This paper presents a robotic team suited for bottom sediment sampling and retrieval in mudflats, targeting environmental monitoring tasks. The robotic team encompasses a four-wheel-steering ground vehicle, equipped with a drilling tool designed to be able to retain wet soil, and a multi-rotor aerial vehicle for dynamic aerial imagery acquisition. On-demand aerial imagery, properly fused on an aerial mosaic, is used by remote human operators for specifying the robotic mission and supervising its execution. This is crucial for the success of an environmental monitoring study, as often it depends on human expertise to ensure the statistical significance and accuracy of the sampling procedures. Although the literature is rich on environmental monitoring sampling procedures, in mudflats, there is a gap as regards including robotic elements. This paper closes this gap by also proposing a preliminary experimental protocol tailored to exploit the capabilities offered by the robotic system. Field trials in the south bank of the river Tagus’ estuary show the ability of the robotic system to successfully extract and transport bottom sediment samples for offline analysis. The results also show the efficiency of the extraction and the benefits when compared to  human-based sampling. Bottom sediments in estuarine bays once stressed by effluent discharges from industries and urban environments were exposed to the deposition of contaminants by physical settling of particulate matter and chemical sorption from water. The process is ruled by physical conditions, such as water turbulence, contact time and sediment surface topography, and by the chemical nature and physical form of contaminants in the water column. All of these variables tend to be spatially complex, leading to heterogeneous distribution patterns of contaminants. In time, the hydrodynamics-induced redistribution of particles will intensify the spatial heterogeneity created by a primary deposition. The presence of inorganic toxicants, like radionuclides and heavy metals, in the mudflats of major rivers is an issue of public concern and environmental relevance, pressing the need for an extensive survey of the intertidal mudflat. Continuous dredging of navigation channels of river estuaries and human scavenging activities, such as intensive clam harvesting, are likely to promote the re-suspension of both surface and anoxic bottom sediments, causing the remobilization of adsorbed toxicants .Currently, environmental monitoring surveys in estuarine mudflats are performed by human experts handling manual sampling tools, such as the manual auger lanyards for extracting cylindrical columns  of soil or sediment to a certain depth. To obtain cores in continental areas, these devices are often endowed with an auxiliary motor. With these manual tools, sediment samples are collected and then carried from the site to the lab for an offline analysis. Walking in the mudflat, handling the sampling tools, transporting the samples and ensuring that these are properly tagged and geo-referenced are just a few of several physically-demanding, costly and time-consuming challenges humans must face in these sampling campaigns. To mitigate these difficulties, this paper presents a robotic team developed to support sampling operations in mudflats and, as a result, to facilitate thorough spatio-temporal sampling campaigns therein.The statistical significance and accuracy of environmental monitoring campaigns depend heavily on the execution of well-prepared experimental protocols, which must be adapted to the characteristics of the sampling site by a human expert. To support the human expert in this process, the UAV provides an aerial mosaic from a set of images acquired right before the mission’s onset with a high resolution camera. The UAV is also used by the expert to supervise the ground vehicle’s operation. Estuarine environments are ruled by intense hydrodynamic processes, which cause considerable morphology changes throughout a single day and, thus, rendering satellite imagery rapidly outdated. By relying on a UAV, the human expert gets access to up-to-date imagery.Current experimental protocols for radiological and heavy metals monitoring in mudflats  are basTo validate the proposed system, a set of field trials was performed in the estuarine intertidal mudflat of the river Tagus . Several bottom sediment samples were collected and processed in the lab. These trials showed the ability of the robotic system to maneuver in several types of terrain that can be found in estuarine environments, which includes dry sand, wet sand, ponds and mud. The results also show the ability of the system to drill efficiently in these terrains and, more importantly, that the collected sediment samples are proper for off-line laboratory analysis. The results also show the benefits of the robotic sampling process when compared to  human-based sampling.This paper is an extended and improved version of a conference paper , and it Environmental monitoring in remote and vast environments is a highly relevant societal problem that is being addressed by several research communities in many disparate ways. Although originally performed by humans, current approaches tend to exploit technology as much as possible. With technology, environmental monitoring can be faster, more accurate and more significant. With the support of technology, it becomes possible to reduce the physical burden of complex surveys and to focus human effort on the design of experimental protocols, their execution monitoring and subsequent data analysis.A common approach for technology-driven environmental monitoring is to exploit remote sensing techniques, either based on aerial vehicles or satellites . HoweveAlthough not yet mainstream in environmental monitoring campaigns, field robots are steadily paving their way towards real-life applications. Robots have already been validated as valuable tools for exploring Mars , subterrTo collect sediment samples, robots need to be equipped with sediment sampling tools and store the collected samples. The high diversity of soils that a robot must face in a sampling campaign renders the design of sampling tools a complex problem. For instance, the same tool must be able to penetrate the soil without revolving it and then must be able to pull the sample without dropping or draining it. The following paragraphs overview previous efforts in devising generic soil sampling tools, that is not specifically devised for robotic operation.Some pneumatic drilling tools have been proposed for operation on unstable soil, including submerged areas, while preserving the structure of the sampled soil . In alteElectrically-driven drilling mechanisms for soil sampling have also been proposed ,29. HoweComplete soil sampling systems that can be towed by a small tractor or other agricultural vehicle have also been proposed ,33,34,35Instead of employing complex drilling tools, it is also possible to consider the use a robotic arm manipulating small sampling containers . HoweverAs an alternative to these previous approaches, we present a complete robotic system for mudflats, equipped with an electrically-driven drilling tool capable of exploiting both linear and rotary motion to reduce soil resistance without revolving the soil. To reduce human intervention, the drilling tool is recharged automatically by a compliant six-DOF robotic arm.The development of the presented robotic system was driven by a particular environmental monitoring case study: radiological and heavy metals monitoring in the mudflats along a major river’s estuary. The goal was to ensure that the robotic tool was able to cope with a realistic and valuable mission, as defined by environmental monitoring experts. Moreover, this design approach allowed roboticists and environment scientists to co-develop the robots and a preliminary experimental protocol for the case study at hand. This experimental protocol defines the sequence of actions that robots and humans must perform, as well as the sampling procedure that must be executed.This section starts by describing the case study and its motivation. Subsequently, the mission workflow is presented. Under a pragmatic perspective, this workflow involves the robots and the human experts. Then, the sampling procedure is detailed. Finally, the way the samples should be stored, conserved and processed in the lab is described. With this multi-disciplinary approach, we expect to contribute towards closing the gap between the robotics and the environmental monitoring communities.The specification, development and validation of the robotic system relies on Tagus River’s estuarine bay as the main case study see . The selOther industries set on both sides of the estuarine bay contributed to the dispersion of toxic metals, like mercury, cadmium or arsenic, and a variety of other different contaminants, including Polycyclic Aromatic Hydrocarbons (PAH) and organometallic compounds that have been previously reported, suggesting a deterioration of water and sediment quality in some critical areas of the Tagus estuary . The conRuditapes philippinarum and its massive population expansion triggered an intense activity of clam harvesting for human consumption without any control of toxicants. The scavenging action of hundreds of clam harvesters and boat clam dredging, scattered over a wide area of shallow and low depth water of the estuarine bay, enhances the remobilization of non-quantified masses of bottom sediments, thus inhibitinginorganic toxicants from reactive processes. Furthermore, the periodical dredging of the navigation channels causes intense remobilization of sediments. Meanwhile, a progressive green coverage of several areas of the mudflat could be witnessed. The sea grass Zostera noltii was then identified in the course of an exploratory sampling initiative and appeared to be the residence substrate for small gastropods. There is also evidence about the presence of squids.The early 2000s was a time for some observable changes in both the presence of human activity and the physiognomy of the estuarine mudflat extending through the shoreline from Barreiro to Alcochete. The introduction of the invasive Asian clam In sum, there are several factors potentiating the presence of radionuclides and heavy metals in the estuarine bay, which, in turn, contaminate the food-chain that ultimately impacts human health. The robotic team herein presented is expected to foster accurate spatio-temporal sediment and biota sampling so that environment researchers can study these phenomena in detail.This section presents a mission workflow (storyline) devised for the case study, taking into account the characteristics of the presented robotic system. The human-robot team reaches the operations site. The robots are unloaded from the transportation vehicle. Based on satellite imagery of the operations’ site, the human expert defines the workspace boundaries. These boundaries will be used by the robotic system to constrain its operation range.The aerial robot takes off and performs a scan to cover the workspace defined by the expert. As a result of these scanning procedure, the UAV builds a high resolution geo-referenced mosaic from a set of mutually-registered aerial images. The mosaic is then presented to the expert, which is now able to discard the satellite imagery, as it is most likely outdated.At the base station, the expert segments potential key features of the environment, such as water ponds and channels, sea grass coverage, salt marsh vertical vegetation, sand banks and all sorts of physical obstacles to the UGV’s navigation. Then, based on this meta-data, the expert specifies a set of transects to be sampled by the robot see .With the information collected in the previous step, the ground vehicle is tele-operated by the expert so as to traverse the transects and periodically sample the terrain while avoiding any peril in its way. At each sampling point, the expert requests the ground vehicle to perform a sediment sampling behavior. If intended by the expert, the aerial vehicle is used to provide aerial images, augmenting the operator’s perception about the mission execution.When the ground vehicle has either its sample containers filled or has visited all of the defined sampling locations, the expert tele-operates the robot back to the base station.Back at the base station, the expert unloads the sample containers into isothermal boxes with cooling pads, which are subsequently brought to the lab for post-processing. If the mission is not complete, the expert loads the ground vehicle with empty sample containers and resumes the mission (return to the previous step).Once the mission in the current operation’s site is complete, the expert washes the ground vehicle with fresh water so as to remove dirt and salt residues. Then, the expert may be called upon to execute some maintenance procedures, such as recharging batteries or re-inflating tires. Finally, the human-robot team leaves the operations site.The mission workflow is defined according to the following enumerated steps:Mission control’s Step 3 requires the expert to define the sampling procedure. This is a critical point in the process from an environmental monitoring perspective. Failing to produce the proper sampling procedure results in a failed mission.A proper sampling campaign must ensure that samples are representative of the spatial diversity of the mudflat. Furthermore, the sampling campaign must also grant that the sampled volume is sufficient for the subsequent off-line analysis to provide significant results. In routine monitoring situations, where the density of contaminants per sample is expected to be low, a possible approach to overcome detection difficulties  is to collect large sample volumes for further concentration of the target analytes. As a consequence, large sample volumes are required if contaminants are to be detected therein. This is an important issue that must be taken into account when selecting the scale of the robotic system.The sampling procedure follows the principle of transect sampling generally described by the International Commission on Radiation Units and Measurements, for the purpose of estimating spatial distribution patterns of radionuclides in large areas with closely-spaced sampling locations . SedimenOne major issue in sampling the bottom sediments of estuarine environments is that the time frame available for sampling is constrained by tidal activity. In general, the sampling procedure must be within a time span of approximately 4 h. Furthermore, the Tagus’s estuary in the Alcochete region, where the field trials took place, is mostly flat, and so, the water level covers/uncovers the mudflat at a very fast pace. Due to all of these reasons, the initial sampling points need to be the ones that are farther from the shore. Then, progressively, sampling can proceed in the direction of the shoreline.At the lab, the cores are unfrozen and sectioned in depth layers , and all sections of a specified depth range are mixed into a composite sample. Composite samples from specified depth layers are oven dried at a temperature of This section details the mechanical, hardware and software designs of the two vehicles comprising the robotic team, devised to meet the requirements of the proposed experimental protocol for robotic sampling of estuarine mudflats.The UGV’s components have been carefully selected to meet the requirements of advanced mobility in estuarine mudflats. This type of environment poses serious difficulties in terms of robot mobility. The robot needs to be able to overcome water streams, move in dry and wet sand and several levels of muddiness and tackle vegetation, rocks and other debris. Moreover, to be able to place the drilling tool in the desired sampling point, it is necessary to have a fine control over the robot’s pose, meaning that the robot needs to be highly maneuverable. In addition to the mobility aspects, the design of the mechanical platform took into account that salt water will wear out materials, that the presence of mud and sand may jam mechanical joints and that the robot will be subject to wide temperature ranges.The robot’s body is made of aluminum and composite materials. The aluminum was the chosen material for the chassis due to its light weight, strength, corrosion resistance, flexibility, thermal conductivity, ductility and cost efficiency. Furthermore, it enables the construction of a fully-sealed fan-less system without overheating issues. To further extend life expectancy and resistance to extreme environments, the robot was coated with a hydrophobic paint. This special cover also repels dust and mud, reducing corrosion, common in maritime environments.The robot is split into a frontal block and a rear block, each actuated by two wheels. These two blocks are attached via a passive longitudinal joint. The passive joint improves the four wheels’ contact with the ground, even on uneven terrain, or when the robot needs to overcome some small obstacles see a. LocomoWith its independent steering and traction motors, the robot is able to move in Ackerman and double Ackerman configurations, as well as to rotate around its own geometric center. This multi-modal locomotion becomes of special importance whenever the robot needs to make fine adjustments to its pose to, for instance, align its drilling tool to a specific sampling spot.The wheels are 60 cm in diameter and are a combination of aluminum rims with motocross tires of 10 cm in width. With this configuration, the robot exhibits a considerable ground clearance, which is key to handle small obstacles in the terrain. All tires are partially deflated to half of the recommended pressure to increase the footprint and reduce the chances of slippage. Each motor is directly coupled to its corresponding wheel through a gear. The absence of chains or belts not only reduces energy loss, but also the number of failure points. An issue often neglected in current robotic platforms is the ease of their transportation and storage. To facilitate transport, each wheel supports hand locking and manual clutch. The longitudinal passive axis can also be locked in the central position.On the top of the mobility platform, the UGV is equipped with a rectangular tube frame, underneath the composite covers. This frame provides the structural strength necessary to hold nine sample containers, a robotic arm to manipulate them and the drilling tool. An actuator in the drilling tool revolves a cylindrical hollow metallic tube with an internal section of 45 mm and a length of 500 mm, which is simultaneously pushed downwards by a linear actuator. To cope with the contingency of drilling in surfaces with buried hard elements , the linear actuator’s current and position is monitored continuously throughout the process. Once the available containers are filled, the sediment samples in the hollow metallic tubes must be removed as a pack, that is without mixing sediments from different depths. To facilitate this task, each hollow metallic tube has two half-hollow PVC tubes , which easily slide out of the metallic tube with the help of the human expert. At each sampling point, it is necessary to store the filled hollow tube in its storage socket and place an empty one in the drilling tool. This process is carried out autonomously with an Universal Robotics UR5 compliant robotic arm. With six-DOF, this robotic arm is able to move the tubes between storage socket and drilling tool without collisions. Moreover, the arm has compliant properties, which is important to ensure the safety of people working in the robot’s workspace. The drilling tool works in a plug-n-play fashion in order to facilitate accommodation and transport. This also allows interchanging sampling tools. For instance, the robot can also be equipped with a tool for dredging seaweed. In this case, the tool also uses a linear motion to push a 1.4 cmField robotics is often limited by the energetic autonomy that robots exhibit. Energy harvesting is an interesting solution to ensure long-lasting operation. However, in our case, given that the robot’s operation depends on the tides, there is little opportunity for battery recharging cycles. As a consequence, the robot must be equipped with sufficient energy for a complete mission. In this sense, energy is supplied by eight lithium ion cells with a total capacity of 100 Ah. Lithium is known by its energy:weight ratio, which combined with the EMUS intelligent Battery Management System (BMS), offers more than 4 h of operation. The BMS extends cell life by keeping voltage, current and temperature parameters within manufacturer ranges.The usability of any mechanical platform relies on the selection of a proper set of components. They are responsible for efficient energy management, for low level motor control, for providing sensory feedback to the control system and for ensuring reliable communications with the base station. The main processing unit combines an Intel Core i7 CPU with a mini-ITX industrial board, ready for extended temperature ranges. Connected to it, two custom-built printed circuit boards, each one with its own micro-controller, are responsible for distributing and controlling supply voltages across all electronic components. These boards also provide internal buses for RS232, CAN and USB intra-robot wired communications. On-board current monitoring of each power output allows for continuous information on devices’ consumption and precise power management, while aiding on early problem detection. A total of 15 analogue and digital I/O ports is available through the two boards for health monitoring sensors . The Universal Robotics UR5 compliant robotic arm includes its own processing unit, which is connected to the UGV’s main computer through an Ethernet connection.The hardware responsible for maintaining wireless communications between UGV, UAV, base station and human-robot interaction devices is detailed in For precise pose estimation, the UGV relies on a Phidgets Inertial Measuring Unit (IMU) and on an Ashtech HDS800 high precision GPS-RTK, which provides centimeter-level accuracy based on GPS corrections obtained from the RENEPnetwork via GPRS. To provide the user with good situational awareness, the robot is equipped with a set of body-fixed Bosch Color Mini Bullet cameras. Three of these cameras are aligned with the forward robot motion; a fourth camera is pointing backwards; and a fifth camera is looking downwards to the drilling tool. To provide higher flexibility to the user, the robot is also equipped with an Axis Communications Q6044-E PTZ camera. The video output of these cameras feeds a Vivotek VS8801 video encoder, which sends video streams using the Real Time Streaming Protocol (RTSP) to the base station through the wireless network. Although autonomous navigation is not addressed in this paper, it is worth mentioning that the robot is equipped with distal sensors for environment perception. A SICK LMS111 2-D laser scanner is mounted on a Robotis tilting unit for delivering complete 3D point clouds. To complement the laser scanner, a pair of PointGrey Dragonfly2 cameras is available for depth estimation. Autonomous behavior based on 3D sensory data has been studied in the UGV’s predecessor, INTROBOT .Both sampling tools  are built based on the same base equipment, simplifying both software and hardware implementations. Namely, 550-W Parker motors are used and controlled by Elmo Gold Guitar controllers. Limit switches in the linear actuators are used to avoid overheating the system in case any control problem arises. Hengstler encoders and a laser pointer are used to report the exact position of the tool with respect to the ground. This allows the system to adjust the drilling height in the face of uneven terrain. Internal power consumption and temperature sensors in the motor controllers inform the control system regarding the mechanical stress being induced in the sampling tool. For instance, a power consumption peak informs the control system about the presence of an obstruction to the drilling process, such as a small buried rock.To promote scalability, adaptability and interoperability, as new requirements are presented to the robotic system, the software layer responsible for controlling the UGV runs on top of the Robot Operating System (ROS) . ROS proAt each moment, the system must gather sensory feedback about the robot’s pose and its surroundings. This sensory feedback comes asynchronously and, therefore, must be associated with a time stamp. ROS provides this functionality. Based on the sensory feedback provided by the wheel odometers, by the GPS-RTK device and by the inertial measurement unit, an Extended Kalman Filter (EKF) recursively estimates the UGV’s optimal pose. Due to the absence of tall buildings or trees in estuarine environments, the GPS-RTK device is capable of producing centimeter-level global localization accuracy alone, in most situations. Nevertheless, wheel odometry helps the EKF to filter out GPS-RTK spurious noise and signal drop-outs. To mitigate motion estimate errors resulting from wheel slippage or blockage, we estimate the motion of the robot using each wheel independently and then fuse the four estimates in a weighted manner. The more similar the motion estimated from one of the wheels is from the estimates obtained from the other three wheels, the higher is its weight in the fusion process. This process exploits the redundancy introduced by the four wheels to disregard deviating motion estimates. Please refer to  for furtControlling the motion of a four-wheeled steering robot in uneven terrain is a complex task, as, ideally, the four wheels should be synchronized and should behave in a compliant way with respect to the environment in the face of transient external forces ,45. As iThe control system also encompasses a node responsible for controlling the drilling tool and another for controlling the robotic arm. While the drilling tool needs to be controlled at the actuator level, the robotic arm is abstracted via the MoveIt! API . With MoThe aerial team member, the UAV, was designed to withstand the difficult operational environment of estuarine environments, where robustness and reliability are key. Taking this into account, a six-rotor configuration with vertical take-off and landing capabilities was chosen see . This coAn overview of the UAV’s hardware architecture, components and connection types is depicted in The UAV’s control system is supported by two computational units, one strictly dedicated to low-level motion control and the other to high-level mission execution functions. Wired communication between controllers is assured by the MAVLink Micro Air Vehicle Communication Protocol via a serial link. The low-level control unit is a VRBrain from Virtual Robotix, interfaced with an on-board IMU based on the MPU6500, a MS5611 barometer and an external GPS device from Ublox. The basic navigation and low-level stabilization is assured by a modified version of the open-source Arducopter platform , whereasThe hardware responsible for maintaining wireless communications between UAV, UGV, base station and human-robot interaction devices is detailed in As satellite imagery is mostly outdated, it cannot be used by the expert to precisely define the sampling mission. For this reason, the UAV, in cooperation with the human operator, is used to produce an up-to-date aerial mosaic of the operations’ site. To perform the aerial survey, the expert needs first to specify the UAV’s workspace by means of three geo-referenced markers on top of satellite imagery see a. These The mosaic creation behavior is then responsible for driving the UAV through a set of waypoints so as to cover the area defined by the expert. These waypoints are automatically defined according to the camera’s Field Of View (FOV), learned from calibration and the required level of inter-image overlap required for a proper image registration process. Concretely, the system uses the FOV of the UAV’s camera to estimate the area covered by each image at different altitudes. Then, given the survey area and image overlap selected by the operator, the system calculates the waypoints required for the aerial mosaic creation. Although this process is automated, the operator can always fine tune the system for a specific application by changing the desired altitude, image overlap and UAV’s velocity. While, ideally, very large areas could be mapped with high detail by flying at low altitude, the resulting large flying missions are hampered by the UAV’s limited energetic autonomy. To prevent the operator from creating infeasible surveys, the system overrides the altitude, speed and image overlap parameters to be able to cover the selected area. For the current hardware configuration, the UAV typically travels at an altitude of 50 m. At each waypoint, an image is recorded and stored in the high-level processing board. Once the flight is complete, all images are transferred in batch modeto the base station so that the mosaic can be built and displayed to the expert. With the updated imagery, the user can define with greater accuracy the sampling points for the UGV. The mosaic is built using the open-source software Hugin Panorama .As defined in the mission workflow of the experimental protocol see , the robThe human expert is allowed to interact with the robotic system through (1) short-range radio frequency (RF) controllers; (2) a wireless tablet; (3) a wireless portable base station; and (4) a diagnostic monitor attached to the robot see . The folThere are two dedicated RF controllers, one for the UGV (a custom Datek device) and another for the UAV (a Graupner MC-32 device), both useful to ensure fine motion control in the line of sight. In the specific case of the UGV, the RF controller can also be useful to drive the robot towards a specific spot that the expert has identified while inspecting the environment by foot. In the UAV case, the RF controller is mostly useful for tuning flight controller parameters and providing basic functionality in case of emergency.Through the tablet, a FieldBook I1 equipped with a touch screen, the user is able to move closer to the operations site while still having almost all of the functionality available at the base station. This flexibility is important as real-life environmental monitoring campaigns often require the expert to freely move in the environment.The portable base station allows the operator to gain access to all graphical user interfaces available to the system, allowing the user to configure and execute the environmental monitoring mission. The base station is composed of a rugged laptop, two analogue joysticks from CTI, a sunlight readable display from Sagitron, a digital video decoder and a wireless router attached to a high-gain antenna (see below). The rugged laptop is a Getac V200 with an Intel Core i7-620LM 2.0 GHz processor and 4 GB of RAM. The joysticks enable the manual control of the UGV’s locomotion (linear and angular velocities) and its pan-tilt-zoom camera individually. The 1200 GB cd/mAPIX API . This APThe distributed nature of the human-robot teamwork demands for heterogeneous wireless networks. This section describes the several elements that compose the communications infrastructure of the proposed system see , that isThe UGV accesses the wireless LAN through two high gain (13 dBi) 2.4-GHz Horizon IH-G12-F2324-V antennas and two high gain (12 dBi) 5.0-GHz Ubiquiti AMO-5G13 antennas . This duThe UAV accesses the wireless LAN with a 2.4-GHz Ubiquiti Picostation 2HPa, which is a radio transmitter with integrated antenna. With this configuration, the UAV is able to communicate with other devices within a line of sight radius of 500 m with transmission rates of up to 54 Mbps. The UAV’s communications range can be expanded by connecting directly to the UGV, serving the latter as a communications relay.The human-robot interaction devices communicate with both the UGV and UAV with the following wireless equipment. The base station is equipped with a Gigabit Edge Router and wireless LAN communications hardware identical to the one employed in the UGV. The UGV’s RF controller communicates with the robot through a 433-MHz custom Datek device with range, in line of sight, of up to 250 m. Communications between the UAV’s RF controller communicates and the robot are maintained through a 433-MHz Scherrer Rx700LR+PSU receiver and a 433-MHz Scherrer TX700Pro transmitter with three power stages, 500 mW, 1 W and 2 W, allowing theoretical ranges of up to 100 km.One of the most important capabilities in complex robotic systems is the ability for the system to pro-actively inform the human operator regarding the system’s overall health. In fact, when the number of components and subsystems increases, the more important it is to identify potential points of failure in the early stages of occurrence during mission execution. A health monitoring supervision system requires two modules: (1) a versatile diagnostic system; and (2) an associated graphical interface. The following describes these two modules.In the robotic system herein presented, a custom implementation of the ROS Diagnostic Stack, which comprises tools to collect, analyze and view diagnostic information , assures that all relevant data are collected and tagged for efficient processing. The system software packages, in particular the ones that communicate with hardware components , are attached to diagnostic routines using the ROS Diagnostic Updater API. These diagnostic routines execute independently at predefined frequencies and are configured individually, enabling access to a set of indicators that are specific for each component or subsystem. Collected diagnostic information is then sent to the ROS diagnostic aggregator, which is responsible for grouping and categorizing data according to pre-set rules.To exemplify, let us analyze the kind of diagnostic information that is produced by three key nodes. The motor control node reports to the diagnostic aggregator, at 1 Hz, the motor controllers’ temperature and current consumption. The Battery Management System (BMS) reports to the diagnostic aggregator, at 0.5 Hz, the overall battery level, the instantaneous power consumption and the voltage per cell, among others. The communications monitor node reports to the diagnostic aggregator, also at 0.5 Hz, the wireless signal strength and noise level, the transmission and receiving rates, among others.Error states are also immediately reported when monitored variables reach some pre-defined thresholds  and then transmitted as asynchronous ROS messages to the base station for human visual inspection. The transmission rate is dynamically chosen so as to cope with bandwidth availability.https://goo.gl/2tx37k).This section presents a set of field trials with the purpose of studying: (1) the robustness of the robotic system in terms of mobility and sampling capabilities; (2) the feasibility of the simplified experimental protocol; (3) the logistic issues involved throughout the sampling process; and (4) the set of potential improvements to be taken into account so as to enable a large-scale monitoring campaign. Videos of the field trials can be found on-line  that the sample in B design the mission workflow, i.e., the set of interactions between operator and robotic team mates . In the


Catheter-related bloodstream infection (CRBSI) is one of the most frequent complications in patients with cancer who have central venous catheters (CVCs) implanted and is associated with substantial morbidity and mortality. Taurolidine is a non-antibiotic agent with broad-spectrum antimicrobial activity, which has been used as a lock solution to prevent CRBSI in some settings. However, little is known about its usefulness in high-risk adult neutropenic patients with cancer. This prospective randomised clinical trial aims to test the hypothesis that taurolidine-citrate lock solution is more effective than placebo for preventing catheter infection in neutropenic haematological patients.This study is a prospective, multicentre, randomised, double-blinded, parallel, superiority, placebo-controlled trial. Patients with haematological cancer who are expected to develop prolonged neutropenia (> 7 days) and who have a non-tunnelled CVC implanted will be randomised to receive prophylactic taurolidine-citrate-heparin solution using a lock technique (study group) or heparin alone (placebo group). The primary endpoint will be bacterial colonisation of the CVC hubs. The secondary endpoints will be the incidence of CRBSI, CVC removal, adverse events, and 30-day case-fatality rate.The lock technique is a preventive strategy that inhibits bacterial colonisation in the catheter hubs, which is the initial step of endoluminal catheter colonisation and the development of infection. Taurolidine is a nontoxic agent that does not develop antibiotic resistance because it acts as an antiseptic rather than an antibiotic. Taurolidine has shown controversial results in the few trials conducted in cancer patients. These studies have important limitations due to the lack of data on adult and/or high-risk neutropenic patients, the type of catheters studied (tunnelled or ports), and the lack of information regarding the intervention . If our hypothesis is proven, the study could provide important solid evidence on the potential usefulness of this preventive procedure in a population at high risk of CRBSI, in whom this complication may significantly impair patient outcome.ISRCTN47102251. Registered on 9 September 2015.ISRCTN, The online version of this article (10.1186/s13063-018-2647-y) contains supplementary material, which is available to authorized users. Catheter-related bloodstream infection (CRBSI) is one of the most frequent complications associated with the long-term use of catheters. Patients with cancer usually have central venous catheters (CVC) implanted for an indefinite period for the administration of chemotherapy and other products and medications and are at increased risk of presenting with this complication, especially during the neutropenic phase. CRBSI in patients with cancer is associated with substantial morbidity and mortality, with excess costs and prolonged length of hospital stay –3. ImporStaphylococcus aureus, are the leading cause of CRBSI in patients with cancer [Gram-positive organisms, mainly coagulase-negative staphylococci (CNS) and h cancer . Howeverh cancer –7. In adh cancer , 9. Thish cancer –12.The most frequent mechanism of infection with long-term, non-tunnelled CVC use is catheter hub contamination during catheter manipulation by health personnel, leading to endoluminal colonisation and infection , 14. BacThe use of lock therapy with different solutions as a method of preventing luminal catheter colonisation is an effective strategy in reducing CRBSI . Lock thVancomycin, ethanol, and taurolidine have been the most frequently studied lock solutions in patients with cancer, with very variable results, according to the significant aforementioned differences in the methodology of the studies and the population involved –29. Of nThis prospective, multicentre, randomised, double-blinded, placebo-controlled trial aims to test the hypothesis that lock solution with taurolidine-citrate-heparin is more effective than placebo for preventing CVC catheter infection in high-risk neutropenic haematological patients.This study aims to determine whether taurolidine-citrate-heparin lock solution is more effective than heparin alone for the prevention of CVC infection in neutropenic haematological patients.Bacterial colonisation of the CVC hubs.Incidence of CRBSI, CVC removal, adverse reactions, and 30-day case-fatality rate.This study is a prospective, multicentre, randomised, double-blinded, parallel, superiority, placebo-controlled trial 1:1). Patients will be randomised after completion of chemotherapy. Patients who were expected to develop prolonged (> 7 days) neutropenia will be randomised to receive a prophylactic solution of taurolidine-citrate-heparin using the lock technique (study group) or a solution with heparin alone using the same technique (placebo group). The study will be conducted in accordance with the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) recommendations , Additio:1. PatieThis study includes adult patients with haematological malignancies admitted to the haematology wards who are receiving chemotherapy, who are expected to develop prolonged neutropenia (> 7 days), and who have a non-tunnelled CVC implanted.The study will be conducted in Institut Català d’Oncologia L’Hospitalet , a 100-bed capacity hospital for adult patients, a referral cancer centre for a population of one million people. University Hospital of Bellvitge (HUB), an 800-bed-capacity tertiary hospital for adult patients. Both hospitals are located in the same health complex and are part of the Institute of Biomedical Research of Bellvitge (IDIBELL). Hospital Universitario Virgen del Rocío (HUVR) is a 1700-bed capacity hospital that caters to half a million inhabitants in the province of Seville.Patients admitted in the haematology wards of the two participating centres receiving myeloablative chemotherapy and who are expected to develop prolonged neutropenia (> 7 days) will be followed up daily by the attending physicians and by a research nurse specifically commissioned to carry out this study. Screening swabs from the catheter hubs and the catheter insertion site will be collected previous to randomisation by the usual nursing personnel; then, the catheter samples will be sent to the microbiology laboratory in order to identify patients with positive cultures (exclusion criteria). Patients with negative screening cultures will be followed up daily by the research nurse in charge and/or the investigators of the study, to assess whether they meet all the inclusion criteria and none of the exclusion criteria. A blood test will be performed every 2–3 days according to the protocols of each centre to document the development of neutropenia. Patients with neutropenia who met the inclusion criteria will be asked to sign the informed consent. The research nurse in charge and/or the investigators of the study will explain the study to the potential participants and ask them to sign the informed consent.Patients aged 18 years or older who are admitted to the hospital because of leukaemia, lymphoma or to undergo haematopoietic stem-cell transplantation (HSCT)Carriers of multiple-lumen, non-tunnelled CVCs, including peripherally inserted central catheters (PICCs)Patients who are expected to develop neutropenia (< 500 neutrophils/μL) lasting more than 7 daysA patient could be included twice if two independent periods of neutropenia occur with at least 1 month interval between them. This interval will be calculated from the final visit (30 days from the end of the intervention) to the new inclusion. In this cases, patients will be randomised again.Patients with a (non-flexible) semi-rigid or tunnelled CVC or totally implantable venous access portsPatients who have been found to have a positive culture of the hubs or the insertion site of the CVC based on the results of the baseline screening culturesPatients in whom the CVC is indispensable for the use of continuous infusion medicationPatients receiving systemic antibiotic treatment ≥ 48 h prior to study entryPatients with an active infectionIn this randomised, double-blinded study, neither the patient nor any of the health staff will know which product is assigned to each patient.A unique list of coded numbers containing the randomised solutions will be generated in the Clinical Research Unit. The randomisation will be performed in each block and stratified. The stratum will contain three groups: patients with leukaemia, patients undergoing an autologous transplantation, and patients undergoing an allogeneic transplantation. The random number generator of SAS version 9.2 will generate the three lists. Once generated, the list will be kept in the pharmacy file, in the pharmacy department of ICO Hospitalet with restricted access to the blinded personnel involved in the trial. When a patient from any of the two participating centres meets the inclusion criteria and signs the informed consent, an email with the recruitment sheet of the patient will be sent to the pharmacy department of ICO Hospitalet, where the randomisation will take place, by the investigator from the pharmacy department in charge of the study. The assignment will be made sequentially, and each patient will be assigned a randomisation number.The taurolidine-citrate-heparin and the heparin-placebo solutions will be prepared in each pharmacy department, and they will be dispensed in 2.5-mL syringes identified with the name of the drug, defined as heparin/taurolidine-citrate, to maintain the blinding. Citrate is an anticoagulant substance without antimicrobial activity, which accompanies taurolidine as an antiaggregant agent. Both lock solutions are colourless and odourless, which makes further masking unnecessary. The blind will be open before the statistical analysis, but only when the trial has concluded and the definitive data will be registered in the database. Only in cases with suspected serious adverse events will the blinding be open.Once the randomisation has been performed, a syringe containing 2.5 mL of the assigned lock solution  will be injected, by usual haematology ward nurses, through each catheter lumen three times a week, approximately every 48-72 h. The solution will be allowed to dwell for approximately 2 h. In all cases, the solution will be aspirated with a syringe and discarded. Before the lock technique, cultures of the hubs and the CVC insertion site will be taken in order to assess the bacterial colonisation of these sites. The research nurse in charge of the study will supervise all the proceedings related with the intervention. Also, the nurse in charge will periodically provide a report to the principal investigator of the study, informing whether the interventions have been performed properly.Multilumen, non-tunnelled CVCs will be inserted into the vein by a specifically trained staff, under the standard precautions. At the time of the catheter placement, the insertion site will be disinfected with 2% chlorhexidine in an alcohol base and then covered with a transparent dressing, except in patients with allergy to this type of dressing, in whom sterile gauze will be used. Also, when there is any sign of inflammation at the insertion site, the dressing will be replaced by a chlorhexidine-impregnated dressing. The care of the CVCs will include the replacement of the dressings every 6–10 days, or before if necessary. Before any manipulation, the catheter hubs will be disinfected using an external clean with 70° alcohol. If the catheter is sealed, the hubs will be disinfected every 6–10 days. If any of the catheters lumens become sluggish or occluded, flushing with urokinase will be performed. Any change of these procedures will be recorded in the database. The manipulations of the CVCs will be carried out under maximum barrier measures.Positivity of any of the cultures taken from the CVC hubsRecovery of neutropenia: > 500 neutrophils/μLRemoval of the catheter for any causeEpisode of CRBSINeed to use the CVC for the administration of a medication in continuous infusion that cannot be stopped for any reasonDischarge from the haematology ward for any reason, despite not having recovered from the neutropeniaDeathPatients will continue to receive intervention until one of following events occurs:Once the patient concludes the study for any of the previous events, a 30-day follow-up will take place to assess possible complications until that date. The intervention will be stopped in patients in whom significant colonisation of a CVC hub is detected, but they will continue to be followed-up to check whether they develop CRBSI. Whenever the catheter is removed on suspicion of infection, the hubs and tip of the catheter will be sent for microbiological culture.A patient will be considered to have discontinued the study when the patient withdraws the informed consent, dies, or is lost to follow-up. The trial will be considered finished when all the recruited participants have completed the 30-day follow-up period. Figure Withdrawal of consentViolation of maskingViolation of the inclusion/exclusion criteriaSerious adverse reactionIn accordance with the current revision of the Declaration of Helsinki and the applicable regulations, a patient has the right to withdraw from the study at any time and for any reason, without this causing any harm to the medical care by their physician. The withdrawal of full consent from a study means that the patient does not wish to continue participating in the study. Any patient withdrawing their consent will be removed from the treatment and/or the study observations immediately after the date the patient requests it.The following data will be collected: sex, age, type of underlying disease, other comorbidities, date of hospital admission, HSCT, type and date of HSCT, immunosuppressive therapy, concomitant medication, duration of neutropenia, type of CVC, days from CVC insertion to inclusion in the study, use of prophylactic antibiotics or antifungal agents, systemic antibiotic therapy, number of catheter locks per episode, results of the cultures of the hubs and CVC insertion site, any episodes of infection, episodes of CRBSI, the need for CVC removal and reason, development of complications, adverse reactions, overall case-fatality rate, CRBSI attributable case-fatality rate, and date of discharge.Significant colonisation of any catheter hub will be defined as the presence of any microbial growth in the qualitative culture of the swabs. CRBSI will be defined as the detection of growth of bacteria or fungi in at least one blood culture obtained from a peripheral vein, associated with clinical signs and symptoms of infection , and no apparent source for BSI other than the CVC. In the case of common skin-colonising microorganisms, such as CNS, two positive sets of blood cultures will be necessary to make the diagnosis of CRBSI. CRBSI attributed to luminal colonisation will be defined as the isolation of identical organisms from the catheter hub and from cultures of separate percutaneously drawn blood specimens; the organism’s identity will be proven by molecular typing for patients with BSI due to CNS. Overall, the 30-day case-fatality rate will be defined as death by any cause within the 30 days following the end of the intervention.For the assessment of the primary endpoint , swabs of the inner surfaces of the catheter hubs will be obtained by the nursing personnel before randomisation and three times weekly (approximately every 48-72 h) thereafter for culture, until the end of the intervention. All swabs are made of viscose, and only the swab of the catheter insertion site has a transport media (Stuart media). One swab is used for each lumen The cultures of the collected swabs will be processed in the microbiology department. The results of the cultures will be reviewed by the participating investigators and will be written down in the data collection form and in the specific database.Incidence of CRBSI: swabs of the insertion site and catheter hubs as well as blood cultures  will be obtained when CRBSI is suspected. In the case of catheter removal, two 5-cm segments, a proximal subcutaneous segment and the tip, will be sampled using the sterile technique for culture. The incidence of CRBSI and the CRBSI attributed to luminal colonisation will be calculated for each group. The differential time to positivity will not be used to diagnose CRBSI. The development of CRBSI will be assessed during intervention and up to 30 days after the last intervention. The rate of CRBSI will be presented as the number of episodes of CRBSI per 1000 admission daysCVC removal: whenever it is necessary to remove the CVC because of occlusion, thrombosis, dislodgement or suspicion and/or documentation of infection, the tip is going to be sent for culture to the microbiology department, and the reasons for removal will be annotated in the data collection form and the database by the participating investigators. The need for CVC removal will be assessed during intervention and up to 30 days after the last interventionAdverse reactions: any adverse reaction potentially associated with the administration of the lock solution will be annotated in the data collection form and the database by the participating investigators. Adverse events will be identified and classified according to the severity and the potential relationship with the lock solution. They will be assessed during the interventionOverall (30-day) case-fatality rate: all patients included in the study will be followed up for a total of 30 days after the end of the intervention by the participating investigators. Death by any cause will be annotated in the data collection form and the databaseThe assessment of the secondary endpoints will be performed as follows:The research nurse in charge of the study will visit the patients daily in order to collect all the data related to the study. Also, the nurse will assess the potential development of any adverse event, and will collect all the information in the database. Adverse events will be classified according to their severity and causality. Only the serious adverse events, and those considered to be related to the use of the product of the study will be reported to the study promoter and to the regulatory authorities.According to our previous experience , we estiThe analysis of the data will be carried out during the last 6 months of the project when all fieldwork has been completed. The database will then be analysed using the stepwise logistic regression model of the R software (R v. 3.2.5). The analyses will be made by intention-to-treat . To handle missing data multiple imputation will be done under the missing at random assumption.For the analysis of the primary endpoint, the frequency of colonisation of the CVC hubs in both treatment groups will be compared. For the analysis of the secondary endpoints, the frequency of CRBSI will be compared between the two groups, as well as the need for CVC removal for any cause and overall case-fatality rate (30 days). Exploratory subgroup analyses will be conducted by underlying haematological condition  and by centre. Due to the fact that a patient could have been included twice, the potential impact of independence violation will be assessed repeating the main analysis using patient as a cluster.t test and a chi-square test. A multivariate analysis will be performed to verify the absence of confusion by other variables. In addition, free time of colonisation of the catheter and BSI will be analysed using Kaplan-Meier curves, and they will be compared between both groups by means of the log-rank test (Mantel-Cox test). A value of p = 0.05 will be considered statistically significant.The statistical analysis will be carried out using a Student’s Samples from the catheter hubs and insertion site will be obtained using sterile swabs and plated in blood agar plates. A qualitative culture of these samples will be performed, and they will be considered positive when the growth of a microorganism is detected. After catheter removal, the terminal portion (5 cm) of the catheter tip will be cultured using the roll-plate technique (Maki’s technique) . A bacteIn the case of CRBSI due to coagulase-negative staphylococci (CNS), if the same species are isolated in any of the catheter hubs or tip and in the blood cultures, the strains will be preserved frozen for further analysis. CNS isolates from blood and catheter samples will be sent to the microbiology laboratory of HUB. Species identification will be confirmed using MALDI-TOF . Finally, isolates will be studied by pulsed-field gel electrophoresis after restriction with SmaI in order to determine the intrapatient genetic relationship following the criteria described by Tenover .In compliance with the standards of Good Clinical Practice, the promoter will carry out the monitoring tasks of the study following the approved monitoring plan. Among others, the tasks of monitoring will include the assessment of the correct application of the inclusion and exclusion criteria, the assessment of the quality of the data collected, the maintenance of the blind, the development and reporting of any adverse events, and the maintenance of the confidentiality of the patients. The need for a Data Monitoring Committee (DMC) was waived by the Ethic Committee because of the low impact on the safety of the patients in the study. Also, no interim analyses were planned.Results will be reported at conferences and in peer-reviewed publications. The first publication will be based on data from the two participating centres and will be analysed as stipulated in the protocol with the statisticians’ supervision. Any formal presentation or publication of data collected from this study will be considered as a joint publication by the participating investigators and will follow the recommendations of the International Committee of Medical Journal Editors.This large prospective, multicentre, randomised, double-blinded, placebo-controlled trial aims to test the hypothesis that lock solution with taurolidine-citrate-heparin is more effective than placebo for preventing CVC catheter infection in high-risk neutropenic haematological patients and, consequently, CRBSI.S. aureus nasal carriage, and application of prophylactic antibiotic ointment on the insertion site [Various strategies to reduce the incidence of CRBSI have been evaluated, including the use of antibiotic-impregnated catheters, strict hygienic measures, eradication of ion site . Howeverion site , 14.A wide variety of antibiotic lock solutions have been evaluated for clinical use, with the largest body of data available for vancomycin and gentamicin . In thisPatients with cancer who have a CVC implanted are at special risk of developing CRBSI. Infection rates of 2.66 per 1000 line-days have been found in adult haematology/oncology and bone marrow transplant units . RecentlTaurolidine is a non-toxic agent with antimicrobial activity against Gram-positive bacteria, Gram-negative bacteria, and fungi. It reduces biofilm formation and does not develop antibiotic resistance because it acts as an antiseptic rather than an antibiotic . InteresThe use of taurolidine-citrate as a lock solution has been associated with mild and self-limited adverse events when used as flushing, with an unpleasant taste in the mouth as the most common side effect.In this study, we aim to assess the efficacy of taurolidine-citrate as a catheter lock solution in preventing CVC bacterial colonisation and, consequently, CRBSI in neutropenic haematological patients. If our hypothesis is proven, the study could provide important solid evidence on the potential usefulness of this preventive procedure in a population at high risk of CRBSI, in whom this complication may significantly impair patient outcome.Recruitment of patients started on 1 January 2013, and it will be completed by 30 June 2018. Protocol version number 5.0. Date: 27 July 2016.Additional file 1:SPIRIT 2013 Checklist: recommended items to address in a clinical trial protocol and related documents*. (DOCX 36 kb)


Xenopus embryonic tissue. The model predicts that mechanical interactions can generate mesoscopic patterns within the monolayer that exhibit long-range correlations in cell shape. The model also suggests that the orientation of mechanical and geometric cues for processes such as cell division are likely to be strongly correlated in real epithelia. Some limitations of the model in capturing geometric features of Xenopus epithelial cells are highlighted.Using a popular vertex-based model to describe a spatially disordered planar epithelial monolayer, we examine the relationship between cell shape and mechanical stress at the cell and tissue level. Deriving expressions for stress tensors starting from an energetic formulation of the model, we show that the principal axes of stress for an individual cell align with the principal axes of shape, and we determine the bulk effective tissue pressure when the monolayer is isotropic at the tissue level. Using simulations for a monolayer that is not under peripheral stress, we fit parameters of the model to experimental data for  Many essential aspects of cell behaviour are controlled, both directly and indirectly, by mechanical cues . For exaThe geometric properties of cells are governed by cell adhesions and cytoskeletal mechanics . The mecTheoretical models of epithelial mechanics fall into a number of classes, including cellular Potts , cell-ceXenopus embryonic epithelia, using cell area over polygonal classes as a measure. Of particular interest is the manner in which mechanical effects constrain the spatial disorder that is intrinsic to epithelial monolayers, which we characterize using simulations, highlighting the appearance of spatial patterns reminiscent of force chains in granular materials. We also discuss the role of the stress acting on the monolayer’s periphery in determining the size and shape of cells.In this article, working in the framework of a popular vertex-based model describing a planar monolayer of mechanically  identical cells, we derive expressions for the stress tensor at the cell and tissue level, and use these results to understand the relationship between a cell’s shape and its mechanical environment, showing that the principal axes of the cell’s stress and shape tensors align. We parameter-fit simulations to images of Xenopus laevis frog embryo. Animal cap tissue was dissected from the embryo at stage 10 of development (early gastrula stage) and cultured on a 20 mm in vivo structure when cultured externally for the time period of our experiments (up to five hours). This system has the advantage of closely resembling in vivo tissue whilst also giving the ability to control peripheral stress on the tissue. For this work, a 0.5 mm uniaxial stretch was applied to the PDMS substrate, which ensured that it did not buckle under gravity or the weight of the animal cap. This small stretch was found to have no measurable effect on cell geometry (data not shown) and we therefore assume that there is negligible peripheral stress on the tissue. The apical cell layer of the animal cap tissue was imaged using a Leica TCS SP5 AOBS upright confocal microscope , given by .Vectors defined relative to a cell centroid are labelled by a greek subscript, We adopt a well-established and widely used vertex-based constitutive model  represThe analogous force for a vertex model lacking the  term in  is givenThe tangential forces show how each edge is a composite structure with tension contributions from two adjacent cells. The factor of cell via . For verWe use this relationship below when considering the boundary conditions at the edge of a monolayer.When the system is out of equilibrium, the net force at each internal vertex isFor a tensor em gives (3.18)\dWe use this weak formulation to derive the stress tensor of the monolayer, taking the stress to be uniform over each cell. The forces acting on cell ument}),  motivateThis reveals conservative (elastic) and dissipative (viscous) contributions to the stress. The former isIf the cell is in equilibrium and under zero net torque, then nt} (see ), ensurimetry of  is confimetry of  requiresmetry of  as is equivalent to that of linear elasticity when considering small perturbations about the unstressed state  and tissTaking imposing , we const}using  and . , we mayment} in  then \dometry of  allows tscipy library.The majority of computational modelling was performed in Python, with some processes sent through C where Python struggled with performance. The cells were described as an oriented graph using the graph-tool module for Python . The algSimulations were performed in a square box of side ondition  within aSimulations for ocument} ; in contocument} .The inherent disorder in equilibrium monolayers is illustrated in  enforce . The varizations  and c reocument}  reveals  systems . In part systems . In cont systems , the corocument}  and b: nell area  and d. M classes  is much ariation .In addition to the model parameters ment} in ) also inXenopus laevis animal cap explants introduced in Of the features described in Using simulations of monolayers under Evaluating  across ahe model . For beshe model  tend to Despite matching area distributions well, the circularity distribution is over-estimated across all polygonal classes . Figure nt} (see ), maintant} (see ) in ordent}, see ) may be We have investigated a popular vertex-based model of planar epithelia, addressing features associated with cell packing rather than division or motility. We focused on a simple version of the model, neglecting refinements such as representations of internal viscous forces , internaocument} , which iment} in  characteXenopus laevis tissue, by maximizing a likelihood function derived from the mean area per polygonal class, suggests The distinction between individual cell stress pression  for the nstraint  on the apressure  is imporpressure  for the pressure  for the onolayer  and 9. Tonolayer . We alsoy packed . The buly packed .The present descriptions of the stress tensor are appropriate for small-amplitude deformations close to equilibria, and in future should be extended to account for irreversible cell rearrangements (such as T1/T2 transitions) that endow the material with an elastic-viscoplastic character, as well as accounting for cell division. Kinematic and geometric quantities (such as the texture tensor) characterizing large deformations of cellular materials have been developed that are based on connections between centres of adjacent cells , the duaDrosophila melanogaster embryo (Xenopus system. Robust evidence of force-shape chains in real epithelia would raise interesting questions about the role of mechanical feedback on patterning of cell division.While the monolayer can be stress-free at the bulk scale, individual cells can have non-zero tterning , stiffertterning  and D1a,r embryo  motivate


The two-point discrimination (TPD) test is one of the most commonly used neurosensory tests to assess mechanoperception in the clinical settings. While there have been numerous studies of functional sensibility of the hand using TPD test, there have been relatively not enough reports on TPD in the orofacial region.  The aims of the present study were to determine the normal values of TPD in the six trigeminal sites  and to investigate the effect of the site, sex, and test modality on the TPD perception. Forty healthy volunteers consisting of age-matched men (20) and women (20) with a mean age of 27.1 years were recruited. One examiner performed the TPD test using a simple hand-operated device, i.e., by drawing compass with a blunt or sharp-pointed tip. The static TPD with a blunt-pointed tip (STPDB), moving TPD with a blunt-pointed tip (MTPDB), and static TPD with a sharp-pointed tip (STPDS) were measured. The predictors were the site, sex, and test modality, and the outcome variable was the TPD value. Three-way ANOVA was used for statistics. The analysis showed a significant effect of the site, sex and test modality on the TPD values. Significant differences between the test sites were observed with the descending order from the forehead and cheek>mentum>upper lip and lower lip>tongue tip and index finger. Women showed lower TPD values than those of men. The STPDS measurements were consistently lower than those of the STPDB and MTPDB. The normal values of TPD in this study suggest that the cheek and forehead were less sensitive than other regions evaluated and women were more sensitive than men. The STPDS was the most sensitive test modality. Routinely conducted tests for the assessment of altered sensation include three levels of tests, i.e., spatiotemporal perception, contact detection and nociception or temperature28. Various traditional techniques such as the Semmes-Weinstein nylon monofilaments for pressure perception, tuning forks for the vibration thresholds and two-point discrimination (TPD) tests have been used for measuring the sensitivity of mechanoreceptors26. Above all, TPD is widely used by clinicians due to its simplicity9. Weber first introduced TPD in 1853 and defined it as “the distance between compass points necessary to feel two contacts”13. Dellon, Mackinnon and Crosby7 (1987) have reported the TPD tests have interobserver reliability. While Jerosch-Herold9 (2000) thought that TPD lacks sensitivity. Such controversy comes from a lack of standardized protocol for determining end-point distance of TPD13. Moberg14 (1990) also stated that valid and repeatable results of TPD test depend on exact protocol and proper tools. Despite controversy regarding the test reliability, TPD is one of the most commonly used clinical tests due to its simplicity for evaluation of peripheral nerve injury and sensory recovery after nerve damage or repair13. Furthermore, there are not enough tools for clinicians to assess tactile acuity in the clinical setting. In this respect, TPD test is still a valuable technique and should not be underestimated as a exploration tool for functional sensation.The primary stimuli for tactile sensation are touch, pressure and vibration applied to skin, and mechanoreceptors are sensitive to the skin deformation caused by mechanical pressure26. It is well established that spatial acuity varies from one body site to another24. Notably, oral region, such as the lip and tongue and finger have superior spatial acuity, i.e., the sensory neural pathways innervating these regions are specialized for spatial information processing24. Therefore, it is no wonder that damage on these sensory nerves is likely to bring a prominent loss of sensory acuity24. Accordingly, accurate measurement of orofacial spatial resolution deserves the attention of clinicians.There are various factors that can influence two-point discrimination values including test site, sex, test modality, age, device, and applied force26. Static and moving TPD with blunt tip is usually tested using the Disk-Criminator and the Aesthesiometer is used for static TPD with sharp tip26.The modality of touch in TPD could be classified into three: static two-point discrimination with blunt tip, moving two-point discrimination with blunt tip, and static two-point discrimination with sharp tipWhile there have been numerous studies of functional sensibility of the hand using TPD test, there have been relatively not enough reports on TPD in the orofacial region. In addition, it is not easy to use the various test tools such as Disk-criminator and Aesthesiometer for different test modalities in the clinical settings. Thus, the purpose of this study was to determine the normal values of two-point discrimination using a simple hand-operated device in the orofacial region and compare the sensitivities of two-point discrimination by the test site, sex, and test modality. The mandibular nerve-innervated area, compared to other regions, was hypothesized to show spatial acuity in the TPD perception. Additionally, we hypothesized that women are more sensitive to TPD than men and the TPD test with moving or sharp tip affects the TPD perception.An advertisement on the experiment was posted in the dental hospital and dental school. The exclusion criteria of this study excluded those who had neurologic disorders, chronic pain, sleep disorders, and systemic diseases such as uncontrolled hypertension or diabetes. Of 52 volunteers from the dental school, we excluded volunteers who had temporomandibular disorder, sleep disorder and numbness after orthognathic surgery. A total of 42 subjects  remained after the exclusion process. To match sex ratio, 20 subjects among 22 women were randomly selected. A total of 40 healthy volunteers from the dental school of Dankook University  aged 21 to 37 years  participated in the study. This study was performed in accordance with the Helsinki Declaration and the University Institutional Review Board approved the study (IRB No H-1303/004/003). Written informed consent was obtained from all subjects after full explanation of the objectives and procedures of the study.11 to calculate the sample size for a power of 80% and a two-sided significance level of 0.05. We performed a pilot test on the cheek and forehead of five participants. Then, we estimated the difference in means and standard deviation of the two sites and calculated the standardized difference. Assuming that the difference in means is 1.7 and the standard deviation is 1.9, we would require approximately 20 patients.As a pilot test, we used Lehr’s formulaThe test sites were defined as the three major sensory branches of the trigeminal nerve region corresponding to the ophthalmic branch (V1), the maxillary branch (V2) and the mandibular branch (V3). In these three branches, six coordinates were selected for the experiment. These were the mentum ; the vermilion of the lower lip; the vermilion of the upper lip; the tip of the tongue; the mid-point of the cheek and the forehead (2 cm above the midpoint of the brow). The index fingertip was chosen randomly between the left and right side and was tested to examine the sensory sensitivity of the subjects and for comparison with the orofacial region. The testing was performed starting with the index finger, then proceeding to the six orofacial test sites in random order, selecting alternatively from the right and left side. To select the test site randomly, the examiner put the papers on which test sites were written in a box and picked a paper before the test. The test sites were chosen according to the site written on the selected paper.Three modalities of TPD tests were performed bilaterally at randomly selected trigeminal test sites. The static two-point discrimination with blunt-pointed tip (STPDB), the moving two-point discrimination with blunt-pointed tip (MTPDB), and the static two-point discrimination with sharp-pointed tip (STPDS) tests were performed in the order mentioned here. There was a rest period of about 1 minute between the tests using three modalities. For MTPDB testing, the tips of the device were moved in a proximal-distal direction with a length of 3 mm. In the case of the index fingertip, only the STPDB and STPDS tests were performed.The two-point test was performed using a simple hand-operated device, i.e., by drawing compass with blunt or sharp-pointed tip . The intThe stimulus intensity was chosen to be that which the subject could perceive as constant touching or moving without the perception of discomfort or pain for the STPDB and MTPDB tests; i.e., skin blanching itself was not used as control. The STPDS test was performed using a similar procedure to the STPDB and MTPDB tests, but the subjects could perceive mild discomfort due to the sharpness of the tip applied.The contact time was approximately 1.5 s. The two points of the tool were applied at the same time and perpendicularly to the test surface. The inter-stimulus interval was approximately 5 s for the STPDB and MTPDB tests and 7 s for the STPDS test.The first distance of the tips, which was large enough for the subject to clearly perceive correctly, was determined at the preliminary testing. The initial TPD test distance was 20 mm for the forehead and cheek, 10 mm for the mentum, 6 mm for both lips and 5 mm for the tongue tip and the index finger tip. If the subject could not correctly perceive the initial distance, a longer distance was set for the initial distance. A threshold was determined using a descending stimulus magnitude and one point was inserted intermittently during the descending series to avoid the subject’s expectation of the continuous decrease in distance between the two points. If the subject answered correctly in response to these changes, the distance decreased in intervals of 1 mm. This testing pattern was continued until the subject answered incorrectly, and the experimenter returned to the next longer distance. The series was terminated when a correct answer for the next longer distance was followed by two incorrect answers on two subsequently shorter distances. This final correct answer was chosen as the end-point for the TPD test. When the subjects continuously had inconsistent responses with the repeated measure of the TPD tests at the given test site, the subjects were excluded from the corresponding test.Two series of TPD testing for three modalities were performed to determine each TPD value and the mean values of two consecutive measurements were calculated. The subjects were given three alternatives for the answers; i.e., the subject was asked to say “one” if the subject felt one point and “two” if two separate points were felt. If the subject said “I can’t discriminate one or two”, it was regarded as an incorrect answer.The above tests were carried out in a quiet room at room temperature by one investigator. The orofacial sensitivity tests were conducted with the subjects in the supine position in the dental chair. The subjects were asked to keep their eyes closed throughout the test procedure.We defined the test site, sex, and test modality as predictors and the two-point discrimination values as the outcome. Before the data analysis, the normality of the data was evaluated using the Kolmogorov-Smirnov test and data were not normally distributed. Thus, log-transformation of data was applied to perform the further statistic calculation and correct the possible heteroscedasticity. All variables were continuous, and the mean threshold values and standard deviations were calculated from the raw data. The side differences at each test site were analyzed by paired t-test.post hoc analysis was calculated for multiple comparison. The upper limits of normality for a given sample were calculated using the 95% prediction interval (1.96 SD). A 95% upper limit of the confidence interval for the population mean of the TPD test values was calculated according to UCL=μ+SEMxt0.05, in which UCL is the upper confidence limit, μ is the sample mean, SEM is the standard error of the mean, and t0.05 corresponds to the percentage point of the t-distribution with (n-1) degrees of freedom which results in a two-tailed probability of 0.05. Statistical tests were performed at the 5% significance level. All statistical calculations were performed using the Statistical Package for the Social Sciences .To test for the effects of site, sex, and test modality on two-point discrimination, data were analyzed using three-way analysis of variance (ANOVA). When the differences were significant, Tukey Healthy young adults consisting of 20 men and 20 women were tested. There were no significant age differences between male and female subjects . In the STPDB test, inconsistent responses were recorded for the forehead and cheek for two men and one woman and the tests were excluded . Tests pThe analyses showed no statistically significant effects of the side on the TPD test values; the means of the right and left side at each site were used for the TPD test threshold values. Descriptive statistics such as mean values and standard deviations were calculated for the test sites and sex in the three different test modalities. Using these data, the 95% upper limits of normal two-point discrimination thresholds in a given sample and the upper confidence limit were also calculated . The meapost hoc analysis, P<0.05). Sex differences were also significant and women showed lower two-point discrimination values than those of men . Test modality showed a significant main effect on the values of the TPD . The STPDS test measurements were consistently lower than the STPDB and MTPDB test measurements . Interactions between two factors or three factors had no significant effect on the TPD thresholds.Three-way ANOVA showed that there were significant differences in the two-point discrimination thresholds depending on the test site, sex, and test modality . SignifiThe main findings of this study are as follows.(1) This study showed that there is superior-inferior gradient for spatial acuity in the orofacial region;(2) Women were more sensitive than men in the TPD perception;(3) The static TPD with sharp tip seemed to be the most sensitive modality for TPD test.9. It is well known that the spatial discrimination ability for touch varies according to the body location26. Weinstein27 (1968) found that the fingertip and face had exquisite tactile sensitivity compared to other body sites. Stevens and Choo19 (1996) assessed spatial acuity over 13 body regions and showed the superior acuity of the fingertip, lip and tongue. Consistent with these findings, our study exhibited site differences for tactile sensitivity, i.e., the tongue tip was the most sensitive for all TPD modalities as predicted and showed the same range of sensitivity as the index fingertip. The hairy skin, such as the forehead, cheek and mentum, was less sensitive than the glabrous skin including the tongue tip, index finger and both lips.Two-point perception tests typically express spatial acuity and reflect the density and receptive field size of the low-threshold mechanoreceptors1.Psychophysical and microneurography techniques in humans and non-human primates have comprehensively identified the causal relation between stimuli and perception, and the sensory afferents corresponding to perception. Slowly adapting type I fibers (SA I) innervate highly sensitive areas of the skin and exhibit high spatial acuity for tactile stimuli, and the innervation density of SA I afferents is relatively higher in glabrous skin than in hairy skin. On the other hand, rapidly acting (RA) type I fibers have higher sensitivity for movement rather than spatial resolution17. Using microneurography, Trulsson and Essick22 (1997) showed that the major population of low-threshold mechanoreceptors in the tongue are RA and SA units. Similarly, Vallbo and Johansson23 (1984) found high density of RA and SA I units in the fingertip. High unitary densities with outstanding spatial resolution in glabrous skin such as tongue tip and fingertip imply that the combined roles of SA I and RA I afferents may enhance tactile acuity. In contrast, predominance of SA afferent has been reported in hairy skin on face, lips and oral mucosa9. Trulsson and Essick22 (1997) interpreted this finding as a functional adaptation of the mechanoreceptive innervation.It has been reported that the body regions with high sensitivity have a large density of sensory spots and a low two-point threshold18. The relatively large area of cortical representation of oral region suggests great importance for oral function in human18.In addition to peripheral factors, high sensitivity of the oral region might be attributed to the cortical representation of oral sensation. It has been demonstrated that the representation of oral sensation over the primary somatosensory cortex is more widely distributed than those for the other body area13. It is well known that controlled stimulus magnitude is one of the prerequisites of psychophysical methods for the assessment of somatosensory function20. Moberg14 (1990) recommended very light force, 10 to 15 g, which corresponds to the force producing first “blanching” in the skin. Whereas Dellon, Mackinnon and Crosby7 (1987) used another method: “just sufficient pressure is utilized for the subject to assess the stimulus”. In fact, Bell-Krotoski and Buford2 (1997) indicated that application of force with handheld instrument produces variations and needs to be controlled for test reliability. This lack of repeatability of the force applied may inhibit the reliability of the TPD test. On the other hand, previous studies showed that spatial discrimination in the skin is relatively insensitive to the force applied25. Vriens and van der Glas25 (2002) reported that the force levels observed were always at an extremely suprathreshold stimulus intensity and, therefore, the thresholds of TPD were almost invariant in relation to the difference in the force applied. Our study used Dellon’s description for the force applied because there was some difficulty in identifying the very first blanching of the skin. In fact, one of the hardest things that the author experienced during the experiment was applying the two tips of the device with even force or synchronously on the skin surface. The subjects could succeed in the TPD test by recognizing uneven contact, i.e., by recognizing the heavier and lighter application force rather than discriminating between two discrete points. Additionally, false TPD occurred if the two tips of the device were applied to the skin at different times. Thus, we should be careful as to the balance as well as the amount of force applied for reliable measurements despite the inherent uncontrolled force in any handheld tests.There are still two unsolved major problems. First, the TPD test with a handheld instrument does not control for applied pressure13. For example, should the test start with the smallest distance using an increasing method or the widest distance using a decreasing method from the initial distance, and how many correct answers should be used for the value of the TPD? It is widely known that the method of limits leads to systemic errors in estimating thresholds due to response biases, i.e., habituation and expectation20. Thus, this study adopted the descending method of limit with intermittent and random insertion of testing stimuli from one to two points as previously reported in Dellon’s study7 to reduce the subject biases. Detailed descriptions of the test procedure should become mandatory. In the future, these methodology shortcomings should be clarified.The lack of a standardized protocol to perform TPD tests is another major problem26. Peters, Hackeman and Goldreich16 (2009) hypothesized that this sex difference in somatosensory perception might result from physical differences between men and women. The study showed that tactile perception improves with decreasing finger size and women, on average, have smaller fingers than men. Considering the Merkel cells around the bases of sweat pores29, higher density of sweat pores in smaller fingers – which had been proved in Peters’s study – suggests that Merkel receptors are packed more densely in women16. Similarly, it is well known that Meissner corpuscles are more densely distributed in smaller fingers15. Considering previous studies in the finger, high tactile sensitivity of women in this study suggests increased mechanoreceptor density in orofacial region of women compared to men. However, there are also other studies with contrasting results17. Considering that site differences were significant for the TPD values in many previous reports despite the different devices, subjects and protocols of those studies, these inconsistent results for the influence of sex on TPD values might imply that sex is not as powerful as site as predictor of TPD values.Sex, as well as the site, influenced the outcome of the TPD tests in the current study. Our findings are in line with previous studies of the face as well as of the hand26. Considering that the significant differences between the STPDS test and the two other modalities were higher in the forehead and cheek than in the lips and tongue tip, the STPDS test rather than the others could be a better modality in the V1 and V2. In particular, this exquisite discriminative ability of the STPDS test would be beneficial in the early detection of sensory recovery in patients with nerve injuries because nociception is commonly regained earlier than touch perception in the course of sensory recovery.Two-point perception was evaluated using three different testing modalities, and static TPD with sharp tip was found to be the most sensitive modality. The difference between the TPD modalities with blunt and sharp tip is the pressure applied. Stimuli with a sharp tip will increase the pressure and might activate the nociceptors4. Dellon4 (1978) reported that the sensation of moving touch is recovered not only sooner but also to a higher degree than that of constant touch. This suggests that the MTPDB test might be useful in evaluating the extent of sensory recovery, like the STPDS test, in clinical settings.While static two-point discrimination with blunt tip called Weber test is a classic TPD test that evaluates the slowly adapting fiber/receptor system that detects constant touch, the moving two-point discrimination test evaluates the function of the rapidly adapting afferents as a detector of transient touch, i.e., movement21. Tamura, et al.21 (2003) indicated that the TPD process is related to evaluation of the distance between the stimuli relative to that of the preceding two-point stimulus, as the conditioning stimulus, as well as the assessment of absolute distance between the stimuli. Thus, the results of our study suggest that the balance between PNS and CNS processing for evaluation of the TPD might be different between hairy and glabrous skin in the orofacial region. In a different point of view, PNS factors might be more influential on the TPD in glabrous skin rather than in hairy skin. Thus, the TPD values should be interpreted with caution in the forehead, cheek and mentum in comparison with the oral region.In the present study, an interesting finding was that most of the subjects often showed inconsistent responses even under the same stimulus condition. In particular, inconsistent responses were prominent in hairy skin on the forehead, cheek and mentum rather than in glabrous skin such as on the lips and tongue. Although it is unclear what actually caused this inconsistency, two factors could be possible explanations. First, the uncontrolled force applied may induce inconsistent responses. However, if we consider that these inconsistent responses were prominent in hairy skin, especially in the forehead and cheek, uncontrolled force alone is not enough for full explanation of these variable responses in hairy facial skin. Second, the neuropsychological aspect of the TPD should be considered. The TPD threshold is influenced by the central nervous system (CNS) as well as by several factors in the peripheral nervous system (PNS)Risk of selection bias should be considered as a study limitation. The participants of this study were not representative of the population because they were young students from a dental school. Thus, our normative TPD values cannot be applicable to all ages and the results of this study should be interpreted as a preliminary study.To our best knowledge, this study was the first to perform TPD test using a drawing compass with blunt or sharp-pointed tip as a simple hand-operated device. The device used in this study, compared to the Disk-Criminator and Aesthesiometer, is simple and affordable enough for use in a clinical setting. In addition, we tested all three trigeminal branches with various TPD modalities.The normal TPD values presented in this study suggest heterogeneity of spatial acuity and sex difference in the orofacial region. The cheek and forehead have lower sensory accuracy than other regions evaluated and women were more sensitive than men in TPD perception. Static TPD with sharp tip would be beneficial for the assessment of sensory recovery as well as abnormal sensation in patients with somatosensory abnormalities. Although the TPD test is not recommended as the only tool for evaluation of sensory impairment or sensory recovery, the TPD test using a simple handheld device would provide much more trigeminal sensory information if clinicians use various TPD test modalities with the understanding of the normative values.


Rett syndrome (RS) is a neurological disorder, affecting mainly females, caused by MECP2 mutations usually resulting in severe physical disability. Due to the physical challenges faced by the individual with RS and her family, her rehabilitation program should support her throughout different daily activities, contexts, and surroundings. Rehabilitation interventions to reverse physical impairments include exercise of various types and different physical modalities. Nevertheless, in the vast majority of cases, hands-on therapeutic intervention opportunities are available for the client through a minute part of her waking hours. Hence, a supplementary system is required in order to engulf the child with a comprehensive network of support. Supplementary intervention can support physical impairment by introducing adaptive techniques, environmental modifications, and assistive technologies. The therapy program of an individual with RS should include the use of assistive technology when such devices improve the user's performance. The term “supplementary management” relates to the fact that this intervention may be performed by nonprofessionals with the supervision of a qualified therapist. Such an intervention can further support the therapeutic goals of the child, at a time when direct intervention is not supplied. The present article will review the available literature on the topic of assistive technology, incorporating the clinical knowledge of the author in the field of RS.