tubulin_Zstack_cisplatin.zip, actin_tubulin_Zstack_untreated_control.zip, actin_tubulin_Zstack_zinc.zip, actin_tubulin_Zstack_docetaxel.zip. Files included in these ZIP archives are named as follows: "cellline_treatment_FOV". Files are 3-layer 16bit tiff files with layer sequence as follows: F-Actin (Phalloidin)/b-tubulin/Hoechst 33342. The dataset contains 242 FOVs of three cell line types/three treatments + one control, files are Z-stacks made of 50 slices. The dataset of atomic force microscopy (AFM) is included in one ZIP archive "AFM_YoungModulus_SetpointHeight.zip", which includes data on Young modulus and Setpoint Height of cell lines 22Rv1, PNT1A and PC-3 and treatments zinc, docetaxel, cisplatin (+control), i.e. identical like for confocal microscopy. The file naming is as follows: "AFM_cellline_treatment_FOV_Youngmodulus.tif" for Young modulus and "AFM_cellline_treatment_FOV_setpointheight.tif" for setpoint height. The data are filtered 32-bit tiff images, where the pixel value correspond to cell stiffness (young modulus) in Pa or setpoint height in m