Nurses and surgeons must identify and handle specialized instruments with high temporal and spatial precision. It is crucial that they are trained effectively. Traditional training methods include supervised practices and text-based study, which may expose patients to undue risk during practice procedures and lack motor/haptic training respectively. Tablet-based simulations have been proposed to mediate some of these limitations. We implemented a learning task that simulates surgical instrumentation nomenclature encountered by novice perioperative nurses. Learning was assessed following training in three distinct conditions: tablet-based simulations, text-based study, and real-world practice. Immediately following a 30-minute training period, instrument identification was performed with comparable accuracy and response times following tablet-based versus text-based training, with both being inferior to real-world practice. Following a week without practice, response times were equivalent between real-world and tablet-based practice. While tablet-based training does not achieve equivalent results in instrument identification accuracy as real-world practice, more practice repetitions in simulated environments may help reduce performance decline. This project has established a technological framework to assess how we can implement simulated educational environments in a maximally beneficial manner. Designing training protocols that effectively prepare people for rare or dangerous situations is not a trivial task. Skills and knowledge required by perioperative nurses cannot always be obtained through repetition-based practice, which can expose patients to unnecessary risk and is not feasible for less common procedures. Perioperative nursing education, like the majority of other nursing domains, often depends on text/image-based memorization of surgical instruments as a method of self-directed learning outside the operating room –4. This seven empirical studies assessing the validity of tablet-based education, yet there is a growing market push for their implementation in the classroom [Recent advancement of digitally-simulated training environments  provide one promising avenue to expand healthcare training. In addition to reducing the risk to patient populations, and increasing the opportunities for practice of rare procedures, digitally-simulated training environments allow greater accessibility to training than real-world practice, while maintaining some of the interactive components not present during text-based study. In particular, tablet-based training can afford trainees an opportunity to practice procedures easily at home, during commutes, or even as a refresher immediately before performing an actual task. Yet, while simulated environments have been implemented with great success in multiple training situations—semi-realistic multisensory simulations have been in use for nearly fifty years see –10, the lassroom –16. ThisA critical consideration for the development and implementation of tablet-based simulations is that they do not demand the haptic interaction required of real-world tool use, and thus may not train the same neurocognitive mechanisms . When inGiven the increasing push for the integration of tablet-based simulation training into healthcare education, it is imperative that we assess its educational value relative to our existing training methods. In the current study, we directly compare real-world performance of an interactive procedure following pseudo-surgical instrumentation training  through 1) tablet-based simulation, 2) text-based studying, or 3) real-world practice. In order to accurately assess the current methodologies available in the field, we modeled training protocols in both self-guided training environments on educational tools that are currently commercially available and marketed for the training of perioperative nurses , 15. ThuForty-six subjects were recruited to participate in the experiment from undergraduate courses in the Department of Psychology at the University of British Columbia. All participant gave written informed consent prior to beginning the experiment. Notably, this population displays a high degree of similarity across sex, ethnicity, and age with students in an undergraduate healthcare provider degree program , 24. DueSurgical Instruments: A pocket guide [In an effort to emulate the types of instruments encountered by perioperative nurses in the operating room, 105 unique handheld items were chosen as “pseudo-surgical” instruments. Pseudo-surgical instruments were chosen to ensure the safety of all researchers and participants, and ranged in size from 30–219 mm along the longest axis. To ensure that no prior knowledge of the item could influence results, each instrument was given a novel name, modelled after the structure of those in Wells’ et guide . As is tSurgical Instruments: A pocket guide [TM surgical instrumentation platform for tablets, PeriopSIM [To assist the training condition in a manner analogous to current text-based learning protocols, a study guide of text and photo based flash cards was developed for each of the three pseudo-surgical instrument sets  modeled et guide . This steriopSIM .To optimize power and eliminate confounds due to individual differences between participants we used a within-subject design. Participants were trained to recognize each of the three pseudo-surgical instrumentation sets in a distinct training environment: text-based study, tablet-based game-play, and real-world item/surgeon interaction, described below. To ensure that no order or practice effects could emerge in the group data, the assignment of instrumentation-set to training environment was randomized between participants, as was the order in which each participant would experience each learning environment. Participants were afforded 30 minutes in each training environment to learn the names, and where applicable, the procedural order of the pseudo-surgical instruments.In the text-based condition, participants were given a set of flash cards corresponding to one of the pseudo-surgical instrumentation sets to assist instrument identity learning. They were instructed to read through and memorize all the names of all pseudo-surgical instruments in the time provided, with the explicit understanding that they would be tested on their ability to identify the items following training. To reflect realistic study environments, no additional instructions were given until after the training period; the participant was entirely responsible for sustained attention to the study material in this training environment. Furthermore, no information about the order in which the tools would be requested  or handling and manipulation of the items, virtual or otherwise, was provided during text-based study, consistent with information given in currently available instrumentation study guides .The tablet-based simulation training environments employed in this study were modeled after a commercially available application used to train perioperative nurses on surgical instrumentation . After bIn the real-world condition, instrumentation training using real-world interaction was conducted to directly emulate the task demands of a perioperative nurse in the OR. Following a specific predefined procedural order, the experimenter requested 21 of the 35 presented instruments, each of which were to be placed in the experimenter’s open hand. After placing the instrument into the palm of the experimenter, the participant received verbal feedback on whether the instrument was correct. Correct instruments would be taken by the experimenter and placed on an empty workspace while incorrect instruments were taken back by the participant and placed back on the ‘prep table’ . For theTo assess the efficacy of each training environment for real-world procedural learning, after each half-hour training period, participants completed an instrument identification task with tangible pseudo-surgical instruments emulating objects and protocols experienced in real-world operating rooms. Here, participants were presented with instruments identical to those on which they had been trained  and were required to pass individual instruments to the experimenter upon request as quickly and as accurately as possible. If the response was incorrect, participants were required to continue passing instruments until the correct object was identified. Experimenter feedback during the test phase was limited to a verbal indication of accuracy .effectiveness. Additional ratings of the tablet- and text-based training methods were collected assessing the participant’s motivation to use each training task, as well as their belief in the likelihood that they would continue to use each method in a classroom setting. All ratings  were collected on a visual analog scale ranging from “not at all” to “very” that was divided into 100 points for subsequent analysis.Finally, to assess long-term retention of instrument names and procedural ordering for each trained instrumentation set, participants were invited back into the laboratory one week after initial training. While the order of assessment was identical to the training order from week one, participants were not given any time for re-training to familiarize themselves with the instruments prior to this testing session. All test sessions were video recorded for further data processing. Following all experimental tasks , participants completed a subjective evaluation of the training protocols, rating each method for its perceived The behavioural measures of interest from both week one and week two testing sessions were accuracy of instrument identification , average number of errors , and response time to instrument hand-off . All dependent measures from the real-world training and test sessions were extracted from the video recordings. Each video session was viewed by two experimenters (video raters) with explicit knowledge of all instruments, protocols and request orders required. Importantly, while raters had knowledge of the experimental design, and the items contained in each set, they were blind to the specific instrument set-training protocol pairing during data extraction for all test and retest video sessions. All video raters were trained to identify the onset of a trial , the end of a trial , and the procedural order and naming system for all instruments to assess accuracy and errors. A custom semi-automated video segmentation program was used to assist video data extraction which allowed for manual tagging of trial onset and offset, as well as trial outcome  for automated calculation of response time and for session averaging. As an interclass correlation analysis performed on the test procedure video sessions  found excellent internal consistency between video raters , data from Rater 1 was used for all subsequent analyses.pMM values presented have been corrected for multiple comparisons using a Bonferroni correction. Additionally, a Greenhouse-Geisser correction has been performed to address any instances where an assumption of sphericity for these data could not be made. Non-significant effects were further investigated through a series of two one-sample t-tests (TOSTs) for statistical equivalence [pTOST values presented have been corrected to control for family-wise error rates [pTOST < 0.05 indicative of statistical equivalency between conditions [As each participant completed training (and subsequent testing) in all three training protocols in a fully within-subject design, a 3  X 2 (time: immediate vs delayed retention test) repeated-measures (rm) ANOVA was conducted on each of the outcome measures using IBM-SPSS . Main efivalence –29 usingivalence . All pTOor rates , with pTnditions .To investigate more dynamic learning processes in both the real-world and tablet-based training environments, and gain additional insight into the strengths and weaknesses of each, task performance during training sessions was analysed using non-parametric local regression (LOESS). In order to assess the course of learning over time in each training protocol, we examined a measure of peak performance achieved in each learning environment as well as the latency to that peak by both task repetition and time spent in the learning environment. Instrument identification accuracy data acquired during the learning periods were divided into ten distinct bins for both procedural repetition (1 bin = 1 task repetition), and training time (1 bin = 3 minutes), resulting in four distinct conditions . To determine the accuracy and latency of peak performance in each condition, LOESS curves  were fit = 6.224, p = 0.003). Real-world training (pMM = 0.010 and pMM = 0.032 respectively). No differences were identified between tablet- and text-based training (pMM = 0.732). An equivalence test marginally supported this null result, indicating trend-level equivalence for these conditions (pTOST = 0.058).Following completion of all experimental procedures , participants rated all three training environments for their perceived effectiveness. A 1 X 3  repeated-measures ANOVA performed on these data identified a significant main effect of training (FMMs > 0.277) and marginal support for equivalence .Ratings of the tablet- and text-based training methods assessing participants’ motivation to study in each training task, and their likelihood of implementing each training tool in a classroom setting, revealed no differences between these training conditions , response time , and number of errors  . Each me = 6.375, p = 0.003) and time  = 122.699, p < 0.001) were observed (pMM = 0.049) or text-based (pMM = 0.006) training. No differences in accuracy were noted between tablet and text-based training (pMM = 1.00), a null finding supported by follow-up equivalency testing (pTOST = 0.018). In addition, a general decrease in performance was observed from immediate to delayed test sessions (pMM < 0.001). No training-type X time interaction was identified in the accuracy data  = 0.043, p = 0.958) with follow-up equivalency testing confirming equivalency between tablet- and text-based training at both immediate and delayed testing , but not for all other contrasts . This pattern of findings was analogous to that observed for the main effect of training type.For accuracy, main effects of both training-type  = 36.752, p < 0.001). Additionally, a trend-level effect of time emerged  = 4.110, p = 0.05). Shorter response times during testing were observed following real-world and tablet-based training compared to text-based study (pMM < 0.001 and p = 0.045 respectively). Shorter response times were also observed following real-world training compared to tablet-based training (pMM < 0.001). Notably, a training-type X time interaction was identified  = 4.574, p = 0.014; pMM = 0.011) but not during delayed testing . Both interactive training protocols  produced shorter response times compared to text-based study during both immediate and delayed test sessions .For response time, there was a main effect of training-type  = 5.072, p = 0.009) and time  = 41.111, p < 0.001). Fewer errors were observed following real-world compared to text-based study (pMM = 0.034) while marginally fewer errors were made following tablet-based training compared to text-based study (pMM = 0.077), with no support of equivalency observed in TOST analyses for this contrast (pTOST = 0.504). No differences were observed between real-world and tablet-based training . As with accuracy, a decline in overall test performance was observed from immediate to delayed testing (p < 0.001). A significant training-type X time interaction  = 6.712, p = 0.006; pMM = 0.001 and pMM = 0.039 respectively. There was also trend-level indication of marginally fewer errors for real-world practice compared to tablet simulations upon immediate testing (pMM = 0.05) consistent with no evidence of equivalency (pTOST = 0.632). No significant differences were identified during the delayed retention test . This null finding for the delayed retention testing was substantiated by equivalency tests demonstrating equivalency between the text-based and real-world training conditions (pTOST = 0.006), and trend-level support for equivalency of tablet-based training with both traditional training methods (pTOST = 0.056 for both comparisons).Analysis of error rates identified significant main effects of both training-type . A series of analyses fit non-parametric local regression (LOESS) curves to the accuracy data as a function repetition/training time. By conducting this analysis in data divided by both the number of procedural repetitions and the amount of time spent undergoing training, the relative strengths and limitations of each protocol can be assessed. From these analyses, the point at which maximum accuracy was obtained in the fitted data, indicative of the transition from a knowledge acquisition task to skilled practice of the task. Together, these analyses contextualize observed differences in test session performance between conditions in the amount of time spent in skilled practice, as well as highlight any potential ceiling effects for each training technique.task repetitions, LOESS curves were constructed for data from both the tablet vs. the real-world training conditions. During tablet-based training, a performance plateau was observed after 6.06 procedural repetitions  = 6.017, p > 0.001).In order to examine how many repetitions it took to reach peak participant accuracy across time, LOESS curves were constructed for data from the training period segregated into ten 3-minute bins. During tablet-based training, peak performance was obtained at 19.2 minutes , and one emerging method . Performance using each of these methods was compared to performance following real-world practice. Notably, participants were significantly more accurate when identifying the instruments following real-world practice compared to both tablet-based simulation and text-based study. Immediate retention tests demonstrated a response time advantage for real-world training compared to simulation training, suggesting a more efficient nurse-surgeon interaction could result. Interestingly, however, this effect disappeared at test points one-week later, suggesting that tablet-based training may be less susceptible to performance decline than real-world training. To investigate the process of learning in each environment, additional analyses identified the level and latency of peak performance for each interactive training method. While the less accurate following tablet-based simulation than following real-world practice. However, in healthcare education, real-world practice is often not a viable method of training. Many procedures are either too resource intensive to be conducted solely as practice [e.g., A substantial gap exists between marketing claims and scientific assessment of tablet-based training for healthcare professionals . In the ce e.g., , 35 or tce e.g., –38 givence e.g., . Thus, ece e.g., , 41 or tce e.g., –4. In thce e.g., . It is ace e.g., , 19 and ce e.g., . Overallvisual experience, it does not alter the haptic experience of the instrument; an additional benefit of real-world item handling well evidenced to enhance learning [A potential avenue to overcome the performance deficit observed for 2D training approaches is the inclusion of interactive 3D visualizations for object and procedural learning –46. The learning , 52. AddBeyond accuracy, an additional marker of perioperative training effectiveness is the efficiency of nurse-surgeon interactions . The curWhile current results indicate instrument interaction efficiency following real-world practice and tablet-based training was similar overall, one notable difference with respect to interaction efficiency emerged. During procedures tested immediately following the training session, response times were faster for real-world practice than tablet training (with both outperforming text-based study); however, when tested following a one-week latency period the observed difference between real-world and tablet-based training disappeared, and these conditions were statistically equivalent. The reduction in training advantage for real-world practice from immediate to delayed retention may stem from the increase in the number of repetitions performed during the tablet-based simulations following the attainment of a performance plateau. Practice repetitions following knowledge acquisition is well evidenced to strengthen learning of both semantic information , 59 and single task compared to text-based study, the latter method may show enhanced generalizability of learned performance, though additional work is needed to assess this claim.A notable difference between simulation training and real-world practice compared with text-based study, which may contribute to differences in interaction efficiency observed in the current work, is the specificity of items called and the ordering of items presented. Both tablet-based learning and real-world practice act as guided training for a single procedure, with presented instruments catered to those required for each specific task. In contrast, text-based study guides often include additional items beyond the required scope of a single surgical procedure. As a consequence, while training through tablet-based simulations may lead to improved performance on a The two interactive training protocols further differ from text-based study in the extent to which multisensory information is presented during training. Beyond the haptic and motor feedback discussed above, in the tablet and real-world conditions, instrument requests during training are made through auditory prompts. To contrast, text-based study of the items required no external or motor feedback, with all novel information regarding the instruments limited to the words and images in the study guides provided. As noted above with respect to haptic information, the inclusion of this additional modality in the real-world training condition may reinforce instrument-specific knowledge , 68 by cInterestingly, current results may also suggest a potential ceiling for tablet-based simulation training protocols (in their current form) that is fundamentally lower than that of real-world practice for the clinically relevant skills. Even with the additional training repetitions performed in tablet simulations, identification accuracy lagged behind that of real-world practiced items in the current work. This effect may be mediated, in part, by the level of fidelity observed in the simulated environment –72. AddiTablet-based simulated training media are being increasingly incorporated into the education curriculum for healthcare providers, yet substantial evidence for their educational efficacy for complex ordered procedures is still lacking. In the present study, the accuracy of pseudo-surgical instrumentation identification, modeled after perioperative nursing procedures, was compared following training through tablet-based simulations, text-based study, and real-world practice. The current results suggest notable benefits from tablet-based simulations, including enhanced object-oriented responding compared to text-based study, and increased practice repetitions compared to real-world practice over equivalent training time. Furthermore, this work demonstrated that knowledge acquired from tablet-simulations can be transferred to real-world tasks both immediately and at a delay, but that it may not replicate the full interactive benefits of practicing with real-world items immediately prior to the tested procedure. Overall, this study highlights the potential of simulated training methods in healthcare education, but suggests that caution should be used in their implementation.S1 Table(DOCX)Click here for additional data file.


Single-cell RNA-seq (scRNA-seq) is a powerful tool for analyzing heterogeneous and functionally diverse cell population. Visualizing scRNA-seq data can help us effectively extract meaningful biological information and identify novel cell subtypes. Currently, the most popular methods for scRNA-seq visualization are principal component analysis (PCA) and t-distributed stochastic neighbor embedding (t-SNE). While PCA is an unsupervised dimension reduction technique, t-SNE incorporates cluster information into pairwise probability, and then maximizes the Kullback–Leibler divergence. Uniform Manifold Approximation and Projection (UMAP) is another recently developed visualization method similar to t-SNE. However, one limitation with UMAP and t-SNE is that they can only capture the local structure of the data, the global structure of the data is not faithfully preserved. In this manuscript, we propose a semisupervised principal component analysis (ssPCA) approach for scRNA-seq visualization. The proposed approach incorporates cluster-labels into dimension reduction and discovers principal components that maximize both data variance and cluster dependence. ssPCA must have cluster-labels as its input. Therefore, it is most useful for visualizing clusters from a scRNA-seq clustering software. Our experiments with simulation and real scRNA-seq data demonstrate that ssPCA is able to preserve both local and global structures of the data, and uncover the transition and progressions in the data, if they exist. In addition, ssPCA is convex and has a global optimal solution. It is also robust and computationally efficient, making it viable for scRNA-seq cluster visualization. Single-cell RNA sequencing (scRNA-seq) technology enables the measurement of cell-to-cell expression variability of thousands to hundreds of thousands of genes simultaneously, and provides a powerful approach for the quantitative characterization of cell types based on high-throughput transcriptome profiles. A full characterization of the transcriptional landscape of individual cells has enormous potential for both biological and clinical applications. However, characterization and identification of cell types require robust and efficient computational methods. Particularly, visualization is crucial for humans interactively processing and interpreting the heterogeneous and high-dimensional scRNA-seq data, because humans rely on their astonishing cognitive abilities to detect visual structures, such as clusters and outliers. Hence, high-dimensional data must be projected (embedded) into a 2D or 3D space with dimension reduction (DR) techniques for visualization.Among different methods proposed for scRNA-seq visualization, the two popular ones are principal component analysis (PCA) and t-distributed Stochastic Neighbor Embedding (t-SNE). PCA projects the high-dimensional scRNA-seq data into the linearly orthogonal low-dimensional vector space thorough variance maximization. Due to its efficiency and conceptual simplicity, PCA has been widely applied to scRNA-seq data dimension reduction and visualization ,3,4,5,6.On the other hand, t-distributed Stochastic Neighbor Embedding (t-SNE) is the most commonly used nonlinear dimension reduction method for cell subtype visualization. t-SNE transforms cell similarities into probability, and incorporates the information of cell clusters into visualization through redefining the probability. It determines the spatial cell maps in low dimension through minimizing the Kullback–Leibler divergence . t-SNE rUniform Manifold Approximation and Projection (UMAP) is a new scRNA-seq visualization software ,15. SimiIn this paper, we propose a semisupervised principal component analysis (ssPCA) method for dimension reduction and visualization of scRNA-seq data. ssPCA is a generalization of PCA and it is nonlinear. It seeks to find principal components that maximize both data variance and cluster dependence, so that cluster (subtype) labels are integrated into scRNA-seq visualization seamlessly. While maximizing the total data variance preserves the global structure in the data, maximizing the cluster dependence captures the local data structure within each cluster. In addition, ssPCA can be solved in a closed-form and does not suffer from the high computational complexity of iterative optimization procedures. Therefore, it is computationally efficient.We propose a semisupervised principal component analysis (ssPCA) approach for the visualization of scRNA-seq data. The proposed approach is based on nonlinear kernel (or similarity) matrices and incorporates cluster labels into the visualization. This optimization problem is involved in two components, including the unsupervised maximization of total variance and the supervised maximization of cluster dependence with the Hilbert–Schmidt Independence Criterion (HSIC). Overall, the proposed approach is, therefore, a semisupervised learning problem. In addition, because cluster labels are required for ssPCA visualization, ssPCA is most useful for the visualization of clusters detected from other scRNA-seq clustering tools. Note that we can easily construct a kernel matrix for ssPCA if there is no similarity matrix available from other clustering software (which is rare).X with n cells and p genes, and a X and cell subtype (cluster) vector X and H is the centering matrix and Given a ariables . With a  studies ,18.X onto a new space, then a kernel is defined as Our proposed ssPCA method for cluster visualization aims not only to maximize HSIC, but also to preserve the data variance. Kernel measures the inner-product in a new feature space. In general, a linear or nonlinear function ustering . The simc cell clusters in i belongs to cluster j, and is 0 otherwise. Then, we define the kernel The kernel for cluster (subtype) labels With the kernel matrices The linear kernel in the low-dimensional space is W is the eigenvectors of k largest eigenvalues. The projection The second term is the unsupervised total variance maximization in the projected space. The variance covariance matrix X, Y, and the low-dimensional projection will be discovered and visualized with ssPCA. If only cluster labels are available (which is rare), we can construct our own kernel Algorithm 1:The ssPCA algorithmX, similarity (kernel) matrix   1. Recode Y, calculate X if   2. Find W, the eigenvectors of k largest eigenvalues.   3. Project to low-dimension with  Given the cluster labels ssPCA is most suitable for visualizing cell clusters detected from other scRNA-seq software, since cluster labels are required for ssPCA visualization. Both the cluster labels and the similarity (distance) matrix used for cell clustering are usually available. In such a case, the similarity matrix is treated as the kernel  . PHATE wOur computational evaluations with four real datasets are based on the cell similarity matrix and clustering labels from three popular scRNA-seq software including SIMLR , SoptSC SIMLR [SIMLR  is denotWhile ssPCA, t-SNE, and UMAP can separate the clusters in different datasets with the similarity matrices from SIMLR, ssPCA performs better in recovering the global cluster structures relative to one another. For instance, with the Buettner scRNA-seq data, ssPCA demonstrates that cluster 2 (green) is close to cluster 1 (red), and far away from cluster 3 (blue), while the relative locations (distances) of the three clusters with t-SNE and UMAP might not mean anything. In addition, with the Pollen data, t-SNE and UMAP display all clusters uniformly on the plane, while ssPCA shows that cluster 7 (light-blue) is far away from the rest clusters. Finally, with the Usoskin data, cluster 3 (light blue) is displayed as several pieces with t-SNE and UMAP, but it is visualized as one entity and adjacent to cluster 1 (red) and cluster 2 (green) with ssPCA. This is reasonable. As demonstrated with the simulation data, ssPCA preserves both global and local data structures with principal component projection, while t-SNE and UMAP only capture the local data structure, and the global structure is not fully preserved. The main reason is that both t-SNE and UMAP are based on the nearest neighbor graph technique which only optimizes the data points close to each other.SoptSC [SoptSC  is a recVisualization of the high-dimensional RNA-seq data is critical for detecting cell subpopulations and revealing biological insights. To date, there are only a few tools including PCA, t-SNE, UMAP, and PHATE available for dimension-reduction and scRNA-seq data visualization. ssPCA provides another viable tool for scRNA-seq visualization and has its advantages. More specifically, PCA projects high-dimensional data into a low-dimensional space through eigenvalue decomposition. PCA is a linear projection method, and it mainly captures the global structure of the data, as demonstrated in t-SNE and UMAP are the two popular local graph algorithms for scRNA-seq data visualization. While t-SNE is the most popular tool currently used in the literature, UMAP produces somewhat similar output with increased speed. However, one key disadvantage with t-SNE and UMAP is that they only preserve the local neighborhood structure in the data. The global structures are not correctly visualized. As demonstrated in PHATE  is a recThe only hyperparameter One interesting finding with the Pollen data is that ssPCA identifies 3 distinct clusters with different kernel (similarity) matrices from different software packages, while 11 clusters were discovered in their original study. Whether this discrepancy is from the novel finding with ssPCA or from the limitation of ssPCA is not known. More investigations are required in the near future. Finally, although the same 4 scRNA-seq datasets are used for We propose a semisupervised principal component method (ssPCA) with HSIC maximization for cell subtype visualization. ssPCA optimizes both local cluster dependence and global principal component projection. It has an analytical solution, and is robust with respect to different values of hyperparameter


Nano-scaled structures, wireless sensing, wearable devices, and wireless communications systems are anticipated to support the development of new next-generation technologies in the near future. Exponential rise in future Radio-Frequency (RF) sensing systems have demonstrated its applications in areas such as wearable consumer electronics, remote healthcare monitoring, wireless implants, and smart buildings. In this paper, we propose a novel, non-wearable, device-free, privacy-preserving Wi-Fi imaging-based occupancy detection system for future smart buildings. The proposed system is developed using off-the-shelf non-wearable devices such as Wi-Fi router, network interface card, and an omnidirectional antenna for future body centric communication. The core idea is to detect presence of person along its activities of daily living without deploying a device on person’s body. The Wi-Fi signals received using non-wearable devices are converted into time–frequency scalograms. The occupancy is detected by classifying the scalogram images using an auto-encoder neural network. In addition to occupancy detection, the deep neural network also identifies the activity performed by the occupant. Moreover, a novel encryption algorithm using Chirikov and Intertwining map-based is also proposed to encrypt the scalogram images. This feature enables secure storage of scalogram images in a database for future analysis. The classification accuracy of the proposed scheme is 91.1%. Nano-scaled structures, wireless sensing, wearable devices, and wireless communications systems are anticipated to support the development of new next-generation technologies in near future. The exponential rise in future Radio-Frequency (RF) sensing systems has demonstrated its applications in areas such as wearable consumer electronics, remote healthcare monitoring, wireless implants, and smart buildings.The advances of low-cost small electronic devices, wireless sensing systems, real-time monitoring, and the Internet of Things (IoT) has played the role of a catalyst that has primarily expanded the horizon of these technologies. In this context, we introduce a privacy-preserving, device-free occupancy real-time occupancy monitoring system for energy optimizing in smart buildings.The smart built environment essentially refers to the man-made environment providing the setting for different human activities including smart cities, large scale buildings, and beyond. It consumes a significant amount of electricity around the world and keeps on increasing day by day . In thisFor example, optimized services provided at shopping malls, hotels, restaurants, and transportation stations can be allotted to improve customer care service. Crowd density control and counting can enhance the indoor evacuation process in emergency cases. Hence, the design of a low-cost, robust, accurate, safe, and secure occupancy monitoring system is of utmost importance that can count the number of occupants while keeping the privacy preserved. Presently, the majority of the occupancy monitoring and detection systems use infrared sensors. However, these systems present a huge number of false alarms when occupants are moving slowly. Furthermore, state-of-the-art occupancy detectors estimate the total number of people in the indoor environment as well. Camera-based systems  are one The Received Signal Strength Indicator (RSSI) obtained from Wi-Fi signals has been used to estimate occupants. The disadvantage of RSSI is that it suffers from coarse grain resolution, is highly susceptible to noise, and presents fluctuations, making it an infeasible solution for occupancy detection. On the other hand, channel state information extracted from Wi-Fi signals provide multiple frequency sub-channels, where one or more frequency carriers can be used to detect a person in an indoor environment. In this paper, we propose a novel low-cost, easily deployable, device-free, privacy-preserving Wi-Fi technology-based occupancy detection system using commercially available, off-the-shelf wireless devices such as Wi-Fi router, network interface card. and an omnidirectional antenna. In this research, two independent modules were implemented: (i) deep learning for occupancy detection; and (ii) chaos-based scalogram image encryption.Numerous researchers have introduced different RF sensing-based occupancy monitoring systems over the past few years ,13,14,15Body centric communication systems need specific privacy and security protocol to ensure the privacy, confidentiality, and data integrity of a person’s information. A supporting body centric communication infrastructure should deploy particular security measures that ensure all aforementioned features . A comprThe received signal strength indicator and the Channel State Information (CSI) extracted are two of the most widely used types of information, applicable in various areas of wireless communication. RSSI measurements only present simple signal strength in the context of signal propagation, which is why this information is inadequate for occupancy monitoring due to its unstable nature. Zou et al.  presenteThe Wi-Fi technology powered by IEEE 802.11 a/a/ac utilizes Orthogonal Frequency Division Multiplexing (OFDM), effectively encountering the multipath propagation effect caused in indoor settings due to physical obstructions such as walls, ceiling, floor, etc. In OFDM, the frequency carrier is divided into multiple  subcarriers where the data stream ,26. The A low-cost, off-the-shelf small wireless device, such as Atheros ar5b225, can be used to extract the CSI information from OFDM subcarriers. Open-source network interface card wireless device drivers reveal the measured CSI for all subcarriers, providing fine-resolution wireless channel information, comprising wireless medium characteristics including multipath fading, reflection, refraction, and shadowing effect.ith subcarrier that carries complex information and is denoted as follows:ith frequency channel, respectively. The phase information extracted from CSI data for single frequency channel ith frequency subcarrier and mi is the frequency channel number. The internal noise of the network interface card and external noise is denoted as Z, and Let (1) Wi-Fi router is deployed closer to the receiving antenna.(2) We avoid using other wireless systems nearby common sources of interference, such as power cables, microwave ovens, fluorescent lights, and cordless phones.(3) We minimize the total number of live devices using the same RF band.(4) Deploying the Wi-Fi router close to the receiving antenna enables the receiver to get the maximum power, thus other nearby devices operating on same frequency do not affect the proposed system.The channel station information recorded using the network interface card connected with the Wi-Fi router is discussed in this section. The CSI data are recorded from the data stream obtained from the Internet Control Messages Protocol (ICMP) data. In principle, the overall recorded CSI data are the same when compared to the ICMP data stream. Nonetheless, it was noticed that marginally fewer CSI data packets were transmitted than ICMP packets. To synchronize the frequency of the recorded data packets, we applied linear transformation function on CSI data recorded in raw form. In theory, the OFDM subcarriers that Wi-Fi signal exploit should carry independent data. However, in practice, the neighboring frequency subchannels carry similar information most of the time. To detect occupancy and obtain separate information from each subcarrier, we applied principal component analysis (PCA) to get independent datasets for each observation. The CSI data stream can be easily incorporated into several independent principle components.We conducted extensive experiments and trials in a hall, as described in t and scale s, as described in Equation  on 1000 CSI data packets. The energy density function Equation .(3)Cc(tEquation :(4)Cc.8.B into three separate matrices, i.e, Reshape 9.Slightly change the initial conditions by adding a value 10.Repeat Steps 6–9 11.C.Combine each channel, The proposed scheme was tested on pick up object scalogram. The original and encrypted scalogram of pick up object are shown in We implemented the autoencoder model in a MATLAB tool, where training, validation, and testing were performed on scalograms generated from Wi-Fi signals. The neural network was trained for 200 epochs with a minibatch size of 90. The performance accuracy of the proposed system was obtained by dividing 20% of the training datasets as the validation set, and the model was evaluated after the completion of each iteration.The adaptive moment estimation technique was used for optimizing the given datasets during the pre-training stage for a fine-tuning learning rate of 0.002. The grid search method was used during the process where optimized values for width and depth overcoming the overfitting problem are shown in This paper presents the application of next-generation body centric communication towards occupancy monitoring that can provide an effective and privacy preserved solution for reducing the energy consumption and carbon footprint. In the proposed model, non-wearable wireless devices such as Wi-Fi router, network interface card, and omnidirectional antennas operating at 2.4 GHz, are used to acquire data. Continuous wavelet transform is applied to the acquired RF signals to obtain Wi-Fi images that are processed using a deep learning algorithm to detect occupancy and to further perform occupancy classification. An unsupervised auto-encoder algorithm is used to classify images corresponding to different human activities of the occupants present in the area of interest. The performance of the proposed system was evaluated in terms of percentage accuracy, providing an overall accuracy of more than 91%. Lightweight image encryption techniques using multi-chaotic maps are proposed to encrypt the scalogram images. This feature enables secure storage of scalogram images for future usage such as improving training and testing accuracy of deep neural model.


To elucidate the populations and conditions where screen-based sedentary behaviors (SB) and internalizing symptoms are coupled, this review synthesized the evidence for factors that may moderate the associations between screen-based SB, depressive symptoms, and anxiety symptoms among youth.Two independent researchers conducted a systematic literature search of the Medline, psycINFO, and Scopus electronic databases in late 2018 for observational studies assessing moderators of the association between screen-based SB and depressive and anxiety symptoms. Studies among children and adolescents were eligible if moderation was assessed by statistical test (interaction) or stratification; and a narrative synthesis of eligible studies was conducted in accordance with PRISMA guidelines.Seventy empirical studies  of 13 different moderating variables of screen-based SB-internalizing symptom associations met the eligibility criteria. Of these, 40 studies were of depressive symptoms, 2 were of anxiety symptoms, and 28 studies assessed symptoms of both. The most consistent evidence of moderation was for screen-type, such that TV viewing was not as strongly associated with internalizing symptoms compared to other forms of screen-based SB. There was also inconsistent evidence for physical activity buffering screen-based SB-internalizing symptom associations and for female sex amplifying screen-based SB-internalizing symptom associations. In general, the body of evidence for anxiety symptoms was more limited than that for depressive symptoms, and were therefore more inconsistent.Screen-type, physical activity, and sex may influence the magnitude of screen-based SB-internalizing symptom coupling; highlighting potential sources of heterogeneity of screen-based SB-internalizing symptom associations. Additional studies aimed at understanding potential mechanistic explanations for the above moderators are needed prior to the development of tailored intervention strategies designed to decouple screen-based SB and internalizing symptoms among youth. Screen-based sedentary behaviors (SB) such as television viewing and computer use have become ubiquitous in children and adolescents, in part because of the increasing availability of youth-friendly digital entertainment . Even moParalleling the rising rates of screen-based SB, the adolescent developmental period is also a high-risk period for the onset of internalizing symptoms and disorders . Recent Previous systematic reviews and meta-analyses have provided evidence that screen-based SB and internalizing symptoms may be associated with one another among youth –14. It iA current gap in the literature is the lack of information on potential moderators of the association between screen-based SB and internalizing symptoms that may be contributing to inconsistencies in previous findings. For example, psychosocial factors associated with puberty may lead to the moderation of screen-based SB-internalizing symptom associations by sex such that screen-based SB may be related to depressive symptoms among girls, but not boys . For girTaken together, the goal of this review was to address this literature gap by providing a comprehensive overview and systematically integrate the results of observational studies assessing moderators of screen-based SB-depressive and anxiety symptom associations. Thus, the aims of this paper were to 1) summarize the moderating variables of the association between self-reported screen-based SB  and depression and anxiety (or depressive and anxiety symptoms) among clinical and nonclinical samples of youth, 2) discuss potential mechanisms of moderation based on the consistency of evidence for particular moderators, and 3) pose suggestions for future research aimed at informing tailored intervention strategies among vulnerable populations.The review was performed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines .Until December 2018, JZ and KI separately queried the Medline/PubMed, psycINFO, and Scopus electronic databases using the following search terms: (sedentary OR sitting OR screen time OR media) AND  AND (child* OR adolescents OR adolescence OR youth). Titles and abstracts were first evaluated. For those identified as possibly relevant, full texts were retrieved and assessed for inclusion in this review. Reference lists of relevant review articles were also examined.Peer-reviewed articles reporting on human studies published in English were included. Studies were included if a screen-based SB-anxiety symptom or screen-based SB-depressive symptom association was estimated (or if group differences were tested), and if moderation was investigated by statistically testing interaction terms or by stratifying analyses  without a formal statistical test for interaction. In addition, studies that reported estimates of associations in a common sample separately across different variants of a construct  were also considered for evidence of moderation. Studies that reported on composite variables comprised of both, screen-based and non-screen-based SB were not included in the current review because screen-based SB-specific associations could not be parsed out. Further, although they may provide specific mechanistic explanations for screen-based SB-internalizing symptom associations, studies that reported on variants within the screen-based SB construct beyond screen-type, such as specific content, were considered outside of the scope of this review. Studies utilizing any measure of anxiety or depressive symptoms, not limited to a clinical diagnosis, and including screening questions were eligible. Disagreement between JZ and KI regarding articles to be included in the review was addressed with discussion until a consensus was met.protective association between screen-based SB and internalizing symptoms among a particular group and a null association among another group, it was considered as evidence of a buffering effect. For example, if screen-based SB were protective against internalizing symptoms among girls and unrelated to internalizing symptoms among boys, then female sex was considered a buffer. Strata of the same variable that showed comparable associations between screen-based SB and internalizing symptoms were considered null. For example, if analyses were stratified by sex, and both sedentary boys and girls experienced greater odds of depressive symptoms , then sex as a moderator was considered null. Percent difference in effect estimates between strata and sample size were also considered when determining the strength of the evidence for moderation on occasions where overlapping confidence intervals were present or when confidence intervals were not provided.Pertinent information from each of the identified full-text studies was extracted, including authors, year of publication, sample parameters (sample size and mean age), study design, measures of screen-based SB and internalizing symptoms, moderators tested, and study results (parameter estimates) which also included significance of these moderators (amplifier/buffer/null). An amplifier was a stratum of a variable that strengthened the association between screen-based SB and internalizing symptoms, while buffers weakened the strength of the observed relationships. On occasions when a moderator resulted in a significantly Consistent with previous systematic reviews , 20, if JZ and KI independently rated the methodological quality of each study using a modified version of an eight-component rating scale , consistFigure n = 20) provided evidence that sex was a significant moderator. Fourteen  of the 20 studies provided evidence for female sex as an amplifier of screen-based SB-depressive symptom associations [inconsistent evidence (46.5% of studies) of moderation of screen-based SB depressive symptom associations by sex; however, among the studies that found sex differences, 70.0% of studies identified female sex as an amplifier of associations.Forty-three studies tested sex as a moderator when investigating depressive symptom-screen-based SB associations, and of these, approximately half  investigated sex as a moderator of the relationship between screen-based SB and anxiety symptoms. Of the 20 studies, 14 studies did not provide evidence for moderation by sex [null (30.0% of studies).Fewer studies  for age as a moderator of screen-based SB-depressive symptom associations.A cross-sectional study in 8029 students aged 10 to16 years old found that compared to the younger children in the sample, adolescents who met screen-time recommendations were less likely to develop depressive symptoms . Howeverinconsistent evidence for moderation by age.Only one investigation assessed age as a moderator of screen-based SB-anxiety symptom associations; this longitudinal and bi-directional study of over 500 young children did not provide evidence for age as a moderator (null interaction term) . Becauseinconsistent evidence for moderation by cultural factors.McHale et al. found that an aspect of Mexican cultural orientation, parental educational value, modified the relationship between TV viewing and depressive symptoms; symptoms of depression and TV viewing were only related to one another among children with fathers with low educational value in this cross-sectional study of 469 youth . Additioinconsistent evidence (50.0% of studies) for PA as a moderator of screen-based SB-depressive symptom associations; however, among the studies with significant findings, 100.0% identified PA as a buffer of associations.Three cross-sectional studies among adolescents found that moderate-to-vigorous PA or vigorous PA buffered screen-based SB-depressive symptom associations , 51, 55.inconsistent evidence for PA as a moderator of screen-based SB-anxiety symptom associations.Only three studies assessed PA as a moderator of the relationship between screen-based SB and symptoms of anxiety. Two found that vigorous PA weakened screen-based SB-anxiety symptom associations , 51, whiinconsistent evidence for peer/social factors as moderators of screen-based SB-internalizing symptom associations.Twelve studies investigated potential moderators that do not fall into the above categories. Three studies assessed how different peer/social factors  may influence the strength of the relationship between screen-based SB and internalizing symptoms. For example, a recent longitudinal investigation revealed a three-way interaction between screen-based SB, sex, and social context in adolescents; girls who played videogames with peers were more likely to experience symptoms of anxiety, while boys who played videogames with peers were less likely to experience symptoms of anxiety . In anotinconsistent evidence for parental factors moderating screen-based SB-internalizing symptom associations due to the limited number of available studies.Three studies investigated parental factors as potential moderators. Parental communication and parental involvement were not significant moderators of screen-based SB-internalizing symptom associations in studies among children and adolescents , 66. Lasnull (0.0% of studies) for socioeconomic status as a moderator of screen-based SB-internalizing symptom associations.Four studies assessed potential moderation by a number of indicators and proxies for socioeconomic status including parental education, household income, and neighborhood-level socioeconomic characteristics, with each study providing no evidence of moderation , 66, 72.Four studies looked at additional variables as potential moderators that do not fall into the above subcategories. Kim and Ahn found that neighborhood divorce rate weakened the positive longitudinal relationship between screen-based SB and depressive symptoms, whereas population size and education were not moderators . In one significant evidence (68.6% of studies) of moderation by screen-type. Below, these individual studies are reviewed in more detail by screen-type.Screen-based SB was conceptualized differently across studies, which may have influenced the strength of the observed associations with internalizing symptoms. Screen-based SB was typically defined as TV viewing, computer use, and videogame playing; resulting in investigators stratifying analyses by screen-type. Twenty-four studies provided evidence for differential associations by screen-type  when looking at screen-based SB in relation to depressive or anxiety symptoms; while eleven studies indicated that type of screen-based SB did not influence the strength of the observed associations. Taken together, there is n = 16, two among clinical samples) found that TV viewing was more weakly related to depressive symptoms compared to at least one other form of screen-based SB, such as computer use and videogame playing [More than half of the studies  of studies found that compared to at least one other type of screen-based SB, TV viewing was not as strongly related to symptoms of anxiety [Thirteen studies  examined TV viewing-anxiety symptom associations among youth. A majority  found that computer use was positively associated with symptoms of anxiety, and that computer use was more strongly related to symptoms of anxiety than other forms of screen-based SB , 86, 87;Eleven of 21 studies  concluded that videogames were more strongly related to depressive symptoms compared to at least one other form of screen-based SB , 86, 87;Seven of twelve studies  found a stronger association between videogame playing and symptoms of anxiety as compared to another form of screen-based SB , 86, 87.In addition to the three main types of screen-based SB , there were some investigations of other screen-types in relation to internalizing symptoms. Six studies assessed how texting/mobile phone use related to depressive symptoms; and of these, only one study found that texting/mobile phone use was more strongly related to depressive symptoms compared to other screen-types longitudinally . AlternaOnly four studies investigated the relationship between other types of screen-based SB and symptoms of anxiety. Specifically, two studies found that texting was more weakly related to symptoms of anxiety as compared to at least one other form of screen-based SB , 69. On Sensitivity analyses of the six studies with high methodological quality were conducted to gain a better understanding of findings across studies that were rated as the least likely to be subject to biases; five were of depressive symptoms , 73, 90 Sex was tested as a moderator in three studies, with mixed findings , 73, 90.The influence of screen-type on screen-based SB-depressive symptom associations was investigated in two studies, and differences by screen-type emerged in both , 73. In In a longitudinal study of middle-schoolers, Kim et al. found that among various neighborhood characteristics, including divorce rate, population size, and education level, only neighborhood divorce rate influenced the strength of the association between videogame playing and subsequent depressive symptoms . Neighboinconsistent evidence for moderating effects by sex, age, cultural characteristics, PA, peer factors , and parental factors . Further, there is no evidence of moderation by socioeconomic status.The aim of this paper was to summarize the evidence for potential moderators of screen-based SB and internalizing symptom associations to better understand the heterogeneity of previous study findings, identify at-risk populations, and to pose future research directions for the field. Screen-type most consistently influenced the strength of the association between screen-based SB and internalizing symptoms. However, the evidence for symptoms of anxiety, specifically, is more limited, and therefore findings should be interpreted with caution. Currently, the literature provides Our results indicate that screen-type influences the strength of the screen-based SB-depressive symptoms relationship; TV viewing appears less likely to be associated with depressive symptoms, compared to computer use and videogame playing. Screen-specific associations highlight that psychosocial mechanisms, which only occur when one is engaged in certain forms of screen-based SB, are likely to explain the link between screen time and internalizing symptoms. For example, being a victim of cyber-bullying may contribute to the positive association between computer/Internet use, specifically, and depressive symptoms , 94. FurBejeweled II)—can improve mood and promote relaxation [reducing internalizing symptoms with certain types of videogames, even among clinical samples [Similarly, only certain forms of videogaming may have negative consequences for youth. A study among children found that violent videogames were correlated with depressive symptoms, while videogames without violent content were unrelated to depressive outcomes . Frequenlaxation , 103; fu samples . Thus, eSex was among the most commonly-studied moderators of screen-based SB-internalizing symptom associations; our review revealed inconsistent evidence for moderation by sex. Inconsistencies in the evidence for sex as a moderator may be due unexplored three-way interactions between sex, screen-based SB , and another third variable, for example age. It is conceivable that sex may be an important moderator of screen-based SB-internalizing symptom associations only during more vulnerable periods in development when sex differences in activity and internalizing symptom levels become more apparent –108, andAcross studies that did find significant moderation by sex, it appears that sedentary girls, but not boys, may be more susceptible to depressive symptoms. Girls have higher rates of depression which may, in part, be a result of biological and cognitive factors such as hormonal changes  and rumiThe few studies investigating PA, including organized sports, as a moderator suggest that it may weaken screen-based SB-internalizing symptom associations. Participation in sports is associated with fewer symptoms of depression and anxiety in some children , which mn = 46), which prevents our ability to draw causal conclusions and limits our understanding of the directionality of associations. Future research should focus on gaining a better understanding of the directionality of screen-based SB-internalizing symptom associations as recent studies indicate that screen-based SB and internalizing symptoms may have a bi-directional relationship [While continuing to test variables such as sex, types of PA, and content with each type of screen-based SB as potential moderators is pertinent to future research, it should be recognized that the current body of literature contains limitations that must be addressed with future, high quality investigations. First, more than half of the studies in this review were cross-sectional  and real-time self-reports  should be used to limit recall bias and provide more detailed information on types and contexts of screen-based SB, as this review suggests these factors can influence associations with internalizing symptoms among youth .Third, the body of evidence for depressive symptoms is much larger than that for anxiety symptoms. Anxiety symptoms are likely related to screen-based SB, but the moderating factors of this association remain unclear, due to the limited evidence available. One potential mechanism for this trend is that the physiologic hyperarousal associated with anxiety symptoms may reduce the likelihood of being influenced by moderators as compared to depressive symptoms, where hyperarousal is not present . SimilarLastly, a majority of the evidence for sex as a moderator is based on stratified analyses without statistical tests for interaction, therefore results must be interpreted with caution. Future research should rely only on formal statistical tests for interaction when assessing moderation. Timely interventions for decoupling screen-based SB and depressive and anxiety symptoms are critically needed, as the prevalence of certain  forms screen-based SB continues to rise , 127; thA strength of the present review is the systematic search strategy used, which yielded a considerable number of studies included in the present review; however, there is the possibility that our search strategy may have missed relevant studies on the topic. Another limitation of this review is that only published articles written in English were included, index terms were not used, and a “grey literature” search was not included; therefore, our evidence may be biased towards positive results due to publication bias. Furthermore, studies that included screen-based SB in addition to non-screen-based SB  as a composite variable were excluded. Similarly, studies examining the nuances within the screen-based SB construct, such as content, weren’t systematically reviewed in the present article.This review summarizes moderators of the screen-based SB-internalizing symptom associations, discusses potential mechanistic explanations, and poses directions for future research. There is consistent evidence that screen-type influences the strength of the association between screen-based SB and internalizing symptoms. Less consistent evidence is available for female sex as an amplifier and physical activity as a buffer, therefore more research is needed on these factors to identify the possibility of vulnerable populations and tailored intervention strategies. Additionally, more evidence is needed for anxiety symptoms in particular. Gaining a thorough understanding of these complex relationships will lead to effective intervention strategies for improving the emotional and physical health of youth to ultimately prevent morbidity and mortality later in life.


The interest in therapeutic cancer vaccines has caught enormous attention in recent years due to several breakthroughs in cancer research, among which the finding that successful checkpoint blockade treatments reinvigorate neo-antigen-specific T cells and that successful adoptive cell therapies are directed towards neo-antigens. Neo-antigens are cancer-specific antigens, which develop from somatic mutations in the cancer cell genome that can be highly immunogenic and are not subjected to central tolerance. As the majority of neo-antigens are unique to each patient’s cancer, a vaccine technology that is flexible and potent is required to develop personalized neo-antigen vaccines. In vitro transcribed mRNA is such a technology platform and has been evaluated for delivery of neo-antigens to professional antigen-presenting cells both ex vivo and in vivo. In addition, strategies that support the activity of T cells in the tumor microenvironment have been developed. These represent a unique opportunity to ensure durable T cell activity upon vaccination. Here, we comprehensively review recent progress in mRNA-based neo-antigen vaccines, summarizing critical milestones that made it possible to bring the promise of therapeutic cancer vaccines within reach. We describe the advantages of mRNA, the mechanisms of mRNA-based vaccines with their clinical development and propose future perspectives.+ T cells, which can then be activated to CTLs . In. In97]. Two approaches of using IVT mRNA in cancer vaccines can be pursued. The first approach is to transfer IVT mRNA encoding TAAs into the patient’s DCs ex vivo . These tIn the second approach, IVT mRNA is injected directly into the patient, circumventing ex vivo production of patient-specific DCs. In this context, the study by Weide et al. , dating In vitro-transcribed mRNA represents an innovative modality to deliver neo-antigens to APCs in principle both in vitro and in vivo. This approach offers many advantages with regard to speed of development, production, scalability, reliability, manufacturing costs and efficacy, thereby positioning mRNA as a prime candidate for preparing cancer vaccines .Neo-antigen mRNA should be delivered to the cytoplasm of APCs, preferably DCs, where it can be translated into protein for further processing and antigen presentation to T cells. Various strategies have been developed for the delivery of IVT mRNA to DCs that have been generated in the laboratory (ex vivo) or that are present within the cancer bearing subject (in vivo). These delivery methods range from the addition of mRNA simply dissolved in a buffer of choice  ,131,132,+ T cells, holding the promise of stimulating broad CTL responses. Co-stimulation needs to be provided together with antigen presentation. Next-generation mRNA vaccines are generated with chemically modified nucleotides; therefore, they lack intrinsic immune activating capacity, signifying the need for adjuvants. Examples of adjuvants that have shown merit in mRNA-based cancer vaccines are monophosphoryl lipid A [+ TH1 cells to ensure full functionality of these CTLs [+ T cells occurs in MHC-II molecules that are loaded in MIIC compartments with peptides that are derived from exogenous antigens. When using mRNA as an approach to deliver antigens, it is imperative to guide the antigen to these compartments through the coupling of the antigenic sequence to MHC-II targeting signals, such as the signal sequence of the invariant chain, lysosomal-associated membrane protein (LAMP) or DC-LAMP [+ TH1 cells affect DCs in several ways ensuring CTL activation, among others through CD40-CD40L mediated DC licensing [+ T cells can exert cytotoxicity and as such can direct cancer cell rejection in the absence of CTLs [+ T cells in tumor-bearing C57BL/6 mice [+ T cells in the neo-antigen directed anti-tumor immune response. This is further supported by the results of recently published neo-antigen vaccine trials that show the presence of neo-antigen specific CD4+ T cells, even though vaccination was performed with neo-epitopes that were predicted to bind to MHC-I [mRNA molecules encoding different antigens can be included in one vaccine ,134, and lipid A , α-Galoc lipid A  and mRNA lipid A ,150. Theese CTLs . Present DC-LAMP ,54,55. Aicensing  and prodicensing , both stto MHC-I ,20,21.mRNA vaccine production starts with the design of the antigen in silico, offering the advantage of rapid production and preclinical evaluation, which in view of neo-antigens could entail evaluation of the correct presentation of neo-epitopes through peptidomics and/or evaluation of the neo-epitope’s immunogenicity. This can accelerate selection of suited neo-antigens for personalized cancer vaccination. Despite a different sequence for each neo-antigen, the production process is standard, reducing the timing and cost for vaccine production due to common manufacturing processes and infrastructure. The production process begins with cloning and linearization of the DNA template to produce multiple copies of the coded mRNA using RNA polymerases .Chemically modified nucleotides are often used as building blocks to lower sensing by PRRs and to enhance translational efficacy ,153. AddTo our knowledge, clinical trials with DCs that have been loaded ex vivo with neo-antigen mRNA have not yet been performed. However, the Flemish-based consortium “Persomed” has set up a roadmap to develop such a vaccine for the treatment of patients with CRC , leveragwww.clinicaltrials.gov showed that 12 clinical trials on direct delivery of neo-antigen mRNA to APCs in situ are currently ongoing, including the study in TNBC (The results of a pioneering study by Sahin et al.  have bee in TNBC . In thesImplementation of NGS and bioinformatics in the neo-antigen identification workflow has led to discovery of the source of neo-antigens, including cancer specific overexpression, alternative exon splicing, intron retention, gene fusions in addition to SNVs and INDELs . MS and Assays that evaluate T cell reactivity have been developed to determine the immunogenicity of candidate neo-antigens. Up-regulation of T cell activation markers such as 4-1BB or production of IFN-γ have been determined as a measure of the T cells specificity for presented neo-epitopes . AdditioClinical trials with neo-antigen mRNA vaccines are mostly in early stages . For theTumors develop mechanisms of resistance when under pressure of a T cell mediated attack. The pioneering study of Sahin et al.  confirmeIt is generally accepted that combining different immunotherapies is likely allowing the immune system to outsmart the tumor. In this regard, neo-antigen mRNA-vaccines that induce T cells against multiple neo-epitopes are likely to benefit from strategies that support the vaccine-induced T cells or that activate other immune effectors with complementary activity. T cells that infiltrate the TME can be confronted with a plethora of immunosuppressive cells and molecules. Therefore, strategies that reverse immune suppression are prime candidates for a combination with neo-antigen mRNA-vaccines. As described above, complete remission has been achieved in a patient with advanced, metastatic melanoma by combining neo-antigen mRNA vaccination with PD-1 inhibition . ActivatCancer vaccines based on neo-antigens hold the promise of improved tumor specificity and immunogenicity compared to cancer vaccines based on TAAs. The IVT mRNA platform could be exploited for immunogenicity testing of candidate neo-epitopes, for example by its incorporation in reporter assays using chimeric receptors, as well as for neo-antigen vaccination itself, being able to deliver neo-epitopes in addition to proteins that support immune activation. Currently, clinical experience with mRNA-based neo-antigen vaccines is scarce. However, data from the first clinical trials are encouraging and warrant further exploration of mRNA as a neo-antigen vaccine platform. The results from different ongoing clinical studies will add to our understanding on the pre-requisites for a potent neo-antigen mRNA-vaccine, potentially shedding light on a preferred route of administration as well as a preferred method of mRNA formulation, lyophilized or dissolved in an appropriate buffer, encapsulated in nanoparticles or already transferred to DCs ex vivo. Overall, results from ongoing clinical trials will catalyze a movement for therapeutic neo-antigen mRNA-vaccines.


In this context, endothelial progenitor cells (EPC) are involved in endothelial tissue repair. However, little is known about their implication in RA-ILD+. Accordingly, we aimed to investigate the potential role of EPC related to endothelial damage in RA-ILD+. EPC quantification in peripheral blood from 80 individuals  patients, and 14 healthy controls) was performed by flow cytometry. EPC were considered as CD34+, CD45low, CD309+ and CD133+. A significant increase in EPC frequency in RA-ILD+ patients, as well as in RA-ILD− and IPF patients, was found when compared with controls . RA-ILD+ patients exhibited a higher EPC frequency than the RA-ILD− ones (p = 0.003), but lower than IPF patients (p < 0.001). Our results suggest that EPC increase may represent a reparative compensatory mechanism in patients with RA-ILD+. The degree of EPC frequency may help to identify the presence of ILD in RA patients and to discriminate RA-ILD+ from IPF.Interstitial lung disease (ILD) increases morbidity and mortality in patients with rheumatoid arthritis (RA). Although the pathogenesis of ILD associated with RA (RA-ILD Althovere ILD ,4. Conse+ remains largely unknown. Given that EPC have been suggested as a marker of endothelial damage in several diseases, the understanding of the role of EPC in RA-ILD+ may be critical for a better knowledge of the vascular pathophysiology that characterizes this disease.The pulmonary microvascular endothelium constitutes a large surface area that displays great growth potential . Accordi+. For this purpose, we compared the frequency of EPC in patients with RA-ILD+ with that observed in the following three groups: RA-ILD− patients, IPF patients, and healthy controls. In addition, the association of EPC frequency with the demographic and clinical features of these patients was determined.Taking these considerations into account, the aim of this study was to investigate the potential role of EPC in the pathogenesis related to endothelial damage in RA-ILD+ patients and three comparative groups , were recruited from the Pneumology and Rheumatology departments of Hospital Universitario Marqués de Valdecilla . RA-ILD− patients fulfilled the 2010 American College of Rheumatology criteria for RA [− patients by the evaluation of high-resolution computed tomography (HRCT) images of the chest by experienced radiologists. Those who fulfilled the American Thoracic and European Respiratory Society’s criteria for ILD were classified as RA-ILD+ [A total of 80 individuals, constituted by 20 RA-ILDa for RA . ILD wasDemographic and clinical features of patients comprising sex, age, smoking history, duration of RA disease, rheumatoid factor (RF) and anti-cyclic citrullinated peptide antibodies (ACPA) status, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), pulmonary function tests (PFTs), HRCT pattern, and immunosuppressive therapies received by RA patients at the time of the study were collected. HRCT patterns of ILD patients were stratified according to the criteria for the usual interstitial pneumonia (UIP) pattern of the Fleischner Society .All the experiments involving humans and human blood samples were carried out in accordance with the approved guidelines and regulations, according to the Declaration of Helsinki. All experimental protocols were approved by the Ethics Committee of clinical research of Cantabria, Spain (2016.092). All subjects gave written informed consent to participate in this study previous to their inclusion.EPC from peripheral venous blood were characterized by simultaneous expression of cell surface markers that reflect stemness (CD34), immaturity (CD133), endothelial commitment , and a low expression of the pan-leukocyte marker (CD45) ,17,18.5 per sample. First, CD34+ and CD45low were gated and then assayed for expression of CD133 and CD309 in the lymphocyte gate. Thus, EPC were considered as CD34+, CD45low, CD133+ and CD309+ cells. EPC quantification was expressed as the percentage of cells in the lymphocyte gate.EPC quantification was analysed by direct flow cytometry, following the recommendations on EPC measurement that were previously described ,17,18. Bn) and percentage (%) for categorical variables. The differences between RA-ILD+ and RA-ILD- patients in sex, smoking status, RF and ACPA status, as well as in therapies received were analysed by chi-square test, whereas the differences in age at study, duration of RA disease, CRP and ESR levels and PFTs were determined by Student´s t-test. Comparisons of EPC frequency between two study groups were performed by Student’s t-test. Relationship of EPC frequency with continuous variables and categorical variables related to disease features was carried out via estimation of the Pearson’s correlation coefficient (r) and one-way ANOVA, respectively. p-values <0.05 were considered as statistically significant. Statistical analysis was performed using STATA statistical software 12/SE .Data were expressed as mean ± standard deviation (SD) for continuous variables, and number of individuals  (% predicted), diffusing capacity of the lung for carbon monoxide (DLCO) (% predicted), and therapies received  (A significant increase in EPC frequency in patients with RA-ILD< 0.001) .+ was also significantly different from RA-ILD− and IPF patients (+ exhibited a higher EPC percentage than the RA-ILD− ones (p = 0.003), but lower than IPF patients (p < 0.001)  .− and IPF showed a higher frequency of EPC than healthy controls    . Moreove< 0.001) .+, RA-ILD−, and IPF patients was analyzed .Since IPF and RA-ILD+, a severe complication in RA patients, were available until the present study. Accordingly, the purpose of this work was to determine the potential role of EPC in the pathogenesis related to endothelial damage in RA-ILD+.EPC are crucial in several inflammatory diseases in which endothelial damage plays a fundamental role ,19,20,21+ patients when compared with healthy controls, supporting an increase in EPC production and mobilization into the systemic circulation. In this regard, we hypothesize that EPC are being recruited at the sites of vascular damage to exert their repair capacities as a compensatory mechanism related to endothelial damage in RA-ILD+ patients. In addition, and in accordance with previous studies in RA and IPF [− and IPF patients in relation to controls, further supporting the compensatory mechanism of these cells.Our results show, for the first time, a higher EPC frequency in the peripheral blood of RA-ILD and IPF ,11, our + patients when compared with IPF and RA-ILD− patients. In keeping with that, patients with the most severe ILD, represented by IPF patients, showed the highest frequencies of EPC. Accordingly, patients with RA-ILD+ exhibited a higher frequency of EPC than those patients with RA-ILD− who did not present any ILD. This suggests a potential value of the quantification of EPC as a complementary tool for establishing a differential diagnosis between these diseases.Interestingly, we also disclosed, for the first time, a significant difference in the EPC frequency of RA-ILDRegarding the association of EPC frequency with disease features, no relationship was observed. In agreement with this, previous reports showed a lack of association of EPC quantification with PFTs and smoking status in IPF patients , as well+ and RA-ILD−, the age was similar between these two subgroups of RA. It was also applicable when RA patients, regardless of their ILD status, were compared with those with IPF. In addition, previous studies have shown that age does not constitute a relevant influence on the number of EPC in peripheral blood in healthy controls [We acknowledge that the present study has some limitations. The difference in the age between patients and controls may constitute a weakness of our study. However, whereas significant differences in the frequency of EPC existed between RA-ILDcontrols . Further+. Moreover, clinical assessment of EPC frequency as a biomarker of endothelial damage may help to identify the presence of ILD in patients with RA and to discriminate RA-ILD+ from IPF.In conclusion, our results suggest that EPC increase may represent a reparative compensatory mechanism in patients with RA-ILDhttps://acrabstracts.org/abstract/endothelial-progenitor-cells-in-the-pathophysiology-of-interstitial-lung-disease-associated-with-rheumatoid-arthritis/) and at the 2020 European E-Congress of Rheumatology (abstract no. SAT0014) .The results of this work were partially presented at the 2019 American College of Rheumatology Meeting in Atlanta, Georgia, USA (abstract no. 57) (View Abstract and Citation Information Online-