To compare the influence of objective and subjective measures of the three learning programs  on resident test performance and study platform preference.Sixty residents from three orthopaedic residencies were included in this study during May 2020. Trauma, pediatrics, and hip/knee reconstruction (joints) were chosen as testing topics. Residents took a standardized pretest of 30 questions per topic, followed by the completion of 50 questions per day for 5 days using one of the three web-based programs, followed by a standardized subject-specific posttest. This cycle was repeated for all the three topics. Bivariate statistics and a mixed-effects linear regression model were used to compare improvements in the scores.P < 0.001): 6.8% higher with OB, 5.4% with RS, and 1.0% with CC. The score improvement with OB was significantly greater than the score improvement with CC (P < 0.001). In total, 100% of residents reported that using OB would improve their score on the orthopaedic in-training examination, compared with 95% with RS and 67% with CC.Across all learning platforms, topics, and postgraduate year classes, posttest scores were 4.4% higher than the pretest score (73.3% vs. 68.9%, OB demonstrated the greatest improvement in scores, followed closely by RS and then CC. Because the Accreditation Council for Graduate Medical Education monitors every residency program's board pass rate, programs have a vested interest in identifying tools to track resident education progress and promote effective learning strategies.2-4For graduating residents in the United States, board certification examinations are very important and are heavily regulated to ensure that competency standards are maintained.2 Established in 1963 by the American Academy of Orthopaedic Surgeons (AAOS), the OITE is administered annually and serves as a tool that allows programs to assess their residents and identify individuals at risk of failing the ABOS examination.5 Over the last decade, several online question banks—including Clinical Classroom (CC), OrthoBullets (OB), and ResStudy (RS)—have been developed with the ultimate goal of promoting long-term information retention through reinforced cycles of information processing and retrieval.6-8 The relative utility of each of these resources in improving OITE test scores among orthopaedic residents has not been investigated.Currently, the orthopaedic in-training examination (OITE) is the primary objective assessment of resident knowledge throughout training and is highly predictive of performance on ABOS part I written examination.9 The primary purpose of this study was to compare objective measures of the top three learning web-based programs OB, RS, and CC for OITE performance. A secondary objective of this study was to use a survey to determine subjective resident preference of these three learning platforms. We hypothesized that, objectively, all three web-based programs would increase a resident's score and that, subjectively, there would be an equal distribution of residents preferring each study platform.Identification of optimal learning strategies and preparation resources available to orthopaedic surgery residents can improve their performance on the OITE, which may translate to superior clinical performance.This was a prospective cohort study of resident orthopaedic surgeons from three US allopathic orthopaedic residences during May 2020. Residents in postgraduate years (PGY) 1 to 4 were included in our analysis. PGY-5 residents were excluded from our study design because of concurrent preparation for ABOS part I written examination during the period in which this study was conducted. Because each residency program had six residents per PGY class, a total of 72 residents met the initial inclusion criteria.Residents were divided into three separate groups , each composed of members from all included PGY classes. All groups were assigned to a 15-day study schedule, which was subdivided into 5-day blocks corresponding to one of the three specific OITE topics. Each block consisted of a 30-question pretest on a single topic, followed by 50 questions completed daily on an assigned learning platform, and finally a 30-question posttest at the conclusion of each block Table . Group sOf the 72 residents who initially met the inclusion criteria, 39 (54%) completed the study plan in its entirety, 12 (17%) residents completed two platforms, 9 (13%) residents completed only one platform, and 12 (17%) residents completed none of the platforms. After excluding the residents who completed none of the study platforms, a total of 60 residents and 300 test scores (150 pretests and 150 posttests) were included in our final cohort.Three topics were chosen for this study: trauma, pediatrics, and joints. These topics represent the most highly tested material on OITEs from 2015 to 2019 Table . Shoulde10 The OB authors typically consist of resident physicians who achieved at least one >90th percentile OITE scores based on PGY class. Each question undergoes a rigorous peer-review process consisting of two independent reviewers with multiple stages of edits and strict tested content criteria.10 The review content is multimodal and includes case-based learning, videos, operative techniques, and discussion forums. OB includes a curriculum across various orthopaedic topics, with the ability to track daily progress through the material and a proprietary machine learning–enabled spaced repetition algorithm, called Anconeus, which provides users with daily learning question reinforcement.10Three learning platforms were evaluated in this study as follows: OB, RS, and CC. OB was developed in 2011 and currently has over 7,000 questions available from multiple sources including 2007 to 2013 AAOS self-assessment examination (SAE) questions, OITE-based questions (OBQ), and SAE-based questions (SBQ). OBQ and SBQ are generated internally from the OB authors.11 However, RS typically explains the rationale for answer choices by citing previously published literature to adequately explain the tested concept.RS was introduced in 2012 and has approximately 5,000 questions available for users. These questions are taken from previous 2012 to 2019 OITEs, 2015 to 2020 SAE questions, and previous Orthopaedic Knowledge Update examinations. Similar to OB, RS also offers residents the ability to select topics for testing and the ability to track the examination history and progress but does not offer a comprehensively structured educational curriculum.12 Although the residents participating in this study had access to OB and RS for several years, access to CC was obtained just before this study.Finally, CC was developed in 2017 and uses adaptive algorithms to reinforce learning based on user input regarding the perceived difficulty level for each question. After every answered question, users rank their subjective level of confidence with one of the four labels: (1) know it, (2) think so, (3) unsure, and (4) no idea. Using this information, proprietary adaptive learning algorithms generate question sets that continually test areas of weakness. CC has approximately 3,500 questions, all generated from well-known experts across various orthopaedic subspecialties. Similar to OB and RS, CC offers users the ability to filter questions by topic.Sixty questions for each topic  were randomly selected from the 2007 to 2013 AAOS SAE examinations found on the OB question database. Each set of 60 questions was divided equally into two sets, a pretest and a posttest. Question difficulty between the pretests and posttests was matched for each set of examinations based on the average percentage of OB users who answered a given question correctly. Considering the large sample size of OB users per question, this method served as a proxy for question difficulty. For participants who used OB as a learning platform, 2007 to 2013 SAE questions were deselected from the question bank in all user accounts so that all practice questions only consisted of OB generated OBQ and SBQ only. This way, we ensured that all pretest and posttest questions were curated from previous subject-specific SAE which were not available in any of the three web-based learning platforms and that no learning platform offered residents an unfair advantage. One PGY-5 resident (S.M.) reviewed the pretest and posttest questions to ensure that the most testable and appropriate topics were included.classmarker.com .13-15 The response data were downloaded directly from the website. All residents who completed the study were given the option to complete this survey. Participation in the survey was voluntary. The questions assessed resident perception of each learning platform's quality, efficacy, and relevance in relation to the in-training examination. These questions also assess what kinds of learning strategy residents were used to study for the in-training examination in the past and what they prefer to use in the future if given limited resources.An anonymous 22-question survey was developed based on surveys used in similar studies and constructed in t-test, paired samples' t-test, and one-way analysis of variance with post hoc analyses were done where appropriate. Because some residents only completed one or two of the platforms while others completed all three, we incorporated a mixed-effects generalized linear regression model to control for (1) inherent differences in test-taking ability between residents, (2) different pretest scores between PGY-classes, and (3) differences in percentages across the study topics. Based on our study design, test scores were nonindependent observations because each resident could account for more than one test score in different topics or learning platforms. In other words, test scores from all three study platforms and all three topics were compiled from tests taken by the same cohort of residents, making these test scores nonindependent of each other. As such, a mixed-effects model was chosen to account for both fixed and random effects stemming from the nonindependence within our data set that was inherent to our study design. Statistical significance was set at P = 0.05. Statistical analyses were done with the assistance of a statistician using STATA version 15.0 .A power analysis was done demonstrating that 150 pretests and 150 posttests were needed to detect a 5% difference between pretest and posttest scores with 80% of power. For bivariate analyses, independent samples' P < 0.001; Figure P < 0.001), but there were no significant differences in comparison between any other study platforms , followed by trauma (4.3%) and joints 2.9%) .On unadjusted analysis, there were no significant differences in score improvement between the PGY classes across all study platforms .At the completion of the study, 32% of all residents (23) responded to the survey. When asked to choose a single study resource, 52% (12) of residents chose OB, 48% (11) chose RS, and 0% (0) chose CC. On average, residents rated OB as 9 of 10, RS as 8 of 10, and CC as 5 of 10. In total, 100% of residents (23) thought that studying using OB would improve their score on the OITE, compared with 96% (22) with RS and 65% (15) with CC. When asked to rank the ease of use, 52% of residents (11) ranked OB first, compared with 43% (10) with RS and 5% (1) with CC . Positive comments for each learning platform included “OB has best explanations and well-designed Anconeus spaced repetition tool,” “ResStudy has the most recent OITE questions and is most similar to OITEs,” and “CC has multiple types of questions, not just multiple choice, along with their Recharge spaced repetition tool.” Criticism of each learning platform included “OB only has older SAE questions and no OITE questions,” “ResStudy has no repetition learning and does not provide explanations for all questions,” and “CC is not similar to OITE questions and has limited number of questions compared with other platforms.”Free response resident feedback was collected to comprehensively evaluate the likes and dislikes of each learning platform .Residents were also asked to provide preferred learning platforms and additional study resources used to prepare for the 2019 OITE  within a given program is required to maintain program Accreditation Council for Graduate Medical Education.12 In our survey, residents noted this difference between CC questions and OITE questions. In contrast to CC, RS uses questions from recent OITEs and SAEs and is thus more representative of an OITE for the question style and content.11 Although OB does not incorporate any actual previous OITE questions, OB offers OBQ and SBQ questions which are specifically designed by the OB authors to mirror OITE questions.10 Because our posttests were generated from a random sample of 2007 to 2013 SAE questions, CC questions may not have approximated standardized exam questions as accurately as OB or RS, leading to poorer posttest performances.The differences in effectiveness between the three study platforms may be explained by how well each study platform's practice question approximates OITE questions. CC, which was associated with the lowest score improvement, contains questions derived from panels of experts, rather than from previous OITEs.8 In a randomized controlled trial of 65 pediatric and emergency medicine residents, Larsen et al.6 demonstrated that study participants who focused on practice questions had markedly greater long-term retention of information than those who passively reviewed the material. However, the way in which practice questions are incorporated into a learning style can add to the effectiveness of any learning strategy and may explain, in part, why the largest score improvement was seen in OB. OB uses a proprietary repetition algorithm which continually tests areas of weakness in a systematic way that promotes active recall and long-term retention. In several studies, this type of “spaced-repetition” has been demonstrated to reinforce neural patterns and improve recall.12 CC also incorporates an automated repetition algorithm that serves to improve long-term retention. RS does not currently include a repetition algorithm, which was noted as a shortcoming of the platform based on survey comments. Ultimately, it is likely that both the similarity of OB practice questions to OITE questions and the robustness of its Anconeus spaced-repetition algorithm collectively contributed to its effectiveness and preference as a study tool compared with CC in our resident population.All three study platforms included in our study used practice questions as the primary study strategy, which has been shown to be effective for long-term information retention.17 Because of stay-at-home orders during the pandemic, we were unable to strictly enforce the completion of practice questions across learning platforms or the poststudy questionnaire among our resident cohort. As such, only 39 of 60 (65%) of residents completed the study plan in its entirety, and 23 of 60 (32%) completed the poststudy questionnaire, which may limit our ability to draw notable conclusions from the data set. Moreover, although we requested residents to use their assigned learning platform, we were unable to verify this during each block of the 2-week study period. Finally, because participants were aware that they were being observed during the study period, they were susceptible to the Hawthorne effect, which may have influenced their performance on pretests and posttests. Additional investigation focusing on the effect on actual OITE and/or ABOS part I scores in relation to each platform may be valuable. Additional studies may also evaluate which learning platforms or components have the greatest effect on establishing a foundational knowledge base and potentially superior clinical performance.The results of this study must be interpreted in context of its limitations. First, our pretest and posttest questions were derived from OB 2007 to 2013 SAE questions, and although there was no overlap of years with the 2015 to 2020 SAE questions on RS, we cannot be certain that some older SAE questions did not appear on the newer forms, although this situation would be highly unlikely. Moreover, the relatively small sample size of 250 questions per subject topic may limit the significance of our results; however, because of time constraints of didactic learning in the pandemic, we were unable to ask residents to complete more than 50 questions daily. Extraneous testing factors—such as location and concurrent work schedule—may have affected resident performance on pretests and posttests and were not accounted for in our study design. It is also possible that residents' lack of familiarity with CC relative to OB and RS may also have contributed to the findings. In addition, it is important to note that individuals have unique learning styles and may benefit differently from various question-based study platforms. Although question-based study platforms were tested, it is still critical for residents to use other resources for a solid core knowledge base. However, in our study, OB resulted in the best posttest improvement that does not necessarily correlate with a better comprehensive knowledge base. CC promotes using a metacognitive approach to improve critical thinking and complex decision making, with a focus on mastery and reinforcement more so than answering test questions. This overall study design was particularly unique because it involved residents from three different residency programs across a span of 2 weeks. A major factor as to why this was accomplished in May 2020 was because of the cancellation of elective cases and adoption of a modified resident schedule during the first wave of coronavirus disease 2019.OB demonstrated the greatest improvement in scores, followed by RS and then CC. These results provide useful information and insights for residents and residency programs seeking optimal learning resources for OITE preparation.


To investigate the diagnostic and staging value of serum angiotensin-converting enzyme (sACE) in sarcoidosis.  Patients with suspected sarcoidosis treated in the Department of Pulmonary and Critical Care Medicine of the China-Japan Friendship Hospital from 2010 to 2020 were included. The data of sACE, erythrocyte sedimentation rate (ESR), complete blood count (CBC), lung function, bronchoalveolar lavage, and biopsy were collected. The differences between the sarcoidosis group and the nonsarcoidosis group and between different stages of sarcoidosis were compared. The receiver operating characteristic (ROC) curve analysis was used for the diagnostic test of sACE in sarcoidosis. P = 0.001). The level of sACE in sarcoidosis patients with peripheral superficial lymph nodes and multiple system involvement was significantly higher than that in intrathoracic sarcoidosis patients (P = 0.009); the percentage of lymphocytes in bronchoalveolar lavage fluid (BALF) of sarcoidosis patients was 45.39 ± 22.87%, which was significantly higher than that of nonsarcoidosis patients (P < 0.001). There was no correlation between sACE and ESR (correlation coefficient = −0.167). According to ROC curve analysis, when sACE ≥ 44.0 U/L, the sensitivity of sarcoidosis diagnosis was 61.4%, the specificity was 92.9%, and the AUC was 0.819.  A total of 84 cases with suspected sarcoidosis were included, among which 70 cases were confirmed to be sarcoidosis by biopsy. The mean value of sACE in sarcoidosis patients was 56.61 ± 30.80 U/L, which was significantly higher than that in nonsarcoidosis patients (28.07 ± 14.11 U/L,  sACE has a good specificity in the diagnosis of sarcoidosis. sACE values in patients with sarcoidosis with systemic involvement were higher than those with simple intrathoracic sarcoidosis. Sarcoidosis is a multisystemic granulomatous disease of unknown etiology. Pulmonary involvement is most frequent in sarcoidosis patients, while extrathoracic involvement occurs in only 30% of cases –4. It haβ-amyloid peptide (Aβ) 42 into Aβ40 in the human brain, and its conversion activity is mediated by two catalytic domains [At present, increasing research results indicate that serum markers are of great value in disease diagnosis and prognosis prediction, with advantages of simple sample collection, minimal invasiveness, low cost, high sensitivity, and specificity , 10. Cli domains . sACE, w domains . This suThis study prospectively included patients with suspected sarcoidosis hospitalized in the Department of Pulmonary and Critical Care Medicine of the China-Japan Friendship Hospital between 2010 and 2020. A total of 84 cases of suspected sarcoidosis were included, 70 cases of which were histopathologically confirmed. Among them, 9 cases were confirmed as lymphatic tuberculosis, 2 cases as malignancy, and 3 cases as other diseases that cause enlargement of mediastinal and hilar lymph nodes. According to the histopathological diagnosis, 70 patients with sarcoidosis were assigned to the sarcoidosis group, and the remaining 14 patients were assigned to the nonsarcoidosis group. Almost all the cases presented with cough, expectoration, chest tenderness, or shortness of breath, while fever as well as fatigue and weight loss were observed in 18 and 19 cases, respectively.Patients meeting one of the following criteria were considered suspected sarcoidosis: (1) chest radiograph or computed tomography showing bilateral or unilateral hilar or mediastinal lymphadenopathy, with or without pulmonary opacities; (2) on the basis of (1), patients presented with respiratory symptoms such as cough, dyspnea, and chest pain, accompanied by fatigue, malaise, fever, or weight loss; and (3) other conditions that cannot exclude sarcoidosis as confirmed by a respiratory physician.The diagnostic criteria of sarcoidosis were based on the following: (1) consistent clinical and radiographic findings, (2) histopathologic detection of nonnecrotizing granulomas, and (3) exclusion of other diseases.n = 14, 20.00%), stage II group , and stage III group , with no stage IV patients. Furthermore, sarcoidosis patients were assigned to the intrathoracic sarcoidosis group , sarcoidosis-involved intrathoracic organ and peripheral lymph node group , and multisystemic sarcoidosis group  based on the location of nodular involvement. Among extrathoracic sarcoidosis, there were 2 cases of skin involvement, 4 cases of hepatic involvement, 3 cases of splenic involvement, 3 cases of ocular involvement, and 1 case of cardiac involvement.Sarcoidosis patients were staged for the disease according to the Scadding system of staging : stage IWritten informed consent was obtained prior to data collection. The study was approved by the Ethics Committee of China-Japan Friendship Hospital.sACE: the peripheral blood serum was collected from patients to detect sACE using an ultraviolet spectrophotometer.Complete blood count (CBC): CBC, including white blood cell count (WBC), lymphocytes, and hemoglobins, as well as the level of C-reactive protein (CRP), was routinely measured on the collected peripheral blood through a blood analyzer .Erythrocyte sedimentation rate (ESR): ESR was measured by an automatic erythrocyte sedimentation rate analyzer .Serum calcium and adenosine deaminase (ADA): the levels of serum calcium and ADA were detected by a biochemical analyzer .Pulmonary function test (PFT): the pulmonary function of patients, including forced expiratory volume in 1 s (FEV1), forced vital capacity (FVC), total lung capacity (TLC), carbon monoxide diffusing capacity (DLCO), and partial pressure of blood oxygen (PaO2), was detected using a pulmonary function tester .Bronchoscopy: the percentages of lymphocytes and neutrophils in bronchoalveolar lavage fluid (BALF) were detected.All cases underwent chest computed tomography. The results showed 54 cases (77.14%) of symmetrical BHL, 5 cases (7.14%) of unilateral hilar lymphadenopathy, and 64 cases (91.43%) of mediastinal lymphadenopathy. There were 55 cases (78.57%) of pulmonary involvement, 37 cases (67.27%) of small nodules or patchy opacities which were predominantly distributed in the upper and middle lung zones, 18 cases (32.72%) of reticular opacities, and 9 cases (12.85%) of small amounts of pleural effusions.Biopsy specimens were obtained via EBUS-TBNA, transbronchial lung biopsy (TBLB), CT-guided percutaneous fine-needle aspiration, video-assisted thoracoscopic surgery, peripheral lymph node biopsy, skin biopsy, etc. Of the above various sample collection methods, EBUS-TBNA and/or TBLB was performed in 50 cases, skin biopsy or peripheral lymph node biopsy was performed in 13 cases, CT-guided percutaneous fine-needle aspiration was performed in 2 cases, and video-assisted thoracoscopic surgery was performed in 5 cases.t test and analysis of variance were used to compare the differences in variables between groups. Count data, represented as a constituent ratio, was compared by the chi-square test. Pearson's correlation analysis was used for correlation analysis between two groups. The diagnostic test of sACE in sarcoidosis was performed by receiver operating characteristic (ROC) curve analysis. Differences with P values < 0.05 were considered statistically significant.All data were analyzed by statistical software SPSS 21.0. The data were presented in the format of mean ± SD. Unpaired Among the 70 sarcoidosis cases, 56 cases (80.00%) were female. The mean age of the total sarcoidosis cases was 55.94 ± 11.83 years, with 52 cases (74.28%) presenting respiratory symptoms.P = 0.001). The percentages of lymphocytes and neutrophils in BALF of sarcoidosis patients were significantly higher than those of nonsarcoidosis patients (P < 0.001). Although 30 (42.85%) sarcoidosis patients presented with elevated ESR, there was no statistical difference in ESR level between sarcoidosis and nonsarcoidosis patients nor were there any significant differences in age and results of CBC and PFT between sarcoidosis and nonsarcoidosis patients. Forty-six sarcoidosis patients underwent tuberculin skin test (TST), returning 38 (82.61%) negative, 4 (8.70%) weak positive, and 2 (4.35%) positive results. Twenty cases exhibited abnormal PFT results, with 5 cases showing restrictive ventilatory defect, 14 cases showing obstructive ventilatory defect, and 1 case showing mixed ventilatory defect. The damage of PFT was mild in sarcoidosis patients.The mean value of sACE in sarcoidosis patients was 56.61 ± 30.80 U/L, which was significantly higher than that in nonsarcoidosis patients . Data are shown in Fifty-nine cases (57.1%) underwent bronchoscopy, and BALF was obtained from all of them. The percentage of lymphocytes in BALF of sarcoidosis patients was significantly higher than that of nonsarcoidosis patients , while there was no significant difference in age, gender, clinical manifestations, CBC, ESR, and PFT among them. Data are shown in The sACE level in patients with intrathoracic sarcoidosis was 47.05 ± 23.66 U/L, the sACE level in sarcoidosis patients involved both intrathoracic organs and peripheral lymph nodes was 56.47 ± 29.68 U/L, and the sACE level in sarcoidosis patients with multisystemic involvement was 64.94 ± 39.72 U/L. The level of sACE in sarcoidosis patients with peripheral superficial lymph nodes and multisystemic involvement was significantly higher than that in intrathoracic sarcoidosis patients (P < 0.05). The mean DLCO%pred was 86.24 ± 13.17% in stage I sarcoidosis patients, 77.45 ± 12.94% in stage II sarcoidosis patients, and 66.05 ± 17.93% in stage III sarcoidosis patients, with statistical significance (P < 0.05). As to the percentage of lymphocytes in BALF, there was no statistical difference between subgroups of sarcoidosis with different stages (P = 0.963). Data are shown in There was no statistical difference in age, gender, clinical manifestations, sACE, CBC, and ESR between sarcoidosis patients of different types of system involvement. As to PFT, the mean FEV1%pred was 99.67 ± 12.55% in stage I sarcoidosis patients, 81.43 ± 15.60% in stage II sarcoidosis patients, and 93.42 ± 20.49% in stage III sarcoidosis patients, with statistical significance  was 0.819. Thus, sACE ≥ 44.0 U/L may be an ideal cut-off value in sarcoidosis diagnosis. The ROC curve is shown in There was no correlation between sACE and ESR .Sarcoidosis is a multisystemic granulomatous disease with the common respiratory symptoms including cough, dyspnea, and chest pain, often accompanied by fatigue, malaise, fever, and weight loss . SarcoidOf the 70 sarcoidosis cases in this study, 52 cases (74.28%) presented with varying degrees of respiratory symptoms such as cough, expectoration, chest distress, and shortness of breath. Extrathoracic involvement can occur in some sarcoidosis patients, which mainly involves the skin, eye, liver, spleen, and cardiac system, presenting as fever, weight loss, and fatigue. In this study, the peak age of onset is middle-aged, with a predilection for women, which is consistent with the previous report . Over 90The final diagnosis of sarcoidosis is based on histopathology, so histopathology is strongly recommended in patients with suspected sarcoidosis. Peripheral lymph node biopsy or skin biopsy is a simple, safe, and reliable diagnostic approach. For patients without peripheral lymphadenopathy or skin involvement, EBUS-TBNA combined with TBLB is a relatively simple and safe diagnostic method with a high positive rate. In this survey, 50 cases underwent EBUS-TBNA and/or TBLB, and the histopathological diagnostic rate was 71.4%, which was similar to the positive rate of 88% reported internationally.The exact etiology and pathogenesis of sarcoidosis remain unknown. In the diagnosis and prognosis evaluation of sarcoidosis, the ideal marker to indicate diagnostic approach and disease activity monitoring has not yet been discovered . ElevateIn this study, the level of sACE in sarcoidosis patients was significantly higher than that in nonsarcoidosis patients with hilar and/or mediastinal lymphadenopathy. sACE can be an indicator for differential diagnosis of lymphatic involvement in nonsarcoidosis patients. It is reported that ESR is a useful marker of sarcoidosis activity . The levSarcoidosis can involve multiple systems with complex and varied clinical manifestations. Over 90% of sarcoidosis patients have pulmonary involvement. Although extrathoracic presentations as the initial symptoms of sarcoidosis are rare, accounting for 23% of sarcoidosis inpatients, they can be the main clinical manifestations and characteristics in some patients with sarcoidosis. According to a survey of 22 sarcoidosis cases whose initial symptoms were extrathoracic symptoms, 6 cases were identified as peripheral lymphadenopathy, 5 cases as skin involvement, 2 cases as ocular involvement, 3 cases as parotid gland sarcoidosis, 3 cases as arthritis, 1 case as cardiac sarcoidosis, 1 case as hepatic and splenic enlargement, and 1 case as neurologic sarcoidosis. Sarcoidosis has varying clinical manifestations and characteristics, which are related to race, staging, systemic involvement, and granulomas activity. The results of this study indicated that the sACE level was significantly higher in extrathoracic sarcoidosis. sACE levels reflect the varying degrees of granuloma, rather than pure pulmonary involvement, and are correlated with disease activity and extent. The level of sACE tends to be higher when sarcoidosis is active. Thus, sACE can indicate extrathoracic involvement in sarcoidosis.In this study, 44.0 U/L of sACE was defined as the optimal diagnostic cut-off for sarcoidosis diagnosis. We used 44.0 U/L as the diagnostic cut-off and histopathology as the golden diagnostic criterion to make a diagnosis in 84 cases. The results showed that although the sensitivity of sACE ≥ 44.0 U/L for sarcoidosis diagnosis was 61.4%, the specificity of 92.9% was high and ideal. According to data from Judson , the sensACE is an important diagnostic approach for sarcoidosis diagnosis. However, the normal range of sACE should be revised based on analysis of large sample data from multicenter investigations to improve the diagnostic efficiency of sarcoidosis. In addition, the high level of sACE in sarcoidosis patients with extrathoracic involvement suggests that sACE can be used as an important indicator for further screening and assessment of extrapulmonary involvement.


In the present study, a simple and low-cost strategy has been employed to fabricate nanoporous Mn2O3 with an increased surface area for an electrode/electrolyte interface that improved the conduction of Mn2O3 material. The crystal phase and morphology of the prepared material was investigated by X-ray diffraction (XRD), scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy (EDX). The prepared electrode materials were deposited on a nickel foam substrate to investigate the electrochemical properties. The galvanostatic charge/discharge (GCD), cyclic voltammetry (CV), and complex impedance studies confirmed excellent specific capacitance and capacitive behavior of the prepared material. The synthesized Mn2O3/graphene composites exhibited an excellent specific capacitance of 391 F/g at a scan rate of 5 mV/S. Moreover, a specific capacitance of 369 F/g was recorded at a current density of 0.5 A/g using the galvanostatic charge/discharge test. The high porosity of the materials provided a better electrolyte-electrode interface with a larger specific area, thus suggesting its suitability for energy storage applications.A simplified sol-gel method that can be scaled up for large-scale production was adopted for the preparation of manganese oxide nanocrystals. Prepared Mn Over the past few decades, energy storage strategies are one of the key challenges owing to the progressive exhaustion of fossil fuels and a fast energy depletion rate . Renewab2O3 composite electrode on Ni foam, while in the present study effective and simple techniques are used to get the valuable electrochemical results . Mn2O3 is an inexpansive, environmental friendly, naturally abundant material  and the microstructural features by scanning electron microscopy (SEM). The elemental study was performed by an energy-dispersive X-ray spectroscopy (EDX). The main focus of the present study was to investigate the electrochemical performance of prepared composite materials. In the present study, the electrochemical performance demonstrates that Mn2O3/graphene nanocompsites have great potential with a simple and low-cost method as compared to report previous.In the recent past, lot of efforts have been made to synthesize a composite material to exploit the properties of both carbonaceous (high conductivity) and transition metal oxides (TMOs) (high specific capacitance) such as a manganese oxide composite with carbon nanofoams , carbon material . The commaterial . Herein,2O3 nanoparticles by dissolving 25 gm of manganese nitrate (Mn(NO3)2.4H2O) in 100 ml of ethylene glycol. The amount of the solvent was adjusted such that manganese nitrate was completely dissolved. The solution was further heated at 80°C under continuous stirring until a thick gel was formed. The gel burnt slowly into a fine powder by increasing temperature to 100°C. The resultant powder was sintered at 700°C in an electric furnace for 4 hours.The sol-gel method was used  to synth2O3/graphene with the ratio  were prepared by mixing appropriate amounts of the composition with a mortar and pestle for 30 min. Then the mixture was dispersed in deionized water and sonicated for 30 min hour. Afterward, the mixture was poured into an electric motor blender and was blended for 30 min. While blending, nanoporous Mn2O3 micro-particles having higher density than graphite acted as high-energy projectiles hitting graphite clusters and exfoliated them to graphene, thus improving uniformity and mixing of the composites. Afterward, a microwave oven has been employed to dry the obtained composite materials for 1 hour.The composites of Mn2 × 1 cm2) was used as a substrate. The deionized water was added to the mixture of composite material and PVA (10%) to make a thick slurry. This slurry of the composite material was then transferred into a glass tube and sonicated for 15 min. The substrate was inserted into the slurry and sonicated for 10 min such that the pores of the Ni foam (The Ni foam with an area of about (1 cm Ni foam  uniformlα) having a wavelength of 1.54 angstrom as an X-ray source.The materials were explored for structural properties by employing the XRD technique. The crystal structure of the synthesized materials was investigated by employing an X-ray powder diffraction technique  with Cu  the plane of the cubic structure of Mn2O3 by using the Deby–Scherer equation, which was found to be 22.67 nm , atom coordinates , cell geometrical parameters  and , occupation (occ), thermal parameters (Bs), and FWHM parameters . The agreement between the experimental and approximated profiles was checked by the discrepancy factors which comprise the expected R-factor (Rexp), the Bragg R-factor, the weighted profile R-factor (RWP), and goodness of fit (χ2). The XRD Rietveld refinement revealed a cubic unit cell with a space group symmetry of Ia-3 space and computed various structural parameters of the crystal structure for Mn2O3 particles. The summary of the refined results are enlisted in the table.The material was further investigated for the crystalline structure and a quantitative analysis, and the XRD profile of Mn2O3/graphene composite. Here, the black symbol (star) marks the Bragg reflections of the Mn2O3 single phase, while the high intensity diffraction peak appearing at 2θ value of 44.57° represented by green symbol (triangle) indicates the Bragg reflections arising from graphene. Thus, the XRD profile confirms the presence of both phases and assures the successful formation of Mn2O3/graphene composite.2O3 has been examined by an energy-dispersive x-ray (EDX) technique, as shown in 2O3 and discard the presence of other elements; this assures the phase purity of the material and hence these results are consistent with the XRD data discussed earlier  MIRA3 TESCAN, as shown in 2O3 single phase. From the figure, it can be observed that surface morphology of Mn2O3 exhibits a nanoporous structure with agglomerated crystallites. The agglomeration may occur due to sintering of the material at higher temperature of about 700°C and also of magnetic nature of its nanoparticles  as a reference electrode, platinum (Pt) is used as a counter electrode, and Mn2O3/graphene as a working electrode in 1 M K2SO4 as electrolyte.The galvanostatic charge discharge and cyclic moltamogram measurements in a three electrode setup have been used to study electrochemical performance of Mn2O3 nanoparticles in aqueous electrolyte indicative of pseudocapacitive behavior. The pseudo capacitance may arise from an Mn+3/Mn+2 couple. The below-mentioned equation was used to determine the specific capacitance of the cell  give high specific capacitance. Specific capacitances of all three composites were estimated using The cyclic voltammetric study performed at 5, 10, 20, 50, and 100 mV/s scan rates, respectively, for all three composites, as shown in the cell :Ccell=Q2At a higher scan rate, all the charges accumulate at the surface of the electrode material. But at the lower scan rate, ions have ample time for their diffusion in the bulk part of the material to accumulate larger amount of charges and hence results in higher specific capacitance , as show2SO4 electrolyte, as depicted in The GCD analysis for all three given samples A, B, and C  at different current densities in 1 M solution of KA sinusoidal potential is applied to measure the current that passes through an electrochemical cell and an AC voltage is used to measure the electrochemical impedance which depicted the AC current along with excitation potential including its harmonics . The pse|Z|. The data for EIS can be demonstrated by equivalent circuit models  exponent m, pore resistance Rpor, corrosion “double layer” capacitance Ccor, a (CPE) exponent n, and corrosion resistance Rcor of the Mn2O3/graphene composite electrode material  and do not behave ideally  of the synthesized material. The value of R ideally . The imp the CPE .2O3/graphene composite is shown in x-axis, while the phase-shift and absolute values of the impedance (|Z| = Z0) are taken along the y-axis. In the abovementioned figure, the Bode plot of Mn2O3/graphene composite is sketched using the logarithmic scale. By changing the frequency of the as-prepared composite, the magnitude and the phase angle is obtained. The phase angle tells about the behavior of the supercapacitor.The Bode plot of MnThe pseudocapacitve behavior can be clearly evident from the charge discharge cycles which have increased the specific capacitance, as shown in 2O3/graphene composite was synthesized with a simple, low-cost, environmental friendly facile method. Herein, the sol-gel–derived Mn2O3 resulted in a nanoporous structure of prepared materials. The high porosity of the materials provides a better electrolyte-electrode interface with a larger specific area that was desirable for energy storage applications. Composites of Mn2O3/graphene were prepared with sonication plus electric motor blending. The method used is simple and cost-effective that can be scaled-up for industrial scale production. Mixing of composites by sonication and mechanical blending resulted in exfoliation of graphene and uniform mixing of composite electrode materials. The XRD pattern confirmed a cubic structure of Mn2O3 having a crystallite size of about 22.7 nm. The surface morphology of Mn2O3 investigated by SEM exhibited nanoporous microstructures. SEM micrographs of the composite revealed the uniform mixing of Mn2O3 with exfoliated graphene sheets. Furthermore, the electrochemical data of different mass ratios of Mn2O3/graphene composites obtained from GCD and CV showed an improved capacitance in the applied potential window. This report provides an effective and simple route to synthesize Mn2O3/graphene composites with an enhanced electrochemical performance for supercapacitor applications. Sample with an Mn2O3/graphene ratio of 1:3 has highest specific capacitance of up to 391 Fg-1 at a scan rate of 5 mV/s due to a large surface area and high conductivity provided by increased amount of graphene. This research work provides an effective and simple way to prepare manganese oxide Mn2O3/graphene composites with high electrochemical performance for supercapacitors. Samples of Mn2O3/graphene with ratios 1:1, 1:2, and 1:3 have specific capacitance of 271, 321, and 391 Fg-1, respectively, at a scan rate of 5 mV/s. The capacitance of composite exhibited an increasing trend with the increasing graphene concentration. These results lead to the conclusion that composite is useful electrode material for super capacitor applications.An Mn


Over the last decades, severe haze pollution constitutes a major source of far-reaching environmental and human health problems. The formation, accumulation and diffusion of pollution particles occurs under complex temporal scales and expands throughout a wide spatial coverage. Seeking to understand the transport patterns of haze pollutants in China, we review a proposed framework of time-evolving directed and weighted air quality correlation networks. In this work, we evaluate monitoring stations’ time-series data from China and California, to test the sensitivity of the framework to region size, climate and pollution magnitude across multiple years (2014–2020). We learn that the use of hourly  The accelerated industrialization and urbanization of many economically developing countries has led to major air pollution with severe impacts both for human health and the environment  in the segment connecting the Bay Area with Los Angeles.As for the spatial distribution in China, Fig. T according to:i is denoted by series i to j for Following an analysis framework proposed and applied for the study of environmental complex systems , and computed as the average of absolute values of correlations from the permuted data. That way, correlations appearing less in the randomized version do not generate links.As in our predecessor studies, the weighted adjacency matrix of the correlation network of week The strength of the correlations can also be quantified by standardizing the correlations i and j defined asTo quantify the influence of a node to its surroundings, we use the weighted directional degree, as introduced in Zhang et al. . For thei and j respectively.t we are evaluating, the communities generated are very stable. Variations in number of communities are minor and only express a merge or split of communities with not significant jumps of nodes between communities. For each network, the algorithm is being run 100 times. For the minority of nodes that may switch communities across runs, we assign them to the community they appear in the most.Community detection algorithms aim to decompose a network into sets of sub-units comprising of highly inter-connected nodes  networks leads to evolving communities with time. To track the changes in structure, we treat our networks as a time-series of static networks, called timeframes with weekly resolution, according to the method proposed by Greene et al. . We aim D we deploy the heuristic threshold-based method introduced by Greene et al. Greene et al.  to account for the seasonal dependence of -3. Over the next two days, -3. The increases were abrupt, causing sharp peaks in the time-series raising the concentrations to four times the average up until that moment during the week. In our example, the majority of generated correlations are positive. Figure In Fig. entclass1pt{minimaNetworks are formulated under both methods as defined in the Data and Theoretical Background section. For the method of the maximal value of the cross-correlation function, all links receive absolute values above the critical correlation threshold  formulates less densely connected networks. The Z-scores, which are computed through standardization of the correlation function, share the same shape PDF }PC Fig. e, but whBased in our analysis with hourly data at a weekly scale and focusing in haze events, we conclude the Z-score approach overcomes the artificial correlations due to persistence and auto-correlations the time-series. These phenomena are frequent in environmental and climate related variables  wind flows were strong northern over the California coast while Southern California’s were weak and of varying directions  of 2020 versus networks from the same dates in 2014 to 2019. This comparison helps us draw conclusions under similar meteorological conditions (same dates across different years) but with significantly reduced emissions from human mobility because the lockdown. Figure To determine regions with systematic pollution transport behavior, the Louvain algorithm of network community detection was applied to identify partitions in the dynamic AQ networks formulated under the Z-score method. We apply the method in China where more monitoring stations are available and pollution haze events are attributed to less unpredictable and variable events such as the California wildfires. This study emphasizes on long-lived and persistent dynamic communities that appear for several consecutive weeks and persist across our years of evaluation. With The southeastern community (blue) appears to be the least persistent of the four according to Fig. mentclass2pt{minimWe identify the core nodes of the four dynamic communities as the nodes appearing in more than half of the timestamps (weeks) the DC appears. That translates to 21, 23, 17 and 18 nodes for the red, green, blue and yellow dynamic communities respectively. As shown in Fig. Our work has shown that the development of directed and weighted air pollution correlation networks exploiting hourly We deem that the Z-score method, based on standardization of the correlation function, is more insightful than the maximal value of the correlation function when it comes to analyzing haze events with sharp peaks. It overcomes the artificial correlations due to persistence and auto-correlations within the time-series records. As shown in Fig. We extend the Z-score network analysis framework via the Louvain method of community detection in China. Results demonstrated distinct communities presenting consistent air pollution transport patterns and high interconnections between their cities across all years of evaluation. In more detail, four communities prevail as the most persistent grouping the provinces of Shangha–Jiangsu, Shandong, Heibei–Beijing–Tianjin–Liaoning, and Hunan–Jiangxi–Guangdong. The four dynamic communities align geographically with the BTH, Yangtze River Delta, and the Pearl River Delta which correspond to the regions in China with the highest concentrated populations attracted from the river basins and constitute the nuclei of the highest industrial output. Interestingly, identifying the core nodes of the four communities, we showed that they are sufficient to reproduce the key temporal and spatial patterns of the analysis. Apart from significantly reducing the complexity of the original networks, this offers potential as a pre-processing method for AQ forecasting and prediction of extreme pollution events as well as informing the re-positioning of stations to monitor areas with no records. Overall, the network partitioning allows to disentangle spatial patterns, identifying regions where pollution transport is more persistent.


Chile, Argentina, Costa Rica, Puerto Rico, Colombia, and Brazil had overall rates between 13 and 17 per 100,000 during the same period.7 Similarly, Asian countries such as Thailand and Korea have reported an increase in mortality rates from 50% to 260%, respectively.8 In the same direction, Center et al4 have reported a rapid increase in mortality in most Central and Easter European countries.On the other hand, many developing countries in Latin America such as Colombia, Costa Rico, and Ecuador have experienced increasing PCa trends from 1993 to 2002, and countries such as Brazil, Colombia, Cuba, and Ecuador have reported an increase in mortality rates.9 have reported that advanced-stage rates in East Africa have been much higher than those of African Americans in recent years. As an example, Osegbe10 reported that 64% of patients with new PCa in a Nigerian hospital died within 2 years of diagnosis. It has been estimated that 57,048 deaths associated with PCa will occur in Africa by 2030. This represents a 104% increase in the number of PCa deaths in Africa over the next 20 years.11Although the cancer-specific mortality has been reported to be the lowest in Asia and Northern Africa, Chu et al12A total of 28,000 deaths of men of all ethnicities in Africa because of PCa as reported were more than four times the 6,500 that occurred among Caribbean men of all ethnicities and exceeded deaths from any other cancer in Africa.13There are multiple reasons aside from variation in tumor biology for the increased mortality rates in developing countries, which include lack of a screening program, diagnosis at an advanced stage, limited access to local and systemic treatments, most affected being urban poor or from rural and remote populations having poor or no healthcare coverage, lack of affordable transportation to health centers, limited number of critical therapeutical tools such as radiation therapy, missing specifically trained healthcare staff including physicians with not a full training in oncology, and lack of information and guidelines. Many guidelines have addressed the gold standard in the diagnosis and management of PCa such as the National Comprehensive Cancer Network guidelines, but in general they do not provide specific, detailed, and adapted information and guidance for low-resources areas.14The first Global Prostate Cancer Consensus Conference for Developing Countries (PCCCDC) was organized around state-of-the-art lectures and presentations and discussed evidence relevant to 12 key topics and subtopics related to the management of PCa in general and in limited-resource regions . Four polling sessions were scheduled during the 2-day conference for panelists to respond to questions regarding these topics. Only physicians who participated in all four sessions are included in the final consensus results. Several of the questions were based on the questions from the Advanced Prostate Cancer Consensus Conference (APCCC) 2017, the conference that stimulated and inspired the PCCCDC.The full panel for this consensus paper consisted of 99 multidisciplinary cancer physicians including urologists, medical oncologists, radiation oncologists, radiologists, and pathologists from developing countries in Latin America, Africa, Middle East, Asia, and Eastern Europe. The panel members were selected on the basis of recent work and their willingness to be present at the first PCCCDC. The draft of recommendations and clinical questions were selected previously by a committee composed of specialists in the fields. The committee of radiologists was led by Douglas Racy, MD; the committee of radiotherapy was led by Robson Ferrigno, MD; the committee of urology was led by Arie Carneiro, MD, Murilo Luz, MD, and Gustavo Guimarães, MD; the committee of oncology was led by Fernando Cotait Maluf, MD, Felipe Moraes, MD, and Andrey Soares, MD.14 The conference included presentations and debates from participants who reviewed evidence relevant to the above questions. On the last day of the conference, all questions were presented with options for answers in a multiple-choice format. The questions were voted on publicly but anonymously. In some cases, discussions and revoting occurred. Each question had five to seven answer options including two nonanswers . Each question was deemed consensus if 75% or more of the full panel selected a particular answer. The two nonanswers were provided for quality control. Unless stated otherwise, it is assumed that for the specific recommendation , therapies are approved and available, no treatment contraindications exist, and no clinical trial is currently ongoing. If consensus is not achieved, then the Steering Committee may opt to leave a clinical question unanswered and state, “Consensus could not be achieved.” All the questions can be accessed in the Data Supplement. For the questions that refer to an area of limited resources, the recommendations consider cost-effectiveness and the possible therapies with easier access. In addition, recommendations are for nonfrail patients and for patients with prostate adenocarcinoma (unless stated otherwise). Their answers are annotated and discussed in the following manuscripts that address screening, diagnosis, and staging tools, and treatments for the topics are stated in Table The consensus was developed on the basis of a modified Delphi approach Fig . In a moThe panel for this consensus paper consisted of 99 multidisciplinary cancer physicians including urologic surgeons, medical oncologists, radiation oncologists, radiologists, and pathologists from developing countries in Latin America, Africa, Middle East, Asia, and Eastern Europe. The selection of panel members was based on special expertise in PCa, recent work in this field, and their willingness to attend the first PCCCDC. All the participants and medical specialties are included in the Data Supplement.14 Following each question, there were five to seven answers options including the option to abstain and/or to identify that they felt unqualified to answer. These options allowed for physicians to opt out of questions that they may not contend with in their specific specialty. Questions were designed to include recommendations of therapies or treatments that are approved and available, and no treatment contraindications exist, unless otherwise stated in the question. An answer was deemed to have reached consensus if 75% or more of the full panel selected a particular answer. For the questions referring to an area of limited resources, the clinicians were instructed to consider both cost-effectiveness and access.The questionnaire was developed by a panel of seven experts to provide relevant real-world physician recommendations for nonfrail patients (as defined by the Eastern Cooperative Oncology Group performance status 0-2) and for patients with prostate adenocarcinoma (unless otherwise stated). A total of 321 questions were constructed to investigate (1) screening, (2) diagnosis, (3) staging tools, (4) treatment, and (5) follow-up of PCa and the impact of limited resources on those treatment recommendations by panelists. For some topic, the questions were based on the APCCC 2017.The series includes seven articles compiling all the questions applied on the PDCCCD. The main objective of each article will be discussing the question raised and evaluating the voting panel and literature discussion related to the questions. The first article includes questions of the consensus on screening, diagnosis, and staging tools in PCa. Benefits on screening and early detection of PCa and limitations of screening diagnostic tools in countries of limited resources will be addressed. The second and third articles will include the consensus statement on treatment of localized PCa of low, intermediate, and high risk. In these articles, interventions for localized disease including active surveillance will be discussed, considering life expectancy and disease risk. The fourth article considers biochemical recurrence in castration-sensitive PCa. Challenges in the treatment of this disease rely on preventing or delaying the onset of metastatic disease; morbidity and mortality will be discussed in the article. The fifth manuscript will include questions on nonmetastatic castration resistance PCa, importance of PSA doubling time, and risk assessment. The sixth article will include questions about metastatic castration-sensitive prostate cancer, and the last article will include issues of metastatic castration-resistant prostate cancer. Discussions about staging, sequencing strategies, and limitations of access in areas of limited resources will be dissected.To provide valuable information for physicians and healthcare professionals who work in areas with limited resources, we sought to provide a global consensus including screening, diagnosis, staging tools, treatment, and follow-up for various stages of PCa for developing countries. The information provided in the consensus posing different scenarios will help to choose, when the gold standard options are not available, the best option possible considering a resource-limited scenario. We hope that the series will help physicians who work treating patients with prostate cancer in areas of limited resources to establish the best possible standard of care for the patients in these countries and help in guiding and educating responsible groups in these countries of the limitations faced by these professionals in the treatment of PCa.


L1 transposons occupy 17% of the human genome and are widely exapted for the regulation of human genes, particularly in breast cancer, where we have previously shown abundant cancer-specific transcription factor (TF) binding sites within the L1PA2 subfamily. In the current study, we performed a comprehensive analysis of TF binding activities in primate-specific L1 subfamilies and identified pervasive exaptation events amongst these evolutionarily related L1 transposons. By motif scanning, we predicted diverse and abundant TF binding potentials within the L1 transposons. We confirmed substantial TF binding activities in the L1 subfamilies using TF binding sites consolidated from an extensive collection of publicly available ChIP-seq datasets. Young L1 subfamilies  contributed abundant TF binding sites in MCF7 cells, primarily via their 5′ UTR. This is expected as the L1 5′ UTR hosts cis-regulatory elements that are crucial for L1 replication and mobilisation. Interestingly, the ancient L1 subfamilies, where 5′ truncation was common, displayed comparable TF binding capacity through their 3′ ends, suggesting an alternative exaptation mechanism in L1 transposons that was previously unnoticed. Overall, primate-specific L1 transposons were extensively exapted for TF binding in MCF7 breast cancer cells and are likely prominent genetic players modulating breast cancer transcriptional regulation. The normal growth and function of cells rely on the precise and coordinated expression of complex gene networks. Transcription is a primary step in gene expression and is regulated by functionally interacting transcription factors (TF), which recognise and bind specific cis-regulatory elements in genomic DNA ,2,3. OveMany human TFBSs are located within transposons, which in total occupy 45% of the human genome ,10. TranAIM2 and APOL1 [Exaptation is the process where transposons contribute cis-regulatory elements for the regulation of host genes and has been described as a common phenomenon in various biological and disease contexts in the human genome ,17,18. And APOL1 ,21. In mnd APOL1 . Furthernd APOL1 . The genThe most abundant class of transposons are the L1 transposons, with more than 500,000 copies colonising 17% of the human genome ,24,25. TL1 subfamilies that expanded during the primate radiation are denoted as “L1PA#”, where higher numbers indicate repeats of increasing age ,26. L1HSMET chimeric transcript, which is associated with enhanced malignancy [SYT1 (L1PA2), TYRP1 (L1PA3), REL (L1PA4) and MRE11A (L1PA5) [Generally, human transposons, including L1 transposons, are heavily suppressed by epigenetic mechanisms and post-transcriptional silencing in normal somatic tissues . The loslignancy ,44,45,46 (L1PA5) ,47.We previously investigated the TF binding and promoter activity of an individual L1PA2 transposon in triple-negative breast cancer cell lines . Upon fuGiven the relatedness and structural similarities between L1 subfamilies, we hypothesised that the capacity for genomic regulation observed in the L1PA2 subfamily was a conserved feature amongst primate-specific L1 transposons and that sequence variations amongst L1 subfamilies, resulting from mutations and truncation of the L1 5′ UTR , would lTFs exert regulatory roles by interacting with sequence features in genomic DNA or their binding motifs. Although putative binding motifs do not always define functional binding sites, they are indicative of potential TF binding activities. We scanned individual transposons of primate-specific L1 subfamilies for occurrences of known human TF binding motifs. A total of 114 out of 401 TFs from the HOCOMOCO core database were identified to have putative binding motifs in over 10% of transposons in each subfamily . HierarcUsing the previously established “TEprofiler” pipeline and consolidated TFBSs from the GTRD and ChIP-Atlas databases ,49,50, wWhile ESR1 binding was highly prevalent in L1 transposons of various evolutionary ages, the youngest L1 subfamilies  displayed a distinct TF binding repertoire compared to the more ancient L1 subfamilies (L1PA4-L1PA12), where the youngest subfamilies were frequently bound by cancer-associated TFs, including MYC, FOXA1 and E2F1, as well as the nuclear receptor NR2F2 and chromatin remodelling-associated CTCF . These TThe distinction in TF binding between young and more ancient L1 subfamilies was apparent when considering all L1 transposon copies in the human genome and could be attributed to varying structural integrity and sequence variations . When coWe next sought to investigate the distribution of TFBSs in different L1 subfamilies. As expected, TFBSs clustered in the 5′ UTRs of relatively young L1 subfamilies, and the TF-binding activity of the 5′ UTR was corroded by heavier 5′ truncation in the more ancient subfamilies. However, in the older L1 subfamilies, the 5′ UTR-lacking transposons contributed TF binding activities via their 3′ ends, which likely corresponded to their 3′ UTRs . SimilarWe next sought to explain the variability in TF binding observed in the intact L1 5′ UTRs. We hypothesised that the differential TF binding between young and more ancient L1 subfamilies was due to variations in the DNA sequence features that the TFs recognised and interacted with. To test that hypothesis, we scanned the L1 5′ UTRs for the known motifs of ESR1, which bound all L1 subfamilies at a similar frequency, as well as FOXA1 and E2F1, which frequently bound young L1 subfamilies but showed fewer binding activities in ancient L1 subfamilies. We used motif models from the JASPAR database , which iTo visualise differences between subfamilies at the locations of these known binding motifs, we used a multiple sequence alignment of the L1 subfamily consensus sequences. The consensus sequences were derived from full-length individual transposons in each subfamily . SequencFor ESR1, motif scanning in individual L1 5′ UTR identified several regions that could account for binding. These regions exhibited different degrees of sequence conservation amongst the L1 subfamilies but showed a general trend of increasing differences between subfamilies that were further apart in their evolutionary ages . For botThe replicative nature of transposons has made them an exceptional vector for dispersing TFBSs of functionally associated TFs. We thus examined the pattern of TFBS co-localisation within the primate-specific L1 subfamilies. The degree of TFBS co-localisation was inversely correlated with the evolutionary age of L1 subfamilies, where binding site co-localisation was a common event in young L1 subfamilies, yet gradually diminished as the evolutionary age of L1 subfamilies increased . Transposons are often heavily suppressed by epigenetic mechanisms in normal somatic tissues, which is loosened in epithelial cancers, allowing the transcriptional activation and functional exaptation of transposons ,54,55,56We next examined each TF individually for their dependency on L1-derived TFBSs. We identified a number of TFs, including CTCF, ESR1, MYC and JUN, to bind L1 transposons almost exclusively in the MCF7 cells while showing minimal L1-binding activity in the MCF10A cells . This caThere is growing evidence supporting the widespread exaptation of transposons as a means of human gene regulation. Transposons are ubiquitous in the human genome and contain cis-regulatory elements that can be recognised by human transcriptional regulatory proteins ,14,15,16L1 transposons are the most abundant family of human transposons, occupying 17% of the total human genomic sequences . There aStudies have shown extensive exaptation of L1 transposons for human genome regulation. They contribute abundant TF binding activities, primarily via their 5′ UTR, to a diverse range of TFs ,28,42. LWith a focus on breast cancer, we developed the “TEprofiler” analysis pipeline for identifying cell type- or tissue-specific transcriptional activities within transposons . Our anaIn our analysis, we showed while sequence truncation was a common event observed in all primate-specific L1 subfamilies, younger subfamilies generally contained more full-length elements, which harboured intact 5′ UTRs . We predThe rapid developments in high-throughput, parallel sequencing technologies have enabled the identification of genome-wide cis-regulatory elements. ChIP-seq technology has been widely applied to identify global TF binding activities, and an extensive collection of ChIP-seq-identified TFBSs has been compiled and made available by public databases, such as the GTRD and ChIP-Atlas databases ,50. UsinIn MCF7 cells, L1 transposons were found to be bound by a total of 99 TFs, including ESR1, MYC, FOXA1 and E2F1, which have well-established and critical roles in cell growth and division and have been linked to breast cancer development ,65,66,67Upon closer examination, it was discovered that the loss of 5′ UTR in the ancient L1 subfamilies was compensated by alternative binding sites in the 3′ end, possibly in the 3′ UTR . The seqIn contrast to ESR1, the other TFs, such as E2F1, MYC and FOXA1, showed a differential binding frequency between the young  and the more ancient L1 subfamilies, as their binding activity was more prevalent in young L1 transposons . This diCis-regulatory elements of functional association show a pattern of co-localisation in the genome, where their genomic locations are often found in close proximity to each other . InteresTranscriptional regulation occurs in a tissue-specific and context-specific fashion, where the interactions between regulatory TFs and the genomic DNA may change drastically from one cell state to another. One mechanism for exerting the context-specificity of transcriptional regulation in diseases is differential TF binding, observed as loss or gains of TF binding activities ,74. We pIn this study, we investigated the exaptation of primate-specific L1 subfamilies for TF binding events in a breast cancer model. Our analysis pipeline demonstrated broad utility and identified abundant TF binding activities in the L1 transposons. Despite having a shared origin, the remnants of L1 mobilisation in the human genome demonstrated great structural and sequence diversity, owing to 5′ truncation as well as sequence variations that resulted from ancestral recruitment of different 5′ UTRs and accumulated mutations. These sequence variations predisposed L1 transposons to diverse TF binding events, where L1 subfamilies of similar evolutionary age displayed resemblance in TF binding profiles. Younger L1 subfamilies harboured substantial and comparable TF binding activities in their 5′ UTRs and contributed to TFBS co-localisation and the combinatorial regulation by functionally interacting TFs. Interestingly, the TF binding capacity of ancient L1 subfamilies was not disadvantaged by 5′ truncation. Instead, these transposons contributed abundant TF binding activities via alternative binding sites in their 3′ ends, suggesting the presence of raw sequence materials in the L1 3′ UTR that could be exapted for TF binding over evolutionary time. Despite the structural differences, these L1 subfamilies showed highly consistent, cancer-specific TF binding activity, where they contributed to differential TF binding between the cancer cell model and the near-normal cells. Overall, L1 transposons exhibited widespread exaptation in breast cancer cells through more diverse mechanisms than previously anticipated. Given their abundance in the human genome and the conserved co-binding patterns of oncogenic TFs, these primate-specific L1 transposons likely play a prominent role in modulating global transcriptional regulation in cancer, leading to alternations in the cancer transcriptome.Transposons and TFBSs that mapped to alternate chromosomes were excluded from all analyses in this study. All genomic locations were in hg38 unless otherwise stated.The genomic locations of primate-specific L1 transposons (L1HS and L1PA2 to L1PA12) were retrieved from the UCSC RepeatMasker table ,75. FullThe GTRD and ChIP-Atlas databases contained a large collection of TFBSs generated by compiling publicly available ChIP-seq datasets ,50. TFBSThe L1HS, L1PA2 and L1PA3 transposons were investigated for their epigenetic states in MCF7 cells using publicly available ChIP-seq and DNAse-seq datasets  ). Brieflp-value < 1 × 10−4 [To predict the TF binding profiles of transposons, known TF binding motifs in the human core mononucleotide collection were acquired from the HOCOMOCO (version 11) database, which contains 401 motif models . Individ1 × 10−4 . For eacFor each cell line, TFBSs overlapping L1 transposons were identified using the “TEprofiler” pipeline . Brieflyp-value < 1 × 10−4 [L1 5′ UTRs bound by ESR1, FOXA1 and E2F1 were scanned for binding motifs. Briefly, TF motif models were acquired from the JASPAR 2020 database for ESR1 (MA0112.2), FOXA1 (MA0148.4) and E2F1 (MA0024.2) . DNA seq1 × 10−4 .The full-length consensus sequences of primate-specific L1 subfamilies were acquired from Khan et al. . Multiplp-value < 0.0005 We next investigated the events of binding site co-localisation for 10 frequently binding TFs  in individual L1 transposons using the “TEprofiler” pipeline . BrieflyExaptation of transposons for the regulation of human genomes has been described and studied in a diverse range of biological and disease contexts. In this study, we demonstrated that the L1 transposons from related evolutionary lineages were exapted to contribute substantial TF binding activities in a breast cancer model. Primate-specific L1 transposons contained abundant “raw” cis-regulatory sequences that predisposed them to TF binding and conferred functional TF binding events in MCF7 breast cancer cells. While intact L1 5′ UTRs remained a prominent reservoir of TF binding activities, 5′ truncated L1 transposons were found to contribute abundant TF binding activities through alternative binding sites in the 3′ ends. This suggests that the mechanisms of transposons exaptation are likely more dynamic than previously anticipated. Current methodology and bioinformatic resources available for investigating transposon-derived transcriptional regulation tend to focus more on young, structurally intact L1 transposons and will likely lead to underestimation of L1 regulatory potential. We find it peculiar that there is a large increase in the number of functional binding events within the youngest L1 subfamilies. This pattern suggests a potential selective advantage for pro-tumorigenic transcriptional events during recent L1 propagation, at least in this breast cancer model. Overall, our findings demonstrate a correlation between evolutionary relatedness and functional conservation in human transposons and reveal primate-specific L1 transposons as prominent genetic players in breast cancer transcriptional regulation.


The growing demand for lightweight and multifunctional products in numerous industrial fields has recently fuelled a growing interest in the development of materials based on polymer matrices including graphene-like particles, intrinsically characterized by outstanding mechanical, thermal, and electrical properties. Specifically, with regard to one of the main mass sectors, which is the automotive, there has been a significant increase in the use of reinforced polyamides for underhood applications and fuel systems thanks to their thermal and chemical resistance. In this frame, polyamide 6 (PA6) composites filled with graphene nanoplatelets (GNPs) were obtained by melt-compounding and compared in terms of thermal and mechanical properties with the neat matrix processed under the same condition. The results of the experimental tests have shown that the formulations studied so far offer slight improvements in terms of thermal stability but much more appreciable benefits regarding both tensile and flexural parameters with respect to the reference material. Among these effects, the influence of the filler content on the strength parameter is noteworthy. However, the predictable worsening of the graphene sheet dispersion for GNPs contents greater than 3%, as witnessed by scanning electron images of the tensile fractured sections of specimens, affected the ultimate performance of the more concentrated formulation. In the last decades, the addition of nanoscale fillers to enhance specific properties of polymers has triggered intensive research activities in science and engineering. Nanoscale fillers such as nanoclays, carbon nanotubes, and graphene appear ideal to develop new multi-functional materials ,2, but tAmong the aforementioned nanofillers, it is worth noting that graphene enjoys a marked competitiveness, especially in terms of cost-effectiveness. It can be derived at high purity from graphite, an abundant natural resource, using relatively convenient approaches; other graphitic carbon nanofillers such as carbon nanotubes and carbon nanofibers, instead, usually require expensive and complicated equipment, as well as high energy consumption. Over time, the increased availability of graphene has meant that many researchers, already engaged in studies on polymer nanocomposites containing nanoclays or nanotubes, have increasingly turned their attention to the graphene-based ones for which dispersion issues have been successfully faced through functionalization ,5, thermPolyamides 6 (PA6) are thermoplastic resins with interesting mechanical properties in terms of resistance and toughness such as to fully fall within the family of so-called techno polymers. Currently, they are widely used in a wide range of industrial sectors, among which the automotive one is noteworthy, where the potential offered by PA6-based materials are strongly fuelled by the continuous attempt to replace metal parts with plastic materials aimed to reduce weight and costs.The interest of experts has so far highlighted the need to further improve the performance of PA6 compounds to meet specific and particularly demanding requirements ,10,11, aIn this frame, special attention was paid to the development of new materials obtained by modifying PA6 by the inclusion of graphene-like fillers such as graphene nanoplatelets (GNPs) ,13,14,15For example, Mayoral et al.  investigGomez et al.  considerLee et al.  reportedThis contribution, as part of a larger project aimed at the development of novel materials potentially usable for the production of energy storage components intended for the growing market of electrical vehicles, reports the results of a preliminary study carried out on compounds based on an injection moulding grade of PA6 and lamellar graphene, supplied by an Italian start-up. To obtain easily scalable solutions, the examination focused on samples, including various amount of the carbonaceous filler (up to 5 wt%), prepared and potentially processable with technologies traditionally used in the reference mass sector.Melt compounded samples were transformed into plates by compression moulding from which specimens of suitable size were obtained for the subsequent static-mechanical tensile and flexural tests. The experimental evaluations revealed the effect of the GNPs content on the main mechanical parameters such as modulus and ultimate strengths and, therefore, highlighted their performances compared with the pure matrix processed under the same conditions. Scanning electron microscope observations of tensile-fractured surfaces of the specimens provided information about the effective dispersion level of the filler, actually achieved under the applied process conditions, supporting mechanical behaviour detected for studied materials.3, HDT: 50 °C) as the matrix and graphene nanoplatelets (GNPs) purchased as G2Nan at Nanesa S.r.l.  as the reinforcing phase. The latter, having a density of 2.2 mg/cm3, a Young’s modulus equal to 1 TPa and a tensile strength of 5 GPa, is available in the form of flakes with diameters between 10 and 20 µm and thickness between 5 and 20 nm.The research was focused on compounds constituted by a polyamide 6 (PA6) resin supplied by Ravago Group under the trade name Ravamid B NC  by setting the screw speed at 40 rpm and the screw temperature profile 25–240–250–250–230–220 °C from the hopper to the die.Two-millimetre-thick plates were prepared by compression moulding at 250 °C, using a Collin GmbH  model P400E press and applying the following pressure profile over time: 3 min at 0 bar, 1 min at 2 bar, and subsequent maintenance of the pressure during the cooling phase of the sample up to room temperature to prevent any shrinkage of the material, which, in this last phase, would generate, among other things, undesirable aesthetic defects of the products.The thermal analysis of the compounds and of the pure PA6 matrix, chosen as a reference, was carried out by differential scanning calorimetry using a DSC instrument from TA Instruments, model Q2000.The tests were carried out at a scanning speed of 10 °C/min on samples of 8–10 mg, applying a typical step-by-step procedure: heating from room temperature to 250 °C, isothermal stasis at 250 °C for 1 min, cooling from 250 °C to room temperature, and subsequent heating again up to 250 °C. A thermal rate of 10 °C/min was applied in all non-isothermal stages. The mean values and standard deviations of these parameters are the result of at least three tests performed on each compound. In this analysis, as in the following ones, the compounds are compared with the pure matrix processed under the same conditions.mT) and melting enthalpy . Furthermore, the average values of the melting enthalpies were used to estimate the degree of crystallinity of the materials studied in accordance with the following simple expression:cX is the crystallinity degree, fX is the weight fraction of the filler, and The processing of the thermograms was mainly limited to those relating to the second heating run to exclude any influences of the previous thermomechanical history (processing) of the material on the thermal parameters such as temperature , which allows the recording of the weight loss of the material examined as a function of temperature. In this regard, all the extruded samples were heated from 25 to 600 °C in nitrogen and from 600 to 800 °C in air, with a heating rate of 10 °C/min.5%) and to the maximum rate of thermal degradation (Td), the maximum rate of weight loss (R), and the percentage of residue at the end of the test. The repeatability of the results was verified by performing at least three measurements for each material.The processing of typical diagrams percentage of weight loss vs. temperature and their derivative graphs provided some characteristic data of the tested material such as temperatures corresponding to a weight loss of 5%  were loaded according to a three-point bending configuration. In both cases, at least 5 measurements were carried out for each material to verify the repeatability of the data (modulus and strength) that will be later provided as mean values and corresponding standard deviations.Morphological observations of sections obtained by cryogenic fracture of compression-moulded samples were collected with the aid of a field emission scanning electron microscope (SEM) (mod. FEI QUANTA 200 F)  operating in high vacuum conditions at the voltage of 20 kV. The observed sections were subjected to a preliminary metallization with a gold–palladium alloy in order to make them suitably conductive.Thermogravimetric curves of neat PA6 and its compounds are shown in (a)The temperature corresponding to the 5% weight reduction, reasonably associated with the thermal stability of the materials, is improved by increasing the filler content. This effect is usually attributed to the consolidated ability of graphene to prevent the emission of volatiles and, thanks to its lamellar structure, to hinder the permeation of oxygen (barrier effect) .(b)d) towards higher values, especially for GNP contents up to 3% by weight as well as by the progressive reduction of the peak height (PH) compared to the pure reference PA6 matrix.The inclusion of GNPs also promotes a slowing down of the degradation kinetics as the filler content increases. This effect, attributable to the organization of graphene flakes in rigid mesostructures hindering the progress of degradation phenomena, is manifested by the slight shift of the peak temperature (T(c)The amount of the residue (ashes) at 800 °C increases from approximately 2%, for the neat matrix, up to about 6%, measured for the compound with the highest GNPs content. This trend, although it does not faithfully reflect the added filler content, reasonably shows a monotonically increasing trend. Among other things, these data also depend on the degradation mechanisms of the GNP used, governed, in turn, by the possible presence of residues of surfactants used during the industrial synthesis of the product to favour its exfoliation , degradaGiven that all the materials examined show a one-step decomposition trend, from the mean values of the thermogravimetric parameters obtained from the processing of these curves and summarized in The tensile properties of the examined compounds are compared with those of the neat PA6 in In our case, given the reduction of the degree of crystallinity of the PA6 matrix in the presence of GNPs already discussed above, it can be assumed that the increase in Young’s modulus is mainly determined by the high stiffness of the added graphene flakes. As regards the strain at break, excluding chemical interfacial interactions between the GNP, hydrophilic and non-functionalized, and the hydrophobic PA6 matrix, the reduction of this parameter, very common in many particle-filled polymers, can be reasonably attributed to a lower probability of exfoliation of the graphene lamellae with increasing their content. The segregation of the graphene flakes generates macro-defects acting as stress concentration points, which induce cracks and favour the premature failure of the tested specimen.As regards the tensile strength, apparently unchanged at too low filler content (1% by weight), the results indicated that this parameter is increased for GNP contents up to 3 wt% but reverses its trend at the concentration of 5 wt%.In this regard, it is well known that it is difficult to predict the trend in tensile strength, because it not only depends on variables to which the modulus is also sensitive, such as the size of the aggregates of the dispersed phase and the crystallinity of the matrix, but also is strongly influenced by the polymer–filler interaction. In our case, as the filler content increases, alongside the reduction of the crystallinity of the matrix already discussed above, it is also reasonable to expect a worsening of the dispersion of GNPs with an increase in the size of the graphene aggregates. These considerations, together with the foreseeable poor interfacial interaction, justify the reduction of the tensile strength over the concentration range 3–5 wt% ,24.Representative flexural stress–strain curves of PA6/GNPs compounds and reference material (neat PA6) are shown in In short, the experimental tests have shown mean values of the flexural stiffness (modulus) visibly increasing with respect to the neat matrix at least up to a content of GNPs equal to 3% by weight; further increases in filler concentration do not seem to significantly affect this parameter.The flexural strength of compounds, on the other hand, is always higher than the neat PA6 one. In particular, this parameter shows a trend similar to that described for the tensile strength: a nearly constant value at low filler content (1 wt%), an increase of about 30% for the compound at 3 wt% of GNPs, and a subsequent reduction with the further increase in the concentration of graphene up to an average value of about 54 MPa, still 18% higher than the one characterizing the pure matrix.In flexural loading, the upper part of the specimen undergoes compressive stresses while the lower part of the same is subjected to tensile stresses. That said, for the 3%-by-weight GNP-filled system, the significant increase in both stiffness and tensile strength can justify the benefits found also in terms of flexural strength offered by the side of the specimen opposite to the one subjected to the concentrated bending load.For the more concentrated compound, despite the further increase in tensile stiffness, the reduction of both tensile and flexural strength was attributed to a worsening of the dispersion of graphene flakes with a higher probability of inclusion of voids in the sample.The microscopic observation of neat PA6  shows thRepresentative SEM micrographs of the tensile fracture surfaces of the compounds see , on the In summary, formulations based on a polyamide 6 matrix and containing 1, 3, and 5 weight percent of a commercial graphene nanoplatelets have been prepared by melt mixing and characterized in terms of thermal and mechanical properties as well as through morphological observations of the tensile fracture surfaces.The inclusion of GNP hinders the crystallization of the polyamide matrix, which shows an evident reduction in the degree of crystallinity, especially for contents up to 3% by weight of filler. Further additions of GNP seem to have no major repercussions on this parameter, which, however, does not reverse this trend.Graphene nanoplatelet compounds, in addition to confirming the improvement in thermal stability widely consolidated in the literature, slightly increase the maximum temperature of the derived signal and progressively reduce its height without changing its shape.Tensile and bending stiffness of compounds increase with the GNP content, but the strength parameter, for both types of loading, shows a non-monotonous trend. Specifically, a non-monotonous trend was detected for this parameter essentially dominated by the inevitable aggregation phenomena of graphene sheets for contents above a threshold value usually dependent on a multitude of factors such as aspect ratio of the filler, matrix viscosity, interfacial issues, and so on. This behaviour, also favoured by the foreseeable increase in viscosity of the host molten matrix, was supported by SEM micrographs of sections of specimens broken under tensile tests.In short, the main results obtained so far show that:


The selectivity of electrochemical sensors to ascorbic acid (AA), dopamine (DA), and uric acid (UA) remains an open challenge in the field of biosensing. In this study, the selective mechanisms for detecting AA, DA, and UA molecules on the graphene and graphene oxide substrates were illustrated through the charge population analysis from the density functional theory (DFT) calculation results. Our substrate models contained the 1:10 oxygen per carbon ratio of reduced graphene oxide, and the functionalized configurations were selected according to the formation energy. Geometry optimizations were performed for the AA, DA, and UA on the pristine graphene, epoxy-functionalized graphene, and hydroxyl-functionalized graphene at the DFT level with vdW-DF2 corrections. From the calculations, AA was bound to both epoxy and hydroxyl-functionalized GO with relatively low adsorption energy, while DA was adsorbed stronger to the electronegative epoxy groups. The strongest adsorption of UA to both functional groups corresponded to the largest amount of electron transfer through the pi orbitals. Local electron loss created local electric fields that opposed the electron transfer during an oxidation reaction. Our analysis agreed with the results from previous experimental studies and provided insight into other electrode modifications for electrochemical sensing. One of the open problems in the field of biosensing is the simultaneous detection of ascorbic acid (AA), dopamine (DA), and uric acid (UA) within body fluids. The lack of ascorbic acid (AA) can cause scurvy and other diseases , while a2, carbon nitride nanosheets (C3N4), polypyrrole, or ionic liquids that could extensively alter the redox potentials [Carbon-based 2D materials, e.g., graphene , graphententials ,26,27,28In this study, relatively simplified model systems were considered to address the fundamental mechanisms of the oxidation potential shift of AA, DA, and DA molecules. The binding of these analytes to graphene oxide models was elucidated through a series of density functional theory (DFT) calculations. Firstly, representative configurations of GO were selected by the lowest formation energy. Then, geometry optimizations were performed on AA, DA, and DA analyte molecules on pristine graphene, a graphene model functionalized by hydroxyl groups, and a graphene model functionalized by epoxy groups. After that, partial charge analysis was performed on the AA, DA, and DA analytes in cases for free analytes, oxidized forms, and the molecules bound to different substrates. Functionalization of a pristine graphene plane by epoxy and hydroxyl groups could result in the altered charge distribution patterns of the analyte and might affect the tendency of oxidation reactions.−6 Ry, and the convergence of geometry optimization loops was set with the force tolerance of 0.001 Ry/Å.All calculations in this study were based on density functional theory (DFT), using the Quantum Espresso 5.3 package . For theGO surface containing GO surface, the pristine graphene substrate, and a free epoxy (O atom) or a free hydroxyl (OH) group. Formation energy is the energy required for each structure to form a configuration. In order to estimate the most energy favorable of surface structures, Monolayer pristine graphene and graphene oxide supercells of the dimension 5 × 5 were created. The vertical slab distance was set up at 20 Å to prevent surface interaction from the above graphene layer under the periodic boundary condition. The formation energy of z-axis) so that the center of the ring was at distances 2.0 Å to 8.0 Å from the substrate, and the potential energy was calculated as a function of vertical distance. The potential energy Then, the optimized structure of each analyte was translated along the vertical axis 5-paraC . SimilarDFT calculations were performed for isolated AA, DA, and UA to understand the molecular basis of the interactions between the proposed graphene oxide surface with AA, DA, and UA analyte molecules. After each DFT calculation, Löwdin charge population on each atom, electron density isosurface, and electrostatic potential were extracted. 3 iso-density surfaces in the right panels of Now, consider the electrostatic potential of AA, DA, and UA molecules mapped onto the 0.005 e/Å3 iso-density surfaces that roughly represent the van der Waals surfaces. Then, the optimized structure of each analyte was translated along the vertical axis (z-axis), and the potential energy was calculated as a function of vertical distance. The potential energy curve was then fit to the Morse potential function, and the adsorption energy can be reproduced from the well depth   between According to our potential energy calculations, the adsorption of AA, DA, and UA on the pristine graphene surface a–c was sLocal partial charge transfer at each atom within an analyte molecule resulted in changes in charge distribution and local dipole moments. As a result, the partial charge around the oxidation sites was affected, which also affected the oxidation potentials. To further quantify this phenomena, partial charge differences of the analytes (i) in oxidized form, (ii) binding with pristine graphene, (iii) binding with GO5-para, and (iv) binding with G(OH)5-paraA were calculated compared to those of bare analytes for each ring member group and shown in Consider the partial charge difference between the atom groups in the analyte molecules before and after binding with the pristine graphene 5-paraA substrates to the charge distribution within the analyte molecules might affect the oxidation. As an analyte molecule became adsorbed on the functionalized substrate, electronegativity of oxygen atoms in the functional groups caused a small amount of electron to transfer from the analyte to the substrate via pi-orbitals of the analyte. This effect was the most prominent for the epoxidized substrates with a relatively uniform negative partial charge of the functional groups and relatively high electronegativity. The spontaneous electron loss from the analyte to the substrate via this physical adsorption resulted in (i) an electric field between the analyte and the substrate that opposed further electron transfer from an oxidation reaction, and (ii) changes in charge distribution and local electric fields that prevented oxidation reactions. According to the partial charge difference analysis between the neutral analyte molecules and their oxidized forms, UA already required the largest local charge transfer to the oxidation site. The largest amount of electron loss from adsorption would only cause a further positive shift for the oxidation of UA. Meanwhile, the lower amount of electron loss from DA and AA adsorption resulted in a greater difference in oxidation potential, which was in agreement with experimental studies using the electrodes modified by rGO.In this study, the mechanisms of oxidation potential shift of AA, DA, and DA molecules on the GO or rGO surfaces were illustrated through changes in the charge distribution within the analyte molecules at different oxidation and binding states. According to our DFT geometry optimizations, the strongest adsorption of UA to both types of functional groups corresponding to the largest amount of electron transfer through the pi orbitals. The local electric field created by the altered charge distribution within the analytes could prevent the oxidation and cause a further positive shift for the oxidation potential of UA from DA and AA. The more electronegative epoxy functional groups contributed to the charge transfer more than the hydroxyls. This understanding of this oxidation potential differentiation through the intrinsic properties of substrates and analyte molecules could be useful for other electrode modification designs of more reliable AA/DA/UA sensors by controlling the electron distribution of analytes via surface functionalization.