Despite the rising incidence of emergency department (ED) visits for sports-related concussion, the frequency and characteristics of youth leaving before being seen are unknown.National estimates of ED visits for sports-related head injuries among youth (10–18 years) were generated for 2006–2017 using the National Electronic Injury Surveillance System. Logistic regression models estimated the odds of leaving without being seen across patient characteristics and time.From 2006 to 2017, 985,966  ED visits were identified for sports-related concussions, of which 5015 (95% CI: 3024–7006) left without being seen.Youth with sports-related concussion must receive timely care and ED improvements may reduce rates of leaving without being seen. Over the last decade, more and more youth have visited the emergency department (ED) for sports-related concussion. At the same time, EDs are becoming more crowded, with long wait times and many patients leaving before being evaluated. An increase in the proportion of youth with concussion who left without being seen (LWBS) is possible. We used national data of ED visits to examine the rate of youth (aged 10–18 years) who LWBS for sports-related concussion from 2006 to 2017. While the rate of LWBS has not changed over time, more nonwhite youth and youth who were not injured at school left before being evaluated. Each year in the USA, an estimated 1.1–1.9 million youth experience sports- and recreation-related concussions , return-Overcrowding is prevalent in EDs across the USA ,9. Long Return-to-play legislation and heightened concussion awareness have contributed to an increase in ED visits for suspected concussions (including among lower acuity injuries) ,11,12. WThis retrospective, cross-sectional study used data from the National Electronic Injury Surveillance System (NEISS), a national probability sample of 100 US hospitals’ emergency departments (EDs), each reporting information associated with product-related injuries . The NEISubjects included were youth, aged 10–18 years, who were presented to EDs with sports-related head injury between 1 January 2006 to 31 December 2017. In this age range (middle and high school), most youth report involvement in at least one organized sport ,15, and Return-to-play legislation applies to youth involved in school sports; the sample was, therefore, restricted to injuries occurring at school or a place of recreation. Injuries coded as the latter were included because many schools train or host sport competitions outside school property. Patient demographic characteristics included sex , age  and race . The NEISS database does not report detailed information on hospital or patient characteristics, such as location, insurance status, International Classification of Diseases (ICD)-10 diagnosis codes, procedures, time of visit or mode of arrival.To examine the potential association between LWBS and time, three time periods were designated based on national adoption of return-to-play legislation. The time period before any US state passed return-to-play legislation (‘prelegislation’) occurred from 1 January 2006 to 31 December 2008. The interim time period spanned 1 January 2009–31 December 2014. The ‘postlegislation’ time period, after every state had adopted return-to-play legislation, was from 1 January 2015 to 31 December 2017.Statistical analyses were conducted using Stata/MP version 14 , using svy commands to account for survey design. National estimates of injuries and 95% CIs were calculated using NEISS sample weights. Demographic and injury characteristics were summarized across cases. The proportion of ED visits that LWBS was calculated as the number of cases with discharge disposition coded as LWBS, divided by total number of concussion-related ED visits in each time period.Bivariate relationships between factors  and discharge status (LWBS vs not) were evaluated using Pearson's design-based chi-squared tests. Multivariable logistic regression models estimated the odds of LWBS for each factor while controlling for co-variates. Results were presented as odds ratios with 95% CIs, demonstrating the relative odds of LWBS, given each co-variate of interest . Time period was included in models as a categorical variable in order to identify potential variation in LWBS over time. As a sensitivity analysis, we created linear spline time functions for each time period (using the Stata function mkspline) and included these in the model  in order to identify potential variation in LWBS across time periods. This study was deemed exempt from human subjects review by the Washington State University Institutional Review Board due to the de-identified nature and public availability of the study data.From 1 January 2006 to 31 December 2017, 985,966  ED visits among youth aged 10–18 years with sports-related head injuries were identified. Most were males (70.8%) whose injuries occurred at recreational locations (away from school) and were associated with football, basketball and soccer .Post hoc power calculation showed sufficient power (100%) to detect this difference in LWBS across time periods.In total, 5015 (95% CI: 3024–7006) ED visits resulted in LWBS, representing an estimated 0.51% (95% CI: 0.38–0.68) of all cases. Cases that resulted in LWBS were more frequently nonwhite or unknown race (versus white), had a diagnosis of internal organ injury to the head (versus concussion) and occurred at a place of recreation rather than at school . Indeed, a study of undergraduate students with history of concussion (mean age at injury: 14 years) reported that a third did not seek care for their injury . CommonlWe found a significant association between LWBS and nonwhite or unknown race, compared with white race. Across regression models, nonwhite youth had two- to three-times higher odds of LWBS than their nonwhite peers . This apparent disparity in ED disposition by race is concerning. Previous research has shown a similar association between nonwhite race and LWBS ,29,30; hEducational efforts can encourage youth with sports-related concussion to receive timely care. Programs, such as the Centers for Disease Control and Prevention's  Head's Up campaign, raise awareness of the importance of timely evaluation and treatment of sports-related concussion. The continuation and expansion of these efforts is necessary, including use of innovative communication and outreach strategies to reach vulnerable populations, such as rural or low-income communities.This study has several limitations. First, the NEISS was designed for injury surveillance purposes and, therefore, it lacks detailed patient information that would be informative when examining LWBS. For example, several known factors associated with LWBS are not captured by NEISS  . The codDespite these limitations, this study adds to the literature by providing national estimates on the incidence of LWBS for youth who present for ED care following sport-related concussion, as well as factors associated with LWBS in this population. This information lays the foundation for future research to prevent LWBS among youth seeking ED care for concussion and examine outcomes after LWBS.Increasing numbers of youth with sports-related concussion are seeking ED treatment for their injuries. From 2006 to 2017, 5015 youth with sports-related concussion left the ED prior to receiving treatment, representing a small percentage of the overall proportion of youth seeking concussion care at the ED (0.51%). It is important to ensure the treatment needs of youth with sports-related concussion are met. To further understand the impact of LWBS on youth with concussion, future research examining outcomes, including physical health, mental health, academic and social outcomes, among youth who LWBS and those who did not is warranted.As Americans increasingly rely on the ED as a primary source of medical care, wait times will continue to rise and crowding will worsen. EDs may not serve the needs of youth who must obtain medical clearance before returning to play following sports-related concussion; instead, they may shift to seek care through primary or urgent care. For minor concussions, this may be an appropriate approach to obtaining timely care. However, for more severe injuries, first seeking care elsewhere may result in delays that could contribute to negative outcomes following concussion.Sports-related concussions are a growing concern in the USA. Increasingly, youth are seeking care in the emergency department following sports-related concussion.This study examined emergency department data for youth with sports-related head injury who left the emergency department before receiving treatment to evaluate changes over time and identify characteristics of youth who left compared with those who did not.From 2006 to 2017, 5015 youth with sports-related head injury left the emergency department before receiving care.Youth identified as nonwhite were significantly more likely to leave the emergency department without being seen, compared with white youth.Following sports-related head injury, it is important that all youth receive timely, high-quality care in order to identify potential complications and ensure a safe and speedy recovery.


Objective: In two independent study arms, we determine the effects of strength training (ST) and high-intensity interval training (HIIT) overload on cardiac autonomic modulation by measuring heart rate (HR) and vagal heart rate variability (HRV).Methods: In the study, 37 well-trained athletes  were subjected to orthostatic tests (HR and HRV recordings) each day during a 4-day baseline period, a 6-day overload microcycle, and a 4-day recovery period. Discipline-specific performance was assessed before and 1 and 4 days after training.Results: Following ST overload, supine HR, and vagal HRV (Ln RMSSD) were clearly increased and decreased , respectively, and the standing recordings remained unchanged. In contrast, HIIT overload resulted in decreased HR and increased Ln RMSSD in the standing position , whereas supine recordings remained unaltered. During the recovery period, these responses were reversed . The correlations between changes in HR, vagal HRV measures, and performance were weak or inconsistent. At the group and individual levels, moderate to strong negative correlations were found between HR and Ln RMSSD when analyzing changes between testing days  and individual time series, respectively. Use of rolling 2–4-day averages enabled more precise estimation of mean changes with smaller confidence intervals compared to single-day values of HR or Ln RMSSD. However, the use of averaged values displayed unclear effects for evaluating associations between HR, vagal HRV measures, and performance changes, and have the potential to be detrimental for classification of individual short-term responses.Conclusion: Measures of HR and Ln RMSSD during an orthostatic test could reveal different autonomic responses following ST or HIIT which may not be discovered by supine or standing measures alone. However, these autonomic changes were not consistently related to short-term changes in performance and the use of rolling averages may alter these relationships differently on group and individual level. Efficient training provides sufficient exercise stimuli to enhance athletes' performance capacities while avoiding sustained non-functional overreaching or underrecovery  with HR-based measures [HR and HR variability (HRV) indices] is an attractive option for testing due to its non-invasiveness and time-efficiency when performed for an entire training group or team. Technological developments within the last few decades have enabled practitioners to use portable devices to obtain accurate beat-by-beat recordings  responses likely differ with training context  . From the original samples of 23  individuals performing ST and 22  individuals performing HIIT, only 19  and 18 , respectively, participants provided a sufficient quantity of resting HR recordings to be included in this investigation (we set a minimum of three baseline recordings and a maximum of one missing recording during recovery). The general subject characteristics are presented in IFT) of at least 16 km/h for females and 19 km/h for males, and a minimum of 5 years of team sport training.The following inclusion criteria were used for the ST group: estimated one-repetition maximum (1RM) for a parallel squat of at least 80% of body mass for females and 120% of body mass for males and a minimum of 3 years of lower-body strength training with at least two strength training sessions per week. The inclusion criteria for the HIIT group were as follows: peak velocity during the 30-15 Intermittent Fitness Test and familiarization trials for training and testing procedures were conducted 1 week before baseline testing. Discipline-specific maximum effort tests were used as criterion measures to assess fatigue- and recovery-related changes in performance. HR(V) measures were recorded daily during the main 14-day study period . An overview of the experimental design is shown in A repeated-measures study was used to investigate the effects of short-term fatigue and recovery on resting HR and HRV measures [HR(V)]. The investigation comprised a 3-day rest period, baseline testing (Pre), a 6-day overload microcycle, and a 4-day recovery period, which included follow-up testing at 1 (Post1) and 4 (Post4) days post-training . OverloaThe analyses presented below were part of an extensive investigation protocol evaluating the ability of different potential surrogate markers to assess fatigue- and recovery-related changes in criterion performance at various overload training camps  using a consistent design. The results regarding the performance tests . The training sessions lasted approximately 90 min. They started with a standardized dynamic warm-up, followed by lower-body exercises, and ended with a combination of upper-body, core, hamstring, and back exercises. Preceding the main training exercises, participants performed specific warm-up sets of 5 to 3 repetitions at 50% and 70% of individual maximum performance, respectively. Exercise intensity was standardized in relation to the estimated maximal dynamic strength (1RM) or maximum effort .IFT) or maximum effort  using a breath-by-breath gas collection system  was employed to measure Participants' maximum voluntary isometric contraction (MVIC) force output for parallel squat and bench press exercises was determined using a Multitrainer 7812-000 testing device similar to a Smith machine  and the corresponding user software . Joint angles were set at 90° using a goniometer and the corresponding testing device position was kept constant throughout the study. Following two submaximal practice trials at ~50 and 70% of participants' MVIC, the participants were asked to produce a 3-s MVIC with initial slow force development. MVIC test performance was defined as the mean force of two attempts separated by a rest of 2 min.n = 38, ICC = 0.96, TE 5.2  and bench press exercises using a Smith rack machine . Squat depth was standardized using an integrated linear transducer that produced acoustic stimuli to mark the turning point of the motion. Participants completed two warm-up sets of 5 and 3 repetitions at 50 and 70% of their individual 5–10 RM, respectively. Using a formula by Brzycki  was determined using a non-motorized treadmill . The participants completed a standardized warm-up prior to the trial. The test consisted of six 4-s maximal sprints beginning from a standing position with passive recovery of 20 s between sprints. The highest velocities measured for each sprint were recorded, and the mean peak velocity was calculated. The reliability of the RSA test was previously investigated in our laboratory and was determined to be high  with a single-subject A-B-A withdrawal design  monitoring with active orthostatic tests displayed altered supine HR(V) measures for ST- and altered standing HR(V) measures for HIIT short-term overload. HR(V) measures remained unchanged in the respective other recording position. These autonomic patterns may not be discovered by supine or standing measures alone. However, HR(V) changes were not consistently related to short-term performance changes, which limits their usefulness as surrogate measures for ST or HIIT performance in overload microcycles. Moreover, the use of rolling averages may attenuate the sensitivity to individual short-term autonomic modulations, despite improving sensitivity at the group level. To provide further guidance for sports practice, future studies should utilize repeated intervention designs  measures in supine rest and following quickly standing up using orthostatic tests can provide unique and more comprehensive insights into athletes' autonomic HR regulation compared to either supine or standing recordings in isolation. Although divergent autonomic patterns might be observed following various training demands, it should be acknowledged that HR(V) measures may not mimic individual short-term performance changes. Furthermore, based on our results, we encourage practitioners to  analyze single-day HR(V) measures when assessing short-term training responses. Finally, day-to-day variability and training response varies substantially between athletes, which further complicates HR(V)-guided training prescription. In summary, we believe that it is still advisable first to gain experience at an individual level through pure observation in order to avoid inappropriate training adjustments due to overly simplistic training-response models.The investigation was approved by the ethics committee of the medical faculty of the Ruhr University Bochum and was conducted according to the guidelines of the Declaration of Helsinki. All subjects participated in the study voluntarily, were free to withdraw without penalty at any time, and provided written informed consent.CS prepared the original manuscript, figures and tables and analyzed the data. CS, AAF, and OH interpreted the results. TW, CR, AAF, OH, LH, and AF assisted with writing and editing the manuscript, figures and tables. TW and CR performed the experiment. OS calculated the heart rate variability indices from raw data files. TW, CR, MK, TM, MP, and AF conceived and designed the experiment.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To report the long-term outcome in lymph nodal–metastatic cervicalsquamous cell cancer after chemoradiation followed by adjuvantchemotherapy.Between 2010 and 2013, five patients were diagnosed with advanced cervicalcancer with clinically involved para-aortic lymph nodes . These patients weretreated with concurrent chemoradiation therapy followed by adjuvantchemotherapy. Concurrent chemoradiation consisted of cisplatin given onceper week concomitantly with extended-field radiation therapy followed byhigh-dose-rate brachytherapy. Adjuvant chemotherapy comprised four coursesof carboplatin and paclitaxel given every three weeks. The primary outcomeswere local and distant failures.None of the patients had local recurrence or distal failure after a minimumfollow-up time of 3 years.Adjuvant chemotherapy after chemoradiation has a probable role in themanagement of lymph nodal–metastatic cervical cancer. This treatment led to a 10%improvement in 3-year progression-free survival. After this, no other trial wasconducted to explore improvements in overall survival.Cervical cancer is an ominous disease when it presents in locally advanced stages. Todate, the standard of care for locally advanced disease is pelvic radiotherapy withconcurrent cisplatin-based chemotherapy. However, distant failure remains a majorproblem that leads to compromised overall survival.2) after completion of chemoradiation therapy. Two ofthese patients have completed 5 years of follow-up, two have completed 4 years offollow-up, and one patient has completed 3 years of follow-up. We present a shortreport of five patients who had lymph nodal–metastatic cervical squamous cellcarcinoma.At our university hospital, we treated two patients in 2010, two in 2012, and one in2013 with four cycles of adjuvant carboplatin (area under the curve [AUC] 5) andpaclitaxel (175 mg/m2/week) and extended-field radiationtherapy to the pelvis and the para-aortic region after three-dimensionalCT-based planning. The pelvis and para-aortic field received a dose of 45 Gy at1.8 Gy/day, followed by a boost to 50.4 Gy to the para-aortic and pelvic sidewall nodes. She then received three high-dose-rate (HDR) brachytherapyfractions, and an 8-Gy dose was prescribed at point A in each fraction. Aftercompletion of chemoradiation therapy and brachytherapy, she received four cyclesof carboplatin and paclitaxel. She completed the whole treatment in June 2010.The follow-up PET/CT scan revealed metabolic complete response at nodal andprimary sites. She is free of any disease at 6 years of follow-up.A 45-year-old woman presented with locally advanced squamous cell carcinoma ofthe cervix. The staging positron emission tomography (PET)/computed tomography(CT) scan revealed a 6 × 8 cm metabolically active cervical mass withpelvic and para-aortic lymph nodes that measured more than 1 cm. She receivedconcurrent cisplatin , histopathology was consistent with cervical squamous cell carcinoma,and resection margins were reported to be positive. Her postoperative CT scanrevealed 4-cm residual disease at the vaginal vault with left parametrialinvasion, multiple pelvic nodes, and a 2-cm enhancing left para-aortic node;also, she had a left pelvic kidney. The treatment plan was as follows: radiationtherapy to the pelvis and para-aortic field with a concurrent cisplatin dose of40 mg/m2 once per week followedby HDR brachytherapy to a total dose of 24 Gy in three fractions to point A.After completion of chemoradiation therapy, the patient received four cycles ofcarboplatin and paclitaxel chemotherapy until January 2012. She was free ofdisease at the last follow-up visit on September 9, 2016.This 48-year-old multiparous woman presented with biopsy-proven squamous cellcarcinoma of the cervix. The staging CT scan revealed a large cervical mass aswell as pelvic side wall and para-aortic lymphadenopathy. Para-aortic nodes weremeasured as more than 2 cm in the short axis. The planned treatment waschemoradiation therapy to the pelvis and para-aortic nodes to a total dose of 45Gy at 1.8 Gy/fraction with cisplatin 40 mg/mThis 54-year-old woman presented with biopsy-proven squamous cell carcinoma ofthe cervix; on examination under anesthesia, the disease was staged as IIIB.However, on a staging CT scan of the chest, abdomen, and pelvis, there weremultiple para-aortic nodes that were more than 2 cm each just below the leftrenal hilum. The disease ultimately was staged as IVB squamous cell carcinoma.The planned treatment was extended-field radiation therapy with concurrentchemotherapy followed by HDR brachytherapy, then four cycles of chemotherapywith carboplatin and paclitaxel. The patient completed treatment in October2012. She was in remission at the last follow-up on October 26, 2016.2 because of grade IIhematologic toxicity. This radiation was followed by a boost to the rightpara-aortic node up to 60 Gy and HDR brachytherapy at a dose of three fractionsof 8 Gy each to point A. After chemoradiation therapy, the patient received fourcycles of carboplatin and paclitaxel chemotherapy until June 2013. On the PET/CTscan and a clinical examination at the last follow-up in June 2016, she was freeof any disease.This 62-year-old multiparous woman presented with cervical biopsy–proven,nonkeratinizing squamous cell carcinoma. Examination under anesthesia revealed a6 × 6 cm cervical mass fixed to the right pelvic side wall, and a stagingCT scan showed a 3.5 × 7 cm nodal mass just superior to aorticbifurcation on the right side. The patient received 45 Gy per 25 fractions ofexternal-beam radiation therapy to the pelvis and para-aortic field withconcurrent cisplatin chemotherapy. In last three cycles of chemotherapy, thedose of cisplatin had to be reduced to 30/mg/mCT simulation was performed with intravenous contrast in all patients. Forimmobilization and reproducibility of set-up, an alpha cradle vac-lock was usedin CT simulation. The superior border of the field was placed at the top of theD12 vertebral body, and the inferior border was 3 cm inferior to the caudal-mostextent of the disease in the vagina. A normal saline–soaked gauze piecewas put into the vagina for a negative contrast on imaging. A radio-opaquemarker was placed at the vaginal introitus, and a plastic tube was placed in theanal canal for facilitation of visualization of organs at risk. Lateral borderswere placed 2 cm away from the pelvic bony margin and 1 cm away from thepara-aortic volume of the nodes, and multileaf collimators were used to shieldnormal tissue. Extended pelvis and para-aortic fields were treated each day withantero-posterior (AP), postero-anterior (PA), and right and left lateral fieldswith 18 MV of photon energy. The radiation planning was performed on Aria 10planning software .Complete blood counts and serum creatinine were checked once per week. All of thefields were treated each day for 5 days per week. Treatment-verification portalfilms were taken once per week for accuracy of treatment fields.In the last week of external-beam radiotherapy, first fraction of HDRbrachytherapy was instituted with tandem and ovoid applicators. Thebrachytherapy procedure was performed under general anesthesia, and carefulgauze-piece packing was performed to displace the rectum and bladder away fromthe applicators. The HDR brachytherapy dose was prescribed at point A accordingto International Commission on Radiation Units and Measurements report 38.3 In aretrospective analysis, Kim et al4failed to show a significant difference in survival in patients who receivedadjuvant chemotherapy in locally advanced cervical cancer. In a phase II trial,Chung et al5 treated patients withextended-field radiotherapy, concurrent chemotherapy, and brachytherapy followed bytwo cycles of adjuvant cisplatin and fluorouracil and demonstrated that thistreatment regimen was feasible. However, we used extended-field chemoradiationtherapy, HDR brachytherapy, and four cycles of adjuvant carboplatin and paclitaxelchemotherapy for our patients; toxicity was acceptable and manageable. Verghese etal,6 in a phase II trial,used cisplatin and paclitaxel concurrent with radiotherapy in an attempt to improvethe outcome for locally advanced cervical cancer in Indian women. The trial failedto show any improvement in response compared with cisplatin alone, and toxicity wasgreater in the combination-chemotherapy arm. At our center, we used single-agentcisplatin concurrent with radiotherapy followed by the combination of carboplatinand paclitaxel as adjuvant chemotherapy, and toxicity was less than grade 3 andmanageable.Advanced cervical cancer is a virulent disease, and the 5-year disease-free survivalrate is dismal. One attempt was made to improve overall survival in locally advancedcervical cancer in a randomized trial that compared adjuvant cisplatin andgemcitabine versus no additional chemotherapy. This trial showed a 10% improvementin the 3-year progression-free survival, but improvement was at the cost of slightlyincreased toxicity seen in the group that received gemcitabine andcisplatin.1 found evidence to use adjuvant chemotherapy afterchemoradiation therapy for locally advanced cervical cancer. Recently, such a trialwas initiated in Australia7 inwhich patients with International Federation of Gynecology and Obstetrics (FIGO)stages IB1 to IVA cervical cancer were randomly assigned to receive four cycles ofadjuvant carboplatin and paclitaxel or to receive no additional chemotherapy aftercompletion of chemoradiation therapy. However, we opted to offer this treatment topatients who had FIGO stage IVB disease (para-aortic lymphadenopathy). Patient case2 had residual 4-cm cervical cancer at the vaginal vault, and she received salvagechemoradiation therapy to the pelvis and para-aortic nodal region. To the pelvicmass, she received a total of 63 Gy at 1.8 Gy/fraction after three-dimensionalconformal radiotherapy. Also, she completed her 5 years of follow-up and is free ofany recurrent disease. There is evidence of adequate local control with radicalradiotherapy of residual disease after inadequate hysterectomy for cervicalcancer.10To date, no randomized trial is available to assess the impact of carboplatin andpaclitaxel as adjuvant combination chemotherapy after chemoradiation therapy inlocally advanced disease. A review by Tangjitgamol et al11and Jelavić et al12reported encouraging results of four adjuvant cycles of cisplatin and ifosfamidechemotherapy after completion of concurrent chemoradiotherapy in an attempt toimprove distant-relapse–free survival. In a randomized trial, Wong etal13 showed improvement inoverall survival after administration of adjuvant chemotherapy with epirubicin at 90mg/m2 compared with pelvic radiotherapy and no additionalchemotherapy.Petrić Miše et al14 Should the FIGO stage IVB disease be segregated into twogroups, patients with widespread distant metastases and those with para-aortic nodesonly. Should the patients with para-aortic disease only have a better prognosis,provided they were treated aggressively. This question may get its answer in thenear future. We hope that the results of the OUTBACK trial  will be able to resolve the question of whether to use adjuvantchemotherapy administration in locoregionally advanced cervical cancer.7This is a unique report of treatment for patients with stage IVB disease (para-aorticlymphadenopathy). In the modern era of oncology, oncologists come across patientswith advanced-stage disease not infrequently, and the treating oncologist, patients,and families often fail to understand the prognosis and goals of treatment, whichcan lead to a poor outcome of treatment.


Hyperglycemia is common and associated with increased mortality after out-of-hospital cardiac arrest (OHCA) and return of spontaneous circulation (ROSC). Mechanisms behind ultra-acute hyperglycemia are not well known. We performed an explorative study to describe the changes in glucose metabolism mediators during the prehospital postresuscitation phase.We included patients who were successfully resuscitated from out-of-hospital cardiac arrest in two physician-staffed units. Insulin, glucagon, and glucagon-like peptide 1 (GLP-1) were measured in prehospital and hospital admission samples. Additionally, interleukin-6 (IL-6), cortisol, and HbA1c were measured at hospital admission.Thirty patients participated in the study. Of those, 28 cases (71% without diabetes) had sufficient data for analysis. The median time interval between prehospital samples and hospital admission samples was 96 minutes (IQR 85–119). At the time of ROSC, the patients were hyperglycemic , with insulin and glucagon concentrations varying considerably, although mostly corresponding to fasting levels . GLP-1 increased 2- to 8-fold with elevation of IL-6. The median glucose change from prehospital to hospital admission was -2.2 mmol/l (IQR -3.6 to -0.2). No significant correlations between the change in plasma glucose levels and the changes in insulin , glucagon , or GLP-1 levels  or with IL-6 (r = (-0.07), p = 0.75), cortisol  or HbA1c levels  were observed. However, in patients who did not receive exogenous epinephrine during resuscitation, changes in blood glucose correlated with changes in insulin  and glucagon  levels, demonstrating that lowering glucose values was associated with a simultaneous lowering of insulin and glucagon levels.Hyperglycemia is common immediately after OHCA and cardiopulmonary resuscitation. No clear hormonal mechanisms were observed to be linked to changes in glucose levels during the postresuscitation phase in the whole cohort. However, in patients without exogenous epinephrine treatment, the correlations between glycemic and hormonal changes were more obvious. These results call for future studies examining the mechanisms of postresuscitation hyperglycemia and the metabolic effects of the global ischemic insult and medical treatment. Hyperglycemia is common after out-of-hospital cardiac arrest (OHCA) and has been associated with a poor outcome . An incrStress-induced hyperglycemia (SIH) is a well-established phenomenon that occurs during critical illness –5. HyperDuring OHCA and resuscitation, the patient is exposed to a global ischemia-reperfusion state that also affects the endocrine organs, such as the liver, pancreas and intestines, possibly leading to diminished hormonal production causing decreased endogenous insulin and GLP-1 levels. Giving exogenous epinephrine during resuscitation from OHCA may also affect the early phase of blood glucose disturbance. Thus, the mechanisms of hyperglycemia in the early postresuscitation phase may differ from those of SIH. Hence, this information could be necessary for designing possible interventions targeting ultra-acute hyperglycemia. The aim of this explorative study was to describe changes in glucose metabolism mediators during the prehospital phase after successful cardiopulmonary resuscitation from OHCA.http://doi.org/10.5281/zenodo.2591961.We conducted a prospective explorative study on biomarkers related to blood glucose metabolism in OHCA patients in the acute phase. The study protocol was approved by the Ethical Committee of Helsinki University Hospital (357/13/03/02/2012), and the study protocol was registered at clinicaltrials.gov (NCT01968148). Written informed consent was obtained from the next-of-kin on the scene. Due to the time-critical nature of the study design, in some cases, an informed verbal consent was asked from next-of-kin before blood sampling if written consent from next-of-kin was extremely impractical to obtain because of environmental factors. The consent was confirmed and signed as soon as it became practical during prehospital care. One additional member of the staff always witnessed the verbal consent. This practice is in line with the Finnish legislation on clinical studies. The studied biomarker samples were collected immediately after ROSC and at hospital admission. The relationship of the change in glucose levels with the studied biomarkers was then analyzed. The study protocol data can be reviewed at We included cardiopulmonary-resuscitated OHCA patients aged 18 years or older regardless of their initial rhythm or suspected cause. Study patients were recruited only on weekdays during office hours (from 8 a.m. to 4 p.m.), when necessary laboratory facilities for handling the study samples were available in the receiving hospitals. We excluded cases without next-of-kin on the scene to provide the consent to participate. The study protocol allowed the clinical staff to exclude a patient in a scenario where patient safety would have been compromised due to a lack of time to carry out the study procedures.Helsinki University Hospital is responsible for organizing and supervising Emergency Medical Services (EMS) for the 1.6 million inhabitants of the Helsinki metropolitan area. EMS has two physician-staffed units. One is a helicopter emergency medicine service (HEMS) unit, and the other is a ground unit. Physician-staffed units are dispatched to all witnessed OHCA cases. This study was conducted in physician-staffed units.Prehospital blood samples were taken from the arterial line or by venous puncture immediately after ROSC and obtained consent. Blood samples for glucose and insulin analyses were drawn into citrate-fluoride and serum-gel tubes, respectively. Blood samples for glucagon and glucagon-like peptide 1 (GLP-1) analyses were drawn into PD P800  tubes, which contained esterase, protease and DPP-IV inhibitors for sample preservation until laboratory analysis. All tubes were covered in icepacks and transported to the emergency department to be delivered immediately to the central laboratory of the hospital. After the patients arrived at the hospital, samples for glucose insulin, glucagon, GLP-1, IL-6, cortisol, and HbA1c analyses were drawn from an arterial line or via venous puncture.A routine central laboratory analysis was conducted for all prehospital samples except GLP-1. Plasma for GLP-1 analysis was separated promptly, frozen at -70 degrees celcius and stored in the laboratory for later analysis. GLP-1 analysis for prehospital and hospital admission samples was performed using an ELISA test  according to the manufacturer’s instructions. The laboratory reference values for the concentrations were as follows: insulin 10–20 mU/l, glucagon <209 ng/ml, GLP-1 1–2 ng/ml, and IL-6 <3.4 ng/l .The exogenous epinephrine dose used during resuscitation was 1 mg intravenously according to European resuscitation guidelines . The numVery few studies with a similar study design have been performed; therefore, to estimate an appropriate sample size for our study, we used the study design of Llompart-Pou JA et al. as a reference . They reContinuous variables are presented as medians and interquartile ranges (IQRs). Categorical data are presented as percentages with 95% confidence intervals, calculated with the modified Wald method. Continuous variables were compared by the Mann-Whitney test and Wilcoxon matched-pairs signed rank test . CompariDuring the study period from January 2014 to July 2015, we recruited 30 cardiopulmonary-resuscitated OHCA patients into the study. Two patients had missing laboratory data and were consequently excluded from the final analysis  were initially hyperglycemic and remained hyperglycemic. Plasma glucose levels showed a decreasing trend in 22 patients and an increasing trend in 6 patients .The measured levels of the hormones and the changes that occurred during the prehospital phase are presented in The glucagon concentration was at a fasting level in the majority of patients. No significant correlations of the change in the glucose concentration with any of the measured hormone levels or with the change in the hormone levels was observed Figs  and 3. HIL-6 concentrations at hospital admission had high internal variation among the study sample (range: 6 to 899 ng/l). Overall, with few exceptions, IL-6 levels were consistently low, but above the postprandial level, with a median of 32 (IQR 22–63) ng/l . CortisoWe additionally analyzed patients (n = 13) for whom exogenous epinephrine was not administered. In these patients, we found median hospital admission blood glucose levels of 8.1 (6.7–10.3) and 9.9 (8.3–12.2) in patients who received exogenous epinephrine, respectively. Exogenous epinephrine administration did not markedly affect HOMA-IR results, indicating a U-shaped pattern of insulin resistance; in half of the cases, insulin resistance increased, and in the other half of the cases, it decreased , during critical illness leads to a better outcome than SIH or hypoglycemia in many patient groups. Hence, interventions to achieve mild hyperglycemia should be considered. By administering exogenous insulin, the altered insulin metabolism in SIH can be normalized, and it has been shown to benefit outcomes ,21. In tGLP-1 is an incretin hormone that causes insulin secretion and suppresses glucagon production in the body . PreviouThere have been promising studies using GLP-1 analogs for critical illness and OHCA instead of using insulin to treat SIH; GLP-1 analogs do not cause hypoglycemic episodes and carry possible beneficial antioxidative effects ,26. WibeThe main limitation of our study was the lack of a previous reference for a sufficient power calculation. For this reason, our sample size is potentially insufficient to elucidate the mechanisms behind glucose disturbance in the ultra-acute phase after OHCA. Due to the availability of laboratory facilities and consecutive slow enrollment, the sample size was limited to 30 cases after the interim analysis. Although the causes were likely multifactorial, we conducted a univariate analysis. This should be taken into consideration when interpreting the results. Other limitations were the inability to measure catecholamine levels, which most likely affect glucose metabolism after OHCA. A high number of confounding factors in glucose metabolism exist. Fasting levels of the hormones or medical conditions of the patients prior to OHCA were unknown, as was the prandial status of the patients. Antihyperglycemic medication, beta blockers, thiazide diuretics, atropine or corticosteroid medications may have affected the results , though Further studies should be conducted with a sufficient power calculation and a healthy control group. A multivariate analysis with a larger sample size should also be conducted to validate the results. In further studies, fasting levels, medical status, and other possible confounding factors should be taken into greater consideration. Finally, intervention studies on early glucose control after OHCA are required.Hyperglycemia was common after successful resuscitation from OHCA. Hormonal responses seemed to differ markedly among patients and seemed to lack specific hormonal mechanisms, except when exogenous epinephrine was not administered during resuscitation after OHCA. Overall, possibly due to global ischemic insult or unknown mechanisms, hyperglycemia in the early postresuscitation period seemed to differ from that commonly linked to SIH.S1 File(DOCX)Click here for additional data file.


Robotic technologies offer a range of functions to augment clinical rehabilitation practice. However, compliance with robot-assisted rehabilitation techniques has not been optimally achieved. Traditional approaches to improving the treatment efficacy are focusing more on the system function, while psychological factors have not been integrated comprehensively. In this perspective paper, eight key factors reflecting three conceptions-robot design, function design, and patients’ expectations have been evaluated and analyzed. Clinical results with 28 therapists and 84 patients indicate that integrating psychological strategies into robot-assisted physiotherapy may promote better trust and acceptance of rehabilitation robots. Robotic technologies offer a range of functions to augment clinical rehabilitation practice. The Lokomat exoskeleton , an estaPhysical disabilities place a substantial physical and mental burden on patients and their families, which may predispose them to depression. Shreds of evidence show that physical independence leads to better cognition and mood , and in One of the most pressing demands of the day is to promote patients’ trust in rehabilitation robotic systems that predispose their willingness to accept robot-prescribed training. A meta-analysis suggests that robot performance  and attributes  are significant contributors to the development of trust in human–robot interaction . Most reThis paper aims to appeal to researchers within the rehabilitation robotics community that immediate and special attention should be given to using psychological strategies to improve human-robot interaction and trust, in developing robot-assisted techniques for physical therapy.Psychological features affect rehabilitative outcomes of patients involved in robotic treatment more than those in conventional rehabilitation . The aimEight factors (numbered as F1 to F8) including: F1-natural and compatible human-robot movement, F2-friendly robotic appearance, F3-attractive interface, F4-adaptable task difficulty levels, F5-intelligent conversation, F6-connecting individuals, F7- performance feedback, F8-accurate expectation, were proposed to enhance patients’ acceptance and trust on rehabilitation robots from the perspective of psychology, as in Considering robot design, rehabilitation robotic systems should have natural and compatible movement with human users, a friendly appearance, and an attractive interface . One wayIn control design, affective computing can be taken to enhance physical and mental engagement. Affective computing integrates psychology, cognitive science, neuroscience, value-centered design, and ethics into engineering and computer science to address human needs. Emotion is fundamental to human survival and functioning, influencing cognition, perception, and activities of daily living. However, technologists have largely ignored emotion and created an often-frustrating experience for people, in part because emotion is intangible and patients’ mental states are difficult to be measured. Developing new technologies that incorporate affect theories to improve human engagement is critical. First, motor learning theory indicates that the learning rate is maximal at a task difficulty level that positively challenges and excites subjects while not being too stressful or boring . Task-diMatching individual expectations with robot abilities is another way to improve people’s psychological state. When robots have limitations in their abilities due to the development lag of science and technology, trust and acceptance can be increased by modifying human users’ expectations . The basAn observational pilot study with 28 therapists and 84 patients with physical disabilities was conducted to preliminarily evaluate the impact of factors from F1 to F8. A questionnaire, as shown in ® Lokomat; (2) NeuroCom® Balance Master; (3) Isomed 2000 System; (4) Imoove 600 System; (5) ReoTM Ambulator; (6) Hocoma Armeo Spring; (7) AMADEO; (8) DIEGO; (9) Rehawalk® System; (10) MOTOmed loop.la; (11) Pablo® Upper Extremity; (12) SaeboReJoyce; (13) E-LINK Evaluation and Exercise System. All participants have experience with operating or using these robotic devices.This study was approved by the Ethics Committee of Shanghai Sunshine Rehabilitation Center (2018083101), and written informed consent had been obtained from all the participants prior to conducting any experiments. All therapist participants are from the Sunshine Rehabilitation Center , with ages over 17 years old and over-five-hours experience in robot-assisted therapy. Patient participants can follow therapists’ instruction and understand the questionnaire well, with ages over 16 years old and over-five-hours experience in receiving robot-assisted therapy. Note that, the Sunshine Rehabilitation Center has the following robots, not limited to: (1) Hocomap < 0.001), F3 (p < 0.01), and F4 (p < 0.05). Moreover, the effect of identity on importance ranking of F5 (p < 0.05) and F6 (p < 0.05) are also significant. Furthermore, therapists rated these five factors as more important than patients did. Besides, a statistical one-way ANOVA analysis was carried out to investigate the effects of three different variables  on the evaluation of these eight factors. As illustrated in In all, eight factors corresponding to rehabilitation robotics design had been preliminarily analyzed and the effects of three variables on the evaluation of these factors had also been investigated. The results demonstrated that the compatible human-robot movement and performance feedback should be promised, and other psychological strategies listed were also recognized to be effective to improve the rehabilitation performance. These results would contribute to the future design of human-robot rehabilitation system for peers in the rehabilitation robotics community to improve the human-robot interaction and rehabilitation efficacy.This study proposed eight psychological strategies (F1 to F8) that may promote better trust and acceptance of rehabilitation robots by human users. It is essential to consider these factors in developing new robot-assisted rehabilitation techniques, especially in mechanical design, functional design, and the training protocol design. Preliminary data collected from 112 human users have positively supported the proposal of bringing psychological strategies into robot-assisted physiotherapy.Note that, two main limitations exist in this study: (1) One is the small sample of therapist and patient participants. This may have made the statistical results of importance ranking not highly convincing as that presented in In general, previous researches on rehabilitation robots  have laid a solid foundation for basic function implementation, and tremendous effort had been spent on improving reliable automation and the panoply of robots, while very little focus has been devoted to optimizing current robot-assisted physiotherapy from the viewpoint of psychology, for example, the factors F4, F5, F7, and F8. Literatures  have evaThe datasets analyzed in this manuscript are not publicly available. Requests to access the datasets should be directed to zhangmm@sustech.edu.cn.The studies involving human participants were reviewed and approved by this study was approved by the Ethics Committee of Shanghai Sunshine Rehabilitation Center (2018083101), and written informed consent had been obtained from all the participants prior to conducting any experiments. All therapist participants are from the Sunshine Rehabilitation Center , with ages over 17 years old and over-five-hours experience in robot-assisted therapy. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The possibility that polymyalgia rheumatica (PMR) can be diagnosed when both ESR and CRP are normal at the time of diagnosis and before therapy with glucocorticoids, has been often discussed in the literature. We present a case report of a 100-year-old Caucasian man referred to our outpatient clinic, complaining of chronic pain in the shoulder and hip girdle associated with normal values of both ESR (21 mm/1st hour) and CRP (4 mg/dL). In the previous four months, several anti-inflammatory drugs and painkillers associated with physiotherapy treatments gave no significant improvement in pain and self-care. After an ultrasound (US) and an 18-fluorodeoxyglucose positron emission tomography associated with total body computed tomography (18-FDG PET/CT) examination, PMR was diagnosed and he started therapy with 17.5 mg prednisone, obtaining a fast improvement in pain and self-care. After 10 months of tapering, he stopped prednisone without relapse. During a 3-year follow-up, no alternative diagnosis was done. When a patient complains of chronic bilateral shoulder and hip girdle pain associated with normal inflammatory indices, it is reasonable to think in the first instance that this person is not suffering from PMR. Moreover, the possibility that PMR may onset in a centenarian person, is exceptional. In our patient, when we piece the puzzle together, the diagnosis of PMR was the most possible one. Polymyalgia Rheumatica (PMR) is a common inflammatory disease affecting older adults. Several investigators consider PMR as the most frequent inflammatory rheumatic disease in Caucasian persons older than 70-years. Its prevalence increases until the age of 90, with a slight decrease thereafter ,2,3. ItsIn May 2015, a 100-year-old Caucasian man was referred to our outpatient clinic complaining of chronic pain in shoulder and hip girdle pain with 4-h morning stiffness. Constitutional manifestations were not present. In the previous four months, several non-steroid anti-inflammatory drugs (NSAIDs) and painkillers associated with physiotherapy treatments gave no significant improvement in pain and self-care. He had an X-ray of the chest, shoulders and pelvic, revealing no pathologic findings. An abdominal ultrasound (US) showed mild hepatomegaly and renal cysts. He did not suffer from psoriasis; no ocular, intestinal or uninary manifestations were present. ESR was 21 mm/1st hour  at the onset and 12 mm/1st hour at the time of our examination. C-reactive protein (CRP) was 4 and 3 mg/dL , respectively. Other laboratory data were all negative. In particular, serum fibrinogen levels were equal to 350 mg/dL ; rheumatoid factor (RF) and anti-protein citrullinated antibodies (APCA) were in their normal range; hemoglobin was equal to 12.8 gr/dL ; transaminases, creatine phospho kinase (CPK), protein electrophoresis, antinuclear cytoplasmic antibodies (ANCA) were in their normal ranges. Occult blood research in the stool was negative and fecal calprotectin dosage was in the normal range. Antibodies to hepatitis C virus and Australia antigen were absent. An US examination showed bilateral long-head-biceps exudative tenosynovitis and subdeltoid bursitis in his shoulders  and trocWhen a patient of greater than 50 years of age complains of chronic bilateral shoulder and hip girdle pain associated with normal inflammatory indices, it is reasonable to think in the first instance that this person is not suffering from PMR. In fact, there are several diseases with these clinical manifestations in which the values of inflammatory indices are normal at the time of diagnosis; among these, there are inflammatory , degenerative, infectious or neoplastic conditions . For morAdditionally, some patients initially diagnosed with PMR may be reclassified as having a different disease during follow-ups, seronegative rheumatoid arthritis being the most common alternative . Late-onUp to now, in absence of a specific diagnostic test, the diagnosis of PMR remains clinical. In the last years, classification criteria proposed by a European League Against Rheumatism/American College of Rheumatology (EULAR/ACR) collaborative initiative highlighted US examination of both shoulders  and hips (showing joint synovitis or trochanteric bursitis) . 18-FDG The reasons why ESR and CRP can be normal in an autoinflammatory disease such as PMR are only speculative. In PMR a non-specific inflammatory reaction is triggered by innate immunity activation . Innate According to a working hypothesis proposed by some investigators, those persons who are genetically predisposed to produce high levels of interleukin-6 (IL-6) during aging, have a reduced capacity to live long healthy lives. The genetic basis could be in a C/G polymorphism allocated at 5′-upstream of IL-6 (−174 C/G locus), with the GG genotype associated with higher levels of IL-6 serum levels and the CC or CG genotype associated with lower levels . These dAccording to our best knowledge, PMR in a 100-year-old is exceptional and studies regarding these suggested elements are very difficult.Lastly, some investigators suggested that PMR with normal ESR and CRP could be an incomplete form of giant cell arteritis (GCA), manifested in the regions in the proximity of axillary, subclavian and/or femoral artery . In GCA,Our case report draws attention to the existence of a non-frequent PMR with normal ESR and CRP at the time of diagnosis. In the clinical practice, this possibility should not stop the physician to include PMR in differential diagnosis. US and 18-FDG PET/CT evaluations might increase diagnostic rates of CRP- and ESR-negative PMR. A rigorous diagnostic work-up  and an adequate follow-up are important ,26.The diagnosis of PMR is possible, even if ESR and CRP have not increased.US and 18-FDG PET/CT evaluations might increase diagnostic rates of ESR and CRP negative PMR.A rigorous diagnostic work-up and a long follow-up  are mandatory to avoid mistakes.The onset of PMR in a centenarian is exceptional. In this age range, the genetic background seems to be important in conditioning laboratory data more than in other periods of life.


To reliably evaluate the acceptance and use of computer-based prostate cancer decision aids (CBDAs) for African-American men, culturally relevant measures are needed. This study describes the development and initial psychometric evaluation of the 24-item Computer-Based Prostate Cancer Screening Decision Aid Acceptance Scale among 357 African-American men.Exploratory factor analysis (EFA) with maximum likelihood estimation and polychoric correlations followed by Promax and Varimax rotations.EFA yielded three factors: Technology Use Expectancy and Intention (16 items), Technology Use Anxiety (5 items), and Technology Use Self-Efficacy (3 items) with good to excellent internal consistency reliability at .95, .90, and .85, respectively. The standardized root mean square residual (0.035) indicated the factor structure explained most of the correlations.Computer-Based Prostate Cancer Screening Decision Aid Acceptance Scale has utility in determining the acceptance and use of CBDAs among African-American men at risk for prostate cancer. Future research is needed to confirm this factor structure among socio-demographically diverse African-Americans.Findings suggest the three-factor, 24-item  Prostate cancer (PrCA) incidence and mortality rates are higher among African-American men than any other racial group . Many soIn order to engage in informed decision making, African-American men need plain language PrCA knowledge information and adequate decision self-efficacy . PrCA knTo determine the quality of a user’s experience while using a technology-based PrCA intervention, some researchers conduct feasibility or usability testing –22. HoweUnified Theory of Acceptance and Use of Technology Scale, that measures acceptance and use of a technology based on each of the aforementioned moderating factors and intention to use technology. In their seminal article, Venkentash et al. [Unified Theory of Acceptance and Use of Technology Scale found that self-efficacy and anxiety are significant predictors of technology use intention [Unified Theory of Acceptance and Use of Technology Scale has been validated for use across several sectors including, but not limited to, mobile banking, social networking, web-based learning environments, decisions support systems, digital learning environments, and retail [While some technology acceptance factors are highly correlated with usability , most th et al.  found thh et al. , self-efntention . Despited retail , 37.Unified Theory of Acceptance and Use of Technology Scale for assessing technology acceptance in health environments [Related to decision support systems and web-based learning environments, there are a growing number of studies that have employed the ronments . These sronments –43. Howeronments –47. Eachronments , these ironments . The addronments . Thereforonments , 46–49. ronments . A few sronments , 51.Unified Theory of Technology Use and Acceptance Scale in this study. A second cancer-related study [Only one recent study  has inveed study , proposeUnified Theory of Technology Use and Acceptance Scale for assessing technology use and acceptance across sectors, including healthcare, the psychometric properties of this scale have not been tested among African Americans. Having a culturally-relevant measure of technology use and acceptance is needed to measure the performance of technology-based interventions that seek to enhance decision making about PrCA screening among African-American men, who experience the highest mortality from the disease [Computer-Based Prostate Cancer Screening Decision Aid Acceptance Scale among African-American men.Although a number of studies report acceptable reliability and validity of the  disease . Given tiDecide, a computer-based decision aid (CBDA) to prepare African-American men for informed decision making about PrCA screening. During the pilot study, participants received a self-administered, paper survey at post-intervention to measure their acceptance of iDecide. Detailed recruitment strategies and a description of iDecide are published in prior manuscripts [This psychometric study used cross-sectional data from a pilot study conducted to assess the efficacy of uscripts . Human sThis study included a purposive sample of 354 African-American men aged 40 and older. Eligible participants were those who (a) self-identified as African American; (b) spoke and comprehended English; (c) had no personal history of PrCA; and (d) had no self-reported history of cognitive decline. Participants were not required to have prior technology use experience. They were recruited from several social and faith-based venues in South Carolina between July 2015 to February 2016. All participants received a written consent form in-person prior to their participation in any study activities. Prior to requesting a signature on the consent form, the form was explained in detail by a member of the research team. This study was approved by the Institutional Review Board at the University of South Carolina.Unified Theory of Acceptance and Use of Technology Scale was adapted to examine the usability and acceptance of iDecide, a CBDA for African-American men [“system”. For example, question Q28 . Subscales  and two of four original moderators  that are hypothesized to be directly related to technology acceptance were retained. Because this scale is specific to CBDAs, the modified scale was titled, the Computer-Based Prostate Cancer Screening Decision Aid Acceptance Scale.The ican men . Specifi28 Table  was adapironment . Scale mComputer-Based Prostate Cancer Screening Decision Aid Acceptance Scale is comprised of 24-items with Likert scale response categories ranging from 1 (strongly agree) to 5 (strongly disagree) and 6 (does not apply). Scoring involves taking the average of all responses, excluding does not apply responses. A higher score indicates greater acceptance and use of the CBDA for PrCA informed decision making. Table Unified Theory of Acceptance and Use of Technology Scale to items of the Computer-Based Prostate Cancer Screening Decision Aid Acceptance Scale.The Computer-Based Prostate Cancer Screening Decision Aid Acceptance Scale emphasizes the influence of the dynamic interplay between the individual and their social and physical environment about whether an individual will adopt a CBDA. To determine the acceptance and use of a CBDA for PrCA informed decision making, seven factors are influential including: (a) performance expectancy , (b) effort expectancy (the amount of effort associated with using the CBDA to retrieve PrCA information), (c) social influence , (d) facilitating conditions , (e) computer anxiety , (f) self-efficacy  attitudes towards technology  and (h) behavioral intention . Each of these factors are moderated by an individual’s age and technology-use experience [Based on the Unified Theory of Technology Use and Acceptance ,the Compperience . CounterComputer-Based Prostate Cancer Screening Decision Aid Acceptance Scale was pre-tested with a convenience sample of two African-American men with high school or higher education. During the pre-test, the men were provided with a survey containing the full battery of 65-items that were used during the testing of the iDecide PrCA screening CBDA. These men completed a hard copy survey and noted if there were questions, words, or concepts on the survey they found difficult to interpret or that might be difficult to interpret for men with low reading levels. After survey completion, they were interviewed by the first author (O.O.) about difficulties completing the survey along with questions about survey formatting . Neither participant suggested changes to the survey.To assess face validity, the Descriptive statistics described the sociodemographic characteristics of African-American men in the sample. Polychoric correlations assessed the association between factors and subscale items. Cronbach’s alpha assessed internal consistency reliability for the total scale and each subscale.Computer-Based Prostate Cancer Screening Decision Aid Acceptance Scale. Performance expectancy, effort expectancy, social influence, facilitating conditions, computer anxiety, self-efficacy, and attitudes towards technology were exogenous latent variables hypothesized to be correlated with each other. Subscale items loading on each factor were also hypothesized to be correlated with each other. The number of participants to item ratio was 14:1, which is above the recommended 10:1 often used to determine a priori sample size for EFA [Exploratory factor analysis (EFA) is a data-driven, exploratory technique and that does not require a priori specification of the relationships between latent and observed variables –58. Thus for EFA .EFA was conducted using preliminary estimates of communalities obtained from the square of the multiple correlation coefficient of each variable. Iterated principal factor extraction with prior communalities set to 1 was used for data extraction followed by Varimax rotation. Factor retention was assessed through parallel analysis, which has been demonstrated as a more accurate assessment of factor retention than other factor retention methods . SpecifiFactor loadings were assessed using item communalities, cross-loadings, and item statistics. A factor with less than three factor loadings was considered weak and unstable  and was n = 2) to 2.54% (n = 9) for scale items. Single and multiple imputation were used to impute missing values for 24 items. Means for each item was compared with and without imputation, which included no imputation, single imputation, and multiple imputation (n = 1000) for missing data, which were similar. Descriptive statistics were analyzed using original data (N = 354). EFA was conducted using original, single, and multiple imputation datasets given the Computer-Based Prostate Cancer Decision Aid and Acceptance Scale is a major adaptation of the Unified Theory of Acceptance and Use of Technology Scale. All data analyses were performed using SAS/STAT® statistical software, version 9.4 [Missing values ranged from 0.56% (sion 9.4 .n = 194). An overwhelming majority were insured  and had a regular healthcare provider . Nearly half were employed  or reported a household income between $20,000–79,999 . Over half  had a high school diploma or attended some college. Most participants report using technology prior to study participation including: television , cellphones , automated teller machines , computers  and/or tablet computers . They also reported using the following technology features: cell phone apps , text messaging , email , and/or the internet .For each item of the 24-item scale, mean responses ranged from 2.2 to 4.46. Table Computer-Based Prostate Cancer Screening Decision Aid Acceptance Scale using the original, single and multiple imputation datasets. Factor loadings were similar for original, single and multiple imputation datasets. Parallel analysis, scree plot , Technology Use Anxiety, had five factor loadings ranging from .52 and .92 for original data, and .41 to .93 for both single and multiple imputation. Factor 3 (F3), Technology Use Self-efficacy, had three factor loadings ranging from .67 to .88 for original and imputed datasets.Table lot Fig. , and theTechnology Use Expectancy and Intention (F1) and Technology Use Self-Efficacy (F3). For single and multiple imputed datasets, five items, Q9, Q10, Q11, Q12, and Q13, cross-loaded on two factors—Technology Use Expectancy and Intention (F1) and Technology Use Self-Efficacy (F3). All cross-loaded items were retained on Technology Use Expectancy and Intention (F1) because all factor loadings were highest on F1 compared to F3 , Technology Use Anxiety (F2), and Technology Use Self-Efficacy (F3). Factor loadings were moderate to high and the total scale and subscales had good to excellent internal consistency reliability. These findings expand the Unified Theory of Technology Acceptance and Use and builds upon prior evidence indicating that technology acceptance and use is a multidimensional construct. To our knowledge, this is the first scale developed to measure the efficacy of a CBDA for PrCA screening. Our findings suggest the Computer-Based Prostate Cancer Screening Decision Aid Acceptance Scale has utility in determining the acceptance and use of CBDAs by African-American men for informed PrCA screening decision making.This study evaluated the psychometric properties of the 24-item, Computer-Based Prostate Cancer Screening Decision Aid Acceptance Scale is inconsistent with the six-factor structure the Unified Theory of Acceptance and Use of Technology Scale [Computer-Based Prostate Cancer Screening Decision Aid Acceptance Scale showed that performance expectancy (Q1-Q4), effort expectancy (Q5-Q8), attitudes toward technology (Q9-Q12), social influence (Q13) and behavioral intention to use the system (Q24) loaded on Technology Use Expectancy and Intention with factor loadings ranging from .44 and .85 for all datasets. The high factor loadings and factor loading pattern suggest that these constructs are far more interrelated than distinctive in a community sample of middle-aged African-American men who used a measure that was adapted for healthcare decision making compared to individuals from business, non-profit, and academic organizations who used the Unified Theory of Acceptance and Use of Technology Scale to assess use and acceptance of a new technology in prior psychometric studies. Our data are also distinct from two healthcare-related studies that conducted psychometric testing [Unified Theory of Acceptance and Use of Technology Scale for assessing healthcare technology acceptance among non-African-Americans. For example, in the assessment of factors influencing Korean healthcare professionals’ adoption of mobile electronic medical records, Kim et al. [Computer-Based Prostate Cancer Screening Decision Aid Acceptance Scale, test moderators, and assess model fit using more fit indices. Overall, the Kaiser-Meyer-Olkin and standardized root mean square residual estimates and small residuals further support the good fit of our 24-item, three-factor model.Although our findings are theoretically consistent with the Unified Theory of Technology Acceptance and Use, the three-factor structure of the gy Scale . Our fingy Scale  validati testing , 50 of tm et al. , but thim et al. , 67, werI have the knowledge necessary to use the CBDA.”) loaded on Technology Use Expectancy and Intention (F1), whereas the other item  loaded on Technology Use Anxiety (F2). Specifically, Q14 is related to whether an individual has the “knowledge” necessary to use the CBDA, whereas Q15 is related to “compatibility” of the CBDA with technologies that an individual currently uses. This suggests that facilitating conditions may not be a distinct construct and possibly conceptually related to technology use and intention overall, especially in the context of informed PrCA screening decision making. Among middle-aged African-American men who are using CBDAs to assist with PrCA screening decisions, facilitating conditions may be different compared to assessment for diffusion of innovation in non-profit, business, and academic organizations. The lack of homogeneity of facilitating conditions for technology use and intention may also explain why the items loaded on two different factors [Computer-Based Prostate Cancer Screening Decision Aid Acceptance Scale in a more heterogeneous sample of African-American men who are at risk for PrCA and women who assist with PrCA screening decision making may provide more evidence about the role of facilitating conditions for technology use and acceptance, especially regarding age, gender, and social environment.As for factor loadings, one of the two items for facilitating conditions . Counter to the Unified Theory of Technology Use and Acceptance Scale, computer anxiety was a salient correlate that may predict whether an African-American man decided to use a specific technology. Prior studies have also identified anxiety as detrimental to intention to use technology For example, among 1204 racially diverse participants, Czaja et al. [The four computer anxiety (Q20-Q23) items loaded onto a et al.  found tha et al.  found thTechnology Use Self-efficacy (F3) with factor loadings ranging from .57 to .88, whereas the remaining item  loaded on Technology Use Expectancy and Intention (F1) with factor loadings at .54 and .52 for original and imputed datasets, respectively. Because African Americans may have less technology use experience and lower technology self-efficacy [Unified Theory of Technology Use and Acceptance Scale [Technology Use Expectancy and Intention may also be attributed to question wording. Similar to a reverse worded item, Q16 contains a negation ‘no one around,’ which is different from other self-efficacy items. Therefore, Q16 may have been misinterpreted because of the negation or vague wording, which increases response bias. Perhaps rewording Q16 to read, ‘I can complete the prostate cancer education program using the CBDA without assistance’, in future administrations and psychometric testing will reduce response bias and provide further evidence of convergent validity  loaded onto efficacy , the idece Scale , it is cce Scale , 70. Thend above ).Technology Use Expectancy and Intention (F1) and Technology Use Self-Efficacy (F3) for both original (Q11-Q13) and imputed (Q9-Q13) datasets. Although all cross-loaded items loaded highest on (see Table Technology Use Expectancy and Intention (F1), cross-loading could be the result of vague and/or confusing question wording. Q9, which reads, ‘Using the CBDA is a good idea’ and Q10, which reads, ‘The CBDA makes learning about prostate cancer more interesting’, may both be related to performance expectancy, social influence, and self-efficacy. Q11, which reads, ‘The CBDA makes learning about prostate cancer fun,’; Q12, which reads, ‘I like using the CBDA,’; and Q13, which reads, ‘People who are important to me will likely support my use of the CBDA’ may all seem vague and conceptually unrelated to technology use and acceptance for healthcare decision making. Given cancer is a grave topic and that the purposive sample of African-American men were at risk for PrCA, asking about whether learning about PrCA is fun may seem awkward or even inappropriate. Perhaps rewording Q9-Q13 during future administrations of the Computer-Based Prostate Cancer Screening Decision Aid Acceptance Scale may improve factor structure.Five items Q9-Q13) cross-loaded on  cross-lon = 201), the device on which our CBDA was administered. Two items  had factor loadings of less than .50 in at least one dataset, which suggests poor convergent validity. Cross-loadings suggest factors may not be conceptually distinct. The modified scale was only pre-tested with two African-American men who may have not been representative of African-American men included in the sample. Lastly, the research team did not test the influence of important moderators that could affect technology acceptance such as faith. Despite these limitations, this study provides valuable psychometric evidence, which can contribute to the future development and evaluation of culturally-tailored CBDAs to facilitate PrCA screening decisions of African-American men who are at risk for the deadliest PrCA globally.Study strengths included a large community sample of African American men, which exceeded the minimum recommended sample size for EFA (> 200). However, the African- American men were from one mid-sized city in a southeastern state and may be more homogenous than a national sample of African-American men. Therefore, psychometric findings reported may not be generalizable to African-American men who reside in other U. S. regions or men of other races and ethnicities. Although participants had moderate experience with technology use, they had the least prior experience with using a tablet computer  is warranted to confirm convergent and discriminate validity of the Computer-Based Prostate Cancer Screening Decision Aid Acceptance Scale can be considered distinct from the Unified Technology Acceptance and Use Scale given that the later was developed to assess technology acceptance and use in business and banking, whereas the former is a major adaption to assess technology acceptance and use for informed PrCA decision making that may have life or death consequences. Thus, the emotion and anxiety evoked by informed PrCA screening decision making as well as the personal nature of the task including involving family members and health care providers suggest our scale is conceptually unique for healthcare decision making. Although preliminary, psychometric evidence from this study suggests the Computer-Based Prostate Cancer Screening Decision Aid Acceptance Scale has a conceptually distinct factor structure, good to excellent internal consistency reliability, and acceptable convergent and discriminant validity. PrCA is highly prevalent among African American men and PrCA knowledge is critical to making decisions about PrCA screening and early identification of PrCA. Given high rates of PrCA mortality among African-American men and the growing development of culturally-tailored CBDAs to assist African-American men with healthcare decisions about PrCA, the Computer-Based Prostate Cancer Screening Decision Aid Acceptance Scale can be influential in the evaluation of technology-based PrCA interventions. Most notably, our scale has robust psychometric proprieties for use among African-American men, who are not well-represented in current studies on technology use and acceptance. Because technology is more accessible than ever before and has been integrated into all aspects of our lives, the Computer-Based Prostate Cancer Screening Decision Aid Acceptance Scale shows promise as playing a key role in increasing PrCA knowledge and assisting in informed PrCA screening decision making among African-American men.In sum, the three-factor, 24-item


Intestinal-type gastric carcinoma exhibits a multistep carcinogenic sequence from adenoma to carcinoma with a gradual increase in genomic alterations. But the roles of microRNAs (miRNA) in this multistage cascade are not fully explored. To identify differentially expressed miRNA (DEM) during early gastric carcinogenesis, we performed miRNA microarray profiling with 24 gastric cancers and precursor lesions . Alterations in the expression of 132 miRNA were detected; these were categorized into three groups based on their expression patterns. Of these, 42 miRNAs were aberrantly expressed in EGC. Five miRNA  showed decreased expression since adenoma. Expression of two miRNA, miR-200C and miR-29a, was down-regulated in EGCs compared to normal mucosa or adenomas. Six miRNA  showed increased expression in gastric cancer compared to normal or adenoma samples. Five representative miRNAs were further validated with RT-qPCR in independent 77 samples. Taken together, these results suggest that the dysregulated miRNA show alterations at the early stages of gastric tumorigenesis and may be used as a candidate biomarker. A recent report by Min et al. showed a gradual increase in genomic alterations, including somatic nucleotide variation, gene fusion, and copy number variation, from LGD to carcinoma3.A subset of gastric cancer  exhibits a gradual development through a multistep carcinogenic sequence from non-neoplastic atrophic and metaplastic gastric mucosa to adenoma with low-grade dysplasia (LGD), followed by adenoma with high-grade dysplasia (HGD) and eventually develops into invasive carcinoma4. MicroRNAs (miRNAs) are abundant non-coding RNA molecules of 18–25 nucleotides that inhibit translation or promote degradation of messenger RNAs (mRNAs) with complementary sequences. miRNAs are estimated to regulate the expression of 30–60% of human genes and are known to modulate cell development, differentiation, proliferation, and apoptosis. Hence, alterations in their expression are associated with human diseases such as cancer6. Depending on its mRNA target, the miRNA may function as a tumor suppressor or promoter of tumorigenesis7.Aside from the accumulation of genetic alterations, epigenetic changes such as DNA methylation and aberrant gene expression by non-coding RNAs are other key players involved in carcinogenesis15. These profiles have the potential to be used as diagnostic biomarkers for early cancer detection and therapeutic targets for cancer prevention. Only a single study has reported the gradual increase in miR-106a, a member of miR-17 family, during the multistep gastric carcinogenesis using real-time quantitative polymerase chain reaction (RT-qPCR)16. However, there are no reports on miRNA profiles in gastric precursor lesions or during multistep carcinogenesis.In colorectal and esophageal adenocarcinomas, miRNA profiles of cancer and their precursor lesions such as Barrett’s esophagus or colorectal adenoma are well describedTo identify miRNA expression signatures through a multistep carcinogenic sequence, we performed NanoString miRNA expression assays in normal gastric mucosa, LGDs, HGDs, and intestinal-type early gastric cancers (EGCs) and subsequently validated five miRNAs using independent sample sets with RT-qPCR. In addition, we used RNA sequencing data from our previous study to investigate the correlation between the expression of miRNAs and their target mRNAs.We performed miRNA expression arrays by using NanoString to identify DEM patterns in normal gastric mucosa, adenoma with LGD and HGD, and EGC Table . We idenPrincipal component analysis (PCA) showed that the cancer and non-cancer samples were separated along the PCA1 axis, whereas the normal gastric mucosa and adenoma samples were differentially located along the PC2 axis Fig. . Unsuper21. The expression of two miRNAs (miR-200C and miR-29a), which are known to be down-regulated in gastric cancer23, decreased in EGCs as compared with normal mucosa or adenomas. In addition, there was an increase in the expression of six miRNAs , which are known to be upregulated in gastric cancer35, in EGCs as compared with normal or adenoma samples  in early gastric tumorigenesis Fig. . Among t36 and esophageal squamous cell carcinoma38, and reported as oncogenic (miR-938) miRNA in colorectal cancers39, but their roles have not been studied in gastric cancer. Unexpectedly, we found that expressions of miR-655 decreased from normal to adenoma and carcinoma, suggesting tumor suppressive role in gastric cancer. Meanwhile, miR-938 was up-regulated in EGCs compared to normal gastric mucosa and adenomas, suggesting oncogenic function in gastric cancer  in hepatocellularFor further experimental validation, we measured the expression of five miRNAs  in 77 independent samples using RT-qPCR, owing to the obvious difference (fold change >4) in their expression between normal and EGC group in the miRNA microarray analysis. We observed a progressive increase in the expression of miR-107 and miR-300 from normal to adenoma and EGC in microarray analysis; these results were subsequently validated in an independent sample set with RT-qPCR. In comparison to the normal gastric mucosa, adenomas and EGCs showed a decrease in the expression of miR-26a and miR-375 in both microarray and qPCR assays Fig. . HoweverTo determine the impact of these miRNAs on target genes, we calculated the correlation coefficient between the expression of miRNAs and their target genes using our previous RNA sequencing data. A negative correlation was observed between miR-26a and EZH2 (r = −0.689), miR-375 and YWHAZ (r = −0.629), and miR-375 and RUNX1 (r = −0.432) Fig. . We also3. Given the importance of miRNAs in the regulation of cell growth and viability, miRNA dysregulation is believed to be closely correlated with the development and progression of gastric cancer4. In the stomach, previous research on miRNA dysregulation was focused on gastric cancer itself40 and only one study has been conducted to explore the role of miRNA during the histologic progression from gastric adenoma to carcinoma without non-tumorous controls, warranting more research16. To our best knowledge, this is the first study to evaluate and compare the expression profile of miRNAs in non-tumor tissue, LGD, HGD, and EGC, using a high-throughput screening array. The aim of our study was to screen alterations in miRNA expressions during the stepwise gastric carcinogenesis and we demonstrated the role of miRNAs during the stepwise gastric carcinogenesis and confirmed their expressions with RT-qPCR.The pathogenesis of gastric cancer involves multistep genetic and epigenetic alterations, which predispose cells to neoplastic transformationet al.16 investigated the miR-106a expression in gastric dysplasia and gastric samples using in situ hybridization. The frequency and extent of miR-106a expression was found to show a gradual increase along the histologic progression from mild, moderate, and severe dysplasia to EGC. However, they failed to evaluate the expression of other miRNAs. In the present study, we identified alterations in miRNA expressions using normal, LGD, HGD, and EGC samples. Overall, the expression of miRNAs showed distinct patterns as compared with the expression pattern of mRNAs. In comparison to the EGC samples, samples from the non-cancerous group , showed similar miRNA expression pattern. Within the non-cancerous group, the normal and adenoma samples were separated based on their expression patterns.Although previous reports have revealed several molecular alterations in gastric cancer such as DNA mutations, copy number variations, mRNA expression changes, and miRNA alterations, most of these studies were conducted by comparing gastric cancer tissues or cell lines with normal gastric samples, without adenoma samples. Zhu NF1, DICER1, FOXO1, and CDK841. We found a gradual increase in the expression of miR-107 with the histologic progression from LGD to EGC. Microarray and RT-qPCR results revealed that the expression of miR-107 was significantly upregulated in HGD and EGC as compared with the normal mucosa .The expression of miR-107 has been reported to increase in the serum and tissues of gastric cancer patients and gastric cancer cell lines. miR-107 acts as an oncogene and regulates gastric cancer development and progression by targeting 34, liver cancer40, and osteosarcoma42. miR-300 displays the potential to be used in the treatment, diagnosis, and prognosis of gastric cancer, as its expression decreased in gastric cancer following chemotherapy or exposure to radiation43.Overexpression of miR-300 promotes cell cycle progression, cell proliferation, and invasion in several cancers, including gastric cancerFOXO133. Although we observed a relative increase in the expression of miR-370 in LGD, it showed a weak correlation with the expression of its target gene, FOXO3.In gastric cancer, miR-370 showed altered expression and acted as either an oncogene or a tumor suppressor. Previous studies have shown that the up-regulation of miR-370 resulted in the progression of gastric carcinoma via suppression of transforming growth factor beta receptor II (TGFβRII) or 44. miR-26a is known to be significantly down-regulated in gastric cancer and suppresses tumor growth and metastasis by targeting FGF9 gene17. Furthermore, miR-26a improves the sensitivity of gastric cancer cells to cisplatin-based chemotherapies by targeting NRAS and E2F245. We found a decrease in the expression of miR-26a from LGD and a strong negative correlation between the expression of miR-26a and its known target EZH2 (r = −0.689).The expression of miR-26a is frequently aberrant in many tumors such as gastric cancer, bladder tumor, breast cancer, oral squamous cell carcinoma, and Burkitt lymphomaJAK2, ERBB2, and YWHAZ48. We found a significant decrease in miR-375 expression in LGD, HGD, and EGC, as evident from the microarray and RT-qPCR results. Moreover, miR-375 expression was inversely correlated (r = −0.629) with the expression of YWHAZ (14-3-3ζ), a member of the 14-3-3 family of proteins, which has been implicated in the initiation and progression of cancers and is a potential biomarker for gastric cancer.Previous studies have demonstrated that miR-375 inhibits cell proliferation of gastric cancer cells by repressing 49. Although they did not show any stepwise elevation during multistep carcinogenesis, we could prove that they worked as tumor suppressive and oncogenic in gastric cancers. These results also prove that those novel miRNAs are important in gastric cancers, but do not work in multistep carcinogenesis, so they were not detected in our DEM miRNA groups.Additionally, we validated two novel miRNAs which have been known as tumor suppressive (miR-655) and as oncogenic (miR-938) miRNA in gastrointestinal cancers other than gastric50. In the present study, we selected target genes that have been known to interact with miRNA in the previous studies by experimental investigation to confirm the relationship between miRNA and target mRNA. Further in vitro experiments using cell lines and larger scale studies are recommended to confirm our results and to analyze the potential effects of those deregulated miRNA.Our study has several limitations. First, the sample size in miRNA array experiments is quite small (n = 24). To overcome this limitation, we replicated our microarray results in 77 FFPE samples and TCGA datasets. Secondly, the effects of altered miRNA on their target mRNA were not investigated. It is widely accepted that miRNAs have multiple -sometimes hundreds- of targets and the main approach to explore connections between miRNAs and their targets has been focused on the most significant target for each miRNAIn conclusion, we found that the upregulated (miR-107 and miR-300) or down-regulated (miR-26a and miR-375) miRNAs show alteration at early stages of gastric tumorigenesis and may be used as candidate biomarkers. Further investigation is needed to elucidate the exact role of these miRNAs in early gastric carcinogenesis.Fresh tumor and non-tumor samples were obtained by forceps biopsy at Samsung Comprehensive Cancer Center. Informed consent was obtained from all individuals who participated in this study. The study protocol was approved by the institutional review board of Samsung Medical Center (IRB 2010-09-020-008) and all experiments were performed in accordance with the approved guidelines and regulations.3. Tumors mixed with components of different histological grades were excluded from the study. Non-tumor gastric samples more than 2 cm apart from the tumor were used as a reference. All non-tumor samples showed intestinal metaplasia. All fresh tissue samples were snap frozen in liquid nitrogen immediately after collection and stored at −80 °C until use. Helicobacter pylori infection status was determined using both urea breath test and histology, while Epstein-Barr virus status was determined by the Epstein-Barr encoding region in situ hybridization.Fresh tumor samples from 14 patients were obtained during endoscopy by forceps biopsy and were used as a test set   samples were collected and used as a validation set . Informed consent was obtained from all individuals who participated in the study, and the study protocol was approved by the institutional review board.51.For the nCounter analysis, 10 consecutive tissue sections (4-μm thick) from archival FFPE tissues of precursor lesions (LGD and HGD) and carcinoma were used. Total miRNA was isolated using the Qiagen miRNeasy Kit  according to the manufacturer’s protocol. Total miRNA samples were analyzed using the nCounter Human miRNA Expression Assay kit  according to manufacturer’s instructions. Briefly, 100 ng RNA was incubated in the presence of miRNA-specific capture and reporter probes and non-hybridized probes were removed, followed by immobilization of the purified hybridized complexes. The abundance of specific target molecules was subsequently quantified on the nCounter Digital Analyzer by counting the individual fluorescent barcodes and assessing the target molecules, as previously described52 were also used to validate the patterns of DEMs and correlation between expression of miRNA and target mRNA expression.To identify the differentially expressed miRNAs (DEMs) among LGDs, HGDs, and EGCs, we calculated analysis of variance (ANOVA) p-value, average of miRNA expression within the same group, and fold change by comparing expression levels among the three groups. DEMs were defined by a p-value < 0.01, average expression >5 in at least one group, and |log2(fold change)| >1. The adjusted p-value was calculated using Benjamini and Hochberg algorithm in R. We next performed a k-means clustering to categorize DEMs into groups using expression patterns. To quantify the mRNA expression level, we counted the number of aligned fragments for each gene using HTSeq-0.6.1 with parameters  according to the Ensembl transcript annotation (GRCh37 version) and calculated the fragments per kilobase per million mapped read (FPKM) values of each gene. Correlation between miRNA and mRNA expression was calculated by Spearman’s correlation using R. miRNA and mRNA expression data from The Cancer Genome Atlas (TCGA) datasetsTen serial paraffin cuts obtained in an Eppendorf tube were deparaffinized in xylene. Total RNA was isolated using RNeasy Micro Kit  according to the manufacturer’s instructions. RNA concentrations were measured using NanoDrop . Total RNA from each sample was reverse transcribed with the TaqMan MicroRNA Reverse Transcription kit . Reverse transcription was performed with the following thermal cycling parameters: 30 minutes at 16 °C, 30 minutes at 42 °C, and 5 minutes at 85 °C (Bio-Rad).The miRNA expression was determined with TaqMan MicroRNA primer/probe sets. All qPCR reactions were performed with 7900 Fast Real-Time PCR System . Gene expressions for hsa-miR-375 , hsa-miR-370 , hsa-miR-26a , hsa-miR-300 , hsa-miR-1260 , and hsa-miR-107 , hsa-miR-655 , and hsa-miR-938  were quantified by TaqMan microRNA Assays (Applied Biosystems) according to manufacturers’ protocol and normalized by U6 snRNA (Assay ID 001973). PCR amplification of target genes and quantification of PCR products were performed by ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems). Differences in the expression were determined by the relative quantification method; the Ct values of the test genes were normalized to the Ct values of the endogenous control U6 snRNA. The fold change was calculated using the equation 2−ΔΔCt.Supplementary information