Disruption of the autonomic nervous system (ANS) can lead to acute coronary syndrome (ACS). We developed a nomogram model using heart rate variability (HRV) and other data to predict major adverse cardiovascular events (MACEs) following emergency coronary angiography in patients with ACS.ACS patients admitted from January 2018 to June 2020 were examined. Holter monitors were used to collect HRV data for 24 h. Coronary angiograms, clinical data, and MACEs were recorded. A nomogram was developed using the results of Cox regression analysis.There were 439 patients in a development cohort and 241 in a validation cohort, and the mean follow-up time was 22.80 months. The nomogram considered low-frequency/high-frequency ratio, age, diabetes, previous myocardial infarction, and current smoking. The area-under-the-curve (AUC) values for 1-year MACE-free survival were 0.790 (95% CI: 0.702–0.877) in the development cohort and 0.894 (95% CI: 0.820–0.967) in the external validation cohort. The AUCs for 2-year MACE-free survival were 0.802 (95% CI: 0.739–0.866) in the development cohort and 0.798 (95% CI: 0.693–0.902) in the external validation cohort. Development and validation were adequately calibrated and their predictions correlated with the observed outcome. Decision curve analysis (DCA) showed the model had good discriminative ability in predicting MACEs.Our validated nomogram was based on non-invasive ANS assessment and traditional risk factors, and indicated reliable prediction of MACEs in patients with ACS. This approach has potential for use as a method for non-invasive monitoring of health that enables provision of individualized treatment strategies. Coronary artery disease (CAD) is the major cause of death worldwide , 2. EvenDuring pathological conditions, there is evidence that disruption of the autonomic nervous system (ANS) potentially affects the growth of atherosclerotic plaques, and these findings have significant prognostic implications for patients with ACS –11. In cA previous report showed that risk stratification using an objective and non-invasive electrocardiogram and a HRV-based tool for 1 month reduced adverse clinical outcomes and improved myocardial infarct score more than thrombolysis, although this study did not perform validation using an external cohort . In addiTherefore, the current study used a systematic and integrated approach that incorporated HRV data with classical risk factors to develop a prognostic model that accurately and non-invasively predicts outcomes in patients with ACS who received emergency coronary angiography.The derivation set consisted of 439 consecutive patients who had ACS and received emergency coronary angiography and successful Holter monitoring for 24 h at Renmin Hospital of Wuhan University from January 2018 to June 2020. The validation set consisted of 241 consecutive patients who had ACS and received emergency coronary angiography and successful Holter monitoring for 24 h at Xiangyang Central Hospital from January 2018 to June 2020 . InternaPrior to emergency coronary angiography, blood biomarkers of inflammation , kidney function  and glucose were measured. Blood was collected in the early morning after fasting to determine the lipid profile {high-density lipoprotein cholesterol [HDL-C], low-density lipoprotein cholesterol [LDL-C], total cholesterol [TC], triglyceride [TG], and lipoprotein a [Lp (a)]}.As previously described, each patient underwent Holter monitoring for 24 h after angiography to evaluate the ANS by analysis of 24-h mean heart rate and variables of HRV –18. An aCoronary angiography was performed immediately after admission for ACS by experienced senior cardiologists who used standard treatment protocols. In accordance with guidelines, loading doses of aspirin, ticagrelor, or clopidogrel were given to patients with ACS and before operation . The perFollow-up, which consisted of telephone calls and/or clinic visitations, was used to determine when an endpoint occurred. The primary endpoint was a major adverse cardiac events (MACEs), defined as the composite of cardiac mortality, recurrence of myocardial infarction, or need for revascularization. Cardiac mortality refers to death caused by any cardiac condition. Myocardial infarction refers to a new myocardial infarction in targeted or non-targeted vessels. Revascularization refers to targeted or non-targeted vessels.t-test was used; for analysis of continuous variables with non-normal distributions, the Mann-Whitney U-test or the Kruskal-Wallis test was used. Analysis of categorical variables was performed using the χ2-test. In all comparisons, a P-value below 0.05 indicated statistical significance.Statistical analysis was conducted using SPSS version 23  and R software version 3.6.1 . Means and standard deviations (SDs) were presented for continuous variables with normal distributions, and medians and interquartile ranges (IQRs) for continuous variables with skewed distributions. For analysis of continuous variables with normal distributions, the Univariate and multivariate analyses were used to identify factors independently associated with outcome (MACEs). Analysis of the statistical significance of each individual variable was performed using univariate analysis. This was followed by multivariate analysis of variables that were significant in the univariate analysis to identify significant and independent predictors of MACEs.Predictive nomograms were constructed using predictors that were statistically significant. R software was used to construct the nomogram using the Regression Modeling Strategies (rms) package. Risk nomograms were analyzed using receiver characteristic curves (ROC) to assess their ability to discriminate MACEs based on calculation of area under the ROC curve . Using tn = 439) and an independent external validation cohort (n = 241). Among all 680 patients, the mean age was 62.9 (± 12.6) years-old and 423 patients (62.2%) were males. The two cohorts were well balanced in baseline characteristics and outcomes except for clinical presentation, LDL-C, in-hospital PCI, several results from the coronary angiography, and HRV (We retrospectively screened 680 patients who had ACS and received emergency angiography and divided them into a development cohort ( and HRV . The oveP = 0.030), diabetes , current smoker , previous myocardial infarction , and LF/HF ratio  (We used multivariable stepwise Cox regression model to identify significant and independent predictors of MACEs in the development cohort . There w= 0.031) .We then used a nomogram model to present the results of the multivariate Cox regression in predicting the 1 and 2-year probability of MACE-free survival . To calcWe determined the discriminatory capacity of the model using receiver operating characteristic (ROC) analysis and calibration plots. In the development cohort, ROC analysis indicated the area-under-the-curve (AUC) was 0.790 (95% CI: 0.702–0.877) for prediction of 1-year MACE-free survival and the AUC was 0.802 (95% CI: 0.739–0.866) for prediction of 2-year MACE-free survival, suggesting the model had good predictive performance . MoreoveWe further analyzed these data using calibration plots to evaluate the consistency of the actual probabilities with predicted probabilities for 1 and 2-year MACE-free survival in the development cohort  and the We then applied DCA to determine the net benefit of the clinical prediction model . In the n = 147), an intermediate-risk group , and a high-risk group  for analysis of 1 and 2-year MACE-free survival We used the total score of each patient in development cohort to stratify patients into a low-risk group  and 2 years . Similarly, the high-risk group had a higher risk of MACEs than the low risk group at 1 year  and 2 years . Patients in the intermediate-risk group had a higher risk of MACEs than the low risk group at 1 year  and 2 years .Kaplan-Meier analysis of the development cohort  with pai2 = 15.791, P < 0.001) and 2 years . Similarly, patients in the high-risk group had a greater risk of MACEs than the low-risk group at 1 year  and 2 years .Kaplan-Meier analysis of data from the validation cohorts  also ind2 = 18.158, P < 0.001) and 2-years . Pairwise comparisons also indicated that the high-risk group had greater risk of MACEs than the low-risk group at 1-year  and 2-years .We also performed a Kaplan-Meier analysis of the overall study population . SimilarOur nomograms for predicting the 1 and 2-year probability of MACE-free survival in patients who had ACS used traditional clinical prognostic variables  and data from non-invasive ambulatory measurements (LF/HF ratio). Thus, we used routinely available clinical variables to develop a nomogram that had reliable discriminative ability, and we validated the nomogram model for predicting the risk of 1 and 2-year MACE-free survival. Our model has potential for use in the risk stratification of patients with ACS who received emergency coronary angiograms. It is possible that inclusion of other predictors in the future may further improve the model.via HRV analysis is a valuable, non-invasive, and easy-to-perform method that has relatively good reproducibility, and may provide useful information for the management of patients with ACS.Acute coronary events may occur in patients who have none of the well-established classical cardiovascular risk factors, suggesting there may be additional coronary risk factors that have not yet been recognized , 10, 11.Our previous study of ACS patients found that automatic evaluation of cardiac ANS function was an effective method for risk-stratified prediction of MACEs when used with GRACE score, in that it provided accurate prognostic information and increased discriminatory ability . In addiEndothelial injury results from the interaction between an imbalanced ANS and inflammation. Failure to maintain lipid homeostasis may result from endothelial injury, and lead to the activation of vascular smooth muscle cells and macrophage infiltration. These pathological events accelerate atherosclerotic plaque formation and cause the deterioration of myocardial ischemia , 26, 27.Our results indicated that it is currently simple and feasible to identify the risk of 1 and 2-year MACEs in patients with ACS. We also demonstrated favorable discriminative ability of the nomogram, based on the C-index and AUC values in the development and validation cohorts. Our internal validation (based on bootstrapping) and external validation also indicated the nomogram had satisfactory consistency in the prediction of MACEs. Our study therefore confirmed that specific clinical variables can be used for individualized predictions of risk and risk stratification for MACEs in patients with ACS. Our results may also contribute to the design and implementation of clinical trials that examine individualized clinical decision-making. Importantly, the rapid proliferation of wearable health technologies, such as smart wearable electronic devices that are equipped with green light-emitting diodes for photoplethysmography, for the self-monitoring of health-related parameters, including heart rate, heart-rate variability, and electrocardiograms, are now available in commercially available smartwatches and smartwatch accessories, with arrhythmia notification provided directly to the user –31. ThusIt is important to note that while our model has significant promise for predicting MACEs in ACS patients who received emergency coronary angiography, there were a few limitations. First, because the sample size was relatively small, the predictive ability of the model needs to be confirmed in large prospective cohort studies. Second, due to the retrospective nature of this study, detailed patient information, such as the GRACE score and the time of ACS onset, was not recorded. Third, although we accounted for several known confounding factors, some unknown confounding factors may have remained that affected HRV. Four, because the two hospitals used different methods for measuring troponin and creatine kinase isoenzyme MB, we did not include these variables in the analysis. Five, because this study was observational, it is unknown if better outcomes could have been achieved from the use of individualized and comprehensive treatment based on other ANS risk models. Six, additional indicators of ANS, such as deceleration capacity, may be also useful as prognostic indicators. Future studies are therefore needed to assess the validity and accuracy of point-of-care testing of cardiac ANS in predicting MACEs after ACS. Finally, for methodological reasons, the ANS can only be assessed using HRV in patients with a sinus rhythm, and the impact of a non-sinus rhythm in patients with ACS remains uncertain.Our observational study demonstrated that a risk nomogram that incorporated four conventional clinical characteristics and a single ANS parameter (LF/HF) was a simple method that clinicians can use to assess the risk of MACEs in patients with ACS. We suggest that future large studies are needed for further validation of our nomogram. Nonetheless, the results indicated that our nomogram appeared to be useful for identifying risk of MACEs in patients with ACS, and thus provide a foundation for the use of mobile health monitoring based on alterations of the ANS.Individual participant data that underlie the results reported in this article, after de-identification can be obtained from the corresponding authors upon reasonable request.The studies involving human participants were reviewed, approved and granted for the study by the Local Ethics Committee . The ethics committee waived the requirement of written informed consent for participation.HJ, XZ, and HF conceived and performed the experiments. JW and JS performed the experiments and wrote the manuscript. XW and TZ provided expertise and feedback. TX, FY, SD, QD, ZL, FG, XL, YW, and LS provided valuable suggestions and edited the manuscript. All authors contributed to the article and approved the submitted version.


Metal-oxide semiconducting materials are promising for building high-performance field-effect transistor (FET) based biochemical sensors. The existence of well-established top-down scalable manufacturing processes enables the reliable production of cost-effective yet high-performance sensors, two key considerations toward the translation of such devices in real-life applications. Metal-oxide semiconductor FET biochemical sensors are especially well-suited to the development of Point-of-Care testing (PoCT) devices, as illustrated by the rapidly growing body of reports in the field. Yet, metal-oxide semiconductor FET sensors remain confined to date, mainly in academia. Toward accelerating the real-life translation of this exciting technology, we review the current literature and discuss the critical features underpinning the successful development of metal-oxide semiconductor FET-based PoCT devices that meet the stringent performance, manufacturing, and regulatory requirements of PoCT. Point-of-care testing (PoCT) describes a diagnostic or prognostic procedure conducted close to or at the site of a patient. It aims to deliver reliable and objective results and, consequently, to improve patient management and/or care ,2. PoCT While many PoCT approaches have relied to date on well-established technologies such as lateral flow assays, there is an unquestionable need for more performance PoCT. For example, quantitative insight into the concentration of one or more biomarkers is required in many cases to establish a reliable diagnostic. This is challenging, especially for analytes present at low concentrations in the samples. Recent advances in nanotechnology and molecular sciences are broadly anticipated to provide implementable solutions to this challenge in the form of nanoscale biosensors, which present significant advantages for the PoCT platform ,14.Conceptually, a biosensor is an analytical device that converts the input variable into a measurable signal using biological recognition elements that integrate within or intimately with a physicochemical transducer element . TypicalWe review the current literature and discuss the key aspects relevant to the development of metal-oxide-based FET for PoCT devices. We first present an overview of the most common wide-band gap metal-oxide semiconductors and discusses important selection criteria for creating successful FET-based PoCT devices. We then describe the main fabrication approaches and challenges associated with integrating metal-oxide-based-FETs. Next, the key yet often neglected issue of sample pre-processing is discussed. Finally, we briefly introduce the regulatory landscape of PoCT technology.Several metal-oxide semiconductor materials have been used as active elements in FETs ,42. The As illustrated in 2O3 FETs  clearance ,173. ForIt is worth noting that new PoCT biosensors where there are no approved/predicated technologies are automatically classified as Class III and must undergo a full pre-market approval process in the US FDA landscape. On the other hand, class III PoCT biosensors with existing predicated technologies can apply to be accessed as class II devices . SimilarThe relatively poor adoption of PoCT can be explained by performance and economic issues compared to more conventional testing in centralized laboratory facilities. In addition, cultural and organizational issues are also commonly identified as barriers to implementation . Finally® test shows high specificity (95%) with noticeably low performance (63%) using serum samples [® test presents good performance (80%) with almost the same specificity as Wondfo® devices (97%) for symptomatic patients only when testing within five days of symptoms onset [The limited performance—real or perceived—of some PoC tests  compared to tests performed within centralized laboratory facilities may impede their adoption and utility. Poor PoCT device performance, particularly “false negative results”, can have serious consequences. Performance issues are also inherently linked to the issues associated with the quality control of PoCT devices. For example, six of eight PoC tests for Hemoglobin A1c yielded clinically unacceptable reproducibility, although certified by the National Glycohemoglobin Standardization Program (NGSP) . The per samples . The CLIms onset . Interesms onset .As noted above, metal-oxide FETs are cost-effective devices derived from the availability of well-established fabrication protocols compatible with scaled-up manufacture. In addition, their performance is high enough to meet the requirements of most PoCT applications. They, therefore, provide an excellent compromise between ultra-high-performance solid-state sensors that typically suffer from limited manufacturability and low-sensitivity sensors that can be readily mass-produced.Despite these advantages, several issues should be considered. A critical challenge associated with the implementation of metal-oxide FET sensors is batch-to-batch performance uniformity. To mitigate this issue, it is essential to consider all factors that affect their performance. For example, it has become evident that the characteristics of metal-oxide semiconducting materials are controlled not only by their structure and geometry but also by the presence of functional defects and their crystal structure. Most importantly, oxygen vacancies play a crucial role in determining the physical properties of metal-oxide FET sensors  and shouImportantly, FET-based PoCT devices should have a small footprint to enable integration with signal processing systems. The inherently small size of FET sensors increases compatibility with wearable technologies, which is currently attracting a huge amount of research. In addition, impedance matching between the subsystem units should be considered, as it affects the signal capture within these units’ signal-to-noise ratio level . One shoFinally, an often-overlooked consideration is the issue of the packaging and shelf-life of FET-based sensors. The environmental stability of the metal-oxide FET itself and that of the biointerfacial layers containing the bioreceptors should be considered and optimized to ensure compatibility with PoC settings, where substantial variations in storage conditions are likely to occur.The economic dimension is a prominent barrier to the adoption of PoCT. In general, the cost per test is higher for PoCT than that associated with conventional batch testing in centralized laboratories. For example, it has been reported that the cost of glucose and blood gas/electrolyte testing is 1.1 to 4.6 times higher for PoCT compared to testing in a centralized laboratory . Along tRegarding the PoCT implementation cost, there are both direct and indirect costs associated with the implementation of PoCT. While direct costs are relatively straightforward to evaluate , indirecFinally, it is worth noting that, in most cases, reimbursement schemes are not adjusted to cover the direct and indirect additional costs associated with PoCT . For insThis review has discussed the most common metal-oxide FETs fabrication routes, material selection and considerations, surface functionalization, and emerging biosensing applications in the PoCT area. Daunting challenges remain, however, to be addressed so that metal-oxide FET technology can be reliably translated into real-life applications.The first challenge is the current significant device-to-device signal variability, which exists even for devices originating from the same fabrication batch. These variations could be significantly reduced by using nanoribbons and thin-film FETs instead of nanowires. These architectures allow much better control over the lateral dimensions and do not affect the device’s performance. Secondly, implementing metal-oxide FETs in the PoCT application area requires careful choice of fabrication procedure, sample collection/processing integration approaches, and readout methods. Therefore, striking the right balance between the privileges and shortcomings of each process and minimizing user involvement.Despite challenges, significant progress has been made in metal-oxide FET-based biosensors recently. With their ultralow LODs, exquisite sensitivities, quantitative, and label-free sensing, metal-oxide FET promises to play a significant role in the bio-diagnostic field.


N = 721) of young adults .Although the association between problematic use of the internet and eating disorders (EDs) in young adults has been previously established, its underlying mechanisms have not been completely clarified. It has been suggested that exposure to idealized very thin and toned body images  on social media might lead to increased feelings of body dissatisfaction which, in turn, can represent a trigger for EDs. We have tested this hypothesis in a sample , muscle dysmorphia (MD), and EDs were used. A mediational model analyzing the direct and indirect effects of SMA-related symptoms on ED-related symptoms through the mediating role of MD-related symptoms was performed controlling for confounding factors .B = 0.213; p = 0.022) and that this association was mediated by MD-related symptoms .The model showed that the total effect of SMA-related symptoms on ED-related symptoms was significant (Our findings support the possibility that MD-related symptoms play a relevant role in mediating the association between SMA severity and ED pathology.Level III, evidence obtained from well-designed cohort or case–control analytic studies. The increasing relevance of the internet in everyone’s life, and especially in the lives of adolescents and young adults, is raising interest on potential health-related sequelae associated with its use. In relation to mental health, recent studies have observed that dysfunctional and excessive use of internet, a condition often conceptualized as internet addiction or as problematic use of the internet (PUI) , 2, is sA link between PUI and mental diseases has been consistently observed in relation to eating disorders (EDs) . EDs areIn relation to the association between PUI and EDs, Hinojo-Lucena et al. recently performed two meta-analyses (involving data from over 8000 and over 5000 students) showing that both EDs  and ED-related symptoms  were significantly higher among individuals with PUI than in those without , thus confirming our mediation hypothesis, and (ii) the direct effect of SMA-related symptoms on ED-related symptoms was not significant , thus suggesting that the association between SMA-related symptoms and ED-related symptoms was mediated by MD-related symptoms.Results of the mediational model are reported in Table The present cross-sectional study on 721 young adults showed that (i) SMA-related symptoms were significantly associated with both MD- and ED-related symptoms, (ii) the relationship between symptoms of SMA and EDs was mediated by the severity of MD-related symptoms, with relevant confounding factors  being controlled for Table ; Fig. 1.At the clinical level, the present findings contribute to raise awareness on the risks for mental health related to excessive use of SM . AccordiIn relation to MD, the present study suggests that preoccupation over body image and physical activity, with symptoms related to drive for size , appearance intolerance , and functional impairment  , is assoThese results are consistent with evidence suggesting that excessive exposure to SM is associated with ED pathology, and that the preoccupation over physical appearance and muscularity, which can be triggered by certain SM contents , plays a relevant role in such phenomenon –23, 25.The present cross-sectional study on young adults showed (i) that SMA-related symptoms were significantly associated with both MD- and ED-related symptoms, and (ii) that the relationship between symptoms of SMA and EDs was mediated by the severity of MD-related symptoms, with relevant confounding factors being controlled for Table ; Fig. 1.Such findings add specificity to this field of research as the data of the present research have been collected through quantitative measures, while several previous studies on the topic used qualitative approaches. Further, the present research was specifically focused on psychopathological underpinnings related to SMA, and not to PUI in general. This is especially relevant as within the PUI construc, there are a wide range of phenomena markedly different from SM use, such as video gaming, pornography, cybersex, and online gambling . Consistn = 721) was adequate, as indicated by a priori power analysis; (iii) we used extensively validated assessment instruments; (iv) we statistically controlled for potential confounders.The present study has several limitations, among which: (i) despite we used an hypothesis-driven statistical approach specifically designed to infer causal relationships between associated variables, definitive causality cannot be established as a complete study of mediation processes would require a longitudinal approach, while we used a cross-sectional approach; (ii) self-reports, non-validated by a clinical interview, have been used, whose results may be affected by several biases  ; (iii) wIn conclusions, our findings provide pieces of insight on the complex relationships between SMA, MD, and EDs. It is possible that, while SM use is not detrimental for mental health per se and it can even be integrated in telepsychiatry approaches , 60, a d


Distraction-based therapies, such as virtual reality (VR), have been used to reduce pain during acutely painful procedures. However, distraction alone cannot produce prolonged pain reduction to manage sustained postoperative pain. Therefore, the integration of VR with other pain-reducing therapies, like guided relaxation, may enhance its clinical impact.The goal of this pilot study was to assess the impact of a single guided relaxation–based VR (VR-GR) session on postoperative pain and anxiety reduction in children. We also explored the influence of pain catastrophizing and anxiety sensitivity on this association.A total of 51 children and adolescents (7-21 years) with postoperative pain and followed by the Acute Pain Service at Cincinnati Children’s Hospital were recruited over an 8-month period to undergo a single VR-GR session. Prior to VR, the patients completed 2 questionnaires: Pain Catastrophizing Scale for Children (PCS-C) and the Child Anxiety Sensitivity Index (CASI). The primary outcome was a change in pain intensity following the VR-GR session . The secondary outcomes included changes in pain unpleasantness and anxiety.P<.001) and at 30 minutes (P=.04) after the VR session, but not at 15 minutes (P=.16) postsession. Reductions in pain unpleasantness were observed at all time intervals . Anxiety was reduced immediately (P=.02) but not at 15 minutes (P=.08) or 30 minutes (P=.30) following VR-GR. Patients with higher CASI scores reported greater reductions in pain intensity (P=.04) and unpleasantness (P=.01) following VR-GR. Pain catastrophizing was not associated with changes in pain and anxiety.The VR-GR decreased pain intensity immediately (A single, short VR-GR session showed transient reductions in pain intensity, pain unpleasantness, and anxiety in children and adolescents with acute postoperative pain. The results call for a future randomized controlled trial to assess the efficacy of VR-GR.ClinicalTrials.gov NCT04556747; https://clinicaltrials.gov/ct2/show/NCT04556747 Ineffective postoperative pain management, defined as surgery-related pain resulting from tissue injury and muscle spasm, has long-term consequences, including increased morbidity, poorer physical functioning, longer recovery, and increased costs . DespiteAlthough multimodal analgesia protocols focus on regional analgesia and nonopioid medications, opioid usage remains ubiquitous in pain management. Children and adolescents are at particular risk of long-term opioid abuse—as few as 5 days of use increases this risk . Other sThere remains a critical need for novel, nonpharmacologic pain management strategies, like virtual reality (VR). VR provides an immersive, multisensory, 3D environment that modifies the experiences of reality and creates a “sense of presence.” This “sense of presence” makes VR an excellent distraction-based therapy . The useAlternative interventions utilizing traditionally delivered mind body–based therapies, like relaxation and slow breathing, reduce anxiety and pain in children undergoing surgery . CombiniThis study aimed to pilot the use of a single session of VR-GR in children after surgery to assess the association between VR-GR and pain intensity in addition to exploring the effect of pain catastrophizing and anxiety sensitivity on this association. We further assessed the association between VR-GR and reductions in pain unpleasantness and anxiety. We hypothesized that one VR-GR session would be associated with transient reductions in pain intensity, pain unpleasantness, and anxiety, with the greatest within-patient changes in children having high baseline pain catastrophizing and anxiety sensitivity.A single-center, prospective, pilot study in a broad pediatric postoperative population experiencing moderate to severe pain was designed to preliminarily assess the correlation between a single VR-GR session and reductions in acute postoperative pain and anxiety, to explore the association between these within-patient changes and pain catastrophizing and anxiety sensitivity, and to show the feasibility of using this technology in a postoperative setting . AdditioThe study recruited 51 children and adolescents who underwent surgery and were followed by the Acute Pain Service at Cincinnati Children’s Hospital Medical Center between July 2019 and March 2020. The study was approved by the Institutional Review Board (IRB #2018-2892) and conducted per the rules and regulations for ethical research. It was registered with ClinicalTrials.gov on September 21, 2020 (NCT04556747). A written consent (and assent for patients >11 years) was obtained from all participants and the study adhered to the CONSORT  guidelines. The patients were recruited and enrolled in the study after surgery following hospital admission, and they were not compensated for participation.Patients were suitable for recruitment if the Acute Pain Service was managing their pain. The patients managed by the Acute Pain Service experience high postoperative pain unmanageable solely by the surgery team. These patients necessitate multimodal analgesia and management with patient-controlled analgesia (PCA) and regional or neuraxial analgesia.The eligibility criteria for inclusion were: 7-21 years old; able to read, speak, and write English; and undergone surgery resulting in significant postoperative pain necessitating care by the Acute Pain Service.Patients were excluded if they did not fall within the age criteria; unable to read, speak, or write English; had a history of developmental delay, neurological conditions , or uncontrolled psychiatric conditions; or had head or neck surgery that precluded the use of VR.Prior to the VR-GR session, we collected the patient’s age, sex, race, surgery type, and American Society of Anesthesiologists (ASA) status. The ASA status is a 6-category classification system that assesses a patient’s general health before surgery; the sickness of a patient increases with the category number. Class I and II are healthy or have mild/moderate systemic disease; class III and IV patients have severe disease that may limit activity or threaten life.The primary outcome was the change in pain intensity after VR-GR. Secondary outcome measures included changes in pain unpleasantness and anxiety.Pain intensity, pain unpleasantness, and anxiety were measured before and after the VR-GR sessions using the numerical rating scale (NRS) . A scripThe Pain Catastrophizing Scale for Children (PCS-C), a validated, self-reported questionnaire assessing pain catastrophizing tendencies ,21, was The Child Anxiety Sensitivity Index (CASI) questionnaire was completed before the VR-GR as described above. The CASI has been used in VR studies in adolescents aged 10-21 years , validatPatient experience was measured with the study team–generated questionnaire. The participants ranked how much they agreed with statements on a 4-point Likert scale. The parent(s) of the participants were asked to fill out a similar survey to understand their perspective of the VR experience. The surveys, included as All patients used the Starlight Xperience VR device, a commercially available headset supplied by the Starlight Children’s Foundation . It is aThe patient visits occurred on postoperative day (POD) 1 or 2. After determining eligibility and obtaining consent, the patients completed the PCS-C and CASI questionnaires and rated their baseline pain intensity, pain unpleasantness, and anxiety levels on the NRS.Following these assessments, the patients were oriented to the VR headset and given a tutorial on the device application. They were instructed to remove the headset for discomfort, nausea, or dizziness. After completing the 10-minute session (based on the standard duration of relaxation sessions), the VR device was removed. Pain intensity, pain unpleasantness, and anxiety were recorded immediately, at 15 minutes, and at 30 minutes after the experience. The patients and parents then completed the experience questionnaires.P value of .05 was the cutoff value for statistical significance. Additionally, statistical significance with the Bonferroni adjustment for multiple comparisons for the primary outcome (change from the baseline pain intensity at three time-points after VR-GR) was assessed. The first-order autoregressive, AR(1), covariance structure was used in all the mixed-effects models. The missing data were examined, and all available data were used in statistical analyses.All statistical analyses were performed using SAS 9.4 (SAS Institute). A Descriptive statistics were calculated for all baseline variables and change from the baseline for outcome variables. Mean (SD) and median (IQR) were used for the continuous variables, while frequency and percentage were used for the categorical variables.t test or signed-rank, as appropriate) at individual time points. The changes from baseline were analyzed with mixed-effects models where time intervals , pain catastrophizing, and anxiety sensitivity were used as the categorical fixed effects.Pain intensity, unpleasantness, and anxiety after the VR-GR session were compared to the baseline within each patient using paired tests (The Pearson or Spearman correlation coefficients were derived to test the association between the traits (pain catastrophizing and anxiety sensitivity) and outcomes .Mixed-effects models were used to examine the association of PCS-C and CASI on the changes from the baseline of pain intensity, pain unpleasantness, and anxiety, where time intervals  were used as the categorical fixed effect.We enrolled 51 patients over 8 months. All patients completed pain and anxiety assessments at baseline and immediately following VR-GR; 100% (n=51) completed the pain assessment at 15 minutes, and 98% (n=50) completed the anxiety assessment at 15 minutes; and 88% (n=45) completed the assessments at 30 minutes following VR-GR. The patients did not receive any analgesic medications during participation in the study. Missing data resulted from limitations in the clinical environment, including patients undergoing imaging studies, receiving care from the care team, or falling asleep.The patients were primarily adolescent, male, and Caucasian . Of the P<.001) (Patients had moderate pain catastrophizing and average anxiety sensitivity . FurtherP<.001) .Pain catastrophizing and anxiety sensitivity were not associated with changes in pain intensity or pain unpleasantness.P<.001) following the VR-GR session and remained significant at 15 minutes , and at 30 minutes   .P<.001), and remained significantly decreased at 15 minutes , and at 30 minutes  following VR-GR.VR-GR was also associated with a change in pain unpleasantness B. The WiP<.001); it remained lower at 15 minutes  but not at 30 minutes.We also found an association between VR-GR and anxiety C. The WiP=.04) and pain unpleasantness . Pain catastrophizing was not associated with changes in pain and anxiety following VR-GR . Additionally, the older participants had a smaller decrease in pain intensity  than the younger patients. No other variables  were associated with changes in outcomes.When assessing the association between changes from the baseline and covariates, we found that the Caucasian patients had a smaller decrease in pain unpleasantness from the baseline than the non-Caucasian participants—difference in least squares mean  believed they felt “calmer and less anxious after having used VR” and that VR “made it easier for (them) to tolerate (their) pain.” Parents reported a similar positive overall experience when asked the same questions. The parents  who completed the questionnaire would recommend VR; 93% (n=41) believed that VR made their child calm, and 84% (n=37) believed that VR helped their child tolerate pain. No patient experienced any self-reported side effects during the VR session.VR-GR may be beneficial in pediatric postoperative pain management. Our pilot study assessed the association between a single VR-GR session delivered after surgery and changes in pain intensity, pain unpleasantness, and anxiety in children and adolescents with acute pain following surgery. The most significant changes were observed for pain unpleasantness, with lesser reductions in pain intensity and anxiety. More considerable reductions in pain intensity and unpleasantness after VR-GR were associated with higher anxiety sensitivity. Pain catastrophizing did not appear to be associated with changes in these outcomes. The qualitative assessments suggested that the patients had positive experiences with VR-GR with no reported side effects.Although pain management strategies increasingly incorporate multimodal analgesia, the percentage of patients experiencing severe postoperative pain did not change in the last 20 years . It was Few studies have used VR for acute postoperative pain in adults , and nonMind-body therapies, like relaxation and guided imagery, decrease anxiety and help manage pain in children undergoing surgery . UnfortuThis study was the first to integrate a mind-body therapy with VR to help pediatric postoperative pain. The study’s goals were to preliminarily assess the use of VR-GR to decrease acute postoperative pain and anxiety and determine whether pain catastrophizing and anxiety sensitivity influence the VR-GR effect in a broad pediatric population. The “Mindful Aurora” application taught patients to slow down their breathing and relax. A single VR-GR session was associated with immediate, acute changes in pain intensity, pain unpleasantness, and anxiety. Reductions in pain unpleasantness were observed at all intervals . Anxiety reduced immediately and at 15 minutes following VR-GR. An association was observed between higher CASI scores and greater reductions in pain intensity and unpleasantness following VR-GR; however, such an association was absent with pain catastrophizing.This study had several limitations. The study design did not include a control group, which restricted us from determining a causal relationship between the use of VR-GR and pain and anxiety reduction. It is possible that interaction with the study team could influence changes in pain and anxiety. Additionally, we did not collect data on analgesic use in the study population or standardize the timing of the postoperative visit. This study was designed to test the feasibility of the VR-GR technology, obtain pilot data in children hospitalized after surgery, and assess a possible association between VR-GR and pain and anxiety reduction. The results of this study support the need to conduct a randomized controlled efficacy trial comparing the use of VR-GR to active control, data collection on the timing and use of all analgesic medications, and standardizing the timing of the VR-GR sessions. The study used a single, short VR session in the postoperative period. Although we saw small changes in pain intensity, pain unpleasantness, and anxiety, these changes did not reach any clinical significance. Previous literature report that a reduction in pain intensity of 2 or more points on the NRS or a 30% reduction in pain is considered clinically significant . We recoTo summarize, our study demonstrates the successful use of VR-GR in children after surgery. A single VR-GR session was associated with transient decreases in pain and anxiety. Future research is needed to investigate the effect of VR-GR in reducing pain and anxiety in the postoperative setting compared to a control.


Atrial fibrillation (AF) is a common arrhythmia that can lead to stroke, heart failure, and premature death. Manual screening of AF on electrocardiography (ECG) is time-consuming and prone to errors. To overcome these limitations, computer-aided diagnosis systems are developed using artificial intelligence techniques for automated detection of AF. Various machine learning and deep learning (DL) techniques have been developed for the automated detection of AF. In this review, we focused on the automated AF detection models developed using DL techniques. Twenty-four relevant articles published in international journals were reviewed. DL models based on deep neural network, convolutional neural network (CNN), recurrent neural network, long short-term memory, and hybrid structures were discussed. Our analysis showed that the majority of the studies used CNN models, which yielded the highest detection performance using ECG and heart rate variability signals. Details of the ECG databases used in the studies, performance metrics of the various models deployed, associated advantages and limitations, as well as proposed future work were summarized and discussed. This review paper serves as a useful resource for the researchers interested in developing innovative computer-assisted ECG-based DL approaches for AF detection. Atrial fibrillation (AF) is the most common heart rhythm disorder. It is seen mostly in the elderly but even young people who do not have underlying heart disease may suffer from it. Although AF itself is rarely lethal, it increases the risk of AF-related complications like heart failure and thromboembolism, which lead to increased morbidity and mortality . AF is aAF can be classified as paroxysmal, persistent, or permanent AF ,7. ParoxThe diagnosis of AF requires electrocardiographic (ECG) documentation of the arrhythmia on at least one lead . ParoxysSystems developed for automatic recognition of AF primarily exploit two key features of AF on ECG signals: absent P wave and/or irregular RR intervals. As such, accurate detection of P or R wave peaks is critical. The low amplitude P wave is especially susceptible to interference from ECG baseline drift and artifacts , which mTraditional machine learning algorithms are commonly used for ECG signal analysis ,17. The We aimed to survey articles that have been published in international peer-reviewed journals on AF detection using DL, focusing on those that address the AF problem directly rather than generic studies of arrhythmia classification that included AF as one of the classes. The studies utilizing only DL in the detection of AF were examined in this study. Performance comparisons were made by considering the data sets, input formats, deep models, and classification approaches used in these studies. As a result, it is expected that researchers have knowledge in deep learning-based studies to be conducted for AF detection. Limitations of the studies and suggestions for future works for AF detection using DL were also discussed.During the development of the search strategy, frequently used keywords in recent studies for the detection of AF were filtered. A review of the literature was conducted by searching the most used keywords  and deep learning models  and the search strategy was restricted to the last 5 years. The keywords “Atrial fibrillation and deep learning”, “Atrial fibrillation AND deep neural networks”, “Atrial fibrillation AND convolutional neural networks OR CNN”, “Atrial fibrillation AND LSTM”, and “Atrial fibrillation AND Neural networks” were used to search Google Scholar, Mendeley, and ScienceDirect databases for relevant articles. In total, 24 articles were selected that had been published in the following journals: Computers in Biology and Medicine ,41,42,43From a detailed analysis of the various methods used in the articles, we constructed a general approach that is illustrated in An ECG signal often contains noise and artifacts that arise from the device used to collect the signal or the environment in which the signal is being collected. Various pre-processing techniques can be applied to denoise ECG signals, including Fourier cosine series operation to remove baseline wander and high frequency components , elliptiThe ECG signal can be configured in various formats—single-lead ECG, multi-lead ECG, heart rate variability (HRV), spectrogram, or fused features—for input into DL models for AF detection .Single-lead ECG: Single-lead input is commonly used in the published studies [ studies ,56,60,61 studies  and whicMulti-lead ECG: Studies using this data input generally have access to standard 12-lead ECG signal recordings [cordings . As the cordings  excludedcordings  trained ECG segment size: Different segment sizes of single- and multi-lead ECG recordings have been used in the studies. Single-beat [gle-beat ,54, fivegle-beat ,59, and gle-beat ,47,49 argle-beat  comparedHeart rate variability: HRV, which measures RR interval variations over a specified finite time duration, reflects the state of the autonomic nervous system [s system ,70 and hs system  segmentes system .Ebrahimzadeh et al.  extracteSpectrogram: One-dimensional signals like ECG RR intervals but not the ECG morphology can be converted to spectrograms [trograms ,81,82 thtrograms ,44,57. Xtrograms  used shoFused Features: Architectures with two or more different input types have been used for AF detection to improve model performance. In the work of Fan et al. [n et al. , both RRn et al.  used rawn et al.  proposedn et al. , the pron et al.  and cohen et al. , which rDeep neural networks (DNNs), the most basic form of DL, have similar structures to the traditional multilayer perceptron (MLP) that is obtained by cascading models with multiple hidden layers. Learning is achieved by abstracting data inputs into the DNN’s many layers. Cai et al.  proposedTo learn, CNN models automatically extract hierarchical features from simple to complex using convolution by applying high-dimensional filters on the input data. They have been used successfully in problems involving two-dimensional images ,86,87,88Recurrent neural networks (RNNs) are a type of artificial neural network developed to solve temporal problems, particularly those with sequential inputs ,90 such Long short-term memory (LSTM) models proposed by Hochreiter et al.  are wideThere are theoretical synergies between CNN models’ representation learning and LSTM models’ sequence learning that can be combined to yield powerful DL models where features obtained from CNN layers are fed to LSTM layers in sequence. Andersen et al.  proposedAF, SR, AFL and ventricular fibrillation ,56;AF, SR, and others ;AF, SR, and AFL ;AF, SR, others and noisy ,51,53,57AF, SR, and non-AF ; andAF, SR, AFL, and junctional rhythm .The class to which ECG signal input belongs is determined at the final layers of DL models. Classification can either be binary—AF versus non-AF—or multi-class. In binary classification, the AF class may include AFL  and the CNN DL can perform automatic feature extraction effectively. Wang et al.  used theThis review chronicled research and development efforts to improve AF detection methodology through continual experimentation with network layer configuration and parameters. The observations contribute to the future design of DL models that are computationally efficient and yet can yield optimal results. Due to the blackbox nature of many DL models, there is a dearth of information on why a particular model should become successful or not. This constitutes an important impediment to clinical acceptance of new AF detection models , and a fIt is important to distinguish AF from SR on the ECG. With manual interpretation, multi-lead ECG signals are more accurate than single-lead signals for AF diagnosis. For example, if the P wave in Lead II is positive and the P wave in precordial Lead V1 is negative or biphasic on a background of regular or equal RR intervals, SR is highly likely . In theoStroke and stroke-related complications can be prevented with early diagnosis of AF and initiation of oral anticoagulant therapy.AF-induced electrical and/or mechanical remodeling of the heart can be averted with rhythm and/or heart rate control.AF-associated heart failure can be prevented and/or ameliorated with specific heart failure drugs.AF-associated hospitalizations and healthcare expenditure can be reduced through optimal preventive management.The chief motivation for developing AF screening systems is to detect AF accurately and reliably so that:Few public ECG databases are available for DL model training, which require a high volume of input data to develop accurate and robust models.Paroxysmal AF, which exacts similar stroke risk as persistent and permanent AF, may escape detection on 12-lead ECG and/or short-duration ECG monitoring.Related arrhythmia like AFL that are morphologically distinct from AF and yet also carries similar stroke risk as AF has only been included in selected studies.Possible limitations of AF screening include:AF has effective preventive and therapeutic strategies and meets the criteria for cost-effective disease screening. Randomized controlled trials are incipient currently but interest is growing apace. When developing a DL model, consideration of the feasibility for implementation in cloud-based applications for real-world, real-time monitoring is imperative. Wearable technology provides low-cost and practical data input options for arrhythmia screening, and DL models are an efficient framework for signal analysis and interpretation.In this study, we discussed 24 papers on DL methods developed for automatic detection of AF on ECG-based signals. Most of the studies used CNN models, which yielded good results with ECG as well as HRV signals. This study can serve as a guide for researchers interested in designing optimal DL models for AF detection with the least computational costs. Aside from the limitations of deep learning methods , another significant limitation of the study is the absence of a systematic search method. On some general journal search engines, the keywords determined were used to conduct a search. In future studies, this search strategy and journal search engines can be expanded to conduct a more systematic review.


Adolescents and young adults (<25 years) working in agriculture are at greater risk of injury than youth working in other industries. Supervisors play an important role in protecting these young workers who lack workplace experience and whose bodies and brains are still developing. A theoretically based approach was used to develop an online training for supervisors of young agricultural workers. The training addresses an expanded view of occupational safety that not only addresses injury prevention, but also focuses on health promotion and worker well-being using a Total Worker Health approach. A pre-post/post study design was used to evaluate the training. Questionnaires included demographics, workplace characteristics, knowledge, beliefs about protecting young workers, and supervisors’ communication behaviors. One-hundred-eighty-two participants completed all parts of the efficacy trial. A post-test administered immediately after completing the training, indicated that supervisors had greater understanding of the risks to young workers and at 3-month follow-up were more likely to engage in communication behaviors to protect the safety and health of young workers. Positive changes in when, how, and under what circumstances supervisors talk about safety and health occurred. Establishing patterns of protective behaviors in the workplace can have lifelong impact, particularly among young workers. Youth begin working in agriculture work at younger ages and in more hazardous jobs than youth in other industries . In the http://www.cdc.gov/niosh/twh, accessed on 31 July 2021) [Interventions that focus on protection from work-related hazards and address health and well-being are more effective than addressing just one ,17,18,19ly 2021) ,22,23. YTo protect this vulnerable population, an online training using a TWH approach for supervisors of young agricultural workers was developed using several theoretical perspectives to increase its likelihood of efficacy . The goaRecognizing the range of settings where young agricultural workers are employed, “supervisor” included employers, parents, and educators. Adults who hire, teach, or train young workers (less than 21 years of age) were invited to participate in the study. A project website was created with information describing the study and contained a link to the study materials. Recruitment began in October 2017 and continued through August 2019. A multipronged approach was used to recruit participants that included conference presentations and recruitment at events, use of an agricultural list-serv, and targeted emails and social media posts through partner organizations and commodity groups.An online training for supervisors of young agricultural workers was developed using a TWH approach and several theoretical perspectives to increase its likelihood of efficacy . The theThe supervisor training begins by describing the threat to young workers in agriculture. This includes developmental differences , the impact of inexperience, risky behaviors that tend emerge in young adulthood , and the difficulties of balancing work, school, and home which impact stress and fatigue. Efficacy was addressed by providing and demonstrating specific skills supervisors can use to reduce threat. These include health communication skills to ensure the delivery of effective occupational safety and health training , with a Questionnaires were used at pre-test, immediate post-test, and at a three-month follow-up. The pre-test questionnaire collected information on demographics, workplace characteristics, beliefs about protecting young workers , and supervisors’ communication behaviors . A 25-item knowledge pre-test was administered immediately prior to beginning training. Upon completion of each module within the training, changes in knowledge were assessed (immediate post-test). Attitude change was assessed after all training modules were completed. Three months after completing the training, participants were invited to complete a follow-up questionnaire (3-month post-test). This questionnaire included workplace characteristics, knowledge, beliefs, supervisors’ communication behaviors, and open-ended questions on training impact. All questionnaires were pilot tested during the development of the training .A pre-post/post design was used to evaluate training efficacy. All study procedures occurred online. Prospective participants were directed to the project website with a link to study materials. Following online consent, participants completed a pre-test questionnaire lasting approximately 15 min. The pre-test questionnaire collected information on demographics, workplace characteristics, beliefs about protecting young workers , and supervisors’ communication behaviors . Supervisor beliefs were assessed using a 5-type Likert scale ranging from “strongly disagree” to “strongly agree.” Participants were then directed to the training module, where they completed a 25-item knowledge pre-test prior to beginning the training. The training took approximately 45 min to complete and included knowledge checks throughout. The knowledge checks were used to assess post-training knowledge (immediate post-test). Next, participants completed an immediate post-test questionnaire. Participants received $50 for completing the pre-test questionnaire, training, and immediate post-test questionnaire. Three months after completing the training, participants were invited to complete a follow-up questionnaire, approximately 20 min in length. This questionnaire included workplace characteristics, knowledge, beliefs, supervisors’ communication behaviors, and open-ended questions describing how they applied the training. Participants received an additional $50 for completing the 3-month post-test questionnaire. A list of all measures can be found in Descriptive statistics were calculated for all measures to characterize demographic and occupational items. McNemar’s test was used to examine behavior changes  at baseline and 3-month follow-up. Linear mixed effects models were used to examine the means of variables at different time points regarding changes in beliefs. Data analysis was completed with SAS 9.4 . An alpha of 0.05 was used for testing significance.The analyses that follow include participants that completed all parts of the study.Three-hundred-ninety-two respondents consented to participate in the study. Of these, 319 completed the pre-test questionnaire, 236 completed the training, 204 completed the immediate post-test questionnaire, and 185 completed the 3-month follow-up questionnaire. One-hundred-eighty-two participants completed all parts of the study. At each successive step, absolute response rates were 81.0%, 74.0%, 86.4%, and 90.7%, respectively. Cumulative response rates were 81.0%, 59.9%, 51.8%, and 47.0%, respectively. Participants represented 31 states, 2 U.S. territories (District of Columbia and Puerto Rico), and 3 provinces in Canada  .The majority of participants were female and agricultural education teachers. On average, participants had worked in agriculture nearly 18 years. Two-thirds had at least five years of experience supervising young agricultural workers . At pre-Pre-test knowledge scores were high (ollow-up .The teach-back method is effective in training workers under the age of 21) also included the option “I don’t know what the teach-back method is.” Agreement with the belief statement increased for all items from pre-test to immediate post-test and changes were sustained at the 3-month follow-up. Only two beliefs did not show significant changes following the training . The teach-back method  when they start a new task, (2) when they are hired, and, (3) after an injury or near miss between the pre-test and the 3-month post-test . Similarn = 172) of participants provided comments; of these, 91% (n = 156) indicated they were more prepared to effectively supervise young workers after taking the training. Of the supervisory practices addressed in the training, teach-back was the most popular, with 23% (n = 40) of participants referencing it in their comment. One participant shared the following story of how they used the teach-back method to ascertain a young worker’s understanding of a task:1. “I had a worker who seemed to know what they were doing. Then when I asked them to talk me through the steps and show me, they didn’t know what to do. They said they were embarrassed to admit they didn’t know. Now they know the proper procedure.”At the 3-month post-test, participants were presented with optional open-ended questions asking how the training helped them and to share a story about a young worker they supervised. Ninety-five percent  of young agricultural workers. Adolescents and young adults <25 years) working in agriculture are at greater risk of injury than youth working in other industries ,2,3. Sup5 years wA pre-post/post study design was used to evaluate the training among supervisors. One-hundred-eighty-two participants completed all parts of the efficacy trial. Participants included agricultural educators, farmers and producers, supervisors, and safety and health professionals. The overall reaction to the training was positive. Positive changes in knowledge, beliefs, and supervisors’ communication behaviors were observed.In this efficacy trial, training intervention produced significant and sustained changes in beliefs and supervisors’ communication behaviors that are likely to impact the occupational health and safety of young, vulnerable, agricultural workers. Although knowledge increased at immediate post-test, it was not sustained at 3-month follow-up. At pre-test, knowledge was high, which may reflect the years of supervisory experience of our participants. These findings indicate that immediately after completing the training, supervisors had a greater understanding of the risks to young workers. At 3-month follow-up they were more likely to engage in behaviors to protect the safety and health of young workers than they were prior to taking the training. Positive changes in when, how, and under what circumstances supervisors talk about safety and health with their young workers occurred. Establishing patterns of protective behaviors in the workplace (including family farms) can have lifelong impact, particularly among young workers. While additional research is needed, these findings indicate that an online training targeting supervisors is a promising approach to changing safety and health behaviors in agricultural settings.https://cultivatesafety.org, accessed on 31 July 2021). They are also promoted through the Great Plains Center for Agricultural Health as part of other online modules addressing agricultural safety health and integrated into the curriculum of Agricultural Safety and Health courses at the University of Iowa. These materials can be used by anyone who assigns tasks in the workplace, provides training, develops policies to address safety, provides supervision, or has regular and frequent opportunities to communicate with workers. Although designed for employers, the training may also be relevant to parents who have children working on a family farm as well as high school agricultural teachers, 4-H leaders, and FFA advisers.Dissemination activities included promoting the training through multiple channels to a broad audience. Materials were disseminated through the Agricultural, Forestry, and Fishing Centers of Excellence funded by the National Institute for Occupational Safety and Health (NIOSH AgFF) as well as through programs addressing agricultural safety and health, employers, commodity groups, extension, and agricultural educators. They are listed as a resource as part of the Cultivate Safety materials  and the Institute for Public Health Practice  websites to house the intervention and other materials developed will ensure that the evidence-based intervention remains freely accessible to key audiences.In addition to the online training, The development of material involved the integration of health promotion, health protection, TWH, and evidence-based communication approaches to provide supervisors the tools to increase the likelihood of impacting worker behavior. Behaviors emphasized in the training  are transferable to many safety and health risks. These behaviors are not limited to specific agricultural operations, but rather fit all workplaces.The efficacy trial demonstrated changes in knowledge, beliefs, and supervisors’ communication behaviors. These short-term and moderate-term (3 month) impacts are essential to longer-term changes in morbidity and mortality ,35. We sStudy participants included those with many years of experience supervising young workers in agriculture. Interestingly, changes in beliefs and behaviors were observed among these more experienced supervisors. Although designed primarily for employers, the most eager study participants in the efficacy trial were high school agricultural education teachers. This is a population that is ripe for intervention and dissemination as intermediaries. They have access to a large number of youth and can build expectations about workplace practices and build confidence in youth to talk about safety and health. Future research should target this influential population. Promoting safety and health can lead to changes in the workplace that will not only reduce injuries, but also promote health and well-being on and off the farm.This project was unique in its comprehensive approach to addressing the combination of both preventing workplace injuries  and promoting healthy lifestyle behaviors, utilizing a Total Worker Health approach. Moreover, the addition of workplace policies expanded the intervention to address multiple levels of influence in the Social Ecological Model: supervisors and workplaces. The Extended Parallel Process Model was used to address elements in the intervention necessary to change supervisors’ behaviors. An efficacy trial demonstrated training effectiveness, specifically positive increases in knowledge, beliefs, and supervisors’ communication behaviors.


Analyses of IGHV gene mutations in chronic lymphocytic leukemia (CLL) have had a major impact on the prognostication and treatment of this disease. A hallmark of IGHV-mutation status is that it very rarely changes clonally over time. Nevertheless, targeted and deep DNA sequencing of IGHV-IGHD-IGHJ regions has revealed intraclonal heterogeneity. We used a DNA sequencing approach that achieves considerable depth and minimizes artefacts and amplification bias to identify IGHV-IGHD-IGHJ subclones in patients with prolonged temporal follow-up. Our findings extend previous studies, revealing intraclonal IGHV-IGHD-IGHJ diversification in almost all CLL clones. Also, they indicate that some subclones with additional IGHV-IGHD-IGHJ mutations can become a large fraction of the leukemic burden, reaching numerical criteria for monoclonal B-cell lymphocytosis. Notably, the occurrence and complexity of post-transformation IGHV-IGHD-IGHJ heterogeneity and the expansion of diversified subclones are similar among U-CLL and M-CLL patients. The molecular characteristics of the mutations present in the parental, clinically dominant CLL clone (CDC) differed from those developing post-transformation (post-CDC). Post-CDC mutations exhibit significantly lower fractions of mutations bearing signatures of activation induced deaminase (AID) and of error-prone repair by Polη, and most of the mutations were not ascribable to those enzymes. Additionally, post-CDC mutations displayed a lower percentage of nucleotide transitions compared with transversions that was also not like the action of AID. Finally, the post-CDC mutations led to significantly lower ratios of replacement to silent mutations in VH CDRs and higher ratios in VH FRs, distributions different from mutations found in normal B-cell subsets undergoing an AID-mediated process. Based on these findings, we propose that post-transformation mutations in CLL cells either reflect a dysfunctional standard somatic mutational process or point to the action of another mutational process not previously associated with IG V gene loci. If the former option is the case, post-CDC mutations could lead to a lesser dependence on antigen dependent BCR signaling and potentially a greater influence of off-target, non-IG genomic mutations. Alternatively, the latter activity could add a new stimulatory survival/growth advantage mediated by the BCR through structurally altered FRs, such as that occurring by superantigen binding and stimulation. Chronic lymphocytic leukemia is a relatively common, yet broadly divergent disease , 2. SomeThe IGHV-mutation status used in the clinic is usually determined by standard Sanger DNA sequencing , and rarIn addition to corroborating that intraclonal V-region diversity exists, the latter study highlighted several important considerations that could be incorporated into future studies of the process . These eIn light of this background, we have addressed the frequency and degree of intraclonal IGHV-IGHD-IGHJ heterogeneity in CLL, the extent that the subclones exhibiting this contribute to the clonal burden, the characteristics of the post-transformation mutations, and the association of intraclonal IGHV-IGHD-IGHJ diversity with clinical outcome. To tackle these issues, we used an NGS approach of considerable depth that incorporates Unique Molecular Identifiers (UMIs) to minimize technical artefacts and PCR amplification bias  to studyin vivo; the fraction of mutations bearing SHM marks is considerably less than in the parental clone and these are inconsistent with selection for enhanced antigen binding; and finally some of the features of the post-transformation mutations might be useful adjuncts to the standard U-CLL vs. M-CLL IGHV mutation status prognostic approach.Our study extends previous findings and documents that: the intraclonal IGHV-IGHD-IGHJ diversification process is virtually ubiquitous and takes place with equal frequency and extent in both U-CLL and M-CLL patients; certain subclones expressing new IGHV-IGHD-IGHJ mutations can be significantly expanded This study was approved by the Institutional Review Boards of Northwell Health and of Dana Farber Cancer Center. Written informed consent was obtained in accordance with the Declaration of Helsinki. PBMCs were isolated from untreated CLL patients by density gradient centrifugation , suspended in in RPMI1640 medium with 50% FBS, and stored in liquid nitrogen until used.+CD5+ B cells were sorted directly into tubes containing Dynabeads Oligo(dT) (ThermoFisher) lysis buffer and stored at −80°C.PBMCs from 45 CLL samples collected at Northwell Health were incubated with V500 anti-CD19 (BD Biosciences) and PE-cy7 anti-CD5 (Invitrogen). After excluding dead cells using Sytox Blue (ThermoFisher), live CD19For the 15 CLL PBMC samples collected at Dana Farber Cancer Institute, B cells were isolated using RosetteSep negative selection prior to density gradient centrifugation if the white blood cell count was less than 25,000/ml or the absolute lymphocyte count was less than 20,000/ml.Library preparation and sequencing were performed as described  and illuhttps://www.ncbi.nlm.nih.gov/Traces/study/?acc=PRJNA673787&o=acc_s%3Aa).The product was quantified with Qubit (ThermoFisher) and 1–10 ng was used to add the Illumina Index with the Nextera XT kit (Illumina). Pooled libraries were sequenced with MiSeq Illumina . Raw data are available at SRA . Pairwise multiple comparisons were carried out for differences among the four groups without adjustment for multiple testing. All results were considered statistically significant if P <0.05.A Cox proportional hazards regression analysis was used to test mutation, complexity and their interaction (mutation × complexity) in the model. The “interaction” between mutation and complexity measures whether the effect of complexity in the For 27 samples, we sequenced four or eight replicates, each containing 25,000 cells from the same blood draw , our lib+ B cells in a CLL patient is very low, we were able to perform a “manual” assessment of the clonal attribution to correct possible false positives, false negatives, and PCR artifacts such as chimeras that might have escaped error correction. The phylogenetic tree for each CLL clonal family was inferred with the R package Alakazam (https://igraph.org).For each sample, we identified the CLL clonal family with ChangeO , using aAlakazam , and theC/GYW) hotspots, we compared the ratio (h:n) of mutations targeting hotspots (h) to non-hotspots (n) to the expected proportion (number of hotspots/number of G:C sites) using a binomial test (C/GA) and Polη (WA/TW), where the expected proportion used A:T sites in denominator); these are similar to Signatures 2 and 13 and to Signature 9, respectively, as defined in the Catalogue of Somatic Mutations in Cancer (COSMIC) database  or bear). When examining Polη-related mutations, many fewer samples exhibited significant targeting (3.4%) . So, the dominant identifiable mutational signature found in the post-CDC samples is AID-related. Nevertheless, this represented only ~25% of the samples.Notably, although there were some sequences bearing APOBEC and the other COSMIC signatures, none of the samples as a whole bore significant features consistent with these mechanisms (not shown). As expected, AID and Polη signatures were identified. Twenty-nine percent of the samples exhibited significant AID targeting, with no significant differences between U-CLL and M-CLL , we also used an extended database containing 2,084 CLL IGHV-IGHD-IGHJ CDC sequences. When employing the larger data, still no samples bore significant evidence for APOBEC and COSMIC Signatures 1 and 5.Next, we carried out similar analyses to determine if the mutational targeting found in the parent CDCs was like that found in the post-CDC samples and how these compared with those in non-productive IGHV-IGHD-IGHJ rearrangements found within the same patients. Notably, for the non-productive sequences, AID mutations accounted for 33% of the CDC mutations  and Polη for 20%  and of the IGHV-mutated cases in the extended CDC set . When analyzing the degree of IGHV-IGHD-IGHJ complexity to the post-CDC mutations, the High complexity samples had a significantly lower Ts : Tv than the Low complexity samples . So, whereas the CDC samples bearing IGHV mutations have Ts : Tv similar to that expected, the post-CDC samples as well as the non-productive sequences have ratios very significantly lower than these.Since AID induced mutations usually result in more base transitions (Ts) than transversions (Tv), usually ~1.5 Ts to 1 Tv –27, we aIn summary, these analyses suggest that the majority of mutations occurring in the post-CDC period are much less attributable to AID based SHM based on their type and targeting and the repair mechanisms employed than those mutations that developed in the CDC.P value. The selection strength measure is analogous to the standard R:S ratio, with positive values indicating greater than expected R mutations, and negative values indicating fewer than expected R mutations. In an adaptive immune response, therefore, an increase in R:S would be expected in the VH CDRs, leading to a structural change in these regions and implying positive selection by antigen and a decreased R:S would be expected in the VH FRs, implying negative selection and hence avoidance of structural changes in the Ig as a whole. Thus, true underlying selection for antigen is reflected by a structurally intact Ig with positive selection strength values in CDRs and negative values in FRs.Finally, we asked if there was evidence for antigen selection in the post-CDC mutations using the BASELINe algorithm that detects and quantifies mutation selection using large datasets such as those generated here by NGS Figure 6-CD27-  B cells, as controls . This pattern resembled that of MZ B cells. However, the selection strength in the FRs was like the mMem, sMem, and DN subsets. In conspicuous contrast, there was a striking difference for the post-CDC mutations, both High and Low complexity, in that there was a markedly reduced and negative selection in the CDRs and positive selection strength for changes in the FRs. Finally, the IGHV-IGHD-IGHJ non-productive rearrangements bear the least evidence for VH CDR selection for mutations but a selection for VH FR mutations somewhat less than that for the post-CDC mutations.Together, the R:S in the VH CDRs and FRs of the post-CDC mutations are categorically inconsistent with selection for enhanced antigen binding as a reason for the distribution of non-synonymous mutations.Since the presence or absence of significant numbers of IGHV gene mutations has proven to be an excellent prognostic indicator, we asked if intraclonal differences in IGHV-IGHD-IGHJ sequence measured throughout the entire rearrangement might provide additional information. For the 61 cases that displayed intraclonal heterogeneity, neither the percentage of intraclonal IGHV-IGHD-IGHJ variants nor the percentage of the PSCs, including or excluding the CDC, correlated significantly with time to first treatment (TTFT) (P = 0.22). Accordingly, when the interaction term was deleted from the model and the cases were divided into the U-CLL and M-CLL subgroups, there was, as expected a significant difference in TTFT between U-CLL and M-CLL  had a significantly shorter TTFT compared to both the M-CLLLow (P <0.0102) and M-CLLHi (P <0.008) complexity groups . See When the clinical course (TTFT) of the cohort was analyzed using Cox regression, no significant interaction between mutation and complexity was observed (P = 0.7) for OS. Although the estimates of the hazard ratios for U-CLLHighvs. U-CLLLow and M-CLLHighvs. M-CLLLow appeared to differ numerically , the insignificant P-values, wide confidence intervals, and lack of pairwise differences between groups do not support a mutation × complexity interaction for OS.Additionally, there was no significant mutation × complexity interaction , with an average of 540 clonal variants observed per sample.In addition to the surprising frequency at which intraclonal heterogeneity occurred, the size that the PSC can achieve in a given patient was also unexpected. When comparing the frequency of individual PSCs to the other intraclonal sequences (excluding the CDC), this reached 9% and was much larger (up to 88%) in some patients. Additionally, when using the absolute lymphocyte count in a patient’s blood at the time of sample collection to calculate numerical size, the PSC was almost always ≥10 cells/µl and could reach >3,600 cells/µl. In a healthy person, the latter level of clonal B cells would be consistent with monoclonal B-cell lymphocytosis , a documvia the microenvironment prior to transformation.Finally, the presence, frequency, and extent of post-transformation IGHV-IGHD-IGHJ mutations occurred equally in U-CLL and M-CLL. This indicates that the initial mutation load does not affect the ability of CLL clones to develop IGHV-IGHD-IGHJ diversity after leukemia develops. The finding also suggests strongly that the lack of mutations found in a U-CLL clone is not due to an inherent inability to carry out SHM. Rather, it strongly implies that the absence/scarcity of IGHV mutations in the normal B lymphocyte that converted to a CLL cell was dictated by signals delivered to the cell These findings also suggested that when individual IGHV-IGHD-IGHJ subclones are found in larger numbers, exemplified by the PSCs, these cells bear a biologic advantage. We, therefore, analyzed the characteristics of the mutations occurring in the post-CDC setting, trying to define the mutation machinery responsible for the downstream mutations. We focused not only on mechanisms involved in targeting the IG loci, and thereby responsible for the generation of antigen-binding diversity in normal B lymphocytes, but also on mechanisms that could account for mutations found outside IG loci in CLL clones. The former included AID and error prone Polη, and the latter APOBEC and Signatures 1 and 5 in the COSMIC database. Although certain clones significantly expressed AID and/or Polη characteristics, none significantly exhibited signatures in the IGHV-IGHD-IGHJ region consistent with the actions of APOBEC or COSMIC Signatures 1 and 5. The approach also revealed that the percent of the post-CDC mutations within a given sample that could be assigned to the actions of AID and Polη was decreased very significantly when transitioning from the CDC to the post-CDC settings, suggesting that most of the mutations occurring post-leukemic transformation were not the consequences of an AID/Polη mechanism(s).Because of these findings, we carried out similar analyses on the CLL sequences in the CDC set. Since not all the CDC samples bore IGHV somatic mutations (49 of 62), we also scrutinized a larger database of CLL clones comprised of 2,084 sequences. Both the CDC set directly related to this study and the expanded CLL set revealed the same features as the post-CDC mutations, with samples exhibiting significance for AID and Polη (AID > Polη), and none for the mutation mechanisms that affect non-IG loci in CLL. However, the percentage of mutations attributable to AID and Polη was very significantly higher in both CDC sets than for the post-CDC mutations, confirming the impression that the influence of AID/Polη mechanisms decreased substantially after leukemic transformation. When including comparisons of mutations detected in clonally expanded, non-productive IGHV-IGHD-IGHJ rearrangements, these mutations bore less evidence for AID and Poln actions than the CDC but more than the post-CDC mutations.Since AID and Polη are essential and important components of SHM, respectively, we also investigated another feature of SHM, the ratio of mutations that represent transitions to transversions. In the normal SHM setting, the Ts : Tv is ~ 1.5:1. Notably, the Ts : Tv for all post-CDC mutations, productive or not, was considerably lower than this, and when the degree of mutational complexity was added to this analysis the High complexity samples had a more significantly lower Ts : Tv than the low complexity samples. Notably, when restricting the analysis to mutations bearing an AID targeting signature, the Ts : Tv was still below that expected for SHM.Finally, we investigated if the post-CDC mutations were consistent with selection for improved antigen binding, based on comparing the expected with the observed R to S mutation ratio (R:S), using as controls normal mature B cell subsets as well as the non-productive IGHV-IGHD-IGHJ rearrangements identified. This indicated that, unlike the mutations found in memory and DN B cells and to a somewhat lesser extent in the starting CDC and MZ B cells samples, mutations in the productive post-CDC and non-productive IGHV-IGHD-IGHJ rearrangements showed a marked decrease of the R:S in CDRs and a significant increase in the R:S in FRs. These features are the opposite of what is expected for the selection of AID-mediated mutations that improve the affinity of antigen binding and for the preservation of an intact membrane IG and B-cell receptor signaling.Collectively, the analyses of the type and targeting of the post-CDC mutations and the predicted consequences of these on selection for antigen reactivity suggest that many of these mutations do not reflect outcomes seen with typical, AID-based SHM that, in the normal setting, would be subsequently selected for improved antigen binding and associated adaptive protection. The findings also suggest that the mutations that develop after leukemic transformation differ, in multiple ways , from those that occurred before/up to the time of transformation.Thus, are those mutations occurring downstream of the transformation event the result of deranged SHM mediated by AID or of another process? If the former, the derangement might be the consequence of a direct effect on the leukemic B cell occurring upon transformation. Additionally, the discrepancy might reflect a change in microenvironmental influences in play up to the time of transformation compared to subsequently. Certainly, the lymphoid microenvironment in which the normal B lymphocyte that converted into a CLL cell developed and was shaped is different from the one to which the subclones were exposed after leukemic transformation. Simplistically, the former was normal, structurally and functionally, whereas the latter was likely dysregulated in both ways. This change is at least due to the expansion of the leukemic clone, resulting in major degradation of the architecture of lymph nodes , therebyAlternatively, the detected post-transformation mutations could, in part, be physiologic. In this case, the strict avoidance of replacement mutations in the CDRs and the deduced selection for replacement mutations in the FRs could provide the post-CDC subclones an alternative way to receive beneficial interactions, such as allowing superantigen binding to structurally altered FRs. In this regard, Staphylococcal superantigen can bind to FR3  or to a The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: NCBI BioProject; PRJNA673787.The studies involving human participants were reviewed and approved by Institutional Review Boards of Northwell Health and of Dana Farber Cancer Center. The patients/participants provided their written informed consent to participate in this study.DB and NC conceived the project and designed the experimental approach. DB, CT, SV, and TM performed experiments and data analyses. MC supplied IGHV-IGHD-IGHJ sequences from normal human B cell subpopulations. JB, SK, SF, KRR, JK, JB, and SA provided clinical samples and clinical correlations. AM, FG, SB, MC, FM, and MS offered interpretations and conceptual insights. All authors contributed to the article and approved the submitted version.DB received funding from the European Union’s Horizon 2020 Research and Innovation Programme under the Marie Skłodowska-Curie grant agreement No 794075. JB was supported by funding from the NIH’s National Cancer Institute through the R01 grant CA 213442. NC and KRR thank the Karches Family, The Nash Family Foundation, The Marks Foundation, and the Jean Walton Fund for Leukemia, Lymphoma, and Myeloma Research for their support of the Feinstein Institutes’ CLL Research & Treatment Program. MS and TM are supported by a Multi-PI grant 1R01AI132507-01A1 from NIAID.JB has served as a consultant for Abbvie, Acerta, Astra-Zeneca, Beigene, Catapult, Dynamo Therapeutics, Eli Lilly, Juno/Celgene/Bristol Myers Squibb, Kite, MEI Pharma, Nextcea, Novartis, Octapharma, Pfizer, Rigel, Sunesis, TG Therapeutics, and Verastem; received honoraria from Janssen; received research funding from Gilead, Loxo, Sun, TG Therapeutics, and Verastem; and served on data safety monitoring committees for Invectys. NC has received research funding from Verastem, Argenx, and Janssen.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.