The impairment of immune functions associated with malnutrition may be one reason for the high mortality in children with severe acute malnutrition (SAM), and thymus atrophy has been proposed as a marker of this immunodeficiency. The aim of this study was to identify nutritional and clinical correlates of thymus size in children with SAM, and predictors of change in thymus size with nutritional rehabilitation.In an observational study among children aged 6–59 months admitted with SAM in Uganda, we measured thymus area by ultrasound on hospital admission to treatment with F75 and F100, on hospital discharge and after 8 weeks of nutritional rehabilitation with ready-to-use therapeutic food, as well as in well-nourished healthy children. We investigated anthropometric, clinical, biochemical and treatment-related correlates of area and growth of the thymus.2 in malnourished children, and 3.5 cm2 in healthy children (p < 0.001). Most anthropometric z-scores, hemoglobin and plasma phosphate correlated positively with thymus area. Thymus area correlated negatively with caretaker-reported severity of illness, plasma α-1 acid glycoprotein, and C-reactive protein >5 mg/L. At follow-up after 8 weeks, median thymus area had increased to 2.5 cm2 (p < 0.001). Increase in thymus area during treatment was associated with simultaneous increase in mid-upper-arm circumference, with 0.29 cm2 higher increase in thymus area per cm larger increment in MUAC (p = 0.03). Children whose F-75 had partially been replaced by rice porridge during their hospital admission had less increase in thymus area after 8 weeks.Eighty-five children with SAM with a median age of 16.5 months were included. On admission 27% of the children had a thymus undetectable by ultrasound. Median thymus area was 1.3 cmMalnutrition and inflammation are associated with thymus atrophy, and thymus area seems positively associated with plasma phosphate. Substituting therapeutic formula with unfortified rice porridge with the aim of alleviating diarrhea may impair regain of thymus size with nutritional rehabilitation. This calls for research into possible effects of phosphate status on thymus size and other immunological markers.ISRCTN55092738.The study is based on data from the FeedSAM study,  Severe acute malnutrition (SAM) in children is a life-threatening condition , and manEven though malnourished children are known to have thymus atrophy, it is unknown how this is modified by clinical factors, such as edema, anemia, electrolyte disturbances, Human Immunodeficiency Virus (HIV), other infections or inflammation in general. Furthermore, although previous studies have documented that thymus atrophy is reversible when malnutrition is treated , 6, littThis study was an observational study among children admitted for in-hospital treatment of SAM between October 2012 and January 2013. The study was nested within the FeedSAM study, investigating physiological changes in children hospitalized with SAM, primarily change in plasma phosphate (P-phosphate). The FeedSAM study was registered in the ISRCTN registry with the number ISRCTN55092738.Mwanamugimu Nutrition Unit at Mulago Hospital is the main treatment center for children with complicated SAM in Uganda. At the time of the study, all children received in-patient treatment based on the Ugandan National Protocol for the Integrated Management of Acute Malnutrition, using World Health Organization (WHO)-recommended milk-based diets, F-75 and F-100 , as well as empiric parenteral antibiotics, usually ampicillin and gentamycin . DehydraInclusion criteria of children were: age 6–59 months; admission on weekdays for treatment of SAM, defined as either weight-for-length z-score (WLZ) < −3, using WHO Growth Standard , or mid-At admission, we obtained information about the child’s current symptoms and history using a structured questionnaire. Caretakers were asked to rate the perceived severity of their child’s illness on a visual analogue scale (VAS) from 1 to 10. Vital signs were noted , as well as edema and oral thrush. To assess appetite, we noted whether the child was able to consume all of the first served therapeutic feed.Body weight was measured daily on a digital scale, to the nearest 100 g. Length and MUAC were measured to the nearest one mm, using an infant length board, and measurement tape, respectively. For analysis, anthropometric z-scores were computed using WHO Growth Standards . In ordeStudy staff monitored children daily (on weekdays), recording weight, frequency and consistence of stools, type and amount of feed given, whether ReSoMal was given, and whether a naso-gastric tube was used for feeding. Children were classified as having diarrhea when passing three or more loose or watery stools per day.The same investigator (MJHR) measured thymus area at three time-points in all the children: One of the first days after admission , a few days before discharge from hospital, and at follow-up, approximately 8 weeks after admission Fig. . The larBlood samples relevant to this study were collected from a peripheral vein at three time points: Sample one at admission before starting refeeding; sample two approximately 48 h after starting refeeding; and sample three, at the day of discharge to outpatient treatment. On all three occasions, 1 ml was collected in heparinized evacuated tubes, and on admission and discharge, 5 ml was also collected in a Cell Preparation Vacutainer® with citrate .o, and shipped to Denmark on dry ice, where C-reactive protein (CRP) and α1-acid-glycoprotein (AGP), were measured at University of Copenhagen, Department of Nutrition, Exercise and Sports, using ABX Pentra® 400 . Heparinized plasma from all three time points was frozen to −20 Co for up to 2 months, and inorganic phosphate (P-phosphate) was measured at Ebenezer Ltd Clinical Laboratory in Kampala , using molybdate UV method (Cobas Integra® 400 Plus).At admission and discharge, hemoglobin level was measured in heparinized full blood, using HemoCue® . Citrate plasma was frozen at −80 CApparently healthy children, with WLZ > −1 and aged 6–59 months, were recruited among children of hospital staff and siblings of hospitalized children and examined once. Thymus area assessment, physical examination, anthropometric measurements and blood sampling was done in controls, as described for the study patients.Data were entered into EpiData  and analyzed using Stata version 12 . Normally distributed variables were expressed as means ± standard deviations (SD), and variables that did not follow a normal distribution were expressed at medians and interquartile ranges (IQR). Two-sample t-tests were used to evaluate differences in means except in case of non-normally distributed outcomes where Mann-Whitney rank-sum- tests were used. Chi-square tests were used to compare proportions, except when the expected numbers were less than five, in which case Fisher’s exact test was used.2 may be less accurate, we assigned all children with an undetectable thymus and children with measured area <1 cm2 to an unknown low value <1 cm2, and such left—censored measurements received less weight in the analysis as compared to accurately observed measurements, using the command “tobit” in Stata. Thymus area did not follow a normal distribution, and hence was logarithm-transformed (base 10); estimates were subsequently back-transformed. The analysis was adjusted for age and sex.To identify correlates of thymus area on admission, while including children with an invisibly small thymus, we used an analysis of covariance (ANCOVA) allowing for left-censored measurements from children with an invisibly small thymus. Since area measurements <1 cm2 in order to calculate the change in thymus area over time.ANCOVA was also used to evaluate predictors of growth in thymus area at discharge and follow-up, defined as the change in thymus area (Δ thymus area = thymus area at follow-up – thymus area at admission). These analyses were adjusted for age, sex, number of days since first scan and thymus area on admission. Children with an undetectable thymus on admission were assigned a thymus area of 1 cmAs sensitivity analyses, we firstly analyzed correlates of thymus area and of predictors of growth in thymus area while only including children with a visible thymus, and without left-censoring of low values. Secondly, to assess if the associations were explained by body size, we analyzed correlates of thymus size while adjusting for body weight after loss of edema.Of 120 children included in the FeedSAM study, 85 (71.7%) were included in the sub-study of thymus size measurement Fig. . Of the p = 0.02), and had higher AGP  (Table p = 0.01). Respiratory symptoms (high respiratory rate or cough) did not differ in children with and without a detectable thymus.In 22 (26%) of 85 children scanned on admission, the thymus was not visible by ultrasound. Children with an undetectable thymus had been rated more sick by their caretakers, were less likely to complete their first therapeutic feed  Table . P-phospOf the 85 children scanned at admission, 54 were scanned at discharge, meaning that 31 were lost before discharge: 13 children died, 13 self-discharge before recommended, and five were discharged when the person performing the scans was not present at the unit. Before follow-up, another 20 children were lost from the study; 5 because they did not show up at follow-up, and 15 because the examiner was not present on the day of follow-up, leaving 34 with complete data at all three time points of the study , but among those where it could be measured, thymus area was not different from those remaining in the study.A greater proportion of those lost to follow-up were boys, while they did not differ significantly in terms of anthropometry, age, HIV-infection or acute-phase reactants. More of those lost to follow-up had an undetectable thymus on admission .Among malnourished children with a visible thymus, thymus area ranged from 0.6 to 5.4 cmβ = 0.73, 95% confidence interval (CI): 0.60; 0.90), meaning that thymus area was 27% lower per each g/l increase in AGP. Children with CRP > 5 mg/l had a 39% smaller thymus area than those with lower CRP . The thymus was 27% smaller in HIV infected children, approaching significance (p = 0.06). Thymus area correlated with hemoglobin, and marginally with P-phosphate on the day of admission. On day two after admission, the correlation between P-phosphate and thymus area was stronger, with a 66% bigger thymus area per mmol/l higher P-phosphate .Thymus area correlated positively with most anthropometric indicators, including body weight, MUAC, weight-for-age z-score, WLZ, and length-for-age Table . CaretakIn the first sensitivity analysis, only including children with a visible thymus, we found overall similar associations. In the second sensitivity analysis, adjusting for body weight, age and sex, the associations were also similar, except there was no longer any significant association with WLZ or weight-for-age z-score (not shown in tables).2 (p = 0.006) at discharge, and to 2.5 cm2 (p < 0.001) at follow-up (Table p < 0.001). While the thymus was undetectable in 22 (26%) children on admission, this was the case for seven (13%) of 53 children at discharge, but in no children on follow-up, or in healthy controls.Children were admitted for a median of 16 days. Median thymus area increased from 1.3 to 1.6 cm2 higher increase in thymus area per cm higher increase in MUAC at discharge , and at follow-up . Children given unfortified rice porridge in hospital had a 0.67 cm2 smaller increase in thymus area at follow-up . The sensitivity analyses showed similar associations, only including children with a visible thymus on admission.Few factors were associated with increase in thymus area during nutritional rehabilitation Table . HemogloThymus atrophy has previously been reported among malnourished children, based on autopsy studies  and in uUsing thymus area as a marker, we found that thymus size was positively associated with most anthropometric indicators of nutritional status. Similar observations have been done among apparently healthy children in Guinea Bissau , Gambia As children admitted with SAM are often both infected and malnourished, it is not surprising that 27% of children in our study had an undetectable thymus on admission. Similarly, an autopsy study of severely malnourished children reported how their thymus was reduced to “an irregular strand of fibrous tissue” . UltrasoThymus size was positively associated with hemoglobin level, and P-phosphate, which to our knowledge, has not previously been reported. The greater association with phosphate on day two may be explained by the fact that most ultrasound scans were done one or two days after admission, and therefore closer in time to the second blood sample. Infections may cause both anemia and hypophosphatemia , and thup = 0.06). It has previously been reported that predominantly well-nourished children with HIV have a smaller thymus [The thymus was smaller in HIV-infected children, although not statistically significant (r thymus . Our finWe found no association between nutritional edema and thymus size. Thymus atrophy is known to occur in children with kwashiorkor , and oneBreastfed children did not have a larger thymus, in contrast to previous studies . This coAlthough thymus size recovered with nutritional rehabilitation, it was still significantly smaller than in well-nourished children. This could be because the children’s nutritional status was still poorer that the healthy controls, or because immunological recovery may take longer than anthropometric recovery, as previously suggested .Few factors predicted change in thymus area. The only anthropometric indicator, in which growth was associated with thymus growth, was MUAC. MUAC reflects muscle mass, and this suggests that recovery of thymus size correlates with recovery of muscle mass. Similar observations were done in a study from Bolivia, where zinc supplementation accelerated both growth in MUAC and growth in thymus size .Interestingly, children in whom F75 had been replaced by unfortified rice porridge at some point during in-patient treatment had reduced thymus growth at follow-up. Rice porridge was given to about half of the children, during their stay in hospital, when children had diarrhea that worsened after starting refeeding. Confounding by indication is an obvious possibility, but most other indicators of disease severity, such as observed diarrhea, reported severity of illness or capillary refill time were not associated with reduced thymus growth. Rice porridge contains very little vitamins and minerals, virtually no fat, and most of its energy is derived from carbohydrates. In this same cohort, we previously reported that giving rice porridge was associated with lower increase in phosphate over the first two days of nutritional rehabilitation , and alsthymic index, obtained by multiplying the diameter by the area [There are a number of limitations to this study: First, its observational design means that we cannot draw firm conclusions about causality. The exploratory approach and the use of multiple testing also means that our findings may only be used to generate hypotheses which should be confirmed in other studies. Second, the most commonly method used previously to measure thymus size is the the area . Howeverthe area , and ourA significant proportion of children hospitalized with SAM have no detectable thymus by ultrasound, and these children appear to be sicker than those with a visible thymus. Thymus size is positively associated with anthropometric indicators of nutritional status, with hemoglobin level and phosphate in plasma, and negatively associated with acute phase reactants in plasma. Growth in thymus size at follow-up seems to be associated with increase in MUAC. Children whose F-75 is partially replaced by unfortified cereal may have poorer recovery of thymus size. Our results emphasize the importance of phosphorous in the diets for malnourished children, and suggests further research into the role of phosphate for thymus size and function in children .


Our aim was to adapt the original software toward an HTML-based solution, thus making it affordable and applicable in any simulation setting.Hands-on training in point-of-care ultrasound (POC-US) should ideally comprise bedside teaching, as well as simulated clinical scenarios. High-fidelity phantoms and portable ultrasound simulation systems are commercially available, however, at considerable costs. This limits their suitability for medical schools. A Linux-based software for Emergency Department Ultrasound Simulation (edus2TM) was developed by Kulyk and Olszynski in 2011. Its feasibility for POC-US education has been well-documented, and shows good acceptance. An important limitation to an even more widespread use of edus2, however, may be due to the need for a virtual machine for WINDOWSWe created an HTML browser-based ultrasound simulation application, which reads the input of different sensors, triggering an ultrasound video to be displayed on a respective device. RFID tags, NFC tags, and QR Codes™ have been integrated into training phantoms or were attached to standardized patients. The RFID antenna was hidden in a mock ultrasound probe. The application is independent from the respective device.Our application was used successfully with different trigger/scanner combinations and mounted readily into simulated training scenarios. The application runs independently from operating systems or electronic devices.This low-cost, browser-based ultrasound simulator is easy-to-build, very adaptive, and independent from operating systems. It has the potential to facilitate POC-US training throughout the world, especially in resource-limited areas.The online version of this article (doi:10.1186/s13089-017-0061-4) contains supplementary material, which is available to authorized users. Point-of-care ultrasound (POC-US) has been shown to improve the diagnostic accuracy of the initial clinical assessment of critically ill patients . The useHigh-fidelity phantoms, as well as portable ultrasound simulation systems are commercially available . However®. With these restrictions, specific hard- and software has to be used. The concept was published open source by the authors, including technical instructions and a set of sample videos to start with [In 2011, Kulyk and Olszynski introduced the portable emergency department ultrasound simulator (edus2TM), an easy-to-build, low-cost bedside ultrasound simulator, that allows for integration into high-fidelity simulation scenarios . The macart with . Anotherart with . This soart with . It is, Anonymized point-of-care ultrasound video clips from patients, showing individual and unequivocal pathologies have been recorded and stored in a database. Together with our departments’ IT section, we aimed to create a browser-based application written in Hypertext Markup Language (HTML5). This in turn reads the input from a sensor as a keystroke to start displaying ultrasound video clips. Sensors should be RFID antennas, Near-Field Communication (NFC), or Quick Response Codes™ (QR Codes™). The input of the sensor initiates the particular pre-recorded ultrasound video clip on the respective device  with any operating system . Access to the pre-recorded videos should be possible wherever they are stored . RFID tags were hidden under the artificial skin of a megacode mannequin  or attached to a patient and then masked, as shown in Additional file Digital Content  and on any gadget available .The main findings of this study are (a) it is feasible to convert the edus2 software into an HTML browser-based application, (b) this—in combination with a mock US probe—makes ultrasound training and simulation affordable in developing countries, since (c) our software solution may be used on any operating system (Mac OSConsidering the capabilities of our readily available ultrasound simulator at only a fraction of the cost of commercially available solutions, it might be especially relevant for medical education and training in developing countries. These can be remote environments or poor areas in the developing world with a lack of imaging modalities other than ultrasound –12. Of nThe high adaptability of the system allows for customization according to the requirements of the respective teaching facility. If there is no opportunity to acquire video material by oneself, there are several freely accessible video libraries for this purpose .Due to its low cost and independence of hardware solutions, our simulator allows to be employed for blended learning in our own medical school in a broad manner, which has the potential to spare time and resource. Learners can not only prepare themselves with conventional e-learning material, but also train more aspects of image acquisition, with a unique haptic experience. The latter very much adds to the imaging skills, compared to the sole use of screen-based training choosing videos on the displaying device manually . Given tWhen using QR Codes™ as triggers, which can be downloaded and printed out, no additional hardware, i.e., sets of RFID or NFC tags, has to be distributed. Thus, the system can remotely be made available for download. Some minor technical details are subject to our further investigation and development, such as the design of the application surface. Furthermore, strategies for accurate review and quality control of the teaching material have to be implemented. Programming and indexing tags with their corresponding video clips may be time consuming, as it is for any ultrasound video loop editing for educational purposes. As some mobile devices failed to feed battery power to the RFID antennas, the capability of using different triggering solutions provides a useful backup plan. Finally, the sole triggering of a video clip to be displayed does not encompass probe manipulation and image optimization skills as commercially available systems do, which is another field of further technical development for the system. Furthermore, the video output format of ultrasound machines and the anonymizing function varies. Several different file types and codecs may hamper their universal utilization for an ultrasound simulation library. This can easily be overcome using free file converters. Altogether, despite some minor technical issues, we did not want to withhold this important technical advance from publication, since we are convinced, that it will have a considerable impact on worldwide training in POC-US.We created a universally available HTML browser-based solution for POC-US simulation at very low cost, which makes ultrasound training and simulation broadly affordable also in developing countries. It was easy to set up and use, running independently from the operating system or displaying device. In medical education, this simulator can be adapted easily to specific demands of the respective setting: undergraduate and postgraduate medical education, simulation, skills training, training of whole POC-US protocols and blended learning. Although the system itself is “low cost,” the technique enables incorporation into high-fidelity simulation, and it enables trainees to learn the indications of ultrasound, learn precise image interpretation, and finally integrate the findings into patient management. We expect our simulator to enhance clinical competence, facilitate health care professionals’ education, and enable research purposes in this important emergency care diagnostic tool.Additional file 1. Mp4, video of cell-phone used as mock handheld ultrasound machine.Additional file 2. Mp4, video of a tablet-pc used as mock handheld ultrasound machine.Additional file 3. Mp4, video with NFC-tags and QR-codes used as ultrasound video triggers.


Previous studies have suggested that extensive action video gaming may enhance perceptual and attentional capacities. Here, we probed whether attentional differences between video-game experts and non-experts hold when attention is selectively directed at global or local structures. We measured performance on a modified attentional-blink task using hierarchically structured stimuli that consisted of global and local elements. Stimuli carried congruent or incongruent information. In two experiments, we asked observers to direct their attention globally (Experiment 1) or locally (Experiment 2). In each RSVP trial, observers were asked to identify the identity of an initial target (T1), and detect the presence or absence of a second target (T2). Experts showed a markedly attenuated attentional blink, as quantified by higher T2 detection sensitivity, relative to non-experts, in both global and local tasks. Notably, experts and non-experts were comparably affected by stimulus congruency. We speculate that the observed visuo-attentional advantage is unlikely to be related to mere differences perceptual tendencies , which has been previously associated with diminished attentional blink. Action video games generally refer to games that require fast responses and exhibition of high visuomotor coordination, while maintaining vigilant monitoring of the periphery for unexpected events and simultaneous tracking of multiple objects3. Different game genres account for their own specific skill improvements4. Improvements are typically found in observers who engage in action video-games extensively for a long period of time although the same enhancements can be seen in non-gamers who participate in video games for as few as ten hours, albeit to a lesser degree9. Despite these intriguing results, some have failed to find differences in perceptual (or more broadly cognitive) capacities between action video-gamers and non-video gamers11.Extensive video gaming has been traditionally associated with negative consequences such as poor physical health, low social motivation or even violence. In the past few decades, a considerable number of studies have suggested that video games, especially of the “action” variety, can enhance visual attention, cognitive abilities, perceptual skills, and even improve brain malleability5. For example, Green and Bavelier5 showed that task-irrelevant information presented in the form of flankers is processed to a greater extent by, and correspondingly affected performance on a target task more significantly for AVGs as compared to NVGs. Specifically, while flanker interference generally declined with increases in the difficulty of the target task in NVGs, AVGs continued to be affected even at higher difficulty levels. The authors attributed this difference in flanker interference between the two groups to reflect greater attentional resources in the expert group.One line of studies seems to indicate that action video-gamers (AVGs) have larger visual attentional capacities, a broader visual field and higher visual acuity as compared to non-video-gamers (NVGs)attentional blink. The attentional blink refers impaired processing of the second target within a set of two targets presented in close temporal succession13. That is, in a RSVP stream of items, participants consistently fail to report the identity or presence of the second target if identification of the first is required. This ‘blink’ is particularly pronounced for stimulus onset asynchronies (SOA) of 200 to 500 ms12. Accuracy generally recovers after 500 ms yet there are a few exceptions such as non-blinkers, who never or seldom suffer from the lightest of attentional blinks. More relevant, AVGs show an attenuated attentional blink13. These findings have been echoed in later training studies .Increased attentional capacity in AVGs may also explain better performance in visuo-attentional tasks under transient-presentations. One oft-used paradigm involves rapid serial visual presentation (RSVP) and measures of an 14. The underlying cause of attentional blink, however, is well-debated. The most widely-accepted account relates to a limitation of resources: a two-staged model which emphasises a significant role of target-discriminability15. According to this model, all stimuli enter an initial stage of perceptual and semantic processing15. In stage one, the stimulus activates its stored conceptual representation but this piece of information is volatile, susceptible to decay, and at risk of being overwritten by subsequent stimuli. In stage two, relevant target features initiate processing and trigger a temporary attentional response that leads to encoding of target into working memory16. Only those stimuli that have successfully undergone the capacity-limited stage two would be safe from overwriting and available for report16. In the case of an RSVP stream where the first target (T1) is temporally adjacent to the second (T2), they will be processed together because such short time difference between the targets allows T2 to enter the same temporal window as the first16.As for adults, benefits of action video gaming experience have been observed in children and adolescents17. Initial demonstrations17 revealed that normal observers responded quicker towards global information than to local information, and that incongruent local information did not affect response time of global level identification . That is, participants appear to have a particular sensitivity to global information – a preference that has sometimes been referred to as global precedence or bias in the literature17. Navon suggested that local features can still be processed but not without a deliberate effort17, and in the event of parallel processing, global processing would still finish earlier than local processing18. Other studies have also shown that global elements of attended and non-attended objects are harder to ignore as compared to local elements20. Of immediate relevance, research using hierarchically-structured stimuli has shown that individuals often have varying degrees of perceptual preferences, and that those with a preference towards global information, also demonstrate less attentional blink22. When placed into the context of visuo-attentional advantages in video-game experts demonstrated independently by a number of groups, these data drive an intriguing question: Might attentional advantages in video-game experts be related to greater global precedence as compared to non-experts?A different, but related line of work considers the relationship between attention and spatial selectivity. Navon proposed that perceptual processing occurs in a temporally-organized manner, starting with global  processing before fine-grained analysesHere, our question is two-fold: We ask if the attentional advantage for action video-gamers versus non-gamers, as demonstrated by a reduced attentional blink, holds both when attention is selectively directed at global or local information. We introduce locally and globally congruent or incongruent stimulus configurations while preserving the classical RSVP blink paradigm. Using the same paradigm, we also probe the possibility that attentional advantages, if any, could be simply explained by differing perceptual preferences . We predicted that to the degree with which video-game experts show a reduced attentional blink, they may show a correspondingly greater global precedence. In two experiments, we tested both action video gamers and non-video gamers in a modified attentional blink task using Navon-type figures. We tested for differences in visuo-attentional capacities between the two groups both when asked to make global stimulus judgments (Experiment 1) and local judgments (Experiment 2) under conditions of both coherent and conflicting stimulus information.M = 22.36, SD = 2.98), and were sorted into action video-gamers (AVGs) or non-video gamers (NVGs) based on their answers to a preliminary survey of their video gaming habits and expertise . Four participants were excluded from the study because although they self-identified to be action video-gamers, they in fact could not be classified clearly into either category based on their responses and our criteria. Among the remaining 58 participants, twenty-nine of these observers were categorised as AVGs . In Experiment 2, we tested fifty-five new observers after excluding two observers who could not be classified , who ranged in age from 18 to 35 years old .A total of 119 participants were recruited through advertisements posted on university notice boards, explicitly requesting self-identified video-game players (at least 4 hours per week of game-play) and non-video-game players. Sixty-two of these participants participated in Experiment 1 , and the rest participated in Experiment 2 . In Experiment 1, observers ranged in age from 20 to 30 years old /third (TPS) person-shooter, sports, real-time strategy (RTS), action-role-playing-(RPG), and action-adventure types, which still share common characteristics in terms of real-time combat  and requirements of rapid responses. In this study, we considered action videogames to include first (FPS)/third (TPS) person-shooter, sports, action-role-playing (RPG), and action-adventure subtypes, which use the same action combat mechanics and have similar rapid response requirements. Traditionally, action video games included games such as Call of Duty, Halo, and Unchartered. Several newer titles, however, also fit action video-game criterion:The influx of new video games in the past decade and consequently, changes in visuo-spatial skills required by games on the consumer market allude to the need for new classifications to be used to fully understand the current gaming landscape. Under a revised sub-categorisation scheme6. In Jubeat, the individual is required to tap multiple small grids on a large screen when prompted. These prompts appear in quick succession, in the periphery and/ or at the centre, and can be very distant from one another especially at high difficulty levels. Execution of this game always requires the action of at least two fingers on each hand. These properties render the requirements for this game akin to those for more traditional action video games. Finally, classification for another recent, and very popular game, League of Legends may appear ambiguous; in fact, this game belongs to an action-RTS or multi-player online battle arena (MOBA) game genre which taps on the cognitive systems as traditional action games23. Hence, it was also included here as fulfilling action-video-game criterion.One such game is Jubeat – a title that is usually classified for the consumer as a music game. However, this game requires vigilant monitoring, fast motions, and simultaneous tracking of multiple targets, which well fit the criteria for action games, as adopted by previous action video game-related studies3. Thus, participants who have sporadically engaged in the above gaming genres were still categorised as NVGs in the present study.NVGs were considered as those who had zero or less than an hour of gaming for the past six months. As video games are an integral part of daily life, participants who played video games without fast motions, vigilant monitoring or any other aspect typical of action video-games still qualified as an NVG. Studies have shown that not all video games enhance visual attention. For example, strategic games such as Rise of Nations, slow-pace sport games including Harry Potter: Quidditch World Cup, role-playing games or the famous Tetris which belongs to simple visuo-motor game genre were not found to be beneficial to visuomotor skills and visual attentionM = 12.26 hours, SE = 1.42) than NVGs  . Most importantly, AVGs spent significantly more hours per week playing action-type video-games  than NVGs . There was no difference between the ages of the two groups , nor differences in terms of years of education since primary one (AVG: .With groups classified according to the above criteria, we confirmed that AVGs spent significantly more hours per week engaging in video game playing , or incongruent .Stimuli were Navon-style figures composed of smaller letters arranged in a configuration that corresponded to a different, or same global letter as its local constituents Fig. . These f2), except for those designated “Target 1”, which were highlighted in white  and centered against a mid-grey background (48.81 cd/m2). The number of local elements comprising each global figure, as well as the spacing between each local element was equivalent across all figures. Stimuli were generated and presented in Matlab24 using extensions from Psychtoolbox26. Stimuli were presented on a 24-inch Dell monitor with a refresh rate of 60 Hz. The experiments were conducted in an undisturbed, dimly lit room.For all tasks, stimuli were presented in black  or Experiment 2 . The full experiment lasted approximately 45 minutes.27. Each stream terminated with a prompt asking participants to respond with the identity of T1 and subsequently, the presence of ‘X’ (yes/no) in the letter stream. Participants were given a maximum of three seconds to input their response after each prompt.In this variant of the attentional blink task, participants were asked to judge the global identity of the figures only. Each trial started with the presentation of a central fixation cross (0.5 × 0.5 deg) of two seconds followed by stimuli shown for 15 ms each, with each separated by a 85 ms interstimulus interval. The letter stream consisted of nine letters, among which T1 was always situated at the third temporal position. Since the temporal position of T2 relative to T1 determines the attentional blink, we elected to fix the position of T1, and vary the temporal position of T2. Introducing an additional manipulation for the temporal position of T1 would require a full counterbalancing of trials that takes into account that factor, together with that for T2 position (variable) as well as congruency (congruent/incongruent) – inflating trial requirements  unnecessarily. T2, in the form of letter ‘X’, had a 50% chance of appearing after the presentation of T1. T2 was not presented immediately after T1 , as previous studies have reported a robust lag 1 sparing effect. T2 could instead appear at lag 2 , lag 3  and lag 4 . Note that as we were interested in testing the relative magnitude of attentional blink between the two groups, and not the absolute attentional blink and its recovery period, we elected to use these three lags that are typically encapsulated within the window expected to elicit an attentional blink, in order to maximise our sensitivity to any group-related differencesAs the Navon figures could either be congruent  or incongruent , four figure conditions were presented within a block of trials: (1) T1 and T2 both congruent; (2) T1 and T2 both incongruent; (3) T1 congruent but T2 absent; (4) T1 incongruent but T2 absent. Participants completed a single run, where each condition was repeated 30 times, yielding to a total of 120 trials. Trial order was randomized and participants were allowed to rest briefly after every 40 trials. The entire task lasted 35 minutes. Prior to beginning the experiment proper, participants were given ten practice trials in which targets were shown at 100 ms to familiarize themselves with the task.Stimuli and procedures were identical to those of Experiment 1 except that new participants were asked to judge the local elements of the figures instead.The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.F = 3.76, p = 0.058]. There was no significant main effect of congruency  nor a significant interaction between congruency and group .We first compared accuracies for T1 identification between the two groups by means of a 2 (congruency: congruent and incongruent) × 2 (group: AVGs and NVGs) mixed ANOVA. This initial analysis of T1 identification performance is critical to establish that the attentional blink is measurable . The analysis indicated a marginally significant effect of group, with AVGs showing a subtle advantage for detecting T1  and congruency . There was also a significant main effect of group, reflecting higher sensitivities for AVGs as compared to NVGs . Follow-up Bonferonni-corrected t tests again indicated that performance at lag 3  was significantly worse than that at lags 2  and 4 . Other interactions were not significant.Next, we analysed T2 detection performance (dprime sensitivities), including only trials where T1 was correctly identified . To unpack this idea, consider that an overall large preference for attending to global information would manifest itself in comparable performance between congruent and incongruent conditions . We can quantify this by computing congruency indices - obtained by dividing T1 accuracies of incongruent trials by those of congruent trials. Computed in this manner, an index close to 1 would indicate a lack of interference of conflicting local information, and provide a measure of global precedence (or preference) . A comparison of indices for two groups would provide a measure of the relative interference of incongruent local elements between the two groups. Analyses of these indices indicated that while the indices of both groups were no different from 1 , there was no difference between the indices of the two groups   in AVGs versus non-expertsF = 39.71, p < 0.001], reflecting the fact that T1 identification performance was better for congruent than incongruent trials. Notably, there was no significant main effect of group; that is, AVGs did not perform any better than NVGs in terms of local T1 identification . There was also no significant interaction between stimulus congruency and group .As for Experiment 1, we first analysed the local task data in terms of T1 identification performance, quantified in terms of proportion of accurate responses. A 2 (congruency: congruent and incongruent) × 2 (group: AVGs and NVGs) mixed ANOVA indicated a main effect of congruency  reflecting the fact that T2 detection was generally better for AVGs versus NVGs. There were also significant main effects of lag  and congruency . Neither the lag by congruency interaction nor congruency by group interaction was significant. Further analysis of the main effect of lag indicated that performance at lag 3  was significantly lower than that at lags 2  and 4 . Other interactions were not significant.Next, we considered T2 detection performance only for trials where T1 was correctly identified Fig. . The datM = 0.84, SE = 0.03; NVG: M = 0.81, SE = 0.04]. The indices computed as such were significantly less than 1 for both the AVG  and NVG  groups, but not significantly different between the two groups , again indicating the degree of global precedence is comparable between the two groups.As for the global task, there was no interaction between group and congruency. As for Experiment 1, we illustrate the lack of interaction between group and congruency on T1 performance, by computing congruency indices, obtained by dividing performance on incongruent trials by that on congruent trials (as described earlier) Fig. . Here, aIn sum, AVGs showed overall better performance than non AVGs in terms of T1 identification for the global, but not local task. Considering the attentional blink (T2 target detection), AVGs showed attenuated blinks  for both global and local tasks. Importantly, however, this advantage was uniform across stimulus congruency in both the global and local tasks, and cannot be simply attributable to enhanced ability to detect/identify T1 in AVGs as any potential group advantages were removed already by considering only trials where T1 were correctly identified. These data suggest that the smaller ‘blink’ observed for AVGs is not attributable to overall better global percepts, nor attributable to differing perceptual tendencies - global precedence effects .9, we probed the interaction between visuospatial attentional requirements and video-gaming expertise. We used a modified attentional blink paradigm involving Navon-type stimulus figures that were congruent or incongruent and asked observers to direct their attention globally (Experiment 1) or locally (Experiment 2). This setup allowed us to probe the extent to which expertise-related advantages in visual attention could be attributable to global precedence. Action video-gamers showed a significantly shallower attentional blink, quantified by higher T2 detection sensitivities. These findings are congruent with attentional advantages shown previously for video game experts5. Still, as noted earlier, despite the large body of literature that align with our findings, other studies have failed to find clear attentional advantages in video game experts11. Several factors may contribute to the apparent discrepancy in the literature.In light of independent lines of work showing advantages for action video gamers for visual attentionThe first factor relates to video-gaming habits. The vast majority of habitual gamers in our study engaged purely in action video-games whereas the gaming habits of participants in other studies may centre around strategy-type games – games that may not require the same degree of visuo-motor precision nor attentional requirements.5, in which participants were required to identify numbers from letters27. The task was evidently easier because of increased target-distractor discriminability and longer target exposure time. Probe tasks require recalibration of the input filter, which is not required in a categorical and feature tasks28. Furthermore, varying lags at which T2 would appear may mask any attentional blink effects10.Likewise, performance seems to be highly dependent on the type of attentional blink task. In a recent study, the probe task employed by Green and Bavelier was replaced by a categorical test30. Perhaps more importantly, attentional blink is concerned with attentional capacities which appear to be difficult to alter at will. Furthermore, previous action video game studies that have used covert recruitment31 have produced findings very comparable to those that have used overt advertisement strategies32. Nonetheless, as both groups were recruited in the same manner, any overall enhancements or degradations of performance due to demand characteristics should be comparable for both groups.One last factor that has gained traction in the recent literature in terms of potential influences on empirical work is broader, but perhaps still relevant, and relates to the method of recruitment. In the present study, advertisements included explicit requests for video-gamers and non-video-gamers; hence participants were initially self-identified. Upon arriving in the laboratory, they were then required to complete a formal screening survey in order for us to perform formal classification. We deem overt recruitment as having been important in order to ensure we did not consistently recruit participants that required disqualification, which would likely be the case under covert circumstances since participants with some casual or former intensive gaming history would be excluded from either group. In order to minimise any potential expectation effects, participants were not provided any information as to the purpose or hypotheses of the study, and were also instructed that the screening form constitutes a part of the standard battery of tests for visual screening (i.e. it was conducted together with the linear acuity test). Literature that has looked directly at the effects of knowledge of a study’s purpose has not shown clear trendsdelayed. As we did not seek to test the temporal window needed to achieve full recovery , previous attentional blink work utilising Navon letter figures in RSVP streams have indicated that it takes a significant period for performance to recover34. In one study33, participants attained a T2 detection performance of 60% accuracy at 1 second post-T1 offset, and required 1.52 seconds (post-T1 offset) to reach 80% correct identification of T2.In the present experiments, attentional blink was strongest for both groups at lag 3, recovering somewhat at lag 4 Fig. . Thus, t22. This is, at least in part, due to design-level differences. Dale and Arnell21 demonstrated that global precedence, as retrieved from performance (reaction times) on a global/local task involving Navon-type figures could predict attentional blink magnitude in a second task using standard (non-Navon-type) elements. To quantify global precedence, the authors first computed measures of global interference , and local interference . Global precedence was then computed as the difference between global interference and local interference.The pattern of results obtained when observers were instead asked to direct their attention to the local elements (Experiment 2) were largely similar to those obtained in the global attention task. One notable exception to this was that while AVGs showed a clear advantage for identifying global letter identity, this advantage disappeared when the two groups were instead asked to judge the local elements. As for the global task however, both groups showed significant global precedence , rather than reaction time data, which would be uninformative here as the T1 response probe occurs after the offset of the entire RSVP stream. Still, we attempted to reconstruct a comparable analysis to that of Dale and ArnellWe then computed additional measures of local and global interference using the attentional blink data, by obtaining the difference between Target 2 detection sensitivities in the incongruent versus congruent conditions (given Target 1 was correctly identified). Finally, we entered these data into simple correlational analyses that revealed no evident relationship between either global or local interference with the attentional blink in either group 16. While the standard attentional blink delay and lag sparings have been explained in the context of Chun and Potter’s two-stage model15, we suggest that the Boost and Bounce model35 could perhaps better explain the ‘delayed drop’ observed here at lag 3. The model suggests that a ‘spread of sparing’ to lags that are temporally near T1 is possible because the attentional blink is caused by the subsequent fall from a strong attention boost required for recognising T1. According to this model, targets at lags 2 and 3 can still be processed and enter working memory provided that the upcoming stimulus and T1 both belong to the same stimulus class set37.Critically, video game experts again showed attenuated attentional blink as compared to non-experts in the current study. Across both experiments, both groups performed worst at lag 3, marking a sharp decline from lag 2 – the temporal position that usually elicits the largest blink in conventional attentional blink paradigms39, we were unable to achieve a gender balance between our video-gaming and non-video-gaming participant groups. Complicating evaluations of the effect of such gender imbalance on our effects is the fact that the current literature suggests that males perform better than females in some , but not all tasks that involve spatial attention; males also tend to show more variability in their performances on these tasks40. In the context of video-game expertise, one study found gender differences in spatial attention among NVGs when tested with a UFOV paradigm9. Still, a previous study using an attentional blink paradigm reported negligible effects of gender14. In order to address potential concerns about gender effects lurking in our data, we plotted T2 detection sensitivities separately for males and females  blinks than the local attentional system34. This characteristic of the global system is intriguing as it has been argued that transient visual attention is driven largely by the magnocellular system46. M-cells have larger receptive fields, transient responses, and higher contrast sensitivity47. Therefore, the M-pathway is especially important when presented with fine temporal information48. Could the attenuated attentional blink observed for video game experts be reflective of an enhanced M-system?Finally, it is interesting to speculate on the mechanisms tapped into by our two tasks. Neuroimaging work has shown that global and local attention selectively activate differing areas in the temporal-parietal cortex, with attention to global figures activating the right lingual gyrus, and attention to local features activating the left inferior occipital cortex9, action video game experts exhibit an attenuated attentional blink relative to non-experts. This benefit holds both when attention was selectively directed towards global or local features. Critically, this apparent visuo-attentional advantage is independent of group differences in attaining general task demands . The attentional benefits also cannot be well explained by greater  precedence perceptual tendencies22, as both groups were comparably affected by stimulus congruency. Instead, the observed advantages may be indicative of a fine-tuned selective attention system adept at resolving temporally-transient information, perhaps manifest in an enhanced Magnocellular system.Using a modified attentional blink paradigm employing Navon-type hierarchical figures, we showed that congruent with early studiesSupplementary information


Malnutrition is a leading cause of morbidity and mortality in cirrhosis. There is no consensus as to the optimal approach for identifying malnutrition in end-stage liver disease. The aim of this study was to measure biochemical, serologic, hormonal, radiographic, and anthropometric features in a cohort of hospitalized cirrhotic patients to characterize biomarkers for identification of malnutrition.In this prospective observational cohort study, 52 hospitalized cirrhotic patients were classified as malnourished (42.3%) or nourished (57.7%) based on mid-arm muscle circumference < 23 cm and dominant handgrip strength < 30 kg. Anthropometric measurements were obtained. Appetite was assessed using the Simplified Nutrition Appetite Questionnaire (SNAQ) score. Fasting levels of serum adipokines, cytokines, and hormones were determined using Luminex assays. Logistic regression analysis was used to determine features independently associated with malnutrition.Subjects with and without malnutrition differed in several key features of metabolic phenotype including wet and dry BMI, skeletal muscle index, visceral fat index and HOMA-IR. Serum leptin levels were lower and INR was higher in malnourished subjects. Serum leptin was significantly correlated with HOMA-IR, wet and dry BMI, mid-arm muscle circumference, skeletal muscle index, and visceral fat index. Logistic regression analysis revealed that INR and log-transformed leptin were independently associated with malnutrition.Low serum leptin and elevated INR are associated with malnutrition in hospitalized patients with end-stage liver disease. Malnutrition consists of skeletal muscle loss with or without loss of adipose tissue mass. It is frequently observed in cirrhosis with a reported prevalence as high as 65–90% –3. MultiDespite its prognostic significance in cirrhosis, malnutrition remains underdiagnosed in clinical practice . AlthougAlthough malnutrition adversely impacts clinical outcomes in cirrhosis, early identification of malnutrition in cirrhotic patients remains challenging. Therefore, the goal of this study was to measure biochemical, serologic, hormonal, radiographic, and anthropometric features in a cohort of hospitalized cirrhotic patients to uncover objective biomarkers for identification and monitoring of malnutrition.This prospective, observational cohort study was approved by the institutional review board (IRB) at the University of Pittsburgh. Fifty-two cirrhotic patients were enrolled at the University of Pittsburgh Medical Center Presbyterian Hospital in Pittsburgh, Pennsylvania from November 2013 to June 2014. Informed consent was obtained prior to study inclusion from either the subject or a designated representative. Inclusion criteria included age over 18 years, ability and willingness to provide informed consent, and ability to perform diagnostic tests. Exclusion criteria included any history of solid organ transplantation or hematopoietic stem cell transplant, death or transplantation during the index hospitalization, and discharge with hospice care.Participants underwent a structured clinical assessment within 48 hours prior to hospital discharge. Age, gender, self-reported ethnicity, and past medical history were obtained from the medical record and through subject interviews. Records were reviewed by two hepatologists  to establish the etiology of cirrhosis, hospital admission diagnoses, cirrhosis-related complications, including varices, hepatic encephalopathy, hepatic hydrothorax, and hepatocellular carcinoma, and to record prior transjugular intrahepatic portosystemic shunts (TIPS) procedures. On physical examination, the presence of jaundice was documented, and ascites was graded as absent (grade = 0), visible only by ultrasound (grade 1), visible ascites with bulging flanks (grade = 2), and tense ascites (grade = 3) .All laboratory tests were obtained after an overnight fast. Routine clinical laboratory measurements were performed at the University of Pittsburgh Clinical Laboratories. These included serum glucose, sodium, potassium, chloride, bicarbonate, urea nitrogen, calcium, creatinine, alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin, alkaline phosphatase, total protein, albumin, complete blood cell counts, and international normalized ratio (INR). The MELD score was calculated as follows :MELD = Patients who received at least two renal replacement treatments within one week of enrollment were assigned a creatinine value of 4.0 mg/dl. Child-Turcott-Pugh Score and Class were determined as previously described .Subjects were measured on a standard upright weight scale, and this weight was designated as wet weight. Dry weight was determined as wet weight with 5% subtracted if grade 1 ascites was present, 10% subtracted if grade 2 ascites was present, and 15% subtracted if grade 3 ascites is present. If a patient underwent cumulative paracentesis of over 10 liters during the hospitalization, the post paracentesis weight was considered the dry weight. An additional 5% was subtracted if lower extremity pitting edema was present . Height Dominant arm mid arm circumference was assessed with a flexible measuring tape at the midpoint between the acromion and epicondylar process. Triceps skinfold thickness was measured at the same location using the Lange Skinfold Caliper . Mid-arm muscle circumference was calculated as follows :Mid−armHandgrip strength was assessed using the Jamar dynamometer . Patients underwent three consecutive measurements of grip strength in the dominant hand, and the mean grip strength was then calculated. A patient was classified as malnourished if both mid arm muscle circumference was < 23 cm and handgrip strength was < 30 kg , 14.Subjects were asked to walk 15 feet at their usual pace, and completion times were measured with a stopwatch. The mean walking time of three attempts was calculated. Patients who were unable to walk were assigned the longest measured walking time in the cohort. Finally, appetite was assessed using the Simplified Nutritional Appetite Questionnaire (SNAQ) score ; anorexi2) derived from the two images was reported. Liver protocol MRI does not routinely extend below the L2/L3 level. Therefore, the single most caudal image from non-fat-saturated spoiled echo T1-weighted sequences was chosen for analysis . There were no significant differences in serum sodium, albumin, bilirubin, and creatinine levels in patients with and without malnutrition. Malnourished participants had higher MELD scores , and this was due to higher INR .Clinical and demographic characteristics are summarized in 2/m2 vs. 46.64(43.25–58.84) cm2/m2,p = 0.005] and visceral fat index  were significantly lower in malnourished patients. Visceral fat index was significantly correlated with triceps skinfold thickness, wet BMI, and dry BMI; skeletal muscle index was significantly correlated with visceral fat index, mid arm muscle circumference, wet BMI, and dry BMI  ng/ml in Child’s Class A, 6.62 (2.96–10.39) ng/ml in Child’s Class B, and 8.17 (3.35–20.79) ng/ml, p = 0.741]. Together, these findings suggest that leptin levels reflect nutritional status and insulin resistance in hospitalized cirrhotic patients.Among metabolic markers, only HOMA-IR exhibited significant, positive correlation with leptin. On the other hand, MELD, clinical chemistries, inflammatory cytokines and other adipokines were not correlated. There were no significant associations with markers of appetite or with appetite-regulatory hormones. Finally, leptin levels were not affected by gender [6.50 (3.11–14.77) ng/ml in men vs. 9.172 (3.348–20.098) ng/ml in women, p < 0.1 in univariate analyses were included in a backward stepwise elimination algorithm to generate the final multivariable model (Tables p < 0.1). In multivariable analysis, log-transformed leptin and INR were independently associated with malnutrition, and this relationship persisted after adjusting for race, gender, and age (p = 0.0006).To identify features associated with malnutrition, logistic regression was performed. Variables with l Tables  and 4. W and age . A separMalnutrition frequently develops in decompensated cirrhosis and significantly impacts survival. Yet, the underlying pathophysiologic mechanisms of malnutrition in cirrhosis are poorly understood, and clinicians lack objective biochemical tests to determine the presence and severity of malnutrition in cirrhosis. Thus, there is an urgent need to identify objective biomarkers of cirrhosis-associated malnutrition to enable early diagnosis, to monitor response to treatment, and to improve cirrhosis prognostic scores by incorporating quantitative biomarkers of malnutrition. Therefore, we evaluated biochemical, serologic, hormonal, radiographic, and anthropometric features in hospitalized cirrhotic patients to obtained insights into the pathophysiologic drivers of malnutrition in cirrhosis and to identify potential biomarkers of liver cirrhosis-associated malnutrition.Our study provided several import insights. First, contrary to our expectation, the degree of liver disease severity in malnourished individuals was only slightly worse than in patients with malnutrition. While the two-point difference in average MELD scores between the malnourished and not malnourished group was statistically significant, it is unlikely that this small difference is clinically meaningful, since there was no difference in the prevalence of cirrhosis-related complications between the two groups. While we were unable to measure food intake, we found no difference in appetite, as determined by the SNAQ questionnaire score, between the malnourished and not malnourished groups. These findings suggest that malnourishment is not an inevitable occurrence in decompensated cirrhosis but, rather, represents a disease sub-phenotype resulting from a distinct pathophysiologic pathway.Second, among the several cytokines and adipokines we tested, only leptin differed between patients with and without malnutrition. Leptin is a 15 kD peptide that is produced by adipocytes to regulate energy homeostasis. In healthy individuals, leptin is secreted in proportion to adipose mass and signals via hypothalamic receptors to decrease food intake and increase energy expenditure. Other regulators of leptin production include inflammatory cytokines , glucocorticoids, and insulin , 27. In Third, logistic regression analysis revealed that besides log-transformed leptin, INR independently associated with malnutrition in hospitalized cirrhotic patients. Malnourished patients with end-stage liver disease exhibited slightly higher MELD scores, and this was driven by differences in INR, as median bilirubin and creatinine were similar between groups. INR elevation in malnourished patients may be not only due to impaired hepatic synthetic function, but also related to altered gut microbial flora, decreased fat intake, and reduced intestinal absorption of fat-soluble Vitamin K. Given its sensitivity to nutritional factors, INR may represent a robust marker of malnutrition in chronic liver disease. Since anthropometric measures of malnutrition are difficult to interpret in the setting of ascites and edema, leptin and INR may provide clinical utility as indicators of pre-clinical malnutrition and adipose tissue reserve.A few limitations are noted. First, there are no generally accepted definitions for malnutrition in cirrhosis. In the current study, we used readily available measures (MAMC < 23 cm and dominant hand grip strength < 30 kg) that previously exhibited 94% sensitivity and 97% negative predictive value for identifying malnutrition in cirrhotic patients , 14. UsiIn conclusion, low serum leptin levels and elevated INR were associated with malnutrition in hospitalized patients with decompensated cirrhosis. We propose that serum leptin should be further investigated as a potential biomarker for cirrhosis-associated malnutrition to enable early diagnosis, quantification of severity of malnutrition and monitoring response to treatment.S1 Fig(A) Spearman rank correlation of visceral fat index with triceps skinfold thickness, dry BMI, and wet BMI. (B) Spearman rank correlation of skeletal muscle index with visceral fat index, mid-arm muscle circumference, dry BMI, and wet BMI.(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


ATG5, MAP1LC3A and BAX in the FAP group. There was also a decrease in the protein level of Beclin-1 in the UC and FAP compared to the control group. Although the LC3II levels by immunoblot were higher in the UC group, LC3/p62 co-localization were lower in the immunofluorescence analysis in the UC and FAP compared to the control group. Corroborating these results, there was an increase of p62 by immunoblot in the UC group. These findings indicated a modulation of macroautophagy markers in the IP, which may explain the mucosa inflammation predisposition.Total retocolectomy with ileal pouch-anal anastomosis (IPAA) is the surgery of choice for patients with ulcerative colitis (UC) that are refractory to clinical treatment. Pouchitis is one of the most common complications after this procedure. Defects in autophagy have been reported in inflammatory bowel diseases. However, there are no studies on the IP. Therefore, we studied markers for autophagy in the IP mucosa of UC and FAP patients comparing them to controls with a normal distal ileum. Sixteen patients with IP in “J” shape, asymptomatic and with endoscopically normal IP were evaluated. The control group consisted of eight patients with normal colonoscopy. There was a significant decrease in the transcriptional levels of  Both diseases, despite being different, may require the same surgical procedure. The ileal pouch-anal anastomosis (IPAA) is the elective procedure of choice in the surgical management of refractory UC, and FAP with many polyps in the rectum3. The main complication after this procedure is the pouch inflammation (pouchitis) that can affect up to 45 percent of patients who are submitted to IPAA for UC, and only five percent of the FAP patients who undergo the same procedure4. This suggests that constitutive differences between UC and FAP pouches have a critical role in its pathogenesis.Ulcerative colitis (UC) is a chronic intestinal inflammation that can affect the large intestine and rectum. Its etiology is not completely established. Familial adenomatous polyposis (FAP) is an autosomal dominant disease which affects young individuals and is associated with the formation of multiple polyps in the large intestine and rectum, which invariably implies a greater risk of cancer5. The distinct immunological aspects of the different inflammatory bowel diseases (IBD), specifically UC, which involve impaired innate and adaptive responses, associated to genetic susceptibility, environmental factors, and intestinal microbiota may be involved in the pouch inflammation etiology6.Pouchitis develops only after ileostomy closure, when the pouch mucosa starts to be exposed to the fecal stream8. There are three primary forms of authophagy: macroautophagy, microautophagy and chaperone-mediated autophagy (CMA)9. Although there are no indications of genetic mutations related to the mechanism of autophagy associated to UC susceptibility, the transcriptional and protein evaluation of this mechanism in the ileal pouch mucosa is of fundamental relevance. Alterations of apoptosis in this tissue have already been described previously10 and both signaling pathways, autophagy and apoptosis, are interconnected12. Indeed, differential expression of Beclin1, a relevant protein that is involved in the initiation of autophagy, was already seen in the colon of UC patients4. Moreover, epigenetic alterations, which have recently been related to the etiology of IBD14, can determine transcriptional changes, which in turn help to better understand the mechanisms that predispose patients to the inflammatory process in the ileal pouch justifying its study. Thus, we evaluated molecules involved in the autophagy pathways in ileal pouch mucosa of UC and FAP patients, even in the absence of clinical, endoscopic and histological inflammation, in order to understand if there is underlying modulation in these pathways that could mediate molecular inflammation in the IP.Autophagy is an evolutionarily conserved catabolic pathway that consists of selective degradation of cellular components and a homeostatic mechanism that protects cells exposed to stress situations Among the aspects analyzed by the PDAI, the histological analysis of the biopsies collected in the ileal pouch mucosa of UC and FAP patients demonstrated a small amount of acute inflammatory cells infiltration, absence of crypts destruction, cell architecture preservation and presence of goblet cells, as demonstrated in Fig. lamina propria is shown in the Fig. lamina propria were counted for quantitative analysis, which was analyzed by two blinded observers (N.M.P. and L.B.P.) in a panchromatic objective field of higher magnification 40X.The polymorphonuclear leukocyte count of the PDAI was performed for all patients taking into account clinical, endoscopic and histological aspects (see supplementary figure). All patients evaluated had PDAI < 7. The aim of our study was to analyze patients without inflammation to show if there were underlying molecular alterations in the ileal pouch mucosa, even in the absence of endoscopic and histological inflammation.ATG5 in the ileal pouch when compared to UC 21. The autophagosome can fuse with lysosomes and the proteins are degraded and recycled. On the other hand, microautophagy is mediated by direct lysosomal engulfment of the cytoplasmic components, which is trapped in the lysosome by the membrane invagination22. The recognition of the protein substrate by specific proteins such as chaperones in the cytosol, binding directly to the lysosome, translocating across it, without additional vesicles formation, is the characteristic of CMA23. For this reason, autophagy prevents the accumulation of abnormal proteins, is also involved in the genomic stability, and participates of the removal of intracellular pathogens24. When there is an autophagy deficiency, it promotes cytoplasmic protein inclusions, which are composed of misfolded proteins. The accumulation of deformed organelles can lead to cell injury and diseases25.There are three primary forms of authophagy: macroautophagy, microautophagy and chaperone-mediated autophagy (CMA)26, and all patients had PDAI less than seven points. We showed increased LC3 II levels in the ileal pouch mucosa of UC patients by immunoblotting analysis. However, the decreased levels of beclin-1 and non-degraded p62 observed in Fig. 27. When p62 is accumulated in the cytoplasm, not binding to LC3, it signalizes that the macroautophagy may be deficient, even higher LC3 II levels is detected. Additionally, we did not verify altered CMA markers between the groups.In the present study, the purpose was to study patients without pouchitis to demonstrate if there was underlying modulation of autophagy markers in the ileal pouch mucosa, even in the absence of endoscopic and histological inflammation. For this, we applied the PDAI (Pouchitis Disease Activity Index)ATG5 and MAP1LC3A in FAP group, besides decreased Beclin-1 protein levels by immunoblotting analysis, and finally, decreased number of total LC3 and LC3/p62 co-localized cells verified by immunofluorescence compared to control group. To explain these findings, we decided to evaluate apoptosis related genes. Although there were no statistical differences in BCL2 expression, we found decreased BAX level, which encodes a pro-apoptotic protein, in the FAP group when compared to controls. Decreased apoptosis markers were already described in the ileal pouch mucosa of FAP patients, what may explain the tendency to low cell turn over and possible development of adenomas in this syndrome10. The decreased BAX levels in FAP group can lead to inhibition of autophagy related genes, as ATG5, and may explain the decreased levels of proteins related to autophagy, as we showed in Figs An interesting finding was also the detection of decreased autophagy markers in the ileal pouch mucosa of FAP patients when compared to controls. The transcriptional analysis showed fewer levels of 17. The relationship between TLR4 and immune system cells, mainly macrophages, frequently are associated to increased autophagy in those cells, especially after LPS treatment, which is a TLR4 agonist29. However, there is data from animal experimentation that addresses the role of autophagy in macrophage polarization30. Despite being in the context of obesity, it shows that in the obese animal, which exhibits increased TLR expression, autophagy is decreased in macrophages isolated from the peritoneum. They correlate this impaired autophagy with changes in the m1 and m2 macrophages profile, leading to inflammation. Therefore, in chronic stimuli such as obesity, autophagy is modulated in some tissues 35. There are still no studies in intestinal mucosa addressing the negative regulation of autophagy through TLR activation, which may explain our results in UC ileal pouch mucosa. The mechanisms by which this may happen are still grounds for investigation in other tissues36. In fact, we verified decreased of macroautophagy markers in the ileal pouch mucosa of both, UC and FAP, but the mechanisms to explain may be distinct, analyzing data already published. In FAP, decreased autophagy markers may be related to impaired apoptosis, otherwise in UC, may be mainly due to increased TLR activation.Conversely, the defect autophagy in UC pouches may be explained by other mechanism. Increased levels of TLR4 were already observed in UC ileal pouch mucosa, even in the absence of endoscopic inflammationTherefore, autophagy is relevant to the cell survive, since the accumulation of unfolded and abnormal proteins leads to activation of pro-inflammatory pathways. Those evidences of autophagy markers modulation may explain the prone to inflammation in the ileal pouch mucosa, mainly in UC. However, some limitations should be considered. First, this is a descriptive cross-sectional observational study and did not intend to correlate with the occurrence of future clinical manifestations. For this purpose, we would need a longitudinal study with a larger cohort and long-term follow-up. Second, we did not measure the autophagic flux directly. However, the ubiquitin-associated protein p62, which binds to LC3, was used to monitor autophagic flux indirectly, as we did in Fig. 38, which can help to find out new targets to ameliorate inflammation in the ileal pouch and even in UC. If these findings are confirmed in a longitudinal study, exploring the correlations with clinical settings, then this work provides novel insight into the complex pathogenesis of primary pouch inflammation.This subject deserves further studies and detailed mechanisms39. All the patients in this study had the absence of pouchitis, which was defined clinically, histology and endoscopically, according to the PDAI (Pouchitis Disease Activity Index)26 and the ileostomy closed for more than one year. In the control group (CTR Group), eight individuals with normal colonoscopy examination were included, with a median age of 62.5  years and 37.5% were female. Six biopsies from each patient were obtained from the terminal ileum (control), from the afferent limb and from the ileal pouch (UC and FAP).Mucosal biopsies were obtained from eight patients with non-inflamed IPAA after rectocolectomy for UC , and eight patients with non-inflamed IPAA after rectocolectomy for FAP . Biopsies of the intestinal mucosa of these patients were collected from the ileal pouch (UC and FAP Groups) and from the afferent limb of the ileal pouch (UC-AF and FAP-AF Groups). The postoperative follow-up was 186.5 (13–360) months. The reservoir design was of the “J” type, and the right colon vascular arcade was preserved as a supplementary blood supply to the terminal ileumThis study was approved by the Ethics Committee of the University of Campinas (UNICAMP), all patient signed the informed consent form, and was performed in accordance with the Declaration of Helsinki. The study was carried out at the University of Campinas, IBD Research Laboratory of the Surgery Department, and at the Laboratory of Cell Signaling of the Internal Medicine Department.Biopsies were embedded in paraffin blocks for histological analysis. Sections of 5 μm were cut and stained with hematoxylin and eosin dye. Photomicrographs were taken using a Zeiss Axiophot microscope and Cannon Power Shot G5 digital camera system . Fields of higher magnification (20X) were scanned and random fields were analysed. The histological part of the PDAI was performed.ATG16L1 (Hs00250530_m1), MAP1LC3A (Hs00261291_m1), BECN-1 (Hs00186838_m1), ATG5 (Hs00169468_m1), ULK1 (Hs00177504_m1), BCL2 (Hs00608023_m1), BAX (Hs00180269_m1) and GAPDH (NM_002046.3). RT-PCR amplification consisted of an initial denaturation step (50 °C for 2 min and 95 °C for 10 min), 40 cycles of denaturation (95 °C for 15 s), annealing (53 °C for 20 s) and extension (72 °C for 20 s), followed by a final incubation at 60 °C for 1 min. All measurements were normalized by the expression of GAPDH gene, considered as a stable housekeeping gene40.Biopsies from the mucosa of the terminal ileum and from the UC and FAP patients  were snap-frozen in liquid nitrogen and stored at −80 °C until use. Total RNA was extracted using Trizol (Invitrogen), according to the manufacturer’s instructions. RNA purity and concentration were determined by UV spectrophotometry at 260 nm. RNA was reverse transcribed using oligo (dT) primers and reverse transcriptase . The reaction mixture (20 µl) was incubated at 42 °C for 60 min, then for 10 min at 70 °C, and cooled on ice. RT-PCR was performed on resulting cDNA, using the manufacturer’s protocol, in a 25 µl reaction volume per capillary. Gene-specific primers (TaqManTM - Applied Biosystems™) were TM Real-Time PCR System (Applied Biosystems). The optimal concentration of cDNA and primers, as well as the maximum efficiency of amplification, were obtained by five-point, two-fold dilution curve analysis for each gene. Real-time data were analyzed using the Sequence Detector System 1.7 (Applied Biosystems). Reagents for Real-time PCR analysis were from Invitrogen  and Applied Biosystems .Real-time PCR analysis of gene expression was performed in a STEP ONETM BCA Protein Assay Kit. Catalog number 23225). Aliquots of the supernatants containing 50 μg total proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes and blotted with indicated antibodies as described in the results. Specific bands were labeled by a chemiluminescence reaction  and quantified by optical densitometry (Un-Scan-It Program). We have applied Ponceau staining to check equal loading of gels and membrane transfer (see supplementary information)42.Biopsies were snap-frozen in liquid nitrogen and stored at −80 °C until use. For total protein extract preparation, the fragments were homogenized in solubilizing buffer at 4 °C  with a Polytron PTA 20 S generator  operated at maximum speed for 30 sec. Insoluble material was removed by centrifugation (12000 rpm at 4 °C for 40 min). The protein concentration of the supernatants was determined by BCA method . HEPES, phenylmethylsulfonyl fluoride, aprotinin, dithiothreitol, Triton X-100, Tween 20, glycerol, and BSA (fraction V) were purchased from Sigma Chemical Co. . Nitrocellulose paper  and the reagents for chemoluminescence protein labeling in immunoblots were purchased from Amersham . The primary antibodies against Beclin (ab-16998), p62 (ab56416 or ab91526) were from AbCam, Cambridge, MA, USA; and LC3 (#2775) from Cell Signaling, Boston, MA, USA or LC3 (ab48394) from AbCam, Cambridge, MA, USA, and Hsc70 (sc7298) from Santa Cruz Biotechnology, Santa Cruz, CA, USA. The protein molecular weight was assessed by the PageRulerTM from Fermentas .43.Histological sections of 5μm were also performed for immunofluorescence procedures of samples included in paraffin blocks. The preparation of slides was performed (deparaffinization and hydration), followed by antigen retrieval. The tissue was incubated in primary antibody anti-LC3 (M115–3) from MBL Internacional Corporation Woburn, MA, USA with a dilution of 1:500 at 4 °C overnight and after with secondary antibody conjugated with Goat Anti-Mouse IgG H&L (Cy3®) preadsorbed (ab97035) from AbCam, Cambridge, MA, USA, with a dilution of 1:500 at room temperature for 1 hour. Three fields for each sample were captured and analyzed through the Leica confocal LAS AF Lite Version 2.6 Software . All cell type stained in the cytosol for Cy3® were considered positive for quantitative analysis, which was performed by ImageJ2, by percentage of LC3 per total tissue area in a panchromatic objective field of higher magnification 40X44. The tissue was incubated in primary antibody anti-LC3  and anti-p62  with a dilution of 1:500 at 4 °C overnight. The secondary antibody was Alexa Fluor® 488  in a dilution of 1:1000 at room temperature for 1 hour or Goat Anti-Mouse IgG H&L (Cy3®) preadsorbed , with a dilution of 1:500 at room temperature for 1 hour. DAPI was used for nuclear staining. Three fields for each sample were captured and analyzed through the Leica confocal LAS AF Lite Version 2.6 Software . All cell type stained in the cytosol for Alexa Fluor® 488 and Cy3® were considered positive for quantitative analysis, which was performed by ImageJ2, by the percentage of co-localized pixel volume in a panchromatic objective field of higher magnification 40X43.Indeed, we evaluated the autophagy by assessing LC3 and p62 co-localizationAll results were reported as means ± SEM. Data were analyzed by non-parametric Test, comparing all groups. The level of significance was set at p < 0.05.This study was approved by the Ethics Committee of the University of Campinas (UNICAMP), all patient signed the informed consent form, and was performed in accordance with the Declaration of Helsinki.Ethical approval by the ethic board of the University of Campinas (UNICAMP) and consent of patients are included in the original publications.All data generated or analyzed during this study are included in this article and in the additional files.Electrophoretic gels and blotsPouchitis Disease Activity Index (PDAI) of all patients included in the studyPonceau-S staining of the Western blot membranes used as loading controls