Personalized Hydration Strategy to Improve Fluid Balance and Intermittent Exercise Performance in the Heat.

Sweat rate and electrolyte losses have a large inter-individual variability. A personalized approach to hydration can overcome this issue to meet an individual's needs. This study aimed to investigate the effects of a personalized hydration strategy (PHS) on fluid balance and intermittent exercise performance. Twelve participants conducted 11 laboratory visits including a VO2max test and two 5-day trial arms under normothermic (NOR) or hyperthermic (HYP) environmental conditions. Each arm began with three days of familiarization exercise followed by two random exercise trials with either a PHS or a control (CON). Then, participants crossed over to the second arm for: NOR+PHS, NOR+CON, HYP+PHS, or HYP+CON. The PHS was prescribed according to the participants' fluid and sweat sodium losses. CON drank ad libitum of commercially-available electrolyte solution. Exercise trials consisted of two phases: (1) 45 min constant workload; (2) high-intensity intermittent exercise (HIIT) until exhaustion. Fluids were only provided in phase 1. PHS had a significantly greater fluid intake (HYP+PHS: 831.7 ± 166.4 g; NOR+PHS: 734.2 ± 144.9 g) compared to CON (HYP+CON: 369.8 ± 221.7 g; NOR+CON: 272.3 ± 143.0 g), regardless of environmental conditions (p < 0.001). HYP+CON produced the lowest sweat sodium concentration (56.2 ± 9.0 mmol/L) compared to other trials (p < 0.001). HYP+PHS had a slower elevated thirst perception and a longer HIIT (765 ± 452 s) compared to HYP+CON (548 ± 283 s, p = 0.04). Thus, PHS reinforces fluid intake and successfully optimizes hydration status, regardless of environmental conditions. PHS may be or is an important factor in preventing negative physiological consequences during high-intensity exercise in the heat.

Janus Membrane-Based Wearable pH Sensor with Sweat Absorption, Gas Permeability, and Self-Adhesiveness.

Wearable biomedical sensors have enabled noninvasive and continuous physiological monitoring for daily health management and early detection of chronic diseases. Among biomedical sensors, wearable pH sensors attracted significant interest, as pH influences most biological reactions. However, conformable pH sensors that have sweat absorption ability, are self-adhesive to the skin, and are gas permeable remain largely unexplored. In this study, we present a pioneering approach to this problem by developing a Janus membrane-based pH sensor with self-adhesiveness on the skin. The sensor is composed of a hydrophobic polyurethane-polydimethylsiloxane porous hundreds nanometer-thick substrate and a hydrophilic poly(vinyl alcohol)-poly(acrylic acid) porous nanofiber layer. This Janus membrane exhibits a thickness of around 10 µm, providing a conformable adhesion to the skin. The simultaneous realization of solution absorption, gas permeability, and self-adhesiveness makes it suitable for long-term continuous monitoring without compromising the comfort of the wearer. The pH sensor was tested successfully for continuous monitoring for 7.5 h, demonstrating its potential for stable analysis of skin health conditions. The Janus membrane-based pH sensor holds significant promise for comprehensive skin health monitoring and wearable biomedical applications.

Wireless wearable potentiometric sensor for simultaneous determination of pH, sodium and potassium in human sweat.

This paper reports on the development of a flexible-wearable potentiometric sensor for real-time monitoring of sodium ion (Na+), potassium ion (K+), and pH in human sweat. Na0.44MnO2, polyaniline, and K2Co[Fe(CN)6] were used as sensing materials for Na+, H+ and K+ monitoring, respectively. The simultaneous potentiometric Na+, K+, and pH sensing were carried out by the developed sensor, which enables signal collection and transmission in real-time to the smartphone via a Wi-Fi access point. Then, the potentiometric responses were evaluated by a designed android application. Na+, K+, and pH sensors illustrated high sensitivity (59.7 ± 0.8 mV/decade for Na+, 57.8 ± 0.9 mV/decade for K+, and 54.7 ± 0.6 mV/pH for pH), excellent stability, and good batch-to-batch reproducibility. The results of on-body experiments demonstrated that the proposed platform is capable of real-time monitoring of the investigated ions.

All-printed wearable biosensor based on MWCNT-iron oxide nanocomposite ink for physiological level detection of glucose in human sweat.

Wearable sensors for sweat glucose monitoring are gaining massive interest as a patient-friendly and non-invasive way to manage diabetes. The present work offers an alternative on-body method employing an all-printed flexible electrochemical sensor to quantify the amount of glucose in human sweat. The working electrode of the glucose sensor was printed using a custom-formulated ink containing multi-walled carbon nanotube (MWCNT), poly (3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOPT: PSS), and iron (II, III) oxide (Fe3O4) nanoparticles. This novel ink composition has good conductivity, enhanced catalytic activity, and excellent selectivity. The working electrode was modified using Prussian blue (PB) nanoparticles and glucose oxidase enzyme (GOx). The sensor displayed a linear chronoamperometric response to glucose from 1 µM to 400 µM, with a precise detection limit of ∼0.38 µM and an impressive sensitivity of ∼4.495 µAµM-1cm-2. The sensor stored at 4 °C exhibited excellent stability over 60 days, high selectivity, and greater reproducibility. The glucose detection via the standard addition method in human sweat samples acquired a high recovery rate of 96.0-98.6%. Examining human sweat during physical activity also attested to the biosensor's real-time viability. The results also show an impressive correlation between glucose levels obtained from a commercial blood glucose meter and sweat glucose concentrations. Remarkably, the present results outperform previously published printed glucose sensors in terms of detection range, low cost, ease of manufacturing, stability, selectivity, and wearability.

Synergistic enhancement of wearable biosensor through Pt single-atom catalyst for sweat analysis.

Real-time monitoring of biological markers in sweat is a valuable tool for health assessment. In this study, we have developed an innovative wearable biosensor for precise analysis of glucose in sweat during physical activities. The sensor is based on a single-atom catalyst of platinum (Pt) uniformly dispersed on tricobalt tetroxide (Co3O4) nanorods and reduced graphene oxide (rGO), featuring a unique three-dimensional nanostructure and excellent glucose electrocatalytic performance with a wide detection range of 1-800 µM. Additionally, density functional theory calculations have revealed the synergetic role of Pt active sites in the Pt single-atom catalyst (Co3O4/rGO/Pt) in glucose adsorption and electron transfer, thereby enhancing sensor performance. To enable application in wearable devices, we designed an S-shaped microfluidic chip and a point-of-care testing (POCT) device, both of which were validated for effectiveness through actual use by volunteers. This research provides valuable insights and innovative approaches for analyzing sweat glucose using wearable devices, contributing to the advancement of personalized healthcare.

[Non-invasive glucose measurements : where do we stand?] / Mesures du glucose non invasives : où en est-on ?

The technologies used to measure blood glucose have significantly evolved the past few years, especially with the introduction of continuous interstitial glucose measurements, simplifying the management of the disease. More recently, there has been a lot of interest regarding some potential revolutionary methods, such as smartwatches, and glucose measurements in sweat, saliva, and even tears. In this article, we review the different technologies that are under development, and notice that although promising, they rest imprecise. False measurements can have fatal consequences for our patients. Nevertheless, these innovations are promising and have the potential to change the daily life of people with diabetes in the future.

Les technologies utilisées pour mesurer les glycémies des personnes présentant un diabète ont beaucoup évolué ces dernières années, avec notamment l'introduction des mesures interstitielles en continu, rendant le contrôle glycémique plus aisé. Depuis peu, il y a un intérêt croissant, notamment dans les médias, autour de potentielles méthodes révolutionnaires via des montres intelligentes, la sueur, la salive et même les larmes. Dans cet article, nous répertorions les différentes technologies en cours d'investigation et notons que plusieurs d'entre elles restent imprécises, empêchant leur utilisation pour nos patients diabétiques, chez qui des mesures incorrectes peuvent avoir de graves conséquences. Néanmoins, ces nouveautés sont prometteuses et ont le potentiel de changer le quotidien des personnes présentant un diabète dans le futur.

Sensing patches for biomarker identification in skin-derived biofluids.

In conventional clinical disease diagnosis and screening based on biomarker detection, most analysis samples are collected from serum, blood. However, these invasive collection methods require specific instruments, professionals, and may lead to infection risks. Additionally, the diagnosis process suffers from untimely results. The identification of skin-related biomarkers plays an unprecedented role in early disease diagnosis. More importantly, these skin-mediated approaches for collecting biomarker-containing biofluid samples are noninvasive or minimally invasive, which is more preferable for point-of-care testing (POCT). Therefore, skin-based biomarker detection patches have been promoted, owing to their unique advantages, such as simple fabrication, desirable transdermal properties and no requirements for professional medical staff. Currently, the skin biomarkers extracted from sweat, interstitial fluid (ISF) and wound exudate, are achieved with wearable sweat patches, transdermal MN patches, and wound patches, respectively. In this review, we detail these three types of skin patches in biofluids collection and diseases-related biomarkers identification. Patch classification and the corresponding manufacturing as well as detection strategies are also summarized. The remaining challenges in clinical applications and current issues in accurate detection are discussed for further advancement of this technology (Scheme 1).

Anti-inflammatory effects of elexacaftor/tezacaftor/ivacaftor in adults with cystic fibrosis heterozygous for F508del.

Inflammation is a key driver in the pathogenesis of cystic fibrosis (CF). We assessed the effectiveness of elexacaftor/tezacaftor/ivacaftor (ETI) therapy on downregulating systemic and immune cell-derived inflammatory cytokines. We also monitored the impact of ETI therapy on clinical outcome. Adults with CF, heterozygous for F508del (n = 19), were assessed at baseline, one month and three months following ETI therapy, and clinical outcomes were measured, including sweat chloride, lung function, weight, neutrophil count and C-reactive protein (CRP). Cytokine quantifications were measured in serum and following stimulation of peripheral blood mononuclear cells (PBMCs) with lipopolysaccharide (LPS) and adenosine triphosphate and analysed using LEGEND plex™ Human Inflammation Panel 1 by flow cytometry (n = 19). ASC specks were measured in serum and caspase-1 activity and mRNA levels determined from stimulated PBMCs were determined. Patients remained stable over the study period. ETI therapy resulted in decreased sweat chloride concentrations (p < 0.0001), CRP (p = 0.0112) and neutrophil count (p = 0.0216) and increased percent predicted forced expiratory volume (ppFEV1) (p = 0.0399) from baseline to three months, alongside a trend increase in weight. Three months of ETI significantly decreased IL-18 (p< 0.0011, p < 0.0001), IL-1ß (p<0.0013, p = 0.0476), IL-6 (p = 0.0109, p = 0.0216) and TNF (p = 0.0028, p = 0.0033) levels in CF serum and following PBMCs stimulation respectively. The corresponding mRNA levels were also found to be reduced in stimulated PBMCs, as well as reduced ASC specks and caspase-1 levels, indicative of NLRP3-mediated production of pro-inflammatory cytokines, IL-1ß and IL-18. While ETI therapy is highly effective at reducing sweat chloride and improving lung function, it also displays potent anti-inflammatory properties, which are likely to contribute to improved long-term clinical outcomes.

Multifunctional laser-induced graphene circuits and laser-printed nanomaterials toward non-invasive human kidney function monitoring.

Faced with the increasing prevalence of chronic kidney disease (CKD), portable monitoring of CKD-related biomarkers such as potassium ion (K+), creatinine (Cre), and lactic acid (Lac) levels in sweat has shown tremendous potential for early diagnosis. However, a rapidly manufacturable portable device integrating multiple CKD-related biomarker sensors for ease of sweat testing use has yet to be reported. Here, a portable electrochemical sensor integrated with multifunctional laser-induced graphene (LIG) circuits and laser-printed nanomaterials based working electrodes fabricated by fully automatic laser manufacturing is proposed for non-invasive human kidney function monitoring. The sensor comprises a two-electrode LIG circuit for K+ sensing, a three-electrode LIG circuit with a Kelvin compensating connection for Cre and Lac sensing, and a printed circuit board based portable electrochemical workstation. The working electrodes containing Cu and Cu2O nanoparticles fabricated by two-step laser printing show good sensitivity and selectivity toward Cre and Lac sensing. The sensor circuits are fabricated by generating a hydrophilic-hydrophobic interface on a patterned LIG through laser. This sensor recruited rapid laser manufacturing and integrated with multifunctional LIG circuits and laser-printed nanomaterials based working electrodes, which is a potential kidney function monitoring solution for healthy people and kidney disease patients.

Recent advances in biosensors based on metal-oxide semiconductors system-integrated into bioelectronics.

Metal-oxide semiconductors (MOSs) have emerged as pivotal components in technology related to biosensors and bioelectronics. Detecting biomarkers in sweat provides a glimpse into an individual's metabolism without the need for sample preparation or collection steps. The distinctive attributes of this biosensing technology position it as an appealing option for biomedical applications beyond the scope of diagnosis and healthcare monitoring. This review encapsulates ongoing developments of cutting-edge biosensors based on MOSs. Recent advances in MOS-based biosensors for human sweat analyses are reviewed. Also discussed is the progress in sweat-based biosensing technologies to detect and monitor diseases. Next, system integration of biosensors is demonstrated ultimately to ensure the accurate and reliable detection and analysis of target biomarkers beyond individual devices. Finally, the challenges and opportunities related to advanced biosensors and bioelectronics for biomedical applications are discussed.

Fully Integrated Multiplexed Wristwatch for Real-Time Monitoring of Electrolyte Ions in Sweat.

Considerable progress has already been made in sweat sensors based on electrochemical methods to realize real-time monitoring of biomarkers. However, realizing long-term monitoring of multiple targets at the atomic level remains extremely challenging, in terms of designing stable solid contact (SC) interfaces and fully integrating multiple modules for large-scale applications of sweat sensors. Herein, a fully integrated wristwatch was designed using mass-manufactured sensor arrays based on hierarchical multilayer-pore cross-linked N-doped porous carbon coated by reduced graphene oxide (NPCs@rGO-950) microspheres with high hydrophobicity as core SC, and highly selective monitoring simultaneously for K+, Na+, and Ca2+ ions in human sweat was achieved, exhibiting near-Nernst responses almost without forming an interfacial water layer. Combined with computed tomography, solid-solid interface potential diffusion simulation results reveal extremely low interface diffusion potential and high interface capacitance (598 µF), ensuring the excellent potential stability, reversibility, repeatability, and selectivity of sensor arrays. The developed highly integrated-multiplexed wristwatch with multiple modules, including SC, sensor array, microfluidic chip, signal transduction, signal processing, and data visualization, achieved reliable real-time monitoring for K+, Na+, and Ca2+ ion concentrations in sweat. Ingenious material design, scalable sensor fabrication, and electrical integration of multimodule wearables lay the foundation for developing reliable sweat-sensing systems for health monitoring.

No effect of tattoos on local sweat concentrations of select cytokines, cortisol, glucose, blood urea nitrogen, or lactate during exercise.

Due to growing interest in the investigation of exercise induced sweat biomarkers to assess an individual's health and the increasing prevalence of tattoos in the world's population, investigators sought to determine whether local sweat concentrations and excretion rates of epidermal growth factor (EGF), interleukin (IL) -1α, IL-6, IL-8, cortisol, glucose, blood urea nitrogen (BUN), and lactate differ between tattooed and contralateral non-tattooed skin during exercise. Sixteen recreational exercisers [female (50%)] (age = 25-48 years) with ≥ 1 unilateral permanent tattoo [median tattoo age = 6 years, IQR = 5] on the arm/torso completed an outdoor group fitness session. There were no significant differences between tattooed and non-tattooed skin for sweat EGF, IL-1α, IL-8, cortisol, glucose, BUN, or lactate concentrations. There were no significant differences between tattooed and non-tattooed skin for sweat EGF, IL-1α, IL-8, cortisol, glucose, BUN, or lactate excretion rate. Findings suggest that permanent tattoos older than 1 year may not impact local sweat EGF, IL-1α, IL-8, cortisol, glucose, BUN, and lactate concentrations or excretion rates during exercise.Clinical trial identifier NCT04920266 was registered on June 9, 2021.

Microfluidic Sensing Textile for Continuous Monitoring of Sweat Glucose at Rest.

Wearable sweat sensors have received considerable attention due to their great potential for noninvasive continuous monitoring of an individual's health status applications. However, the low secretion rate and fast evaporation of sweat pose challenges in collecting sweat from sedentary individuals for noninvasive analysis of body physiology. Here, we demonstrate wearable textiles for continuous monitoring of sweat at rest using the combination of a heating element and a microfluidic channel to increase localized skin sweat secretion rates and combat sweat evaporation, enabling accurate and stable monitoring of trace amounts of sweat. The Janus sensing yarns with a glucose sensing sensitivity of 36.57 mA cm-2 mM-1 are embroidered into the superhydrophobic heated textile to collect sweat directionally, resulting in improved sweat collection efficiency of up to 96 and 75% retention. The device also maintains a highly durable sensing performance, even in dynamic deformation, recycling, and washing. The microfluidic sensing textile can be further designed into a wireless sensing system that enables sedentary-compatible sweat analysis for the continuous, real-time monitoring of body glucose levels at rest.

Fundamental Study of a Wristwatch Sweat Lactic Acid Monitor.

A lactic acid (LA) monitoring system aimed at sweat monitoring was fabricated and tested. The sweat LA monitoring system uses a continuous flow of phosphate buffer saline, instead of chambers or cells, for collecting and storing sweat fluid excreted at the skin surface. To facilitate the use of the sweat LA monitoring system by subjects when exercising, the fluid control system, including the sweat sampling device, was designed to be unaffected by body movements or muscle deformation. An advantage of our system is that the skin surface condition is constantly refreshed by continuous flow. A real sample test was carried out during stationary bike exercise, which showed that LA secretion increased by approximately 10 µg/cm2/min compared to the baseline levels before exercise. The LA levels recovered to baseline levels after exercise due to the effect of continuous flow. This indicates that the wristwatch sweat LA monitor has the potential to enable a detailed understanding of the LA distribution at the skin surface.

Evidence of ostarine cross-contamination via sweat in 2 athletes sharing the same neoprene hamstring sleeves. An original situation of drug transfer where the anti-doping rule violation was suspended by the sport authorities.

The presence of ostarine, a selective androgen receptor modulator (SARM) in an athlete's sample constitutes one of the most frequent anti-doping rules violation. It is possible to challenge this violation but it is the athlete who has to demonstrate he / she is innocent. The conditions to evidence no fault or negligence are mostly based on 2 points: 1. the athlete or his/her legal representative must present verified circumstances of contamination and the source of contamination must be identified; and 2. there must be verified claims by the athlete about the fact that he / she did not knowingly take the prohibited substance, i.e. that the violation was not intentional. During a 2-weeks period, a male athlete tested two times positive for ostarine in urine (<0.1 ng/ml) and he challenged these results. His hair and nail tests returned negative (LOQ at 0.5 pg/mg). He admitted using two neoprene hamstring sleeves of another athlete who confessed abusing ostarine. This was confirmed in his hair (190 pg/mg), his fingernail clippings (780 pg/mg) and his toenail clippings (45 pg/mg). To document the presence of ostarine in the hamstring sleeves and therefore possible drug transfer, the hamstring sleeves were analysed. Ostarine was identified in 12 different selected pieces (about 1 g) of the sleeves at concentrations ranging from 3 to 142 pg/g. Sport authorities (USADA) agreed that the most likely source of contamination was the hamstring sleeves, thus confirming the scenario of drug transfer and gave the athlete a no fault.

Hygroscopic cooling (h-cool) fabric with highly efficient sweat evaporation and heat dissipation for personal thermo-moisture management.

Moisture evaporation plays a crucial role in thermal management of human body, particularly in perspiration process. However, current fabrics aim for sweat removal and takes little account of basic thermo-regulation of sweat, resulted in their limited evaporation capacity and heat dissipation at moderate/intense scenarios. In this study, a hygroscopic cooling (h-cool) fabric based on multi-functional design, for personal perspiration management, was described. By using economic and effective weaving technology, directional moisture transport routes and heat conductive pathways were incorporated in the construct. The resultant fabric showed 10 times greater one-way transport index higher than cotton, Dri-FIT and Coolswitch fabrics, which contributed to highly enhanced evaporation ability (∼4.5 times than cotton), not merely liquid diffusion. As a result, h-cool fabric performed 2.1-4.2 °C cooling efficacy with significantly reduced sweat consuming than cotton, Dri-FIT and Coolswitch fabrics in the artificial sweating skin. Finally, the practical applications by actually wearing h-cool fabric showed great evaporative-cooling efficacy during different physical activities. Owing to the excellent thermo-moisture management ability, we expect the novel concept and construct of h-cool fabric can provide promising strategy for developing functional textiles with great "cool" and comfortable "dry" tactile sensation at various daily scenarios.

Wearable Sensing Device Integrated with Prestored Reagents for Cortisol Detection in Sweat.

Wearable sweat sensors have achieved rapid development since they hold great potential in personalized health monitoring. However, a typical difficulty in practical processes is the control of working conditions for biorecognition elements, e.g., pH level and ionic strength in sweat may decrease the affinity between analytes and recognition elements. Here, we developed a wearable sensing device for cortisol detection in sweat using an aptamer as the recognition element. The device integrated functions of sweat collection, reagent prestorage, and signal conversion. Especially, the components of prestored reagents were optimized according to the inherent characteristics of sweat samples and electrodes, which allowed us to keep optimal conditions for aptamers. The sweat samples were transferred from the inlet of the device to the reagent prestored chamber, and the dry preserved reagents were rehydrated with sweat and then arrived at the aptamer-modified electrodes. Sweat samples of volunteers were analyzed by the wearable sensing device, and the results showed a good correlation with those of the ELISA kit. We believe that this convenient and reliable wearable sensing device has significant potential in self-health monitoring.

Adaptively resettable microfluidic patch for sweat rate and electrolytes detection.

Skin-interfaced microfluidic patch has become a reliable device for sweat collection and analysis. However, the intractable problems of emptying the microchannel for reuse, and the channel's volumetric capacity limited by the size of the patch, directly hinder the practical application of sweat sensors. Herein, we report an adaptively resettable microfluidic sweat patch (Art-Sweat patch) capable of continuously monitoring both sweat rate (0.2-4.0 µL min-1) and total ionic charge concentration (10-200 mmol L-1). We develop a platform with a vertical and horizontal microchannel combined strategy, enabling repeatedly filling sweat and emptying the microchannel for autonomously resetting and detecting. The variation in the emptied volume is designed to be adaptively identified by the sensor, resulting in enhanced stability and an enlarged volumetric capacity of over 300 µL. By integrating with self-designed wireless transmission modules, the proposed Art-Sweat patch shows product-level wearability and high performance in monitoring variations in regional sweat rate and concentration for hydration status assessment.

A wearable flexible electrochemical biosensor with CuNi-MOF@rGO modification for simultaneous detection of uric acid and dopamine in sweat.

BACKGROUND: In health assessment and personalized medical services, accurate detection of biological markers such as dopamine (DA) and uric acid (UA) in sweat is crucial for providing valuable physiological information. However, there are challenges in detecting sweat biomarkers due to their low concentrations, variations in sweat yield among individuals, and the need for efficient sweat collection. RESULTS: We synthesized CuNi-MOF@rGO as a high-activity electrocatalyst and investigated its feasibility and electrochemical mechanism for simultaneously detecting low-concentration biomarkers UA and DA. Interaction between the non-coordinating carboxylate group and the sample produces effective separation signals for DA and UA. The wearable biomimetic biosensor has a wide linear range of 1-500 µM, with a detection limit of 9.41 µM and sensitivity of 0.019 µA µM-1 cm-2 for DA, and 10-1000 µM, with a detection limit of 9.09 µM and sensitivity of 0.026 µA µM-1 cm-2 for UA. Thus, our sensor performs excellently in detecting low-concentration biomarkers. To improve sweat collection, we designed a microfluidic-controlled device with hydrophilic modification in the microchannel. Experimental results show optimal ink flow at 2% concentration. Overall, we developed an innovative and highly active electrocatalyst, successfully enabling simultaneous detection of low-concentration biomarkers UA and DA. SIGNIFICANCE: This study provides a strategy for sweat analysis and health monitoring. Moreover, the sensor also showed good performance in detecting real sweat samples. This study has shown great potential in future advances in sweat analysis and health monitoring.

Sweat as a source of non-invasive biomarkers for clinical diagnosis: An overview.

Sweat has excellent potential as one of the sources of non-invasive biomarkers for clinical diagnosis. It is relatively easy to collect and process and may contain different disease-specific markers and drug metabolites, making it ideal for various clinical applications. This article discusses the anatomy of sweat glands and their role in sweat production, as well as the history and development of multiple sweat sample collection and analysis techniques. Another primary focus of this article is the application of sweat detection in clinical disease diagnosis and other life scenarios. Finally, the limitations and prospects of sweat analysis are discussed.