Metabolic changes in obese pregnant women, such as changes of plasma lipids beyond physiological levels, may subsequently affect fetal development in utero. These metabolic derangements may remain in the offspring and continue throughout life. The placenta mediates bidirectional exchange of nutrients between mother and fetus. The impact of prepregnancy obesity on placental transfer of lipids is still unknown.13C-labeled FFA was evaluated by ex vivo perfusion of human placentae as a function of prepregnancy obesity. Mathematical modeling complemented ex vivo results by providing FFA kinetic parameters.We aimed to examine materno-to-fetal free fatty acid (FFA) transfer by a combined experimental and modeling approach. Flux of 13C-FFA. Clearance of polyunsaturated 13C-docosahexaenoic acid (DHA) was most prominently affected. The use of the mathematical model revealed a lower tissue storage capacity for DHA in obese compared with lean placentae.Obesity was strongly associated with elevated materno-to-fetal transfer of applied Besides direct materno-to-fetal FFA transfer, placental mobilization accounts for the fetal FA supply. Together, with metabolic changes in the mother and an elevated materno-fetal FFA transfer shown in obesity, these changes suggest that they may be transmitted to the fetus, with yet unknown consequences. Consequently, increasing numbers of women in the reproductive age are also affected by this disease. Obesity is defined by an excess of body weight for height and is expressed by a body mass index (BMI) score of >30 kg/m2 . Obesity2 . Infants2 , 6 and s2 , a predi2 . Thus, iFacing childhood obesity as a serious public health problem, it would be necessary to disclose the origin of fetal overgrowth and understand the mechanisms resulting in childhood obesity.In this context, some studies report alterations in maternal plasma lipid profile in obese women such as elevated triglyceride levels , 10, or 13C-labeled FFA across placental tissue of obese and lean mothers. In addition, the mathematical model for placental FFA transfer mechanisms, previously published by Perazzolo et al. [The driving mechanisms underlying the transplacental FFA transfer need to be elucidated. The question arises if and how the placenta is affected and regulated under pathophysiological metabolic conditions, such as maternal obesity. In this study with a sole focus on the placenta, we aimed to examine alterations in placental FFA transfer in respect to maternal prepregnancy obesity. Therefore, we used the ex vivo dual placental perfusion approach to study direct transfer of o et al.  was appln = 8) or BMI ≥ 30 kg/m²  were included in this study. Gestational age of all women was >38 weeks and all delivered by elective cesarean section. According to the medical records, participating women were fasted overnight and did not take any medication. All women were normotensive. Gestational diabetes was excluded by oral glucose tolerance test between gestational week 24 and 28. The study followed the declaration of Helsinki was approved by the ethics committee of the Medical University of Graz (EK No. 24-529 ex 11/12) and written informed consent was signed by all study participants.Women either with a prepregnancy body mass index (BMI) < 25 kg/m²  in a 3:1 medium buffer ratio containing amoxicillin , dextran FP40 , 2 g/L glucose , and essential fatty acid free bovine serum albumin (BSA) .For all placental perfusion experiments Dulbecco´s modified eagle medium  was used. DMEM was mixed with Earl´s buffer  was prepared under a stream of argon to prevent fatty acid oxidation. The albumin to FFA molar ratio was 0.77, which is in the physiological range in the systemic circulation of a pregnant women [Perfusion medium containing nt women , 23. Pre2 and 5% CO2 during the experiment. The fetal circulation was always perfused with fresh medium (open circulation), which allowed us to distinctly follow each FA released from the placenta. A constant fetal artery inflow of 4 mL/min was set up by using a magnetic pump . Volume loss was monitored within the first 30 min, thereafter every hour at the fetal venous outflow port. Each cotyledon displayed at least 95% fetal flow recovery. The backflow pressure in the fetal vascular system was recorded by a micro catheter pressure sensor  inserted into the fetal arterial cannula. Experiments with vessel back pressure lower than 65 mbar were considered as successful [The ex vivo placental perfusion set up used in this study was adapted and technically extended based on the approach of Schneider et al. . Brieflyccessful .2, 20% O2, and 75% N2 by applying a gas exchange device. The maternal flow rate was kept at 8 mL/min. Within the first phase of perfusion experiment, antipyrine  was added to the maternal reservoir and perfusion was operated by open circulations. Antipyrine crosses the placental barrier by passive diffusion and serves as quality control substance for the size of the materno-to-fetal exchange area [Next, maternal circulation was set up by inserting three rounded needles into the intervillous space of the cotyledon. During the experiment, medium in maternal circulation was gassed with 5% COnge area .Samples from maternal artery and fetal vein outflow (perfusates) at the time points 0, 10, 20, and 30 min were collected, centrifuged at 3000 rpm at 4 °C for 10 min, and stored at −80 °C until antipyrine concentrations were determined.13C-FFA mix  and the system changed to closed circuit at the maternal side. Samples of both circuits were collected at 0, 10, 20, 30, 60, and 90 min, centrifuged and stored at −80 °C. Finally, maternal reservoir was switched to media containing 0.5% BSA with an open circuit configuration for a further 30 min. At the end of the experiment, wet weight of the perfused cotyledon was determined and is designated as perfused tissue mass (ptm) in the calculations and statistics section.Thereafter, the maternal reservoir was switched to perfusion medium containing 2), carbon dioxide (pCO2), pH, lactate production, and glucose consumption were measured by a blood gas analyzer . LabVIEW based recording software  was used to register data sets obtained by the blood gas analyzer, the magnetic pumps, and the pressure sensor.In order to assess viability of the perfused cotyledon, maternal and fetal perfusates were sampled  during the experiment every 30 min. Oxygen  in water mixed 1:1 with acetonitrile. Antipyrine peaks were detected at 255 nm and antipyrine concentrations were calculated according to the obtained standard curve.Antipyrine concentrations of maternal and fetal perfusates were determined by HPLC as described by Annola et al. . BrieflyAntipyrine fetal/materno ratio (FM ratio) was calculated and only perfusion experiments with a FM ratio ≥0.3 in 30 min were considered as a successful experiment and further processed.3/methanol (1/1). FFA were determined out of total lipid extracts, as previously published by Fuchs et al. [Total lipids, out of 1 mL perfusion medium, were extracted in methanol/tert-butyl methyl ether/water (1.5/5/1.25) as described by Matyash et al. . Sampless et al.  with som13C-labeled FFA carried 13C isotopes on each C-atom position. The labeling efficiencies were 97% atoms for DHA and 99% atoms for the other FFA, as specified by the manufacturers. In order to verify retention time and mass of the individual 13C-FFA, we performed a trial measurement of each single 13C-FFA by GC–MS, equipped with a quadrupole triple-axis detector , run on negative ion chemical ionization and selected-ion monitoring (SIM) mode (a list of masses given in Supplementary Table 13C-labeled FFA were detected by retention time (according to mixed FFA standards) and mass, after loss (m-1) in SIM scan mode. To quantify specific FFA, areas under the curve (AUC) were calculated by Mass Hunter . Ratios between internal standard (3.75 nmol/mL C15:0 FFA) and each individual FFA species were calculated. Additionally, FFA in perfusates containing 0.5% BSA  were determined, and obtained values (mainly low levels of 16:0 and 18:0) were subtracted from measured FFA concentration of each sample. No corrections were made for natural abundance of 13C since each C-atom carried an 13C-isotope of used labeled FFA and a defined mass shift depending on FFA chain length of +16 , +18 (oleic and linoleic acid), or +22 (DHA), respectively. We assumed equal response factors for all labeled and un-labeled FFA. All reagents were purchased form Sigma-Aldrich, Steinheim, Germany, if not other stated.All 13C-DHA uptake and transfer in comparison with the experimental data. By fitting the model to the data, four parameters were estimated: the maximum uptake flux parameter for the microvillous membrane (vMVM), the maximum delivery flux parameter for the basal membrane (vBM), the syncytiotrophoblast metabolic accumulation rate constant (kacc) and the syncytiotrophoblast metabolic release rate constant (krel). The parameters were determined separately for each of the lean and obese subjects, averaged for each group and tested for significant difference between groups. To test the predictive capabilities of the model, an additional independent experiment was performed. The first phase of the experiment was performed with 13C-FFA mix containing 0.3 µmol/L 13C-DHA . The parameters extracted from the first phase of the experiment (0–120 min) were then used for the prediction of the second phase from 120 min to 210 min.The mathematical model used here has been described in detail previously . BrieflyAntipyrine transfer was calculated as followed.Materno-to-fetal FFA transfer was normalized to mean perfused tissue mass  was calculated as followed.13C-FFA tissue uptake (%) was calculated as followed.13C-FFA absolute quantity in the maternal reservoir at time point 0 min; mr t90: 13C-FFA absolute quantity in the maternal reservoir at time point 90 min; fr t90: 13C-FFA absolute quantity in the fetal reservoir at time point 90 min13C-FFA clearance), non-parametric group comparison Mann–Whitney U test (model parameter for 13C-FFA and study population characteristics) and Kruskal–Wallis with post hoc Dunn´s test . P values <0.05 were considered significant. For statistical analysis GraphPad Prism 7.0  and IBM SPSS Statistics (version 23) was used.Data are presented as mean ± SD. Statistical analysis was performed by two-way ANOVA with Bonferroni post hoc test , oleic acid , linoleic acid , and docosahexaenoic acid  was examined by placental ex vivo perfusion. Stable isotope ion Fig. . The lon13C-DHA transfer data in our previously published mathematical model [13C-DHA presented in one representative plot , the vMVM was significantly (p < 0.01) higher than the vBM by over two orders of magnitude, implying that the effective permeability of the MVM was much greater than from the BM. Comparing the lean control and obese group there was a trend for a difference in the accumulation into metabolism parameter kacc for 13C-DHA, but it was not significant (p = 0.08). The estimated parameter krel for metabolic release of 13C-FFA during the experiment was zero in both cases meaning no relevant release back in the system after sequestration by the metabolic pool in the 90 min experimental period.Therefore, we implemented al model . The matlot Fig. . A robusReliability of obtained ex vivo perfusion experiments is shown by monitoring specific metabolic parameters in accordance with recommended practice  Supplem.n = 15) was classified according to maternal prepregnancy BMI into a lean  and obese group . The obese group was matched for maternal age and gestational age. Neither placental weight nor birth weight or ponderal index was significantly different between lean and obese groups  and obese (n = 6) women . Independent of the group, clearance for all investigated 13C-FFA occurred within the first 10 min, with a maximum between 10 and 20 min and an equilibrated 13C-FFA-clearance after 30–90 min of perfusion. A significant increase in 13C-FFA-clearance across the placental barrier was observed in the obese group  followed by 13C-PA, 13C-LA (20–35%) and lowest enrichment for 13C-DHA with 3–5%  was lower in obese compared to lean placentae, indicating a favorable direct transfer of DHA to the fetal circulation under this specific metabolic condition, which matches our experimental data.In this study we demonstrate that direct transfer of saturated and unsaturated FFA from the mother to the fetus is elevated in obese compared to lean women. However, maximum membrane flux parameters at the MVM and the BM obtained from in silico evaluation were similar between lean and obese placentae, suggesting that other variables than membrane fluxes may account for this effect. Interestingly, the rate constant for the metabolic DHA-accumulation in the model  FA, beside materno-to-fetal transfer, which overall cannot be fully accessed by in vivo studies. In our in silico model The low number of investigated subjects is certainly a methodological limitation of our study. Nevertheless, in comparison to other ex vivo perfusion studies, enrolled tissue perfusions are of high experimental quality  and the To conclude, the importance of this area is illustrated by our findings in which only a small proportion of FFA are directly transferred across the placenta in comparison to the FFA proportion released by the metabolic pool in the same time. The exogenous FFA transfer capacity in the syncytium reaches a maximum in a short time period implying that this process contributes only slightly to the materno-to-fetal FA supply. In contrast, tissue stored FA (mainly in phospholipids) are an efficiently and quickly available source of FA for the release to the fetus. Overall, our data suggest an expanded compartment mediated concept of FA uptake, storage, and delivery to the fetus in the human placenta Fig. . SpecifiSupplemental Data


In particular, the main scientific contribution of this paper is twofold: (i) to present a preliminary new pilot study investigating whether exercises based on the Nintendo Wii Fit® balance board included in a rehabilitation training program could improve physical abilities and Quality of Life (QoL) of patients compared to that of a conventional four-week rehabilitation training program; (ii) to discuss how such a rehabilitation training program could be adopted in the perspective of near future networks of medical IoT-based rehabilitation devices, interconnected with a hospital Cloud system for big data processing to improve patients’ therapies and support the scientific research about motor rehabilitation. Results demonstrate the advantages of our approach from both health and technological points of view.Multiple Sclerosis (MS) is a well-known, chronic demyelinating disease of the Central Nervous System (CNS) and one of the most common causes of disability in young adults. In this context, one of the major challenges in patients’ rehabilitation is to maintain the gained motor abilities in terms of functional independence. This could be partially obtained by applying new emerging and cutting-edge virtual/augmented reality and serious game technologies for a playful, noninvasive treatment that was demonstrated to be quite efficient and effective in enhancing the clinical status of patients and their (re)integration into society. Recently, Cloud computing and Internet of Things (IoT) emerged as technologies that can potentially revolutionize patients’ care. To achieve such a goal, a system that on one hand gathers patients’ clinical parameters through a network of medical IoT devices equipped with sensors and that, on the other hand, sends the collected data to a hospital Cloud for processing and analytics is required. In this paper, we assess the effectiveness of a Nintendo Wii Fit Multiple Sclerosis (MS) is a chronic demyelinating disease of the Central Nervous System (CNS) and the most common cause of disability in young adults . MS show® Plus Balance Board (WFBB) recently showed various benefits regarding patients’ motor rehabilitation. WFBB is an accessory for Nintendo Wii and Wii U video game consoles. It has the shape of a domestic body scale containing four pressure sensors that are used to measure the user’s center of balance. It communicates with the game console via Bluetooth. Several ad-hoc video games were developed so far.Despite advances in MS care, limited long-term benefits in contrasting disability burden and enhancing the social participation of patients were reached so far. In this context, one of the most important challenges in rehabilitation is to maintain gains obtained in “functional independence”. This could be partially achieved, by applying new emerging cutting-edge virtual/augmented reality and serious game technologies among others for a funny, noninvasive treatment that was demonstrated to be quite efficient and effective in enhancing the health status of MS patients and their reintegration into society. In particular, both aforementioned technologies provide artificial sensory feedback, allowing a patient to experience activities and events similar to those of real-life and to develop motor and cognitive abilities in an inclusive 3D environment that reproduces the real world, besides being inexpensive and easily configurable at the patient’s home. In this context, a system that, on one hand, collects patients’ health parameters through a network of medical Internet of Things (IoT) devices interacting with virtual/augmented reality and serious game computer programs aimed at motor rehabilitation, and on the other hand, sends gathered data to the hospital Cloud for analytics is required. In this context, the Nintendo Wii FitThis work has relevance for the development and evaluation of other and cheaper interventions that incorporate technology and personalized rehabilitation training to enhance physical activity levels in patients with MS. It was shown that Virtual Reality (VR) system is useful to improve balance capability in MS patients . NeverthTo present a pilot study investigating whether a novel exercise adopter in a WFBB rehabilitation training program may improve physical abilities and QoL of patients compared to that of a conventional four-week rehabilitation training program. To this end, we consider two groups of patients: the first one performing a rehabilitation training program using WFBB, which we named the Wii group, and the second one performing a Standard Rehabilitation (SR) training program, which we named the SR group. In particular, for each group, we considered both demographic and clinical characteristics. In addition, we also focused on balance performance, walking, and functional independence measures.To discuss how such a rehabilitation training program could be adopted in the perspective of near future networks of medical IoT-based rehabilitation devices interconnected with a hospital Cloud system for big health data processing. On the one hand, this would allow for the analysis of aggregated data by providing clues about how to improve patients’ therapies, and on the other hand, facilitating scientific research about motor rehabilitation.This paper, specifically, study the effectiveness of WFBB rehabilitation training program exercises in terms of physical abilities improvements of MS patients. Moreover, considering WFBB as an IoT medical device, we also evaluate its possible integration in a hospital Cloud context. Specifically, the main scientific contribution of this paper is two-fold:In particular, in our preliminary pilot study, we screened 42 MS patients consecutively, referring to MS Centre at IRCCS “Bonino Pulejo” of Messina . Functional Independence Measure (FIM), Berg Balance Scale (BBS), and Timed 25-Foot Walk test (T25-FWT) were considered at baseline (T0) and at the end of treatment (T1). In our pilot study, we enrolled 42 MS patients: 20 MS patients (47.6%) were assigned to the Wii group and 22 MS patients (52.3%) to SR group. Specifically, we experienced that at T1, both groups improved in FIM total score motor functions subitems. BBS score improved only in the Wii group, proving that Nintendo Wii Fit could be considered as a helpful tool to standard rehabilitation treatment on moderate MS patients with disabilities. In the end, to motivate the feasibility of our approach from a technological point of view in the perspective of near-future scenarios, we highlight the feasibility of interconnecting a network of the considered medical IoT devices to a hospital Cloud system. Security aspects are out of the scope of this paper.The remainder of the paper is organized as follows. Material and methods are discussed in Nowadays, in response to this need, the number of easy and economic rehabilitation training approaches able to regularly involve MS patients is rapidly growing. Recently, the use of interactive video games became increasingly popular with different targets of intervention ,8,12. A The balance board, shown in Sixty-five pwMS referring to our MS Centre at IRCCS “Bonino Pulejo” of Messina, from July 2016 to November 2017, were screened for possible participation in the study. PwMS were enrolled according to the revised McDonald diagnostic criteria were  and gave® balance platform. Patients enrolled in the trial were not allowed to change or start any medication for the entire study period, except for steroids required to treat MS exacerbation. A new MS exacerbation was considered as a dropout treatment. PwMS fulfilling the inclusion criteria were enrolled by a skilled clinician, who also administered consecutive evaluations prior to group allocation. Patients were enrolled in order of recruitment and were assessed at baseline (T0) and at the end of the rehabilitation program (T1) by the clinician who did not share any clinical information with the treating physician and physiotherapists.Moreover, a weight limit of 140 kg was used due to the restriction stated by the producer of the Nintendo Wii Fit® for four weeks, with a total of 20 sessions for Wii group. Participants in SR group received five sessions a week of 60 min for four weeks, individually tailored to meet patient needs, consisting of physiotherapy  and occupational therapy involving fatigue management and functional retraining in tasks of daily living. This study was an open, single-blind, control group prospective study. Patients were randomized in order of recruitment. Those who met all eligibility criteria underwent study assessments were assigned either to the Nintendo Wii Fit balance program (Wii group) or a standard rehabilitation program (SR group). As described elsewhere , we adop® is a video exercise game containing balance games, yoga poses, strength training, and aerobics. Games in the Nintendo Wii Fit Plus® targeting balance were selected by a physiotherapist and ranked to standardize the progression of exercises. During the training, the patient stands on a Wii Balance Board detecting the center of balance. The exercises focus on controlling the games using the patient’s center of balance. The first session started with the games categorized as easier . During all sessions, the PTs encouraged the participants to get more difficult games . The Nintendo Wii Fit PlusThe following outcome measures were collected at each scheduled visit  by two neurologists blinded to the patient’s treatment: EDSS, Functional Independence Measure (FIM), Berg Balance Scale (BBS), and Timed 25-Foot Walk test (25-FWT). The FIM is an 18-item instrument measuring a person’s level of disability in terms of the burden of care. Each item is rated from 1  to 7 (completely independent). Three independent FIM scores were generated by summing item scores: a total score , a motor score , and a cognitive score . The validity of the FIM for use in inpatient and outpatient rehabilitation settings is well established, and its reliability is good ,22. BBS p values less than 0.05 in either direction were considered significant. Analyses were carried out by using the STATA software version 11.2.Given the exploratory nature of this study, no sample size analysis was performed. Data are presented as means and standard deviations. Well-balancing of the two treatment groups were tested by using the Mann–Whitney U for continuous variables. Descriptive statistics were used to ensure comparability of scores between groups at baseline, to describe performance at each phase and to test whether the assumptions for the use of parametric statistics were met. If the assumptions for F or t-tests are violated, equivalent nonparametric statistics will be used. The percentage of patients who improved in the FIM scores of 3 or 5 points  was calcA total of 42 pwMS, 16 women (38.1%), mean age 50.2 ± 10.2 years, mean disease duration 14.2 ± 4.6 years, and mean EDSS 4.8 ± 1.2 (range 4.0–6.0), matched the required criteria. Twenty (47.6%) pwMS were assigned to the Wii group and 22 (52.3%) to SR group. One pwMS of the Wii group dropped out due to side effects during treatment (low back pain) and was excluded from the analyses.p-value. The p-value, also called the confirmatory hypothesis test, was used in our pilot study to understand the validity of the Nintendo Wii Fit Plus® balance based rehabilitation program. It indicates the probability to obtain a result equal or more extreme than the one observed supposed that the null hypothesis t from unknown distribution T. Then the p-value p is what the prior probability would be of observing a test-statistic value at least as “extreme” as t if null hypothesis T is symmetric about zero, we considered the p-value can as in Equation (Equation  for a onEquation  for a twEquation .(1)p=P indicates strong evidence against the A small p-value (>0.05) indicates weak evidence against the A large p-values very close to the cutoff (0.05) are considered to be marginal.A p-values of 0.07 and 0.04 respectively indicate that FIM and FIMmf are different at T0.In our case, ® Balance program group; SR: standard rehabilitation group; BBS: Berg Balance Scale; F25WT: Timed 25-Foot Walk test; FIM: Functional Independence Measure; FIMmf: Functional Independence Measure motor function; FIMcf: Functional Independence Measure cognitive function; ns: not significant. Moreover, both groups’ FIMtot and FIMmf scores, transfers, and locomotion subitems significantly improved between T0 and T1. Neither accidental falls nor any other side effect was reported by patients while performing Nintendo-Wii training.Results depicted in ® balance rehabilitation program showed improvements compared to the four-week standard rehabilitation program. According to ∘, we can observe that the Nintendo Wii Fit® balance rehabilitation program is valid as well as the four-week standard rehabilitation program, but we can observe also a relevant reduction in the BBS score obtained with the Wii rehabilitation program compared to the four-week standard rehabilitation one. Therefore, as the main result, Wii group showed a significant improvement in terms of BBS scores between time points. In both groups’ FIM and FIMmf scores, transfers, and locomotion sub-items, there were significant improvements between T0 and T1. Neither accidental falls nor any other side effect was reported by patients while performing Nintendo-Wii training.In particular, we analyzed *: Wii T0 versus Wii T1; ∘: SR T0 versus SR T1; §: Wii T0 vs. SR T0. Specifically, according to * we can notice that BBS, FIM and FIMmf values showed significant improvement in the Wii group so that Nintendo Wii Fit® Wii® Balance Board® on physical abilities and QoL in pwMS with mild to moderate physical disability (EDSS score < 6.5). After treatment, the Wii group demonstrated superior improvement in balance as per BBS, whilst both groups achieved a significant improvement in activities of daily living and in mobility measured by FIM (FIMtot and FIMmf). The benefits of the Wii exercise training found in our study, especially on balance and mobility outcome measures , could be due to the constant visual information feedback, allowing pwMS to shift the weight on the balance board using the visual display and according to the performed score. Thus, the better scores obtained by the Wii group patients in BBS, could be partially explained by the improvement of proprioceptive pathway [In this study, we investigated the effects of a visual-feedback physical training with a Nintendo pathway ,30,31. A pathway .Falls in MS are considered to be linked with increased postural sway  and asso® training does not improve balance outcome [Moreover, based on mirror neurons theory, watching one’s movements while executing an action could facilitate motor relearning in neurorehabilitation . Indeed, outcome , our res outcome ,38. Inde outcome .In UK pwMS, some authors showed that a home-based use of the Wii offers benefits in terms of its capability to support remote communication. These promising data need to be confirmed in larger sample studies to better evaluate the device efficacy and cost-effectiveness .A recent meta-analysis showed that balance training was identified as being the most recurrent topic in the scientific literature and appears to be the goal of Wii Fit Training. Moreover, Wii Fit was employed to prevent falls and to induce functional improvements in seniors or in subjects presenting neurodegenerative diseases. Concerning balance training, the same review revealed that Wii Fit interventions had a positive impact on BBS and Time Up and Go test (TUG). In line with our study, Wii Fit interventions appear very safe, with very low levels of injuries being reported . Our stuMore homogeneous samples are needed to show whether and to what extent sex may affect the patient’s outcomes. Fourth, the lack of a more complex randomization in selecting the two study groups possibly biased the findings.Finally, we do not have a follow-up evaluation of the maintenance of the visual-feedback exercises’ effects over time. Despite these limitations, our results show that rehabilitation exercises based on an interactive visual-feedback platform by Nintendo Wii Balance Board could be effective in improving balance and physical disabilities in MS patients with mild to moderate physical disabilities. There is not a consensus about the time schedule of treatment, depending above all on the methodological difference of intervention . In our In this paper, we demonstrated that Nintendo Wii Fit has the potential to positively affect exercise training, and it may offer an alternative tool to traditional therapy. This work has relevance for the development and evaluation of other interventions that incorporate technology and personalized rehabilitation training to enhance physical activity levels in chronic degenerative neurological disorders, such as Multiple Sclerosis (MS).Future studies should also explore who may benefit the most from having access to Nintendo Wii Fit-like tools and whether people with more severe balance and mobility problems can safely use Wii Fit without supervision. In this regard, we plan to study big data analytics strategies based on Machine Learning (ML) algorithms ,41,42 aiThe objective of this scientific work was not to promote the Nintendo Wii Fit but to demonstrate that a serious gaming approach can be as effective as a standard rehabilitation one. For the sake of clarity, the latest version of the Nintendo gaming console does not support the balance board. However, home-brew developers allowed the board to be unofficially supported by the Linux kernel 3.7. In addition, such a kernel opens toward the possibility to make Nintendo Wii Fit a remote programmable IoT medical device aimed at tele-rehabilitation interconnected with a hospital Cloud for remote clinical monitoring of patients in the perspective of near future tele-rehabilitation services. Tele-rehabilitation recently emerged as an effective tool for providing rehabilitation care to patients directly in their own home, while also increasing clinical outcomes, positively enhancing patients’ Quality of Life (QoL), and fostering their reintegration into society. Moreover, it allows the hospital personnel to supervise the rehabilitation process of a patient when he/she is at his/her home. Therefore, this work aims at paving the way toward low-cost intelligent IoT Cloud-based Tele-Rehabilitation as a Service (TRaaS)  adopting


Ischemic heart disease is one of the leading causes of deaths worldwide. A major hindrance to resolving this challenge lies in the mammalian hearts inability to regenerate after injury. In contrast, zebrafish retain a regenerative capacity of the heart throughout their lifetimes. Apex resection (AR) is a popular zebrafish model for studying heart regeneration, and entails resecting 10–20% of the heart in the apex region, whereafter the regeneration process is monitored until the heart is fully regenerated within 60 days. Despite this popularity, video tutorials describing this technique in detail are lacking. In this paper we visualize and describe the entire AR procedure including anaesthesia, surgery, and recovery. In addition, we show that the concentration and duration of anaesthesia are important parameters to consider, to balance sufficient levels of sedation and minimizing mortality. Moreover, we provide examples of how zebrafish heart regeneration can be assessed both in 2D (immunohistochemistry of heart sections) and 3D . In summary, this paper aims to aid beginners in establishing and conducting the AR model in their laboratory, but also to spur further interest in improving the model and its evaluation. In response to injury such as myocardial infarction (MI), the mammalian heart may compensate by changing its form and function, however the damaged myocardium exhibits limited regeneration with respect to the replenishment of dead cardiomyocytes . InsteadIn contrast to mammals, the adult zebrafish heart has a marked capacity for self-restoration after injury ,4. This The process of heart regeneration after AR may be divided into different steps. Initially after AR a fibrin clot is formed, whereafter a cascade of immune responses and transient scarring occurs. The peri-/epicardium also starts remodelling ,9,10, anThus, AR in zebrafish is a valuable model for studying cardiomyocyte proliferation and heart regeneration in order to understand and potentially unlock the regenerative response in mammals. The huge interest in the AR model is reflected in our recent systematic overview of AR zebrafish studies where many different research groups globally have improved the understanding of heart regeneration through cardiomyocyte proliferation . HoweverHere we provide a complete protocol for AR in zebrafish, including video footage showing the entire resection procedure and a sham operation. Furthermore, we elaborate on crucial steps such as anaesthesia that increase zebrafish survival and the control design required for interstudy reproducibility. We also suggest and test a new approach to performing subsequent analysis of heart outgrowth taking the whole 3D structure of the heart into consideration. We thus hope this article will ease entry into using the AR in zebrafish as a model for studying heart regeneration.Anaesthesia is not only crucial during surgery, but it also has a major effect on zebrafish revival and survival and thus data outcome . During Thus, based on zebrafish survival and the ability to maintain stage 3 anaesthesia ) may be dissected, embedded and sliced for further analysis via Masson Trichrome staining  zebrafish, which specifically express GFP under the control of the cardiac myosin light chain (myl7) 2 promoter in cardiomyocytes of the myocardium, although the remaining tissue was visualized through DAPI staining .tg(myl7:EGFP) [Outbred wild type (AB) or l7:EGFP)  zebrafis2 small aquariums/tanks for sedation and recoverySmall fishing netMicroscope Large plastic spoonPasteur pipetteFine curved forceps Micro scissors Sponge with pre-cut slit big enough to hold the zebrafishPro-ophta, pre-cut on the middle in a 30° angle/cotton swabTissues for removing (zebrafish) scales from surgical instrumentsTricaine solutions in water from your system/aquarium for anaesthesiaWater from your system/aquarium2O, the pH was thereafter adjusted to 7.4 with 1M Tris-base  in ddH2O. Stock solution was kept at −20 °C for a shorter time until use.Stock solutions of Tricaine were established by dissolving Ethyl 3-aminobenzoate methanesulfonate  in ddHThe zebrafish was placed in either 0.02%, 0.03%, or 0.04% Tricaine in system water for either 120, 150, or 180 s to reach stage 3 anaesthesia for the initial part of the study. For the remaining part of the study 0.03% Tricaine for 120 s was used. Hereafter the zebrafish was tested for loss of tail reflex before it was placed in the pre-cut slit in the sponge .The sedated zebrafish, in the pre-cut slit in the holding sponge, were placed underneath a stereomicroscope  with the head of the zebrafish pointing towards the non-dominant hand of the person doing the surgery. Afterwards the sham or AR procedure was performed as shown in At the endpoint zebrafish were euthanized by placing them in ice-cold water for 3 min whereafter the hearts were removed, embedded in Tissue-Tek  before they were snap-frozen in dry ice-cooled isopentane. Hearts were sectioned in 10 µm thick sections and kept at –80 °C before staining.In brief, sections were fixed in 4% neutral buffered formalin (NBF), rinsed in running tap water, stained in Mayer haematoxylin , rinsed in running tap water and stained in eosin , before they were dehydrated in increasing concentrations of ethanol and mounted with Pertex .Masson’s Trichrome staining was performed as recommended by the manufacturer. Briefly the sections were acclimatized, fixed in Bouin’s solution , incubated with Weigerts Iron Haematoxylin , and then incubated in the Masson’s Trichrome Staining Kit .Sections were fixed at 10 min in 4% NBF (Sigma-Aldrich cat. no.: HT501128), rinsed in tris-buffered saline (TBS), permeabilized in TBS containing 0.5% Triton-X 100 , rinsed in TBS, blocked for non-specific binding in TBS containing 2% BSA  and incubated for 2 h at room temperature with anti-MYH1  and 488-conjugated WGA . Following incubation, sections were rinsed in TBS and incubated with a secondary antibody  before a final rinse in TBS and mounted with antifade mounting medium with DAPI .2O and ScaleCUBIC-1 reagent [Zebrafish were euthanized as above and hearts were carefully dissected, whereafter they were placed in 2–3 drops of phosphate buffered saline (PBS). The atrium was then removed, before the hearts were placed in individual wells in a 24-well plate containing 4% NBF for 24 h at 4 °C. On the following day the hearts were incubated in 1:2 dilution of ddH reagent  for 15 m reagent . If the t-test (p ≤ 0.05. Data are presented as mean ± SD.Comparisons were performed using one- and two-way ANOVA with Tukey’s multiple post-test  or usingt-test  (GraphPa


A noteworthy frame of the literature has maintained the idea that communication in the classroom is dominant in language education, and in the process of language learning, teachers as an important figure may apply several ways to develop interpersonal relationships and social manners, such as teacher immediacy that has been established to support affective and cognitive learning in instructional settings. Therefore, this theoretical review tries to systematically refocus on the existing literature about teacher immediacy and its types, such as non-verbal and verbal, and their significant connections with affective and cognitive education. To this end, this review focuses on social behavior to review the eminence of teacher immediacy in the classroom and unquestionably exemplify their relationship with affective and cognitive learning. As a final fact, this review has been intended to consider the prevailing literature about teacher behavior, and suggestions and recommendations have been presented correspondingly for language teaching stakeholders in the educational setting. Universally, teachers are noted as the main resources in any educational system, and the communication between teachers and their learners has been regarded as an essential part of the instructional cycle of language learning  is not clear all the time. The educational correspondence centers around the role of correspondence in all educating and training settings, in addition to settings across the entire life expectancy  is its consideration of the relational parts of educational correspondence (Johnson et al., Immediacy has been an exploratory concept in the instructional literature and has been related to numerous preferred education consequences, containing affective learning, teacher credibility, the enthusiasm of learners, and cognitive learning (Pogue and Ahyun, In addition, immediacy in the practice of teachers during teaching correspondence identifies with a positive effect and expanded cognitive learning and more positive evaluation of learners made by teachers Bicki, . It simiBesides the abovementioned clarifications demonstrating the significance of the verbal and non-verbal immediacy of teachers, numerous researchers (e.g., Sutiyatno, Based on the review of the literature and in line with the function of teacher immediacy in an instructive setting, many studies have pursued to scrutinize the relationship between these relational manners, namely, immediacy and learner-associated issues such as educational commitment, participation, willingness to take part in classes, cognitive and affective learning, fulfillment, and enthusiasm (Gholamrezaee and Ghanizadeh, Although teacher immediacy has been investigated in various settings and territories and on several interindividual and intraindividual features, as Liu  noted, iBroadly referred to as a characterizing attribute for effective learning in both customary and online learning conditions, communication is at the core of the learning experience Swan, . BesidesTeacher immediacy is characterized as the verbal and non-verbal signals diminishing the teacher-learner physical and/or mental distance (Estepp and Roberts, A series of agencies that can ascertain distinctive behaviors such as smiling, verbal emotion, and a situation of the body has been established by immediacy scholars, and these types of immediacy variables can be taught to teachers to increase the learner-teacher rapport, student enthusiasm, commitment, and cognitive learning (Velez and Cano, Non-verbal immediacy is assumed as extra-language communications guided by teachers to learners intended at creating spiritually positive interactions. They are associated with the affective realm of communication. The non-verbal clues are intermediated through such active teaching behaviors as proper eye contact, the usage of motions, movement about the class, vocal variability, and the practice of humor (Chesebro and McCroskey, This review set forth several implications in language education. Among the manners that teachers are inclined to establish in courses is verbal/non-verbal immediacy that has considerable impacts on the affective and cognitive learning of learners. The suggestions and recommendations of this study become more vivacious in assisting teachers to upturn the commitment and achievement of learners through emerging their immediacy. Successful immediacy courses provide teachers with a noble opportunity to increase more information about the prominence of communication and then encourage their verbal and non-verbal immediacy. By promoting immediacy, constructing behaviors in teachers, the learning of learners, stress tolerance, motivation, satisfaction, and self-image would expand (Chesebro and McCroskey, The review serves to show support for teacher immediacy as an important element of learner success, that is, the goal of language learning. Immediacy in the class assists learners to form a constructive opinion of the ability, credibility, and thoughtful approach of teachers Rocca, . These pBased on the review, it was proved that more learner awareness of immediacy causes more motivation in learners and that better learner motivation brings about more view of cognitive learning, besides higher effective ones. The role of teacher immediacy behaviors is assured in constructing cognitive learning (Frymier and Houser, In addition, when a teacher is highly immediate, it is obvious that they have a positively better influence on their learner motivation, which refers to the affective learning that can be defined as the encouraging principles that learners stick on to the behaviors of teachers in the classroom and it is proved that there is a positive relationship between the immediacy and affective learning of teachers (Frymier and Houser, Similarly, this review is beneficial to those supervisors and administrators who wish to select the best teachers for their language school or institute since the information of teachers, classroom administration capability, and other important teacher features; for example, the capability to create an immediate rapport with learners should be deliberated as a new prominent issue. Furthermore, aiming to aid learners struggling with negative issues such as stress, boredom, burnout, low self-confidence, or other factors (Seifalian and Derakhshan, As the purpose of education is to look for routes to encourage students, possibly teacher training teachers should study the upshot of immediacy, so all the stakeholders of learning progression should be stimulated to assess and contemplate their verbal and non-verbal communication approaches. If teachers propose to assist an ideal classroom situation, they must direct caring and considerate communication messages to all learners. Training of teachers is becoming gradually mutual in different contexts (Gibbs and Coffey, Some lacunas were also found in the existing literature, which needs to be highlighted. First and foremost, there is a dearth of research to investigate in what way the social interaction practices of teachers can be heightened and as it is concluded that based on the review of the related literature, teacher immediacy is deemed to be a crucial element, so future studies, particularly experimental ones, should be carried out to present instructional treatment to a group of teachers on a specific facet of relational interactions to notice how receiving intervention can encourage the social behavior of teachers. Second, most of the previous studies in this area were carried out in general education (Bozkaya and Aydin, The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.