OPEN protocol

The institutions performed the CRS–HIPEC procedure under the same standardized protocol. Extensive debulking with peritonectomy and, when needed, multiorgan resections were performed, as described by Sugarbaker et al.10 (link),11 (link) and all the latter recommendations. The purpose of the cytoreduction was to obtain a macroscopically complete CRS (R1) resection, which means that no macroscopically visible residual tumor was left at the end of the surgical resection. After the cytoreduction, the open perfusion protocol of the abdominal cavity with mitomycin C was performed.17 (link) The inflow temperature of the perfusate was 41–42 °C. As soon as this temperature was reached, mitomycin C was added, 35 mg/m² body surface, in three fractions (one half, one fourth, and one fourth of the total dose) with a 30-min interval. Mitomycin C was used under the same schedule for all first HIPEC procedures. If a patient had undergone a HIPEC before, procedures were done with intraperitoneal oxaliplatin (460 mg/m²), systemic folinic acid (20 mg/m²), and 5-fluorouracil (5-FU; 400 mg/m²). When new institutions started performing CRS–HIPEC, a surgeon of an experienced institute monitored the procedure to ensure that the procedure was performed according to the Dutch HIPEC protocol.

CRISPR/Cas9 target sites in exons of zebrafish genes were identified using CRISPRScan (http://www.crisprscan.org/; Moreno-Mateos et al., 2015 (link)). 5’ and 3’ homology arms of specified length directly flanking a genomic targeted double strand break were generated by annealing two complementary oligonucleotides. The double stranded 5’ and 3’ homology arms with appropriate overhangs were cloned into the pGTag vector BfuAI and BspQI sites, respectively, flanking the cargo. A three-nucleotide buffer sequence lacking homology to the genomic target site was engineered between the donor UgRNA PAM and the 5’ end of the homology arms. This was done in case the UgRNA PAM sequence was complementary to the nucleotides located 5’ to the start of the homology arm, which would increase the 24 or 48 bp homology arm length. Maps for the pGTag vectors and an open source protocol for cloning the homology arms are available at http://genesculpt.org/gtaghd/. The pGTag vectors are available through Addgene (https://www.addgene.org/kits/essner-geneweld/).
To generate 1 kb homology arms for zebrafish cx43.4 and esama, ~2 kb of genomic DNA surrounding the CRISPR target site was PCR amplified from adult WIK finclips using the proofreading enzyme KOD (EMD Millipore), and then sequenced to identify polymorphisms. Primers were designed to sit 1032 bp up and down stream of the cut site according to the Ensemble.org reference genome V11. Primers also contain either BfuAI and BspQI recognition sequences to make the appropriate overhangs for Golden Gate cloning into a pGTag vector or sequence for Gibson cloning into a pGTag vector. PCR was performed with the proofreading polymerase KOD and using genomic DNA from animals homozygous for the most common polymorphisms was used as template. The products were then Topo Blunt (Thermo Fisher Scientific) cloned for sequencing. The homology arms were Golden Gate or Gibson cloned into a pGTag vector containing the same cassette as the previous injections for the target locus. pGTag vectors with 1 kb homology arm vectors were injected into embryos from adults with the matching genomic sequence. Supplementary file 1 Table S5 lists the sequences of all homology arms, sgRNA target sites, and spacers. For each locus injections were done in triplicate, and for those targeting the locus with 1 kb homology arms the following controls were also performed; plasmid only, plasmid with universal gRNA and without genomic gRNA, and plasmid linearized in vitro with genomic gRNA.

The open field apparatus was made out of white polyacrylics and consisted of a white-square arena (50 × 50 × 30 cm). Experimental subjects were carried to the testing room in their home cages at least 30 min before the beginning of the experiment. Mice were placed in the center of the open field and allowed to freely explore it for 10 min. In juvenile mice, the first 5 min were used for the analysis of the following behavioral parameters: time spent in the inner area (central zone, 30 × 30 cm) and in the outer area (periphery) of the open field arena, locomotor activity (total travel distance), average speed, immobility time, grooming activity (including washing or mouthing of forelimbs, hind paws, face, body, and genitals), and number of defecations (number of fecal boli produced). The protocol for the open field test in matured mice was the same as that used for young mice. However, since the elevated plus maze test was performed to evaluate the anxiety status of the mice, parameters that highly depend on manual counting were not analyzed. Before the start of each session and between animals, the open field test apparatus was carefully wiped with a 70% alcohol solution.

For this work, the UPCT autonomous vehicle (UPCT-CICar [23 (link)]), was driven by a human pilot in manual mode. CICar is a real-world prototype, based on a commercial electric vehicle, the Renault Twizy, which has undergone a series of modifications to provide it with the required functionality. The CICar has been equipped with multiple sensors, including LiDAR, cameras, IMU, GPS, encoders, etc., necessary for the vehicle to perform autonomous driving tasks. This platform setup integrates a perception system, a control system, and a processing system on board the vehicle.
Perception System: The purpose of a sensor system is to collect data from the surrounding environment of the AV and send that data to the control system. These sensors measure different physical quantities, which are typically selected to overlap each other, providing the redundant information needed to correctly merge and correlate the information. In our autonomous vehicle, two types of sensors are used to measure the environment: short-range sensors (up to 10 m) and long-range sensors. Installed short-range sensors include a Sick 2D laser ranging scanner and time-of-flight camera. The long-range sensors are a 3D LIDAR scanner and a camera in the visible spectrum. Table 3 and Figure 3 show the different devices involved in data acquisition during the tests, as well as the details of the variables involved in obtaining them.
Driver Biometric System: The drivers’ biometric signal collection system has been carried out using a non-invasive wearable device, bracelet type, called Empatica E4. The Empatica E4 is a wrist-worn top-quality sensor device considered a Class IIa Medical Device according to 93/42/EEC Directive. Empatica E4 device measures the acceleration data (ACC), as well as other physiological parameters, namely the Blood Volume Pulse (BVP), from which the Heart Rate Variability (HRV) and the Inter-Beat Interval (IBI) are derived as well, skin temperature (TEMP) and also changes in certain electrical properties of the skin such as the Electrodermal Activity (EDA). For the creation of our dataset, among the several measurements recorded by the Empatica E4, this signal was considered, since it provides information better suited for activity recognition. A summary of the technical specifications of the accelerometer sensor is detailed in Table 4.
Control System: The main control systems of the Renault Twizy have been automated in order to allow the vehicle to be autonomously controlled. The modified systems are the steering wheel, the brake pedal and the accelerator pedal (see mechanical modification in Figure 3). Despite the fact that all driving will be manual and not autonomous, the system will record the data with two controller drives through a CAN bus. The Compact Rio cRIO 9082 controls the accelerator, brake and steering wheel movements with the CAN-Open communication protocol, as well as I/O signals.
Processing System: Each sensor works with its own sample rate, and in most cases, this is different between devices. The achieve the synchronisation of the data and accurately reconstruct the temporal sequence, time stamps have been generated to synchronise the operating start and finish times. All of this is controlled and synchronised by the on-board processing system.