Conventional diet assessment approaches such as the 24-hour self-reported recall are burdensome, suffer from recall bias, and are inaccurate in estimating energy intake. Wearable sensor technology, coupled with advanced algorithms, is increasingly showing promise in its ability to capture behaviors that provide useful information for estimating calorie and macronutrient intake.This paper aimed to summarize current technological approaches to monitoring energy intake on the basis of expert opinion from a workshop panel and to make recommendations to advance technology and algorithms to improve estimation of energy expenditure.A 1-day invitational workshop sponsored by the National Science Foundation was held at Northwestern University. A total of 30 participants, including population health researchers, engineers, and intervention developers, from 6 universities and the National Institutes of Health participated in a panel discussing the state of evidence with regard to monitoring calorie intake and eating behaviors.Calorie monitoring using technological approaches can be characterized into 3 domains: (1) image-based sensing ; (2) eating action unit (EAU) sensors ; and (3) biochemical measures . We discussed how each domain functions, provided examples of promising solutions, and highlighted potential challenges and opportunities in each domain. Image-based sensor research requires improved ground truth (context and known information about the foods), accurate food image segmentation and recognition algorithms, and reliable methods of estimating portion size. EAU-based domain research is limited by the understanding of when their systems  succeed and fail, need for privacy-protecting methods of capturing ground truth, and uncertainty in food categorization. Although an exciting novel technology, the challenges of biochemical sensing range from a lack of adaptability to environmental effects  and mechanical impact, instability of wearable sensor performance over time, and single-use design.Conventional approaches to calorie monitoring rely predominantly on self-reports. These approaches can gain contextual information from image-based and EAU-based domains that can map automatically captured food images to a food database and detect proxies that correlate with food volume and caloric intake. Although the continued development of advanced machine learning techniques will advance the accuracy of such wearables, biochemical sensing provides an electrochemical analysis of sweat using soft bioelectronics on human skin, enabling noninvasive measures of chemical compounds that provide insight into the digestive and endocrine systems. Future computing-based researchers should focus on reducing the burden of wearable sensors, aligning data across multiple devices, automating methods of data annotation, increasing rigor in studying system acceptability, increasing battery lifetime, and rigorously testing validity of the measure. Such research requires moving promising technological solutions from the controlled laboratory setting to the field. The marked rise in obesity, particularly in the United States, is a complex sociodemographic and public health problem that is largely driven by poor diet, excessive caloric intake, and insufficient caloric expenditure . Weight image-sensing technology ; (2) eating action unit (EAU)–based technology ; and (3) biochemical measures . Although their impact on improving care and health outcomes remains untested, the validity of such devices is a prominent concern among researchers. Prior narrative reviews and surveys have focused on describing existing technologies [To date, 3 types of technology-enabled wearable domains for calorie and nutrient monitoring have emerged: (1) nologies -9 and alnologies , along wprimary aim of the workshop was to discuss the development, evaluation, and use of technology to detect and understand diet behaviors and estimate calorie and macronutrient intake.An expert, consensus-building 1-day workshop, supported by the National Science Foundation and organized by Northwestern University, was held on June 20, 2017, in Chicago, Illinois. The A total of 30 participants from 6 universities and from the National Institutes of Health were selected to participate in the workshop. To capture varying perspectives across multiple fields, participants included population health researchers, such as behavioral scientists, nutritionists, obesity epidemiologists, and intervention developers ; computer scientists ; and engineers in biomedical, material science, and computer technology . primary deliverable was a set of presentations delineating current gold standards for measuring energy intake and an appraisal of the state of research related to calorie- and macronutrient-monitoring technology. Experts within each technology-enabled wearable domain identified new insights and opportunities from these presentations and conversations, which were used to inform the final recommendations presented in this paper. A final review of the recommendations was performed by the authors of this paper.A team consisting of at least 2 participants was organized to lead a discussion about one of the following topics: types of technology-enabled calorie and macronutrient monitoring, potential research gaps and technical challenges to advance the capture of energy expenditure, and methods for how technology can assist conventional subjective diet assessments. Workshop participants were also randomly separated into 2 groups to delineate key topics for future research. Overall, there was consensus regarding the need to refine technology-supported calorie- and macronutrient-monitoring approaches. The Technology-enabled measures can reduce participant burden and increase granularity of diet data collection through automated measures . We haveImage-based sensing systems ,13 that ground truth label, derived from observable data, is the objective verification of particular properties of a digital image, used to test the accuracy of automated image analysis processes. The ground truth of food images includes known, fixed features such as pixels representing food objects in the image (used for food segmentation), food types (used for food recognition), and food portion size in grams (used for portion size estimation). Such information is necessary to train the image analysis system to accurately estimate calorie intake from the image. Several publicly available food datasets contain a substantial amount of food images [ground truth labels to improve the learned models. These ground truth labels should clearly delineate different food items even if they are on a single plate and should include portion size information for each food item.A d images  and provground truth information, a solution is to merge food images sourced from the internet or from nutrition studies with manual annotation from crowd-sourcing platforms. Amazon Mechanical Turk (AMT) has been used for food image collection and annotation tasks [To address the need for constructing large-scale food datasets with food images that provide comprehensive on tasks ,20, althon tasks .Image segmentation is the process of partitioning an image using an algorithm into disjointed and coherent regions on the basis of prespecified features. Food image segmentation is important for multifood images in which subsequent analysis, such as recognition and portion size estimation, depends on having accurate segmentation of each food in the image. Owing to the complexity of food images , accurate food image segmentation is a difficult task and affects the ability of image-based sensing systems to identify food types. Previous studies -26 have Owing to the inefficiency and high expense, some studies have applied graph-based methods to select regions containing foods  or have Research -32 in foRobust and accurate food image recognition remains a challenge because many foods have a deformable appearance and thus lack of rigid structures and because there often exists subtle differences in visual features among different food categories. Factors such as food preparation and personal preferences can also affect the appearance of food ingredients. Deep neural network–based approaches provide opportunities to improve the robustness and accuracy of food recognition systems but depend heavily on well-constructed training datasets and proper selections of neural network architectures. However, there is an opportunity to provide contextual information in the food recognition algorithm, which can include environmental cues and previous diet history. There have been advances in restaurant-specific food recognition ,37,38 whEstimating food portion size from an image is challenging as preparation and consumption impose large variations on food shape and appearance. Several food portion estimation techniques based on reconstructing the 3-dimensional (3D) models of the foods have been developed, which require users to take multiple images or videos or to modify mobile devices -45 to enDespite some promising results from existing approaches, the performance of current portion estimation methods is not yet satisfactory. More recently, several groups ,50 have Depth sensors and dual camera configurations are quickly gaining popularity on consumer mobile devices. More 3D information can be collected without significantly adding to a user’s burden capturing the eating scene. For example, mobile phones equipped with depth sensors enable simultaneous capture of image depth and the RGB color model image. For dual camera systems, at least two images are captured from slightly different angles, enabling multiview 3D reconstruction techniques. The additional information captured by the mobile devices may improve the accuracy of food portion estimation by providing additional 3D information on food objects.Although existing imaging technologies have shown reasonable success in estimating calorie intake and nutrients from images, there is growing interest in capturing proxies to calorie intake with sensor modalities that have fewer wearer privacy concerns. EAUs  are a mechanism to understand calorie intake patterns and behaviors. Accelerometer- and gyroscope-based inertial measurement units ,52 are eCapturing EAUs enables actionable insight, where information generated can be used by wearers, clinicians, and dietitians in a timely manner. They also enable interventionists to test the efficacy of calorie-informed, just-in-time interventions in close proximity to eating episodes. Although the detection of EAUs has shown promise, several challenges prevent these systems from being adopted in clinical and population settings .Several systems have shown promise in free-living populations but fall short of delineating the contexts for when and where their systems succeed and fail, which prevents others from building on previous work to advance EAU systems under challenging scenarios. For example, gesture EAUs are confounded by smoking action units; however, few studies attempt to consider other challenging contextual scenarios that can confound eating behaviors . AttemptAdditional data are needed on the strengths and limitations that lead systems to succeed and/or fail in free-living populations. Understanding the context of success or failure enables identification of strengths and weaknesses of various systems and advances both the hardware and algorithm used to solve these challenges. Studies are beginning to identify the context in which sensor-algorithm pairings fail; however, they lack validity through visual confirmation. With the exception of a few recent studies -62, few Evaluation of an eating detection system necessitates testing against visually confirmed (with video) ground truth . Visual confirmation of eating behaviors is the strongest form of ground truth available for EAUs but also one of the most burdensome on the participant and researcher. Many researchers limit their studies to controlled settings primarily because of the limited robustness of the sensor and the time required to manually label video streams to produce ground truth. There is significant time and cost associated with designing a fail-safe device that can function in free-living environments . HoweverEnsuring a long-lived, minimally intrusive method for capturing ground truth is necessary for capturing realistic data and rigorously testing interventions. As many participants report concerns for bystanders as their main reason for being unwilling to wear a camera all day ,67,68, rSimilar to image processing, EAU-based sensors have shown success in distinguishing between different food categories. Chewing crunchy chips compared with chewing a banana produces very different sensor signals, and researchers are beginning to capitalize on these variations to distinguish between food items and type. Automatically determining solid versus liquid ingestion has provided some utility in identifying sources of ingestion behavior and intervention design, allowing researchers to investigate this phenomenon -71. ThisKnowledge of an individual’s diet may narrow the search space, enabling sensors to automatically distinguish between individuals, given their known diet and food environment. An opportunity exists to categorize foods in a way that would be most useful for researchers and clinicians in improving calorie intake estimates. Although distinguishing between liquid and solid consumption may have research utility, it is unknown what other types of food categories may be distinguishable. Within-subject variability of nutrients is influenced by gender, age, and education . Thus, tThere are concerted efforts underway to characterize the biochemical changes in the body that result from food and calorie intake. Novel classes of biochemical and electrochemical sensing systems could be used to analyze changes in metabolic activity observed in interstitial fluid, saliva, or sweat ,74. In tRecent efforts have focused on biochemical analysis of eccrine sweat using wearable devices -77, whicContinuously monitoring biomarkers in sweat requires highly sensitive techniques for extraction and electrochemical analysis. Researchers have devised strategies to reduce the contamination effects of skin in contact with the device, while increasing sweat collection volume. This new class of wearable biochemical sensors could provide viable pathways for creating noninvasive and remote analysis of diet, wellness, and health . HoweverKey opportunities lie in the design and deployment of biochemical-sensing devices that can endure temperature changes owing to environmental factors and mechanical impact while maintaining signal quality without degradation over time. Beyond device resiliency, comparisons of sweat and blood analyte levels must be tested across healthy and sick populations to determine validity and applicability of on-body sweat sensing.The rich heterogeneous blend of electrolytes, metabolites, and proteins in sweat represents a unique set of noninvasively collected data. These biomarkers have been shown to correspond to the physiologic state and may serve as the basis for understanding cognitive impairment in the field. To date, most studies have focused on characterizing electrolytes and metabolites  using bioassays in controlled laboratory settings. Metabolic biomarkers could change with physical stress and diet during daily activity outside of controlled laboratory settings. The stability of wearable biochemical sensors is thus crucial to maintain over extended time periods in real-world settings.Wearable biochemical sensors that employ onboard dehydrating reagents or buffers that reduce degradation could lead to broad-scale deployment of these systems. Refrigeration is useful in protecting against bioassay degradation, but it requires special instructions and specialized equipment for proper handling and modes of operation. The development of new classes of wearable devices that require limited handling and refrigeration and that can handle enzyme-linked immunosorbent assay and protein-based analysis, using dehydration and freeze-drying steps to promote chemical stability, represents an area of enormous potential for robust remote-based deployment of wearable technologies.Continuous monitoring of sweat biomarkers requires flexible electronics modules, memory storage, and onboard batteries to facilitate data capture, signal processing, and transmission. Significant practical considerations, such as sensor corrosion at the interface with ionic fluids, need for cleaning, and the resulting signal degradation that could occur over time limit the utility of reusable systems. Electrochemical sensing systems consisting of a reusable electronics module and single-use electrochemical sensors provide compelling routes to address these challenges. Single-use system designs may circumvent the challenges of long-term wear, fluid–device interface, and signal degradation. However, disposable devices must be carefully engineered to support sufficiently reduced cost to warrant single-use deployment.Sweat is a corrosive biofluid that engenders significant device cleaning to facilitate reuse of the device. Thus, single-use wearable biochemical-sensing systems address important limitations of reusable systems assuming cost constraints are met. Hybrid designs, in which the reusable module mechanically couples to a single-use biochemical sensor, may mitigate the limitations of reusable and single-use systems. Although hybrid systems tend to cost more, they have significantly greater signal processing and battery capacity for long-term continuous monitoring.Regardless of the type of sensing modality, calorie estimation techniques share a set of common challenges because of the unique role that eating and nutrition play in everyone’s lives. These challenges are related to sensor development, validation, and refinement both in controlled and free-living settings.Although many researchers have studied detecting eating using a single wearable device, several are beginning to combine multiple sensors and context via multiple wearable devices to advance the total accuracy of an eating detection system. Mirtchouk et al  showed tWrist sensors coupled with other sensor modalities  may aid in distinguishing among smoking, eating, and other activities. Examples of multimodal systems include using a jaw motion sensor, a hand gesture sensor, and an accelerometer  and usinA multidevice system brings challenges in coordinating and synchronizing activities and sensing across devices. As each device manages its own internal clock, this network of clocks can become unsynchronized following power failure or reset. Most devices are designed to be standalone and use an internal clock, as opposed to time stamping their data using a nearby smartphone or body sensor unit. Time synchronization in real time has been a long-studied problem; however, automated time-synchronization methods post data collection can enable researchers to test multiple devices simultaneously, without the need to reengineer the device to use a central hub. Without reliable millisecond time-synchronization techniques, annotations from 1 sensor stream  are not transferable to another sensor stream.Once a sensor is deployed in a real-world setting, a supervised learning model, which aims to categorize data from prior known labels or information , is designed to process the data and determine system viability. However, one challenge in building a supervised learning model is providing sufficiently annotated instances or labels to train the model. Prior studies in real-world settings have depended on self-reported annotations ,83-86, wFor a system to succeed in real-world settings, it must be acceptable for the population of interest. Although several surveys have been designed to assess wearability of systems, there is no validated standard survey or approach to assess willingness to wear and use a device in the nutrition context. Current systems deploy devices for a variable amount of time  and then report comfort based on a Likert scale. Habits regarding technology adoption are not properly understood until at least 1 week  . As a ret <12 hours [Long battery lifetime is essential for wearable technology to ensure high sensor sensitivity and recall of eating episodes in free-living populations. It is reported to be the most important feature rated by mobile device users -90. Batt12 hours  when attAlthough technology development serves as an important contribution to the health community, reproducibility of the results is essential to determine proper construct validity, internal reliability, and test-retest reliability to increase confidence in the potential of a system to work in real-world settings. Most existing wearable sensors and systems show success by their principal investigator but have not extended beyond the laboratories in which they were implemented. To prevent bias in reporting, researchers need to disseminate their systems  to other teams to provide independent testing and review. Such rigor in testing of sensing platforms is needed across all sensor modalities.Conventional diet assessments comprise subjective and objective  approaches . AlthougOne primary concern for using subjective diet assessments includes intentional or unintentional misreporting of dietary intake . SpecifiA limitation specific to 24-hour dietary recall and food records is the measurement of acute intake and not the usual diet . MultiplBiochemical measures can also determine nutrient status of the body. However, biochemical concentrations are not true markers of dietary intake and can reflect how the body absorbs, transports, metabolizes, and excretes the nutrient . TherefoTechnology-enabled devices that measure calories and nutrients can also have far-reaching implications in clinical practice. A recent study reported that providers perceive health-tracking technologies as very useful when reviewing patient data, managing medical visits, and facilitating patient–provider communication . A growiSensors from the 3 domains presented  have the potential to identify markers that improve estimates of calorie intake. However, the technologies still require considerable user input from the end user, scientist, or clinician who may have to label or segment images or metrics from such wearables to train a machine learning system. Fully automating technology-enabled calorie and nutrient monitoring would open the possibility to providing highly informed and validated information to augment recall methods and advance estimates of calorie intake for clinicians and patients.Wearable-based sensing modalities focused on biochemical processes offer a solution for understanding food nutrients. However, more technical expertise is needed to merge conformal, low battery, secure, and valid technology with appropriate caloric assumptions. Once a stronger correlation can be drawn between biochemical products analyzed and calories consumed, biochemical-based wearables may provide promise in future automated calorie estimation systems. However, as with most wearable technologies, adherence to wearing the device remains problematic. This may be overcome if the value in such technologies pans out.Although calories and nutrients can be consumed and monitored, it is essential to understand the behavioral choices that drive these decisions and if those behaviors can provide insight into calorie intake. All 3 systems have the potential to provide such information from cortisol levels for stressful eating  to late-night snacking  and beyond. The biggest challenge then becomes how to use the reliable big data collected from these devices to drive an actionable outcome such as lower calorie consumption or identification of eating behaviors that increase calorie intake.It is our view that the defined research opportunities regarding calorie intake monitoring apps are the most promising, which may move the science toward a ubiquitous future of such monitoring. Nonetheless, challenges remain to fully introduce such solutions to have the desired health impact that clinicians and patients alike expect. At present, the next logical step is for scientists to improve the functionality of such devices, for human-computer interaction experts to improve usability, and for clinical teams and behavioral scientists to assess what information can be used to improve health behavior interventions given these new advanced technological tools. This translational, multidomain effort will demonstrate whether calorie intake monitoring enables higher quality of life and thus challenges the public health crisis of obesity.


Summary: External supply of the natural polyamine spermidine can extend life span in model organisms including yeast, nematodes, flies and mice. Recent epidemiological evidence suggests that increased uptake of spermidine with food also reduces overall, cardiovascular and cancer-related mortality in humans. Here, we discuss the possible mechanisms of this intriguing spermidine effect.Polyamines including spermidine play an essential role in intermediate metabolism. Since they are synthesized by higher eukaryotic cells, they are not vitamins. However, the levels of polyamines are profoundly influenced by their external supply, either by oral ingestion with different food items or by the intestinal microbiota that can synthesize polyamines as well .Saccharomyces cerevisiae, the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster) or to the drinking water (as we did for the rodent Mus musculus) is sufficient to extend longevity and to improve health span at multiple levels [Our groups have shown over the past decade that supplementing spermidine by adding it to culture media , best known as EP300 . EP300 aUntil now the literature on the longevity-enhancing effects of spermidine has been limited to model organisms. Now, two prospective population-based studies (summarized in the same paper) report for the first time that nutritional spermidine uptake is also linked to reduced overall, cardiovascular and cancer-related mortality in humans . Both stIn addition to the aforementioned epidemiological results, there are further, though admittedly indirect arguments in favour of a health-improving role for spermidine in human health. Thus, spermidine has been classified as a “caloric restriction mimetic” that has broad health-promoting effects due to its capacity to induce similar biochemical changes as does caloric restriction . Second,The fine mechanism through which spermidine (and aspirin) have such a broad effect on human health have not yet been fully elucidated. Based on current knowledge, these agents may slow down the general clock of the aging process, for instance by a global effect on cellular fitness, thereby mediating a pleiotropic effect on all aging-related diseases. The health-improving effects of aspirin have been initially attributed to its capacity to inhibit thrombocyte aggregation (via inhibition of cyclooxygenase) and hence to act as an anti-coagulant. Since spermidine has not been reported to have similar anti-coagulant activity, we prefer the hypothesis that aspirin may mediate its broad pro-health effects via the inhibition of EP300. As an alternative, yet non-exclusive mechanism, the natural EP300 inhibitor spermidine and its pharmacological equivalent aspirin may both act on different yet distinct cell types including stem cell compartments and differentiated cells engaged in cardiovascular function , anticancer immune surveillance (cancer and immune cells) or neurodegeneration  to reduce the incidence of the major age-related diseases . Future


Insular epilepsy often goes under-recognized and misdiagnosed due to the similarity of its features with temporal lobe epilepsy and the common exclusion of the insula during intracranial electroencephalography (iEEG). Here, we present a case of medically refractory epilepsy in a 43-year-old male with a 12-year history of tonic-clonic seizures. Insular epilepsy cases are often considered for diagnosis in the setting of abnormal insular pathology, such as a low-grade central nervous system (CNS) lesion. This is a unique case of non-lesional insular epilepsy, successfully managed by the resection of the insular cortex. Insular epilepsy is a rare condition in which the insula is the primary or one of two principal epileptogenic zones. Its semiology resembles that of temporal, parietal, and frontal lobe epilepsies, and thus it often goes misdiagnosed and undertreated . ManagemA 43-year-old Caucasian male with a past medical history of hypertension and 12 years of intractable seizures was evaluated for craniotomy and iEEG monitoring to localize and resect seizure foci. Seizure semiology included the patient awakening from sleep, bilateral hand and oral automatisms, head turning toward the left, blinking, and left lower extremity tonic posturing. The patient was completely amnestic regarding these events. Owing to intractable seizures, neurological assessments had revealed worsening neurocognitive decline, which created a poorer quality of life. His failed anti-seizure medications included clobazam, lacosamide, and lamotrigine, with persistent breakthrough seizures one to three times per month.Previous electrocorticography (ECoG) had captured right frontal and right parietal onset seizures, but no depth insular or hippocampal electrodes were placed during this recording. Magnetic resonance imaging (MRI), functional MRI (fMRI), and positron emission tomography (PET) scans were done. The PET scan showed reduced uptake in the right hemisphere and right basal ganglia. fMRI showed a language predominance in the left hemisphere. Brain MRI showed post-craniotomy changes over the right parietal region, without evident parenchymal lesions.The patient was status-post vagus nerve stimulation (VNS) implantation, video-EEG (vEEG), and a prior right-sided craniotomy and grid placement for an intracranial electrodes study. vEEG monitoring revealed tonic-clonic seizures with loss of consciousness. The patient described aura preceding seizure activity as being characteristically “sick - not right feeling.” The initial iEEG study revealed that seizure foci were at the edge of the grid and did not confirm the localization of seizure foci clearly. As the previous iEEG was non-confirmatory, and the patient continued to have recurrent, disabling seizures, current iEEG was considered. Depth electrodes were placed in the right hippocampus and insula (anterior and posterior), in addition to an 8x8 grid over the neocortex.ECoG done during the operation confirmed that seizures were originating from the right anterior insular cortex and rapidly spreading to the right frontal and temporal operculum Figures -3.The patient subsequently underwent a right frontotemporal insula lobectomy without serious complications, except minimal left-sided weakness. The patient was monitored in the intensive care unit (ICU) following surgery, demonstrated improvement in participation and alertness, and reported an improvement in left-sided weakness secondary to right-sided surgery. He has remained on his medication regimen and seen occupational therapy/physical therapy (OT/PT) to improve weakness. At six months post-surgery, the patient reported no seizures and noted an improvement in his left-sided weakness, approaching his near-baseline level.The insular cortices are situated at the bottom of the lateral cerebral fossa bilaterally, deep within the lateral sulcus, which separates the frontal and parietal lobes from the temporal lobe . The deeThe insula is further divided by the central insular sulcus into anterior and posterior lobes, which are distinct in their roles . The antAlthough the exact incidence of insular epilepsy is unknown, the insula as a primary epileptogenic zone is uncommon. In a study of 50 patients with atypical TLE, sEEG demonstrated that, of the 86% of seizures that spread to the insula, only 12% actually arose from it .The differing functions of the anterior and posterior lobes of the insula also suggest that seizures originating from either lobe will produce distinct symptomatology. Cortical stimulation of the anterior lobe characteristically produces viscerosensory symptoms, in contrast to the somatosensory symptoms produced by the stimulation of the posterior insula . The ictThe insula is often overlooked as a primary focus for medically refractory epilepsy. Insular epilepsy is often concurrent and confused with perisylvian seizures, temporal plus epilepsy, hypermotor seizures, MRI-negative frontal and parietal lobe epilepsies, and insular lesions . HoweverInsular epilepsy and temporal lobe epilepsyInsular epilepsy has symptomatology that distinguishes it from TLE, PLE, and FLE and should be explored as an etiology of seizure activity in the following scenarios: TLE-like patients exhibiting laryngopharyngeal discomfort, throat constriction, limb paresthesias, thrashing, or pedaling behaviors; PLE-like patients with oral or wide cutaneous paresthesias; and FLE-like patients with somatosensory aura preceding nocturnal hypermotor seizures .Our patient’s seizures typically occurred during sleep, manifesting as head turning to the left and perioral automatisms, which is usually characteristic of temporal lobe seizures. The insular focus was thus overlooked and was not considered in the previous ECoG. This led to delayed localization in this patient.Insular epilepsy should be considered in any patient with intractable seizures for whom TLE surgery has failed; as it stands, seizure freedom rates after TLE surgery are less than 70% at the three-year follow-up . This suTypical association with CNS lesionsInsular epilepsy is often found in the setting of low-grade CNS tumors in or affecting the insula, most commonly gliomas . The absMethods for detectionScalp EEG does not suffice for the detection of insular involvement, due to its limited coverage: insular epilepsy must be confirmed by intracranial ictal recordings . It is pRecommended treatment and complicationsInsular resection is an appropriate treatment for medically refractory epilepsy localized to the insula or “temporal plus” epilepsy involving the insula . One stuThe greatest complication associated with insular resection is an aphasic disturbance, though the role of the insula in speech processing is unclear . AnotherThis case is reported due to the common oversight of insular involvement in refractory epilepsy. iEEG monitoring often excludes the insula, delaying its recognition as a seizure focus; however, early identification has the potential to limit epilepsy morbidity and prompt action that may eradicate further seizure activity. Surgical resection has the potential to be a definitive treatment, as demonstrated by our patient’s continued seizure freedom.


Schizophrenia and schizoaffective disorder are schizophrenia spectrum disorders that cause significant disability. Among individuals who have schizophrenia or schizoaffective disorder, alcohol use disorder (AUD) is common, and it contributes to worse outcomes than for those who do not have co-occurring substance use disorder. Common neurobiological mechanisms, including dysfunction in brain reward circuitry, may explain the high rates of co-occurrence of schizophrenia and AUD or other substance use disorders. Optimal treatment combines pharmacologic intervention and other therapeutic modalities to address both the psychotic disorder and AUD. Further research on the etiology of these co-occurring disorders and on treatment of affected individuals is needed. Schizophrenia and schizoaffective disorder are heterogeneous psychotic disorders that often cause significant disability, with symptoms that include delusions, hallucinations, disorganization, and cognitive impairment.The lifetime prevalence of schizophrenia is estimated to be about 1%.3Individuals with these psychotic disorders have three times the risk of heavy alcohol use relative to the general population.10The genetic risk for schizophrenia has been fairly well-established. Heritability is estimated to be 80% to 85% for schizophrenia.12Diagnostic and Statistical Manual of Mental Disorders) revealed 17 traits, including schizophrenia, that had significant genetic correlations to alcohol dependence.Several polymorphisms (genetic variations) of the brain-derived neurotrophic factor (BDNF) protein correlate with co-occurring schizophrenia and alcohol dependence but not with alcohol dependence alone, suggesting that these polymorphisms may contribute to a specific vulnerability to these co-occurring disorders.Several theories have emerged to explain the high prevalence of co-occurring schizophrenia and substance use disorder.1Another theory explaining the high rate of substance use disorder among individuals who have schizophrenia is the self-medication hypothesis, which suggests that people use substances to find relief from symptoms or in an effort to decrease side effects that arise from antipsychotic treatments.19In 2018, Khokhar and colleagues reviewed the unifying hypothesis that the co-occurrence of schizophrenia and substance use disorder may relate to a dysregulation of the mesocorticolimbic reward system in the brain.24The neurodevelopmental theory of schizophrenia suggests that an early insult in brain development may lead to onset of symptoms of schizophrenia in late adolescence or early adulthood.Although models of co-occurring AUD or substance use disorder and schizophrenia or schizoaffective disorder continue to evolve, understanding the basis of the co-occurrence may inform treatment approaches, especially pharmacologic treatment for the co-occurring disorders. Moreover, regardless of the model, it appears that AUD or substance use disorder and schizophrenia or schizoaffective disorder are linked. Thus, treatment for such co-occurring disorders must address both the psychotic symptoms and the alcohol or other substance misuse.Given the high rates of co-occurring AUD among individuals with schizophrenia or schizoaffective disorder, as well as the clear evidence that such use can worsen the course of the psychotic disorder, diagnostic assessment for any individual presenting with symptoms of psychosis should include screening for alcohol and other substance use. In emergency departments, consideration of possible substance-induced psychosis is important. In one study, 18.9% of those with a diagnosis of substance-induced psychosis had alcohol as the primary substance.Because alcohol can precipitate psychotic symptoms during acute intoxication, withdrawal, or chronic use, obtaining a detailed history and creating a timeline of periods of psychotic symptoms and substance use can help clinicians differentiate between substance-induced psychosis and a primary psychotic disorder. Individuals with psychotic disorders may not be able or willing to provide these details of their history, particularly during periods of symptom exacerbation. Therefore, collecting information from collateral sources, such as family members, is often necessary. For initial evaluations and assessments of treatment response, laboratory examinations, such as testing for ethyl glucuronide, can provide useful evidence of recent alcohol use.29Treatment for substance-induced psychosis focuses on acute management, often with reduced stimulation, in a supportive, abstinent environment, and sometimes with short-term antipsychotic treatment. Once an individual becomes abstinent and withdrawal has resolved, psychotic symptoms also usually resolve, but 25% of cases may persist, resulting in diagnoses of schizophrenia spectrum disorders.This section reviews the evidence for the efficacy of medications used to treat AUD in individuals who have co-occurring schizophrenia or schizoaffective disorder. In addition, this section includes a review of the effects of antipsychotic medications on alcohol intake among individuals who have these co-occurring disorders.Several studies have examined the safety and efficacy of medications  used to treat AUD in individuals with co-occurring schizophrenia and AUD.33Another study of patients with serious mental illnesses, including schizophrenia, compared naltrexone and disulfiram individually and in combination with placebo.No known studies have assessed extended-release naltrexone for AUD in a population that includes individuals with psychotic disorders. However, given the cognitive and executive dysfunction associated with schizophrenia, this formulation  of naltrexone may have potential benefits for increasing medication adherence.A small, randomized controlled trial examined the use of acamprosate for individuals with psychotic disorders.Theoretically, disulfiram has a risk of worsening psychosis in predisposed individuals because of its action of inhibiting dopamine beta-hydroxylase, but this phenomenon appears to be rare in clinical practice.37Few studies have examined use of other medications for off-label treatment of AUD in individuals with schizophrenia spectrum disorders. For example, the effects of topiramate on alcohol outcomes have not been studied in this population, although it has been used to potentially control weight in people with schizophrenia.Varenicline, which has been approved by the U.S. Food and Drug Administration for the treatment of nicotine use disorder, has been shown to decrease alcohol consumption among participants with AUD.In summary, although few studies have examined the effects of medications  that treat AUD among individuals with psychotic disorders, evidence of the safety and potential benefit is sufficient to encourage increased use in this population see .The choice of medication for treating psychotic or affective symptoms in people with psychotic disorders may have implications for alcohol consumption. First-generation antipsychotic medications do not appear to decrease alcohol use and actually may increase substance use and craving in people with schizophrenia and co-occurring substance use disorder.2 receptor blockade coupled with its noradrenergic effects.46A hypothetical framework has been delineated that supports the use of clozapine to ameliorate the brain circuit dysfunction experienced by people with schizophrenia and substance use disorder and is related to clozapine’s weak dopamine DLong-acting injectable formulations of antipsychotics may help improve adherence or clarify when nonadherence is present, which may be particularly relevant in a dual-diagnosis population. In a randomized controlled trial comparing oral and long-acting injectable risperidone treatment for individuals with schizophrenia and AUD, heavy drinking worsened over time for those in the oral risperidone group compared to those treated with the long-acting injectable formulation.48Lastly, a randomized, open-label, review board–blinded study comparing once-monthly paliperidone palmitate to daily oral antipsychotics examined real-world outcomes for participants, a majority of whom had a diagnosis of co-occurring substance use disorder.2 blockade may have theoretical benefit over typical antipsychotic medications, although evidence in prospective controlled trials is limited. It has been postulated that the unique mechanism of action of aripiprazole  may have beneficial effects for alcohol use.2 receptors, has support from small, open-label trials that showed reductions in alcohol use.Other second-generation antipsychotics that do not have potent dopamine DChronic psychotic illness is often accompanied by cognitive deficits and diminished executive functioning, which may be worsened by the effects of alcohol in those who have co-occurring AUD. Therefore, integrated and tailored care for both the psychotic disorder and AUD can improve access to care, deliver consistent messages about treatment and recovery, provide interventions that support attempts to reduce substance use, and manage behavioral health conditions.53Group therapy using cognitive behavioral therapy, motivational enhancement therapy, or contingency management has a role in treating AUD and co-occurring schizophrenia.29Contingency management involves agreed on, immediate, tangible rewards to reinforce positive behaviors, such as treatment attendance or abstinence that has been verified by biologic measures. Such a management strategy for alcohol abstinence has been shown to be effective for people who have schizophrenia or other serious mental illness and who also have AUD. For example, one study demonstrated that participants who received contingency management intervention were 3.1 times more likely than participants from the control group to have a negative result on a urine test for the alcohol biomarker ethyl glucuronide.More intensive interventions, including assertive community treatment (ACT) and residential programs, may benefit individuals with co-occurring schizophrenia and AUD. ACT is the most widely tested model of community care for people with severe mental illness. ACT consists of an interdisciplinary team  with a low participant-to-staff ratio. This team provides a range of comprehensive services, including community outreach, 24-hour availability for emergency communication, and integrated pharmacotherapy and behavioral treatments for substance use disorder. For people with dual disorders, faithful implementation of and adherence to the ACT model is associated with superior outcomes in substance use, including significantly fewer days of alcohol and drug use.53Alcoholics Anonymous is underused among individuals with co-occurring AUD and psychotic disorders, although this population has unique considerations. People who have psychotic disorders benefit from the education and support they receive by attending and processing 12-step meetings, but people who have acute psychosis may not be able to tolerate these meetings.59Additional research related to co-occurring AUD and schizophrenia or schizoaffective disorder is needed. Environmental factors, including substance use, that contribute to the risk of developing schizophrenia continue to be investigated. Prospective longitudinal markers of neurobiological function in adolescence before onset of psychotic symptoms and alcohol consumption could further elucidate the etiology of these disorders. Moreover, further development of evidence-based interventions to address alcohol and other substance use in adolescents before and during first-episode psychosis is required. Lastly, additional investigations into the efficacy of various treatment modalities are necessary, particularly because individuals with co-occurring disorders often are excluded from clinical trials.


To assist with management and interpretation of human metabolomics data, which are rapidly increasing in quantity and complexity, we need better visualization tools. Using a dataset of several hundred metabolite measures profiled in a cohort of ~1500 individuals sampled from a population-based community study, we performed association analyses with eight demographic and clinical traits and outcomes. We compared frequently used existing graphical approaches with a novel ‘rain plot’ approach to display the results of these analyses. The ‘rain plot’ combines features of a raindrop plot and a conventional heatmap to convey results of multiple association analyses. A rain plot can simultaneously indicate effect size, directionality, and statistical significance of associations between metabolites and several traits. This approach enables visual comparison features of all metabolites examined with a given trait. The rain plot extends prior approaches and offers complementary information for data interpretation. Additional work is needed in data visualizations for metabolomics to assist investigators in the process of understanding and convey large-scale analysis results effectively, feasibly, and practically. Advances in metabolomics technologies have enabled the generation of large-scale metabolomics measures in human studies . AccordiThe most commonly used methods for visually analyzing and representing associations between metabolites and outcomes are borrowed from conventional statistics and other biological fields . MethodsFor development of these visualization methods, we used a metabolomics dataset comprising >500 bioactive lipids assayed by high resolution liquid chromatography-mass spectrometry (LC-MS) in a subset N = 1447 participants  of the Framingham Heart Study offspring cohort. LC-MS was performed according to previously described protocols . In brie https://github.com/biodatacore/2017.09-rainplots. Detailed instructions and the R code for recreating the graphics in this paper is also provided at the site. All analyses and data visualizations were performed using R v3.4.1. Given the biological importance of lipid metabolites with respect to cardiometabolic disease traits, we performed multivariable regression analyses to examine the relation of each metabolite with several clinical traits and outcomes, in the following order: age, female sex, body mass index, metabolic syndrome , prevaleAll approaches to visualizing the results of association analyses demonstrated a range in the degree to which different metabolites were related to the different outcomes of interest. The extent and type of information conveyed varied across the visualization techniques, as summarized in m/z)  . The marm/z) . Althougm/z) . The extFor studies that involve multiple staged experiments or statistical models, several options exist for visualizing analysis results. Our objective was to overcome limitations associated with discerning clear patterns within or between metabolites, especially across multiple plots and outcomes, and with increasing data set size. We therefore combined the visualization concepts offered by the conventional heatmap and previously reported raindrop plot methods;  the lattA ‘rain plot’ approach combines the information from paired heatmaps into a single plot that emphasizes two types of information: (i) between-outcome comparisons, such as the extent to which most metabolites in this panel are associated with certain outcomes  more than others; and, (ii) between-metabolite comparisons, such as the extent to which certain metabolites are associated with an aggregate measure of clinical cardiovascular disease risk  with or without concurrent relations to major component risk factors . Between-metabolite comparisons, in particular, can facilitate identification of potentially important biological differences underlying the observed results of relating multiple metabolites to multiple phenotypes. The rain plot visualization emphasizes two types of comparisons: (1) between-outcome results, and (2) between-metabolite results. For between-outcome comparisons, visually scanning for vertical patterns of large sized or deeply colored droplets  serves to highlight those outcomes that are the most broadly associated with a given panel of metabolites. As shown in As analytical chemistry methods continue to mature, resulting in larger and more complex metabolomics data, there is a growing need for ways to visually understand and interpret the relations of these high-dimensional data with multiple outcomes of interest. Using a dataset of metabolite measures performed in a population scale cohort, we compared several existing techniques that are commonly used for visualizing associations between metabolites and clinical outcomes. To improve these prior methods, we developed and demonstrate the potential utility of a rain plot approach to maximally render the multiple types of information that can be derived from the observed relationships between a panel of metabolites and a set of clinical traits and outcomes. We anticipate that this approach may be further extended and applied to alternate study designs using different types of molecular phenotyping data—as part of the ongoing effort to effectively, efficiently, and feasibly convey the results of large-scale, high-dimensional data analyses ,31.


Obesity is a major public health problem that has reached epidemic proportions in the United States and around the globe. According to data from the Center for Disease Control (CDC), the prevalence of obesity was 39.8% and affected about 93.3 millions of US adults in 2015~2016, with nearly two thirds of Americans being either obese or overweight [This rapid rise in obesity has also led to a concurrent rise in type 2 diabetes, cardiovascular disease (CVD), including heart disease and stroke, as well as cancer, resulting in increased morbidity and premature mortality among affected individuals . The mosAmong the novel potential therapeutic targets, brown adipose tissue (BAT) has generated substantial interest since the discovery of functional BAT in adult humans. Upon stimulation by cold exposure or other stimuli, BAT is activated to promote non-shivering thermogenesis, thereby contributing to increased energy expenditure. The major inducer of BAT growth and activation is the sympathetic nervous system, through the release of norepinephrine . ClassicHigh BAT activity correlates with high resting metabolic rates upon cold stimulation, and with low body mass index and low-fat mass –11. HoweHypothetically, the high metabolic activity of BAT could be beneficial for increasing the clearance and utilization of circulating glucose and lipids, hence potentially ameliorating systemic metabolic homeostasis. This hypothesis is supported by indirect evidence demonstrating that low outdoor temperature is associated with high BAT activity , 20, lowAcute cold exposure was reported to increase basal and insulin-stimulated whole-body glucose disposal in individuals with high BAT mass and activity . SimilarIn addition to studies using cold exposure as a means to increase BAT activity, several studies were performed using pharmacological approaches to activate BAT. Catecholamines are physiological humoral factors that activate BAT through β3-adrenoreceptors. Therefore, studies investigating the role of the acute and chronic effects of β3-adrenoreceptor agonists on glucose and lipid homeostasis have been performed. Acute administration of mirabegron or TAK-677, β3-adrenoreceptor agonists, showed no effect on glycemia ,30. MoreRecent studies have uncovered an additional role for BAT as an endocrine organ . Brown a3), which is elevated in the plasma in response to BAT activation. Thyroid hormones were shown to exert strong cardioprotective effects in both humans and animals [3 production is uncertain, since T3 is produced by many other tissues [The first known endocrine factor secreted by BAT was the active thyroid hormone triiodothyronine . FGF21 exerts beneficial effects on lipid and glucose metabolism in mice and humans, and was identified as a candidate endocrine factor released by transplanted BAT . FGF21 iRecent studies have identified yet another endocrine factor that can be secreted from BAT in response to cold  and caloSeveral lipids have been identified that are released from tissues and act locally or systemically to promote insulin sensitivity and glucose tolerance; as a class, these lipids are referred to as lipokines. Upon cold-induced activation of BAT, free fatty acids (FFA) are released from stored triglycerides (TG) by lipolysis. Therefore, it was hypothesized that there might be thermogenic lipokines that activate BAT in response to cold. Indeed, a study using global lipidomics found that the lipid 12,13-dihydroxy-9Z-octadecenoic acid  was increased in the circulation of humans and mice exposed to cold. Injection of 12,13-diHOME acutely activated BAT fuel uptake and enhanced cold tolerance. Mechanistically, 12,13-diHOME increased fatty acid (FA) uptake into brown adipocytes by promoting the translocation of FA transporters to the cell membrane . On a seFinally, a study suggested that BAT is activated and exerts systemic cardioprotective effects in models of catecholamine-induced injury in mice, leading to decreased myocardial injury, fibrosis, and pathological left ventricle remodeling. The authors suggest this cardioprotective effect could be due to systemic actions of BAT-derived secreted factors, although no specific batokines were identified in this study .We presented the current knowledge suggesting a general beneficial effect of BAT activation toward the reduction in CVD risk. We also discussed BAT-secreted factors with potential direct and/or indirect cardioprotective effects via modulation of systemic metabolism. Studies of different models of cardiac stress would be of value in determining if BAT activity is also induced and whether it is associated with improved cardiac phenotypes. Determining the specific batokines mediating the cardioprotective effects would also offer valuable insights regarding the potential role of BAT on cardiovascular protection. These secreted factors may exert metabolic benefits on adipose tissues, the heart, and /or other peripheral tissues in addition to coordinating the metabolic adaptations during cardiovascular insults, thus potentially resulting in cardio protection.Finally, understanding the mechanisms regulating induction of batokines could result in identification of useful therapeutic targets based on brown fat activation. Future research is warranted to address some of these questions and to determine if BAT and its batokines will yield novel therapeutic options for the treatment of the growing epidemic of obesity and associated metabolic disorders, and CVD.


Finally, zebrafish has been used to model hematopoietic diseases. Recent technological advances in single-cell transcriptome analysis, epigenetic regulation, proteomics, metabolomics, and processing of large data sets promise to transform the current understanding of normal, abnormal, and malignant hematopoiesis. In this perspective, we discuss how the zebrafish model has proven beneficial for studying physiological and pathological hematopoiesis and how these novel technologies are transforming the field.Hematopoiesis is an optimal system for studying stem cell maintenance and lineage differentiation under physiological and pathological conditions. In vertebrate organisms, billions of differentiated hematopoietic cells need to be continuously produced to replenish the blood cell pool. Disruptions in this process have immediate consequences for oxygen transport, responses against pathogens, maintenance of hemostasis and vascular integrity. Zebrafish is a widely used and well-established model for studying the hematopoietic system. Several new hematopoietic regulators were identified in genetic and chemical screens using the zebrafish model. Moreover, zebrafish enables  Over the past three decades, zebrafish has been established as an important model to study various biological processes during development and homeostasis, including hematopoiesis. Many attractive features underpin the success of zebrafish as a model for vertebrate hematopoiesis. Cell-intrinsic and -extrinsic signaling mechanisms in hematopoiesis are well conserved between zebrafish and mammals, with the exception of a few hematopoietic niche components . MoreoveFigure 1). Interestingly, it was recently discovered that HSC-independent T-cells can originate from the AGM and PBI during the embryonic and larval stages of development  form in the posterior blood island (PBI) during a transient intermediate wave. Finally, during the definitive wave, hematopoietic stem cells (HSC) with multilineage capacity originate in the aorta-gonad-mesonephros (AGM) region. The HSCs then translocate to and expand in the caudal hematopoietic tissue (CHT), which is followed by the colonization of the kidney and the thymus  emerge from the ventral wall of the dorsal aorta in vivo . Moreovein vivo  and revein vivo . Multiplin vivo . These uin vivo . Where tcroscopy . Moreovecroscopy  have enaThe development of single-cell RNA-sequencing (scRNA-seq) revolutionized the way we understand hematopoiesis. As most cellular compartments have a certain degree of heterogeneity, with bulk RNA-seq one cannot distinguish between a small transcriptional difference in many cells, and a large transcriptional difference in a few cells. Several insightful reviews describe the different methods used for single-cell RNA-seq .In zebrafish, one of the first methods used to characterize the transcriptome of single cells was massive parallel qPCR, where up to 96 transcripts could be analyzed in great sequencing depth using the Fluidigm system. This method revealed two distinct sub-populations of HSPCs in the CD41-GFP low-expressing stem cell compartment of the adult kidney marrow. Moreover, by using this technique and genetic ablation of T cells, a previously uncharacterized hematopoietic cytotoxic T/NK cell population in zebrafish was uncovered .low population revealed four HSPC sub-populations with different cellular characteristics and potential novel markers for HSCs were uncovered. Importantly, some cells in these subpopulations expressed the thrombocyte differentiation program long before they would have been characterized as thrombocytes, showing that there is an early lineage bias , a method that combines traditional histological techniques with low-input RNA sequencing and mathematical image reconstruction .in vivo across development. Such screens have led to the identification of prostaglandin E2 as a compound that increases HSC production  predisposition syndromes are caused by innate mutations in transcription factors that affect embryonic hematopoiesis, such as Gata2 and Runx1 , suggestin vivo. Gene regulatory elements can be identified in zebrafish using chromatin immunoprecipitation combined with sequencing (ChIP-seq), however, the technique is limited by the low number of zebrafish-specific antibodies currently available and the large amount of input material required . ATAC-seq requires much less input material than ChIP-seq, and has even been used successfully with single cells . This me1  developed in Ido Amit’s laboratory, should be adapted to zebrafish . Moreove1 . This reIn the era of genome-wide technology, gene expression studies should be complemented with proteomic studies, as transcriptional and translational outcomes can sometimes differ. Additionally, the extension of these analyses to metabolomics may uncover another layer of regulation critical for hematopoiesis. Indeed, it was recently shown that dormant stem cell populations have low metabolic activity, and this is required to maintain the hematopoietic system during aging and periods of intense stress . AlthougFigure 2), have opened the way for novel exciting hypotheses on the mechanisms promoting hematopoietic diseases, the role of the niche in normal and malignant hematopoiesis, and the effect of chemical compounds on malignant cells. The high conservation between the zebrafish and human hematopoietic systems means that discoveries in fish may have strong translational potential and important clinical implications for the treatment of hematopoietic diseases.The zebrafish has become an invaluable model system for understanding how HSCs form and are maintained, and how hematopoietic cell differentiation is regulated during embryogenesis and in adulthood. The unique advantages offered by this model system over traditional mouse models regarding the use in chemical screens and the accessibility during embryonic stages allowing easy manipulation and visualization and tracing into adult stages, in combination with recent new technologies (EdP and ET conceived and wrote this manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Use of molecular markers in urine, tissue or blood offers potential opportunities to improve understanding of bladder cancer biology which may help identify disease earlier, risk stratify patients, improve prediction of outcomes or help target therapy.A review of the published literature was performed, without restriction of time.Despite the fast-growing literature about the topic and the approval of several urinary biomarkers for use in clinical practice, they have not reached the level of evidence for widespread utilization. Biomarkers could be used in different clinical scenarios, mainly to overcome the limitations of current diagnostic, predictive, and prognostic tools. They have been evaluated to detect bladder cancer in asymptomatic populations or those with hematuria and in surveillance of disease as adjuncts to cystoscopy. There is also a potential role as prognosticators of disease recurrence, progression and survival both in patients with non-invasive cancers and in those with advanced disease. Finally, they promise to be helpful in predicting the response to local and/or systemic chemotherapy and/or immunotherapy.To date, due to the lack of high-quality prospective trials, the level of evidence provided by the current literature remains low and, therefore, the potential of biomarkers exceeds utilization in clinical practice. Bladder cancer (BCa) is a heterogeneous disease with significant diagnostic, therapeutic and prognostic challenges . The tooMolecular markers detected in urine, tissue or blood offer promising opportunities to improve our understanding of biology of a specific cancer and its micro- and macroenvironment. This could help identify disease earlier, risk stratify patients, improve prognostication and prediction of outcomes and help target therapy. While some areas such as urine-based tumor markers have been more extensively studied, the current guidelines have yet to embrace markers in routine management of BCa . In thisBiomarker research can be categorized, similar to drug-development studies, into initial pre-clinical exploratory studies, clinical assay development and validation studies, small clinical retrospective studies, external validation in larger cohorts , prospective clinical trials and further post-approval studies as well as possible expansion to other clinical scenarios and disease stages , 5. In aThe main goal for marker development is to identify a validated test which can improve clinical decision-making in a cost-effective way. It is, therefore, not sufficient to merely show statistically significant independent association of the marker with the investigated outcome, but to show improved prognostic or predictive accuracy of a multivariable model over already available clinical features alone. Ideally, the integration of the marker should be improved with regard to discrimination, calibration and decision-analysis .Finally, BCa is a heterogeneous disease. Therefore, it is unlikely that one single marker exists that can adequately characterize the potential and behavior of a cancer to allow reliable treatment conclusions. This has led many investigators to evaluate comprehensive pathways rather than single markers , 7. MarkIn patients without history of BCa, there are several scenarios in which urinary biomarkers may play a role. One scenario that has frequently been discussed is the use of biomarkers for screening purposes. So far, the low prevalence of the disease in the general population has been a challenge for developing effective screening strategies . ActuallIdentification of high-risk populations may help overcome these limitations . HoweverA second clinical scenario in which urinary biomarkers may add value is in the risk stratifying patients with asymptomatic microhematuria (AMH). Patients with asymptomatic gross hematuria have a significant risk of BCa (approximately 10%) making urologic workup necessary . In thesWhile these studies suggest a potential benefit to urinary biomarkers in patients with AMH, prospective controlled trials are lacking and urgently needed. Moreover, so far, there is no evidence that the use of urinary biomarkers in a screening/early detection setting has an effect on cancer-specific mortality . TherefoUrinary biomarkers in surveillance setting have been developed to overcome the limitations of cystoscopy and urine cytology. The former is invasive and may miss significant proportion of cancer recurrences, especially CIS, whereas the latter has low sensitivity in low-/intermediate-risk non-muscle-invasive bladder cancer (NMIBC) and suffers from considerable inter- and intra-observer variabilities especially in patients after BCG immunotherapy –21. BiomA urine marker would need a very high sensitivity and negative predictive value (NPV) to replace cystoscopy. One recent survey suggests that patient preference would have sensitivity as high as 90–95% for a marker to replace cystoscopy . OverallDue to the unsatisfactory performances of most single biomarkers, panels of markers have been used to improve sensitivity or alternatively combining markers. One study found that combining two tests among cytology, immunocytology, FISH and NMP22 resulted in sensitivity and negative predictive value of no greater than 89.8% (Immunocyt + NMP22) and 92.1% (FISH + Immunocyt) . If cytoth BCG instillation) and before the 3 months’ cystoscopy (before the first maintenance course) are significantly associated with both cancer recurrence and progression. FISH at 3 months identified 50% of patients who experienced cancer progression within 2 years in half of those with positive test compared to only 3% of those who had normal result [Based on these findings, the European (EAU), the American Urological Associations (AUA) as well as the Society of Urologic Oncology (SUO) do not recommend urinary biomarkers yet for the routine surveillance of patients with NMIBC , 29. Accl result .Another approach toward improving surveillance protocols of NMIBC and optimizing costs is to use reflex testing. In patients with negative/uncertain result of one test, the accuracy of follow-up is significantly increased by adding subsequent highly sensitive biomarkers. Immunotherapy is known to evoke inflammatory changes within the bladder often making reliable assessment of the lower urinary tract challenging. As such, accuracy of cytology used as an adjunct to cystoscopy to increase the detection of CIS or upper tract lesions is hampered by BCG. FISH and ImmunoCyt® were investigated in patients with atypical cytology. UroVysion FISH has 100% sensitivity and 100% negative predictive value in those with negative cystoscopy, yet equivocal cytology . ImmunoCIn summary, in the surveillance of patients with NMIBC, there is insufficient evidence that urinary biomarkers can replace cystoscopy and prospective studies are necessary to demonstrate whether this is safe. Urinary biomarkers, however, could be used to help assess the response to intravesical immunotherapy and as reflex test in cases with equivocal urinary cytology (Expert Opinion).Tissue biomarkers can theoretically be used in NMIBC to predict oncological outcomes such as recurrence and progression as well as the response to intravesical BCG and, ideally, could be used to improve individualized treatment and surveillance based on individualized risk profiles. Moreover, they can be useful to identify the proportion of high-risk NMIBC patients who are likely to develop disease progression to invasive disease, thus requiring consideration for intensified therapy such as early radical cystectomy.To date biomarkers associated with pathways important for tumor growth and spread have been evaluated intensively, such as cycle cell regulators, angiogenesis, apoptosis, signaling proteins and hormones.p53, the product of TP53, the most common oncosuppressor gene mutated in all human cancers, has been associated with features of tumor aggressiveness and correlated with poor oncological outcomes , 33–37. The association between an altered expression of the tumor suppressor retinoblastoma (Rb) and oncological outcomes in BCa is relatively weak and the majority of trials failed to find an association with recurrence and progression . More reThe antiapoptotic biomarker survivin was found to predict recurrence, progression and survival , 42. In Based on the current literature, one can conclude that none of the evaluated tissue biomarkers alone could be used to predict oncological outcomes with sufficient accuracy to change decisions in routine clinical practice. Therefore, it has been postulated that a panel of biomarkers could improve the predictive accuracy over clinical information alone , 50. HowThe development of MIBC involves alterations in multiple homeostatic pathways with profound deregulations within a complex molecular circuitry. Therefore, these alterations can serve as prognosticators of outcomes, predictors of response to therapy, and they may also act as therapeutic targets.Several retrospective studies have reported that nuclear accumulation of p53 is prognostic in MIBC, especially in patients treated with radical cystectomy , 52. HowInactivating mutation of Rb, in conjunction with other cell cycle regulatory proteins, has been shown to be prognostic in MIBC . CombineAs in NMIBC, apoptosis biomarkers such as survivin and Bcl-2 family are associated with outcomes in MIBC . InteresFGFR3 expression, mutations and copy number gain, thereby suggesting that these patients may respond to FGFR inhibitors or its downstream targets. These tumors also showed decreased miR-99a and miR-100 expressions, which in turn downregulate FGFR3 expression [GATA3 and FOXA1. These tumors also harbor increased expression of uroplakins, E-cadherin and members of the miR-200 family. Increased expression of ERBB2 and estrogen receptor-β by these tumors also suggested that they may serve as potential targets for hormonal therapies. Expression signature of tumors in cluster III  were similar to that of basal-like breast cancers and squamous cell cancers of the head and neck and lung, characterized by overexpression of epithelial lineage genes. These findings suggest the presence of distinct molecular subtypes of MIBC with characteristic expression signatures, which may impact prognosis and serve as candidates for selective therapeutic strategies [Finally, tissue-based gene- and transcriptome-level profiling has been used to identify biomarkers that characterize and can potentially predict prognosis in patients with MIBC . Comprehpression . Tumors rategies .Assessment of biomarkers in blood offers several advantages over tissue samples, such as ease of procurement, minimally invasive collection method, and higher sample homogeneity. However, clinical applicability and value of these biomarkers in the setting of MIBC remain to be further tested and validated in large well-controlled multi-institutional studies.Biomarkers associated with cellular proliferation such as insulin growth factor binding proteins (IGFBPs) play a central role in regulating cellular growth, proliferation and transformation. IGFBP-3 is a member of this family that also has its own pro-apoptotic effects. Lower preoperative plasma levels of IGFBP-3 are a significant predictor of lymph node involvement in patients undergoing radical cystectomy and also portended a significantly increased risk of disease recurrence and cancer-specific mortality . SimilarInflammation and immune biomarkers such as interleukin-6 and C-reactive protein (the most widely studied serum marker for inflammation in BCa) have been consistently associated with adverse survival outcomes –72. In lThe presence of circulating tumor cells (CTCs) in peripheral blood may represent an early step in the metastatic progression of BCa. In an evaluation of 55 patients undergoing radical cystectomy, the presence of CTCs was associated with survival. The study noted that patients with one or more CTCs had a shorter time to disease recurrence and shorter CSS than those with none . These fIn BCa, chemotherapy can be administered intravesically  or systemically (for MIBC and metastatic BCa). Prediction of response to chemotherapies could be helpful for decision regarding type and timing of systemic and local therapies.A hallmark of cancer, including BCa, is dysregulation of the cell cycle which results in the sustained signal for proliferation required for cancer development. Several cell cycle regulators and markers of proliferation have been tested as predictors of chemotherapy response such as CyclinD1, CCDN1 and Ki-67. A second hallmark of cancers is their ability to escape apoptosis, a process controlled by caspases. Caspases, in turn, are regulated by several molecules involved in the detection of DNA or mitochondrial damage, including p53 and Bcl-2. p53 has been extensively tested in BCa, both as a prognostic biomarker and as a predictor of treatment response. While initial retrospective studies were promising, randomized trial results showed no role for p53 as a predictor of chemotherapy response .Many chemotherapy agents work by causing DNA damage and, if its DNA integrity is sufficiently disrupted, the cancer cell cannot replicate. Cancer cells that have deficient DNA damage repair mechanisms are unable to fix the damage induced by these chemotherapy agents and are, therefore, more susceptible to being killed by the agents. Proteins involved in DNA damage detection and repair that play a role in BCa chemotherapy response include BRCA-1, BRCA-2, RAD51, PARP1, ERCC1, ERCC2, ATM, RB1, and FANCC. In the study by Plimack et al., response to neoadjuvant chemotherapy (NAC) before radical cystectomy (stage T1 or less at surgery) was associated with an alteration in ATM, RB1 or FANCC . SimilarIt has been known for decades that the mitogenic signals derived from the binding of growth factors to their receptors are crucial for cancer development. Many growth factors and growth factor receptors signal into the cell via transmembrane tyrosine kinases, and these kinases are the targets of several new systemic therapies in oncology . While these three drugs appear to have limited activity in BCa, the possibility of biomarker enrichment for response has been assessed . FinallyBased on the current data, none of the biomarkers has reached the level of evidence sufficient to determine therapeutic approach and resistance to targeted chemotherapies. Ongoing, biomarker-driven trials will shine more light on this important issue.Over the last years, immunotherapies have proven unprecedented activity in BCa after failure of cisplatin-based therapies or in patients not suitable to receive cisplatin-based chemotherapy. Inhibition of immune checkpoints has been shown for several agents targeting programmed death-1 (PD-1) receptor or its ligand (PD-L1) and cytotoxic T-lymphocyte antigen 4 (CTLA-4). Nevertheless, the majority of patients still do not respond to treatment –88, whicThe expression of T-cell coregulatory proteins is altered in a large proportion of BCa with differential upregulation in cancer versus normal urothelium, and an association of B7-H1 with mortality after radical cystectomy in organ-confined disease has been shown . DetectiMolecular subtypes of MIBC have recently been categorized based on gene expression. The Cancer Genome Atlas (TCGA)  describeHigh mutational load may be associated with better response to immunotherapy, particularly for checkpoint inhibitors, with some trials (such as Imvigor 210) showing a correlation between patients with a higher mutational burden and better responses to immunotherapeutic agents . FinallyDespite these promising findings, use of biomarkers to predict the response to systemic immunotherapy remains limited; indeed, a significant proportion of patients with negative biomarkers status still respond to treatment and many patients with positive biomarkers status fail to respond.Despite the plethora of studies investigating the role of urinary, blood and tissue biomarkers in BCa with an ever-rising rate of data, none of the studies have reached the required level of evidence to change clinical practice and, therefore, none is widely used or to be recommended. Except for some specific clinical scenarios where biomarkers can be used as an adjunct in the clinical setting, use of biomarkers in BCa remains experimental and is still not recommended in clinical practice. However, biomarkers are the basis for the individualized medicine and represent undoubtedly the future of BCa treatment.


Post cholecystectomy syndrome is characterized as recurrence of symptoms as experienced before cholecystectomy. In rare cases, a remnant cystic duct is causing these symptoms and occasionally surgical resection is performed. During surgery, visualization of the biliary ducts could be difficult due to inflammation and dense adhesions.In this article, we presented a 36-year old woman with post-cholecystectomy syndrome in which we evaluated the feasibility of near-infrared (NIR) fluorescence imaging using indocyanine green (ICG) for visualization of the remnant cystic and common bile duct during robot-assisted surgery. Intraoperative visualization of the remnant biliary duct and other important structures was feasible, and resection of the remnant cystic duct was successfully performed under fluorescence guidance, without any complications.NIR fluorescence imaging of the biliary ducts using ICG does not prolong the operating time, and could potentially decrease the operation time in difficult procedures, because of easy and fast detection of the biliary tract. Furthermore, it is a non-hazardous and non-invasive technique, as it does not require use of radiation and cannot cause bile duct injury. This case illustrated that ICG NIR fluorescence imaging during difficult robot-assisted surgical procedures of the bile ducts is effective and therefore highly recommended.The online version of this article (10.1186/s12893-019-0567-8) contains supplementary material, which is available to authorized users. Cholecystectomy is the most common surgical procedure for patients with symptomatic cholecystolithiasis. Currently, minimally invasive surgery, either laparoscopic or robot-assisted, is the standard approach. Unfortunately, in some patients symptoms, such as abdominal pain and dyspepsia might persist after surgery, which has been defined as the post cholecystectomy syndrome (PCS) . A remnaA 36-year old woman presented with an acute biliary pancreatitis at a peripheral hospital, after which a laparoscopic cholecystectomy was performed. One year later, she developed several episodes of acute onsets of abdominal pain, which were caused by biliary pancreatitis. Endoscopic ultrasound (EUS) revealed a high suspicion on presence of small bile stones in the remnant cystic duct. Although, Magnetic resonance cholangiopancreaticography (MRCP) did not show either an evident remnant biliary duct or presence of bile stones in either the remnant cystic duct or CBD, which could be explained by the limited image quality due to artefacts. Moreover, it has been shown that MRCP has a 93% sensitivity for detection of bile stones . The a pDa Vinci Xi  fluorescence camera (Firefly™), the liver and CBD could be easily visualized, around 30 min. After IV injection of ICG. After adhesiolysis, the fluorescent remnant cystic duct and the non-fluorescent cystic artery were identified, also confirmed by the placed metal clips during the previous operation  of ICG was injected IV in order to visualize the biliary ducts. The patient was positioned in reverse Trendelenburg, and a small incision was made for introduction of 12 mm trocar at the umbilicus. Consequently, four additional robot trocars (8 mm) were placed under direct visualization. After docking, one experienced HPB surgeon and one surgical resident started the surgical procedure with exploration of the hepatoduodenal ligament until the liver hilum A case report of a patient with a symptomatic remnant cystic duct, who underwent robot-assisted resection under fluorescence guidance, is presented. Good visualization of the remnant cystic duct and CBD was achieved by NIR fluorescence imaging using ICG, which helped to identify both structures and thereby resulting in an easier and safer surgical procedure. Intraoperative identification of a symptomatic cystic remnant duct can be challenging. Adhesions and inflammation could hamper visualization during surgery. As a consequence, BDI could occur, which is a complication with high mortality . During Intraoperative assessment of the biliary ducts can be performed by different imaging techniques, of which IOC has been studied the most. However, the benefit of using IOC has not been proven in randomized clinical trials (RCTs), as level 1 evidence is low and all trials were insufficiently powered , 11. MorOptimal injection times of ICG are still under discussion, although a systematic review showed that most studies used a preoperative administration, which was therefore recommended , 19. We In conclusion, this case demonstrates the use of NIR fluorescence imaging as a valuable tool for surgical guidance during a robot-assisted procedure of resection of the remnant cystic duct. This technique is safe and can easily be implemented in the surgical procedure. Therefore, we recommend to use ICG NIR fluorescence imaging during difficult robotic surgical procedures of the biliary tract, such as resection of a remnant cystic duct and gallbladder surgery in which inflamed surrounding tissue is expected. Nevertheless, a less invasive approach by using ERCP, remains the first line of treatment for patient with a symptomatic remnant cystic duct and therefore robotic surgery should only be performed in difficult cases.


Clinical studies with cellular therapies using tolerance-inducing cells, such as tolerogenic antigen-presenting cells (tolAPC) and regulatory T cells (Treg) for the prevention of transplant rejection and the treatment of autoimmune diseases have been expanding the last decade. In this perspective, we will summarize the current perspectives of the clinical application of both tolAPC and Treg, and will address future directions and the importance of immunomonitoring in clinical studies that will result in progress in the field. The number of patients with autoimmune diseases, severe allergies and recipients of organ or stem cell transplants is increasing worldwide. Currently, many of these patients require lifelong administration of immunomodulatory drugs, which cause generalized immunosuppression and hereby only partially alleviate the symptoms but do not cure the disease. Besides these drugs are inevitably associated with a risk of immediate or late-occurring severe adverse effects . Targeting the fundamental cause of autoimmunity, i.e., loss of tolerance to self-antigens, or inhibiting induction or execution of undesired immunity in transplantation and allergy will provide the next steps forward to avoid general immunosuppression. Accumulating knowledge on mechanisms of immune activation and tolerance has led to the development of tolerance-inducing cellular therapies with regulatory T cells (Treg) and tolerogenic antigen presenting cells (tolAPC), such as tolerogenic dendritic cells (tolDC) and regulatory macrophages (Mreg) , witThe development of cell-based therapies is clinically attractive for many reasons, not in the least through their potential of being of low-toxicity, to simultaneously control many different inflammatory cells and induction of antigen-specific immunity. Since immunological tolerance is a self-reinforcing state , the theIn recognition of the potential of tolerance inducing cell-based therapies and to join forces in the ongoing efforts in the field, A FACTT (Action to Focus and Accelerate Cell Based Tolerance-inducing Therapies (CTT) was initiated through EU COST Action Funding. Our goal was to initiate a network that would coordinate European CTT efforts to minimize overlap and maximize comparison of the diverse approaches across Europe. Now, looking back at 4 years of very active network interactions, we have evaluated our combined current stance in the field and defined avenues to support future directions.Over the past 20 years extensive experimental research has been invested in the generation and characterization of tolAPC, including tolDC and Mreg, with the aim to restore tolerance in autoimmune diseases –10 and tin vitro starting from CD14+ monocytes and share some phenotypic and functional characteristics. Indeed, both tolAPC types express low to intermediate levels of T-cell costimulatory molecules, and secrete low amounts of pro-inflammatory cytokines, indicative of a partially matured APC. Similarly, immature DC (iDC) display minimal expression of costimulatory molecules and little secretion of inflammatory cytokines, demonstrating potential optimal requirements for tolerance induction in vivo in vitro.in vivo mechanism of action of these cells  the immune phenotype and their functional activity (potency). Hence, a better identification of the characteristics that identify the tolerance-inducing properties of tolAPC, irrespective of the conditioning regimen, would be valuable for safe cell therapy delivery into patients. Joint efforts in translating tolAPC into the clinic by harmonizing protocols and defining functional quality parameters have been initiated , 55. A Mse cells .Ex vivo generated tolAPC have the potential to therapeutically induce, enhance, or restore antigen-specific tolerance. Indeed, after loading these cells with exogenous or endogenous antigens, one major advantage is their capability to act in an antigen-specific manner.Targeted regulation of antigen-specific T cell responses would avoid generalized immunosuppression as observed with immunosuppressive drugs and monoclonal antibodies currently in use in the clinics and may thus overcome occurrence of impaired immune-surveillance leading to infections or development of malignancies. in vivo studies demonstrate that antigen loading of tolAPC is indispensable to reach efficient clinical responsiveness following tolAPC therapy. For instance, a beneficial effect of vitamin D3-tolDC loaded with MOG40−55 peptide was demonstrated in experimental autoimmune encephalomyelitis (EAE), whereas no clear beneficial effect on the clinical score of EAE mice was found when mice were treated with vitamin D3- tolDC not loaded with myelin peptides (in vivo. Suitable disease-associated self-antigens responsible for T cell priming have been identified for T1D and multiple sclerosis (MS). However, this is not the case for other autoimmune diseases, such as rheumatoid arthritis or Crohn's disease, for which specific disease-associated antigens are unknown or not tissue specific. Moreover, not all patients uniformly display the same set of self-antigens for a given disease. MS, for example, is associated with a range of self-antigens and auto-antibodies that are differentially expressed among patients and at different points during the disease  where the tolAPC could suppress auto-reactive effector T cell responses is logical. In this context, intra-articular injection of tolDC differentiated using dexamethasone and vitamin Dis score . Similaris score . In someis score . Similaris score . Howeverin vitro generated tolDC and Mreg are currently being used in various clinical trials  with monoclonal antibodies has been shown to induce antigen-specific T cell hyporesponsiveness in experimental models  or in the periphery as induced regulatory T cells (iTreg). Both subsets can induce tolerance toward auto- and alloantigens utilizing a variety of mechanisms including cell-to-cell contacts, secretion of immunosuppressive cytokines and inhibitory molecules , local depletion of IL-2, or through killing of other cells (in vitro under Good Manufacturing Practice (GMP) conditions for therapeutic purposes. Indeed, Treg have become a promising cellular drug that can potentially be used to control disease-causing immune responses.CD4er cells . Treg acex vivo expanded Treg was further confirmed in T1D patients  after hematopoietic stem cell transplantation (HSCT)  Table 2Table 2. patients  (Table 2patients .The ONE Study and ThRIL)   . The ONEudy.org) . The ThRl trials –107.ex vivo cell product expansion  -antigens may offer an advantage for Treg function compared to polyclonal Treg . Adoptivn grafts –115. CARBased on promising results with antigen-specific Treg in pre-clinical models, the use of alloantigen-specific Treg generated by culturing recipient Treg with donor-specific cells, either using activated donor-derived B cells  or donorSince adoptive transfer with Treg alone, particularly with a one-time infusion, may not be sufficient to control the immune response, combined or successive therapies are being tested. One approach is based on evidence that low doses of IL-2 can preferentially increase the endogenous pool of Treg; so far, low-dose IL-2 treatment has been safe in inflammatory conditions such as GvHD after HSCT . In a prin vitro or in a transplant setting  provides a highly automated GMP-grade approach which is easy to standardize across centers. However, the resultant Treg product does generally contain a minor population of CD127+ cells that could jeopardize product purity after expansion. Addition of rapamycin to the culture media has helped to maintain Treg purity and function, without reducing the expansion rate too much , this sorting approach is likely to become the preferential Treg isolation method.It is now accepted that the optimal way to manufacture Treg for clinical use requires an efficient too much . The secin vitro culture results in epigenetic changes in Treg—methylation of TSDR region of foxp3 gene—which in-turn reduces suppressive ability . This significantly changes the legal path for their registration requirements, for manufacturing license and marketing authorization. While the idea of an ATMP in Europe is relatively nascent, and it is continuously evolving through public consultations with interested parties, cellular drugs have a distinct central paneuropean path for registration. While the Committee for Advanced Therapies (CAT) steers this process in Europe to optimize safety for patients, it would be useful to introduce wider rules allowing for introduction of the cells as a routine treatment. To accelerate the whole process, acknowledgment of flexible new types of manufacturing cGMP equipment and reagents could open the way to more widespread ATMP use. Furthermore, measures to reduce manufacturing costs would lessen this major limitation to new trials. When considering cell therapy, scientists, physicians and regulators must keep in mind that ATMP must be affordable for patients and society. Interestingly, scientists are largely responsible for current guidelines, and should revisit those recommendations based on factors such as cost , 132.Since cell-based therapies are becoming more common, it is important to reliably monitor the immune system for both desired and undesired immunological effects. Rigorous immunomonitoring will therefore provide information about the safety of these treatments, ideally at early time points after CTT administration. In addition it will give insight in the therapy-related mechanisms of tolerance-induction and maintenance and may aid in patient-tailoring of therapy. To accurately measure the effects, especially across different trials, it will require introduction of harmonized and validated immunomonitoring assays.+ T cell responses after viral-antigen stimulation is possible by flow cytometry via CD40L expression or cytokine production in a functional assay; these assays could potentially identify non-specific immunosuppressive effects  of the transferred cell subset after treatment suggests the presence of infused cells, it does not distinguish transferred from endogenous cells. Therefore, current techniques for tracking infused cells depend on direct cell labeling strategies, such as indium labeling or deuterium introduction during artments , 142. Une models , 144. Ote models , 146. Adanalysis .in vitro assays also represent a challenging method to standardize, involving different approaches depending of the cell type and function. Nevertheless, several efforts have been made to establish a minimal harmonization of antigen-specific functional assays  , 2. At tImmunomonitoring is an indispensable aspect of current and future tolerogenic cell-based therapies that will provide fundamental information to understand and optimize cell therapies. Monitoring will also aid in identifying biomarkers with the capacity for early identification of therapy responders and non-responders and patient-tailoring of therapy. As part of the overall strategy to increase implementation of ATMPs, it will be critical to harmonize GMP manufacturing protocols, product characterization and immunomonitoring. Minimal information models such as MITAP and MiTREG , 151 wilAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.PT Medical University of Gdańsk–owns a patent related to presented content. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.