N = 17, control group, N = 14) to characterize the longitudinal changes following intervention in intrinsic and extrinsic effective connectivity (EC) between core regions of the DMN. Then, we analyzed the correlation between the changes in EC and SRS-2 scores to establish symptom-based validation. We found that after the 12W-MBTP intervention, the SRS-2 score of preschoolers with ASD in the experimental group was decreased. Concurrently, the inhibitory directional connections were observed between the core regions of the DMN, including increased self-inhibition in the medial prefrontal cortex (mPFC), and the changes of EC in mPFC were significantly correlated with change in the social responsiveness scale-2 (SRS-2) score. These new findings shed light on DMN as a potential intervention target, as the inhibitory information transmission between its core regions may play a positive role in improving SC behavior in preschoolers with ASD, which may be a reliable neuroimaging biomarker for future studies.Neuroimaging has revealed numerous atypical functional connectivity of default mode network (DMN) dedicated to social communications (SC) in autism spectrum disorder (ASD), yet their nature and directionality remain unclear. Here, preschoolers with autism received physical intervention from a 12-week mini-basketball training program (12W-MBTP). Therefore, the directionality and nature of regional interactions within the DMN after the intervention are evaluated while assessing the impact of an intervention on SC. Based on the results of independent component analysis (ICA), we applied spectral dynamic causal modeling (DCM) for participants aged 3–6 years  on August 05, 2019. Autism spectrum disorder (ASD) is a complex neurodevelopmental disability  that emeIn general, SC deficits in individuals with ASD often result in exclusion from social interactions , contribVarious evidence-based ASD interventions aimed at improving SC ability , have beOf note, although a deficit called “social communication” is considered one of the most universal and specific characteristics of autism in the DSM-5 diagnostic criteria , there iSynthesizing the previous literature, a universal consensus has recently been reached that the abnormal functional connectivity between the core regions of the DMN contribute to the SC impairments in ASD . FurtherOverall, research increasingly indicates that, as a core network for mentalizing process , the atyThis original research aims to investigate the underlying neural mechanisms of exercise interventions in enhancing social communication (SC) skills for preschoolers with autism by using the spectra DCM. Therefore, we hypothesized the exercise-induced effects may alter the imbalance of local excitatory/inhibitory in the DMN feedback loop, improving the influence of secondary pathological processes caused by chronically elevated metabolic stress and relieving the symptoms of social deficits in preschoolers with ASD.This research design with a two-factor (time and group) repeated measurements was conducted between October and December 2018 in Yangzhou, China. The current research is mainly aimed at preschool (3–6 years old) with ASD to reduce the deviation caused by age development . ResearcAll participants were outpatients diagnosed by pediatricians in a tertiary hospital based on the Diagnostic and Statistical Manual of Mental Disorders 5th-edition (DSM-5) and assisted by Childhood Autism Rating Scale (CARS) . Potentin = 36) or declined to participate in this study (n = 18) were excluded so that 40 participants were finally eligible and randomly equally distributed into two groups by using simple randomization with a random number table: the experimental group (n = 20) and the control group (n = 20). Of note, participants from the two different places and their parents had no prior social interactions with one another to minimize differential expectancies. Because some subjects were unable to complete the MRI scans post-intervention (n = 6), and inferior images were not allowed for subsequent analysis (n = 3). The final data analysis: a total of 31 participants between the experimental group (n = 17) and the control group (n = 14). Before the research, explain the research including contents and precautions in detail to the parents or guardians of the participants and obtain written authorization. The study was approved by the Ethics and Human Protection Committee of the Affiliated Hospital of Yangzhou University, and complied with the ethical standards of the Helsinki declaration. Meanwhile, this study retrospectively registered with the Chinese Clinical Trial Registry (ChiCTR1900024973) on August 05, 2019.Ninety-four participants were recruited from Chuying Child Development Center and Starssailor Education Institution . Of the initially diagnosed ASD preschoolers, participants who did not meet study criteria  at baseline, the CARS and clinical assessment report were used to evaluate the symptom severity of ASD. The CARS is a behavior rating scale consisting of 15 items for assisting in ASD diagnosis and determine the severity. The SRS-2  is a relBesides routine behavior rehabilitation training according to the standard rehabilitation program set by the institutions, the experimental groups also received an additional 12W-MBTP, while controls maintained a consistent lifestyle and did not participate in other sports-related activities.SD = 7.45) using a heart rate meter (POLAR M430) to keep the activity at a moderate intensity. Most importantly, assessments were performed after each course, and participants were removed from the study if they asked for leave for more than 2 days consecutively or more than 7 days cumulatively.The MBTP was conducted by two certified physical educators, adopting the mode of combining the basic movements of a mini-basketball with sports games to design projects of varying degrees of difficulty and a collective teaching model to facilitate social interactions and communications among participants while parents were strongly inspired to accompany them throughout the curriculum. The 12W-MBTP contents  can be simply summarized as a three-phase stage, with four small parts for each course including first 2 min classroom routine preparation, then 8 min warm-up activities, followed by a 25 min mini-basketball training program, and finally 5 min cool-down activities. More details of the intervention program, please refer to the articles published by our colleagues  and SuppThe participants were deprived of sleep with the consent of the family members or guardians and sedation with 10% chloral hydrate, a safe way for children was administered before the scan to avoid excessive head motion . Two groNeuroimaging data were acquired using a 3.0 T GE scanner  located in the Affiliated Hospital of Yangzhou University. Functional images were collected in 28 axial slices using echo-planar imaging (EPI) with T2* weighted contrast sequence sensitive to blood-oxygen-level-dependent (BOLD) contrast . Each fMRI session lasted 8 min and thus contained 240 volumes. High-resolution T1-weighted structural images were acquired in a sagittal orientation using a three-dimensional magnetization-prepared rapid acquisition with gradient-echo (MPRAGE) sequence .Conventional functional imaging preprocessing was performed using Statistical Parametric Mapping software (SPM12 version 7771) implemented in MATLAB 2013b . The initial ten volumes of each dataset were discarded, then the remaining images were corrected for differences in slice time. Motion correction was performed by aligning each participant’s time-series to the mean image, and 6 motion parameters were calculated during realignment. Participants’ data were excluded if movement in the translational or rotational planes exceeded 2.5 mm or 2.5°, respectively. Functional images were registered to the standard MRI template, unbiased age-specific structural brain atlases specially provided for the Chinese pediatric population , with spTo identify regions for subsequent DCM, ICA was performed by the Group ICA of fMRI Toolbox (GIFT). First, the optimal number of components was estimated to generate 33 independent spatial maps by using a minimum description length approach. Meanwhile, the corresponding components for each subject were calculated via a back-reconstruction step. After visually inspecting all highly relevant components, we determined the peak coordinates of each region of interest in the group level while excluding all components related to head motion, physiological noise, or cerebrospinal fluid fluctuations. Finally, subject-specific coordinates were identified as the peaks in subject-specific ICA maps within 8 mm of the group-level coordinates .To control motion and physiologic noise effects and correct for the influence of interindividual differences, a general linear model (GLM) containing a discrete cosine basis set and the nuisance regressors that included 6 head motion parameters and WM and CSF signals , age, seGenerally, DCM analysis requires the specification of a model space . Due to After performing the first-level analysis, we created a second-level analysis by specifying a design matrix with four regressors, including the effect of time interacting with the group that best described the effects of the intervention of the 12W-MBTP.p-values but instead in terms of posterior probability values (PPs), so connections that survived a non-zero criterion with posterior probability PPs > 0.95 are considered significant.To finally utilize the model, we used Bayesian model reduction (BMR) , an autoFinally, we asked whether any directed connections are related to symptom severity to establish the symptom-based validation. Following our hypothesis, we then analyzed the correlation between the changes in EC and SRS-2 scores. The alterations here refer to post-intervention minus the baseline level unless otherwise specified. The analysis of the correlation was limited to the final model following second-level analysis by PEB. If a correlation was found between them, then the correlations between the connectivity strength and the items of the given SRS-2 sub-score were also analyzed.t-tests for continuous variables and χ2 tests for categorical variables. We conducted two-factor (time and group) repeated measurements ANOVA analysis to evaluate the positive role of the 12W-MBTP in SC on preschoolers with ASD. Once we found significant group × time interaction effects, post hoc tests were performed. We calculated the mean and standard deviations of all variables and adopted the traditional cutoff of p < 0.05 to determine significance. The Bonferroni correction for multiple testing was applied in Pearson correlation analysis, resulting in a corrected α-value of (0.05/5 = 0.01).The demographic analysis were performed using SPSS Version 21.0 , with two-tailed independent sample p = 0.722 and p = 0.396, respectively  = 8.785, p = 0.006]. Follow-up simple effect analysis indicated that the SRS-2 total score of the experimental group at the post-test were significantly lower relative to baseline , whereas such a positive effect was not found in the control group . Notably, a greater SRS-2 total score indicates worse SC performance.For the SRS-2 total score  = 8.258, 9.537, 4.993, p = 0.008, 0.004, 0.033, respectively]. Follow-up simple effect analysis results for the subscales of SRS-2 as follows: (1) the pre-test social cognition score was significantly higher than that at post-test in the experimental group , where no significant change from the post to pre-test was observed in the control group; (2) the post-test social communication score was significantly lower than that at baseline in the experimental group , whereas a higher score was observed in the control group from the post-test to pre-test ; (3) there was no significant difference between baseline and post-test in the experimental group  in terms of autistic mannerisms, whereas the post-test score was significantly higher than that at baseline in the control group . All the above simple effect analysis uses Bonferroni correction to correct multiple comparisons.Group × time interaction effect was only observed in the three subscales , PCC , LTPJ , and RTPJ  in the DMN that specifically engaged by in the ToM process, which were selected as nodes for subsequent DCM.The EC parameters of each specific effect obtained following group-level analysis were listed in For the effect of group × time interaction, parameters best describing the effect included the inhibitory directional connections mediated by PCC between bilateral TPJ (from left to right). Besides, the positive self-connection values observed in mPFC that showed no overall mean effect (PPs = 0) were instead the best discriminative parameters of an interaction effect, which can be explained as the size of the positive effect means an increase in self-inhibition . To further explore these results, the correlation between each of the five sub-items of the SRS-2 and self-connectivity in the mPFC was analyzed. Changes in EC of mPFC correlated significantly with the changes in social cognition and social communication sub-scores , while the correlation with autistic mannerisms  sub-score did not survive the correction for multiple tests. Unquestionably, there was no significant correlation between changes of EC strength  and SRS-2 total scores in the control group, p value was 0.3011, 0.6348, and 0.5344, respectively.As illustrated in Since the absence of assessment about the influence of one region to another region in functional connectivity analysis, it is impossible to reasonably explain the information exchange between brain regions in the context of physical exercise intervention. Therefore, this research aims to investigate the underlying neural mechanisms of exercise interventions in enhancing social communication (SC) skills for preschoolers with autism by using the spectra DCM in the framework of Parametric Empirical Bayesian to characterize the longitudinal changes in intrinsic and extrinsic effective connectivity in DMN core regions. Following the exercise intervention, we discovered noteworthy inhibitory directed connections in the default mode network in the experimental group, which predicted an improvement in SC skills.Differently, previous research focused more on assessing the changes in the effective connectivity of ASD during various task states about psychology, but rarely assessed the impact of endogenous neuron fluctuations under the resting state. However, earlier effective connectivity study by multivariate Granger causality analysis with blind deconvolution have also indicated the impaired mPFC pathway and its association with SC deficits in children with ASD, which is consistent with our findings using resting state data .The mPFC, as the core region of the social brain network , is engaTherefore, the final model of interaction effect found strong evidence of greater self-inhibition in mPFC, indicating a decrease in the gain or excitability of this structure . SimilarInterestingly, E/I ratio in mPFC is related to an SC impaired, and this effect can be improved by increasing the inhibitory function  imbalance  in localfunction . Taken tFrom the significant negative correlations between changes in EC and SRS-2 score   . Not surd (GABA) .Taken together, we speculate that the inhibitory information transmission between bilateral TPJ caused by exercise intervention altered the imbalance of local E/I, improving the influence of secondary pathological processes caused by chronically elevated metabolic stress . Such a Concerning the high-level cluster in the social brain, most core regions (left TPJ instead of right) exhibited a left-favored lateralization pattern of functional connectivity . UnsurprBehaviorally, the classical role of PCC is to quickly switch between different cognitive processes to coordinating external and internal-oriented cognition . After tWe presume the impact of intervention is primarily because of the exercise-induced neuroplasticity caused by the multipath effect. Evidence-based investigations have confirmed continuous moderate-intensity aerobic exercise will increase basal peripheral brain-derived neurotrophic factor (BDNF) concentrations . BDNF isThere are a few limitations of our work must be taken into consideration and addressed by future studies. The lack of ADOS or ADI-R assessment might also be a potential limitation of the study. For preschoolers with autism, it is difficult to successfully recruit more participants on the premise of successful completion of the entire intervention process and two MRI scans. On the other hand, given the male preponderance in the prevalence of autism , the proTaken together, this study provides sufficient evidence that exercise intervention can improve SC skills for preschoolers with ASD. Such inspiring results also emphasize the importance of early and timely intervention for the neurodevelopmental of ASD and improvement of core symptoms and quality of life. By applying spDCM in the framework of PEB to characterize the longitudinal changes in intrinsic and extrinsic effective connections between the DMN, we conclude that the inhibitory information transmission in DMN may be an underlying neurophysiological mechanism for improving SC behaviors among preschoolers with ASD who delay or stunt maturational processes. Finally, these findings provide further factual evidence that the directional effectivity connection between key regions of the DMN has become one of the well-validated targets for exercise intervention to improve SC behaviors, which may be a reliable neuroimaging biomarker or endophenotype in subsequent studies.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by The Ethics and Human Protection Committee of the Affiliated Hospital of Yangzhou University. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.HY and WW drafted the manuscript, performed all administrative tasks required for submission, and contributed to the identification of research topics and preparation of study design. HY and HQ took part in planning, supervision, brainstorming the manuscript, designed the study, and contributed to the statistical analysis and interpretation of results. HY, AC, YD, and ZL designed the intervention method and guided the intervention process. All authors critically revised the manuscript and approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


As COVID-19 roared through the world, governments worldwide enforced containment measures that affected various treatment pathways, including those for hip fractures (HFs). This study aimed to measure process and outcome indicators related to the quality of care provided to non-COVID-19 elderly patients affected by HF in Emilia-Romagna, a region of Italy severely hit by the pandemic.We collected the hospital discharge records of all patients admitted to the hospitals of Emilia-Romagna with a diagnosis of HF from January to May in the years 2019 (pre-pandemic period) and 2020 (pandemic period). We analyzed surgery rate, surgery delays, length of hospital stay, timely rehabilitation, and 30-day mortality for each HF patient. We evaluated monthly data (2020 vs. 2019) with the chi-square and t-test, where appropriate. Logistic regression was used to investigate the differences in 30-day mortality.Our study included 5379 patients with HF. In April and May 2020, there was a significant increase in the proportion of HF patients that did not undergo timely surgery. In March 2020, we found a significant increase in mortality (OR = 2.22). Male sex (OR = 1.92), age ≥90 years (OR = 4.33), surgery after 48 hours (OR = 3.08) and not receiving surgery (OR = 6.19) were significantly associated with increased mortality. After adjusting for the aforementioned factors, patients hospitalized in March 2020 still suffered higher mortality (OR = 2.21).There was a reduction in the overall quality of care provided to non-COVID-19 elderly patients affected by HF, whose mortality increased in March 2020. Patients’ characteristics and variations in processes of care partially explained this increase. Policymakers and professionals involved in the management of COVID-19 patients should be aware of the needs of patients with other health needs, which should be carefully investigated and included in future emergency preparedness and response plans. Timely detection, intervention and rehabilitation are key factors for the successful management of patients who suffer from hip fractures (HFs) –4.To impIn 2020, the COVID-19 pandemic forced governments worldwide to enforce containment measures such as social distancing and home quarantining. These measures had a considerable impact on various treatment pathways , 9, inclAs COVID-19 roared across the world, treatment pathways for HF felt the blow and musculoskeletal facilities were reorganized as a result, patients were at risk of being left without proper care and were exposed to increased disability and mortality –15. SomeAfter the first case of COVID-19 on February 21, 2020, the first wave of the pandemic struck all over northern Italy, including Emilia-Romagna see . Italy’sHealthcare systems’ ability to adapt to the mutating population’s health needs caused by an emergency such as the COVID-19 pandemic can be assessed through several health processes and outcome indicators . AccordiGiven the forced reorganization of healthcare services during the first wave of the pandemic, we aimed to measure process and outcome indicators related to the quality of care provided to non-COVID-19 elderly patients affected by HFs in Emilia-Romagna. Specifically, we analyzed the length of hospital stay (LOS) as well as the rates of and delays in surgical treatment/rehabilitation as process indicators, and 30-day mortality as the main health outcome.The Italian national health service  is a universalistic health system funded through general taxation. Emilia-Romagna is one of the largest regions of northern Italy, with ~4.5 million inhabitants as of 2020. Its regional health system includes 8 local health trusts, 4 university hospitals, 1 general hospital trust, and 4 research hospitals.In 2013, Emilia-Romagna improved the management of patients with HF, reducing delays in surgery and designing specific modalities for post-operative rehabilitation .In this retrospective cohort study, we gathered the hospital discharge records (HDRs) of all patients aged ≥65 admitted to the hospitals of Emilia-Romagna with a primary or secondary diagnosis of HF (ICD-9-CM code 820.xx) between January and May 2020 (study period) and between January and May 2019 (control period). In Italy, 2007 ICD-9-CM is the official system of assigning codes to diagnoses and procedures associated with hospital utilization, and hospital cases are classified into 538 homogeneous and mutually exclusive groups using the Medicare diagnosis-related group (DRG) system, version 24.0.We excluded non-residents in Emilia-Romagna, transfers from other hospitals, polytraumas (DRG 484–487), diagnoses or history of malignant tumors , and cases of COVID-19 using the criteria issued on March 10, 2020, by Italy’s Ministry of Health . We excluded patients who died within 1 day of hospital admission without surgery, and those directly admitted to spinal injury units, rehabilitation hospitals or long-term care facilities.We collected from the health administrative databases of Emilia-Romagna the drug prescriptions of each patient over a lookback period of 3 years to compute the Modified Chronic Disease Score (M-CDS), a drug-based index that has been shown to be a good predictor of 1-year mortality .Process indicators included LOS, HF surgery , and rehabilitation within 30 days of hospital admission, i.e., facility bed-based rehabilitation programs after HF provided in public or private hospitals, intermediate care facilities, community hospitals or nursing homes.Hip-fracture surgery was defined as any of the following procedures registered in the HDRs: closed reduction of fracture without internal fixation ; closed reduction of fracture with internal fixation ; open reduction of fracture without internal fixation ; open reduction of fracture with internal fixation ; total or partial hip replacement .Through data linkage, we were able to track whether post-acute patients entered bed-based rehabilitation programs in public or private hospital units, community hospitals, or nursing home beds in residential care facilities for the elderly.The health outcome under study was 30-day mortality, i.e., all-cause death within 30 days of hospital admission, either inside or outside the hospital.t-test. Differences in 30-day mortality were expressed as odds ratios (ORs) with 95% confidence intervals (CIs), and were adjusted by age, sex and M-CDS via multivariable logistic regression analysis.Numerical variables were summarized as mean ± standard deviation; categorical variables were summarized as counts (percentages). Comparisons of patient characteristics and process indicators between 2020 and 2019 were investigated with the chi-squared or All analyses were stratified by month of the year. If a significant difference was present between 2020 and 2019, we further adjusted the analysis by including HF surgery and LOS as additional covariates in the multivariable logistic regression model.All analyses were performed using SPSS 26.0  and Stata 15 . The significance level was set at 0.05.Comitato Etico Indipendente di Area Vasta Emilia Centro . This retrospective study was carried out in conformity with the regulations on data management with the Italian law on privacy (Legislation Decree 196/2003 amended by Legislation Decree 101/2018). Data were pseudonymized prior to the analysis and each patient was assigned a unique identifier that does not allow to trace the patient’s identity or other sensitive data. Pseudonymized administrative data can be used without a specific written informed consent when patient information is collected for healthcare management and healthcare quality evaluation and improvement . All procedures performed in this study were in accordance with the 1964 Helsinki Declaration and its later amendments. Data used in this research were obtained from the Regional Healthcare Information System, which includes detailed information on the use of healthcare services by all regional patients. The study, based on routine administrative information, was carried out in conformity with the regulations on data management of Emilia-Romagna and with Italian privacy law.This study was approved by the As shown in In In this observational study, we assessed the impact of the first wave of the COVID-19 pandemic on non-COVID-19 patients with HF. Specifically, we investigated whether the health crisis affected the quality of care provided to elderly patients by analyzing delays in surgical interventions, LOS and timely rehabilitation as process indicators, and 30-day mortality as the main health outcome.Our study shows that the pandemic negatively affected non-COVID-19 patients with HF. The quality of care has been undermined by the unavoidable services’ reorganization needed to address the emergency. The proportion of patients undergoing surgery and receiving timely treatment decreased, as well as the mean LOS and the timely use of rehabilitation services. Health outcomes suffered as well: patients with HF experienced an increased mortality rate, particularly in March 2020. In the following months, HF mortality returned to approach pre-crisis levels, demonstrating the adaptation and resilience of the healthcare system.Our analysis shows that the first wave of the COVID-19 pandemic determined a significant reduction in HF hospitalizations in Emilia-Romagna, one of the most severely hit areas of Italy and Europe, although the patient characteristics did not differ between 2020 and 2019. This finding is consistent with other studies –23 and cThe disruption of the healthcare services determined an increase in the percentage of patients with HF that did not undergo surgery and a decline in the proportion of patients undergoing timely surgery within 48 hours of hospital admission, together with a reduction in mean LOS. These changes could be ascribed to the sudden hospital overload experienced during the first months of 2020, which coerced healthcare institutions to enforce prioritization of their services. Many professionals’ skills, such as surgeons’ and anesthesiologists’, were repurposed to attend to COVID-19 patients in dedicated wards. This created service gaps, reducing both the number of physicians and the time dedicated to non-COVID-19 patients . DiminisFurther considering health services’ performance, we found that the number of patients receiving bed-based rehabilitation within 30 days of hospital admission from February to May 2020 was lower compared with the same period of the previous year. The performance of rehabilitative care could have been undermined by the difficulty to reorganize treatment pathways for non-COVID-19 patients. In Emilia-Romagna, many rehabilitation centers experienced COVID-19 outbreaks, preventing them from providing adequate standards of care and safety . As showSince the initial outbreak of the pandemic, several authors reported an excess of mortality for patients with COVID-19 affected by HF –30. Our In keeping with existing literature , our anaIn the following two months of 2020, 30-day mortality was higher than in April and May 2019, but the gap was not significant and narrower when compared to that observed between March 2019 and March 2020. However, during these months the process indicators remained significantly worse than in 2019. These findings underline the Emilia-Romagna healthcare system’s capacity to respond to the initial health crisis and to take effective actions to mitigate the impact of the pandemic.In this study, we found that the quality of care for patients with HF has been undermined by the unavoidable healthcare services’ reorganization needed to address the COVID-19 pandemic . After tAnalysis of complete data related to the whole healthcare system of a wide region is the main strength of this study. Moreover, the Italian experience during the first wave of the COVID-19 pandemic represents an instructive event that has the potential to illustrate a healthcare system’s early response to a severe health crisis. Misreporting and misclassification of COVID-19 cases and deaths is the main limitation of our study and is a common problem highlighted elsewhere in the recent literature , 33. TheThis study addressed the impact of the COVID-19-related healthcare reorganization on healthcare quality and 30-day mortality for non-COVID-19 elderly patients with HF. Our results show a reduction in the proportion of patients undergoing surgery and in the proportion of patients receiving timely surgery and rehabilitation. Mortality increased significantly in March 2020 as compared with March 2019, but differences in patient characteristics and quality of care only partially explained such increase. Further studies are needed to verify additional determinants, in order to identify the strengths and weaknesses of healthcare systems and to develop capacities and capabilities suited to face the upcoming public health challenges.The care and attention required for patients with COVID-19 should not distract from the needs of patients with other critical acute and chronic conditions, which should be carefully investigated and included in future emergency preparedness and response plans.S1 Dataset(XLS)Click here for additional data file.S1 FigDipartimento della protezione civile.Source: (DOCX)Click here for additional data file.


Drosophila model to demonstrate that exercise-training regulates the expression of apoLpp (a homolog of apolipoprotein B) in cardiomyocytes, thereby resisting heart insufficiency and low exercise capacity caused by obesity. The apoLpp is an essential lipid carrier produced in the heart and fat body of Drosophila. In a Drosophila genetic screen, low expression of apoLpp reduced obesity and cardiac dysfunction induced by a high-fat diet (HFD). Cardiac-specific inhibition indicated that reducing apoLpp in the heart during HFD reduced the triglyceride content of the whole-body and reduced heart function damage caused by HFD. In exercise-trained flies, the result was similar to the knockdown effect of apoLpp. Therefore, the inhibition of apoLpp plays an important role in HFD-induced cardiac function impairment and low exercise capacity. Although the apoLpp knockdown of cardiomyocytes alleviated damage to heart function, it did not reduce the arrhythmia and low exercise capacity caused by HFD. Exercise-training can improve this condition more effectively, and the possible reason for this difference is that exercise-training regulates climbing ability in ways to promote metabolism. Exercise-training during HFD feeding can down-regulate the expression of apoLpp, reduce the whole-body TG levels, improve cardiac recovery, and improve exercise capacity. Exercise-training can downregulate the expression of apoLpp in cardiomyocytes to resist cardiac function damage and low exercise capacity caused by HFD. The results revealed the relationship between exercise-training and apoLpp and their essential roles in regulating heart function and climbing ability.Apolipoprotein B plays an essential role in systemic lipid metabolism, and it is closely related to cardiovascular diseases. Exercise-training can regulate systemic lipid metabolism, improve heart function, and improve exercise capacity, but the molecular mechanisms involved are poorly understood. We used a  Obesity is caused by the imbalance between energy input and output and the interaction between environmental factors and susceptibility genes . ObesityDrosophila is an excellent model for studying cardiovascular function. In Drosophila and mammals, most of the genes that regulate cardiac differentiation are conserved and the genetic regulation of cardiac function may also be conserved , are similar to mammalian apoB-containing lipoproteins; their scaffolding apolipoproteins, the apolipophorins (apoLpp), are members of the apoB family, which is conserved throughout the animal kingdom  and Hand-Gal4 (cardiomyocyte-specific drive) were obtained from Xiu-shan Wu . UAS-apoLppRNAi  flies were obtained from the Bloomington Stock Center. Through the hybridization of Hand-Gal4 or W1118 with UAS-apoLppRNAi, flies with apoLpp knockdown of the cardiomyocytes or the whole-body were produced . HFD-fed flies needed to be reared in an environment of 21–22°C to prevent part of the high-fat food from melting and causing the flies to stick to the food and die.UAS-The preparation of the standard medium is as before . The higDrosophila movement device, which accounts for Drosophila’s negative tropism climbing movement by controlling the glass tube’s overturn. Each vial was rotated along its long axis with the accompanying rotating steel tube, which made the flies climb. Most flies continued to respond by climbing throughout the exercise period. The few that failed to climb were actively walking along the inner wall of the vial . The offspring flies emerge at approximately 12 days, and then the female flies that emerge within 12 h are collected. All fruit flies needed to be fed on normal medium for 5 days first and then transferred to fresh normal medium or the high-fat medium on the 6th day for another 5 days. The heart was collected or the whole flies were dissected on flies 11 days old. Note: High-fat medium needed to be placed at 21–22°C, and normal medium at 25°C.Parental flies harboring the appropriate Gal4 driver with parental flies harboring UAS-transgene 60 hearts or 10 whole-body tissues were put into 1 mL of Trizol reagent lysis solution for homogenization and extract RNA. Trizol extracted the organic solvent, and 10 μg total RNA was purified using oligo (dT) were synthesized from total RNA with superscript II reverse transcriptase (Invitrogen). qPCR amplification reactions were performed in triplicate, by mixing 1 μl of RT product with 10 μl of SYBR qPCR Mastermix (TaKaRa) containing the appropriate PCR primers. Thermal cycling and florescence monitoring were performed in an ABI7300  using the following PCR conditions:  × 40. Values were normalized with gapdh. Primers used were as follows:Gapdh F: 5′-GCGTCACCTGAAGATCCCAT -3′R: 5′-GAAGTGGTTCGCCTGGAAGA -3′apoLpp F: 5′-AATTCGCGGATGGTCTGTGT -3′R: 5′-GCCCCTTAGGGATAGCCTTT -3′Based on previous research methods . First, Drosophila, the flies climbed up the sides of the apparatus after being taped down at the bottom. Flies were allowed 8 s to climb after being taped down, and the climbing heights reached by the flies were calculated. A video camera was used to film the fly climbing during the whole process, the 4th, 5th, and 6th climbing images at the end of 8 s were intercepted, and the number of flies reaching the top was counted. Climbing index = number of in the uppermost area/total number.According to the previous method . The cliFor quantification of TGs in whole flies, flies (15 per genotype) were weighed and homogenized in PBS containing 0.1% Triton-X100 in an amount (μl) that was 8 X the total weight of the flies (μg). Then, centrifugation was used and the supernatant was obtained. Fifty microliters of standard or sample was added to the appropriate wells. Blank wells had nothing added. One-hundred microliters of enzyme conjugate to was added standard wells and sample wells except for the blank well; they were covered with an adhesive strip and incubated for 60 min at 37°C. The microtiter plate was washed four times then, Substrate A (50 μl) and Substrate B (50 μl) were both added to each well, mixed gently, and incubated for 15 min at 37°C (while being protected from light). Following this, 50 μl stop solution was added to each well. The color in the wells should change from blue to yellow. If the well color was green or the color change does not appear uniform, the plate was gently tapped to ensure thorough mixing. The optical density (OD) was read at 450 nm using a microtiter plate reader within 15 min. Using the OD value of the measured standard product as the abscissa and the standard product’s concentration value as the ordinate, the standard curve was drawn to obtain the linear regression equation. The OD value of the sample was substituted into the equation to calculate the concentration of the sample.Drosophila with Oil Red O. First, remove the intestines and fat bodies of fruit flies. Fix with 4% paraformaldehyde for 20 min and wash with PBS three times for 10 min each time. Filter the oil red O solution (the staining solution and distilled water are 3:2), then incubate at room temperature for 30 min and wash with PBS three times. The photos were taken under a Leica stereo microscope, and the images were processed using Adobe Photoshop.In order to quantify lipids, we stained the abdomen of Drosophila with the same genetic background. Independent sample t-test was used to identify differences between W1118 > apoLppRNAi flies and Arm-Gal4 > apoLppRNAi or Hand-Gal4 > apoLppRNAi flies. A value of p < 0.05 was considered to be statistically significant.Drawings were made using GraphPad Prism 6 software. Analyses were performed using the Statistical Package for the Social Sciences (SPSS) version 21.0 for Windows . All data are presented as the mean ± SEM. A two-way ANOVA was used to identify differences among the NFD, NFD + E, HFD, and HFD + E groups of Drosophila cardiomyocyte-derived apoLpp in lipid metabolism, we used different degrees of knockdown to verify the differential contribution of apoLpp in the whole-body and the heart. We use the previous high-fat regimen to feed female flies on a NFD and HFD for 5 days , and reduce the whole-body TG level  Exercise-training combined with apoLpp low expression can resist HFD-induced heart functional damage and low climbing ability. These results clarify the previously unknown relationship between apoLpp and cardiac function and exercise capacity and suggest possible ways for apoLpp in an exercise-regulated heart to improve cardiac function and exercise capacity.Obesity caused by HFD affects heart function and exercise capacity. Obesity is an important cause of diabetes, cardiovascular disease, and low exercise capacity. Obesity caused by a HFD diet dramatically affects the incidence of cardiovascular disease. We discussed possible uses of exercise-training to reduce heart function impairment and low exercise capacity caused by HFD. Our main findings are: (1) Under the conditions of high-fat feeding, the low expression of Drosophila is a good model organism for studying heart function. Its heart is a tubular structure that pumps hemolymph in an open circulatory system. Development of the heart occurs through evolutionarily conserved molecular mechanisms. Because of its simple structure and the availability of powerful genetic tools, the Drosophila heart has become a model system for studying the fundamental genetic and molecular mechanisms of cardiac development, function and aging (apoLpp in cardiac function and exercise capacity, as well as the regulatory effect of exercise-training on apoLpp in cardiomyocytes.nd aging . Diet, pnd aging . Exercisnd aging . HoweverapoLpp on heart function is mainly to resist the increase in heart rate and the occurrence of fibrillation. The fibrillation observed in flies is similar to human atrial fibrillation (apoLpp in Drosophila cardiomyocytes. A reduction of apoB after exercise-training has also been found in humans (Drosophila, the effects of exercise-training on lipoproteins are similar to humans, especially in terms of heart function.In humans, apoB is important in preventing coronary heart disease  and is aillation . It is tillation , and corillation . Our resn humans . For exan humans . After en humans . Fitnessn humans . Howevern humans . Exercisn humans , and then humans . Our res1118 group with NFD-fed, exercise-training was able to increase cardiac apoLpp expression and decrease whole-body triglyceride levels. M-mode showed that the incidence of arrhythmias was decreased and shortening fraction was increased in exercise-trained flies, suggesting enhanced cardiac function. Under HFD feeding, apoLpp derived from cardiomyocytes was upregulated about eightfold and Drosophila heart function was impaired and abdominal fat accumulation (apoLpp expression was significantly decreased, exercise capacity was enhanced, and Drosophila cardiac function recovered and no obvious lipid accumulation in the abdomen (apoLpp mRNA, which may have secreted more Apolipoprotein to bind circulating neutral lipids, resulting in significantly higher whole-body TG levels, and exercise-training resisted this effect of HFD-induced apoLpp overexpression, results that are strikingly similar compared to humans.In flies from the Wmulation  with a s abdomen , even exapoLpp expression levels, but whole-body triglyceride levels remained unchanged. In addition, flies showed increased exercise capacity and improved cardiac function, mainly in the form of arrhythmias and reduced incidence of fibrillation. In contrast, the effect of exercise-training on whole-body apoLpp was not significant under HFD conditions, and there were no significant changes in whole-body triglyceride levels or climbing ability, and abdominal fat does not accumulate (apoLpp could resist HFD-induced cardiac impairment and low climbing ability. The exercise-training to increase cardiac apoLpp expression levels without significant changes in whole-body triglyceride levels. Suggests that exercise-training increased lipoprotein activity and increased neutral lipid translocation and utilization to adapt to the changing environment of the body, resulting in increased apoLpp expression but no significant differences in whole-body triglyceride levels, which is consistent with the results of the Arm group under NFD conditions. In addition, the climbing ability of flies did not change significantly compared to its control group but was significantly higher than that of flies in the W1118 group, a result that may be due to the knockdown effect that itself increased climbing ability without the superimposed effect of the imposed exercise-training. In contrast, under HFD conditions, flies in the Hand group showed no significant changes in cardiac apoLpp expression levels, systemic triglyceride levels, or climbing ability after exercise-training, there is no obvious fat accumulation (apoLpp combined with exercise-training can effectively reduce the impairment of cardiac function and low climbing ability caused by HFD feeding. In short, a HFD is harmful to heart function and exercise capacity. It shows the accumulation of apoLpp in the body. Exercise-training and limit apoLpp expression can alleviate this situation, but considering that apolipoprotein B can shuttle in the body, it may also actively excrete lipids out of the organs during a HFD, thereby protecting the organs from lipotoxicity damage. Therefore, the effect of high-fat feeding combined with exercise-training in reducing apolipoprotein B is not absolute. This subtle relationship between them is worth studying.The data also showed that flies in the Arm group under NFD conditions, exercise-training increased whole-body cumulate  which fumulation . In addiDrosophila Apo B for cardiac function and exercise capacity under different dietary conditions. In addition, exercise-training to regulate combined apoLpp low expression against HFD-induced impairment of cardiac function and low exercise capacity.In conclusion, our results demonstrate the important role of The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the Ethics Committee of Hunan Normal University.MD and LZ conceived and designed the experiments and wrote the manuscript. MD, QL, WW, and MZ collected the samples. MD, WW, WP, and MZ performed the experiments. MD analyzed the data. All authors have read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


PRKCDBP methylation as a potential biomarker for non-small cell lung cancer (NSCLC).The occurrence and development of lung cancer are closely linked to epigenetic modification. Abnormal DNA methylation in the CpG island region of genes has been found in many cancers. Protein kinase C delta binding protein (PRKCDBP) is a potential tumor suppressor and its epigenetic changes are found in many human malignancies. This study investigated the possibility of PRKCDBP in the three groups of NSCLC tissues. Promoter activity was measured by the dual luciferase assay, with 5'-aza-deoxycytidine to examine the effect of demethylation on the expression level of PRKCDBP.We measured the methylation levels of PRKCDBP in tumor tissues and 3 cm para-tumor were higher than those of distant (> 10 cm) non-tumor tissues. Receiver operating characteristic (ROC) curve analysis between tumor tissues and distant non-tumor tissues showed that the area under the line (AUC) was 0.717. Dual luciferase experiment confirmed that the promoter region was able to promote gene expression. Meanwhile, in vitro methylation of the fragment (PRKCDBP_Me) could significantly reduce the promoter activity of the fragment. Demethylation of 5'-aza-deoxycytidine in lung cancer cell lines A549 and H1299 showed a significant up-regulation of PRKCDBP mRNA levels.The methylation levels of PRKCDBP methylation is a potential and promising candidate biomarker for non-small cell lung cancer. Clinically, 80%-85% of lung cancer patients are non-small cell lung cancer (NSCLC). The survival rate of lung cancer depends largely on the stage of diagnosis. The five-year survival rate of lung cancer diagnosed at local stage is 54%, but only 15% of patients can be diagnosed at local stage. At present, clinical diagnosis of lung cancer relies on spiral computed tomography (CT) and X-ray, but it is prone to both false negative and false positive cases. Therefore, finding new diagnostic markers for lung cancer is of great significance for lung cancer.The latest data show that one in four cancer patients in the United States is lung cancer. DNA methylation is one of the most studied epigenetics, and abnormal DNA methylation in the CpG island region of genes has been found in many cancers. CpG island mainly occurs in the coding region of eukaryotic genes, and abnormal methylation of CpG island will cause down-regulation of tumor suppressor gene expression or overexpression of oncogene, thereby promoting the development of cancer.The occurrence and development of lung cancer is closely linked to epigenetic modificationPRKCDBP) is a potential tumor suppressor and its epigenetic changes are found in many human malignancies. The PRKCDBP gene is located at the human chromosome 11p15.5-p15.4, which harbors frequent deletions in the tumors of breast cancer and lung cancer, suggesting its possible role in tumorigenesis. PRKCDBP can induce G1 cell arrest, enhance the sensitivity of cells to various apoptotic stimuli, and promote apoptosis. In addition, PRKCDBP transcription is found to be directly activated by tumor necrosis factor α (TNFα) via nuclear factor kappa-B (NF-κB) signaling pathway. TNFα is an important pro-inflammatory cytokine that not only plays an important role in the inflammatory process, but also involves processes such as apoptosis, proliferation, differentiation, wich are often related to the induction and development of cancer. As a transcriptional target of the TNFα-NF-κB signaling pathway, PRKCDBP plays a key role in TNFα-induced apoptosis.The protein kinase C delta binding protein  and distant non-tumor tissues (more than 10 cm from tumor tissues). We collected a total of 119 samples, including 44 pairs of tumor tissues and distant non-tumor tissues, and 31 para-tumor tissues. We collected clinical pathology information for NSCLC patients, including age, gender, family history, and pathological type. The study protocol was approved by the Ethics Committee of the First Affiliated Hospital of Fujian Medical University, and each patient signed an informed consent form.DNA of tissue samples were extracted using EZNA Tissue DNA Extraction Kit  and quantified using a NanoDrop 1000 spectrophotometer . The bisulfite modification was performed according to the instruction of EZ DNA Methylation-Gold Kit . The converted DNA was stored at -20 ℃ for methylation detection.PRKCDBP gene, and the qMSP product was 96 bp in length. The forward primer was 5'-ATA GGTCGGTAAAGGTTT-3', and the reverse primer was 5'-CCACGAACTACTAATAACG-3'. We used a SYBR-green-based qMSP to assess the level of methylation of PRKCDBP in three groups of NSCLC tissue samples. In order to avoid errors in the amount of sample loading, each sample used ACTB as an internal control. The primer sequences of ACTB were as follows: Forward, 5'-TGGTGATGGAGGAGGTTTAGTAAGT-3' and reverse, 5'-AACCAATAAAACCTACTCCTCCCTTAA-3'. At the same time, M-SssI-treated human sperm DNA and nuclease-free water were used as positive and negative controls, respectively, to ensure the accuracy of the experiment. The qMSP product was randomly selected for capillary electrophoresis  and Sanger sequencing to verify the homogeneity of product and the successful conversion of sodium bisulfite. We used the formula to calculate the methylation reference percentage (PMR) value. The relevant formula was as follows: .The qMSP primers were designed to amplify the CpG rich region of PRKCDBP plasmid of the target gene. To avoid the difference in the number of cells, the pGL3-basic plasmid and the pRL-SV40 plasmid were co-transformed as a negative control, and the pGL3-promoter plasmid and the pRL-SV40 plasmid were co-transformed as a positive control. Human embryonic kidney 293T cell line was used for the transformation. After 24 h of transfection, luciferase assays were performed using SpectraMax 190  following the Promega Dual Luciferase Reporting System kit instructions. Luciferase activity was finally calculated using the Dual Luciferase Reporter Assay Systems .The PKCDBP CpG-rich fragment (+181 bp to +581 bp) was selected to construct the pGL3-PRKCDBP was 5'-GCGGGAAGCTCCACGTTC-3', and the reverse primer was 5'-GCTCTGTACCTTCTGCAATCCG-3'. In order to avoid the error in the amount of sample loading, each sample had ACTB as the internal reference. The forward RT-PCR primer for ACTB was 5'-AGCACAGAGCCTCGCCTTT-3', and the reverse primer was 5'-AGGGTGAGGATGCCTCTCTT-3'. A negative reference was also made using nuclease-free water. The real-time PCR system was configured according to the Takara TB Green kit instructions, and the mRNA expression of PRKCDBP was detected using an StepOnePlusTM (96) real-time PCR instrument .Lung cancer cell lines A549 and H1299 were purchased from the cell bank of Shanghai Academy of Sciences, and cells were treated with 5'-aza-deoxycytidine. RNA was extracted by TRIzol one-step method before and after treatment, and product cDNA was obtained using Takara reverse transcription kit. The forward RT-PCR primer for PRKCDBP methylation levels of tumor tissuses and para-tumor tissues from 51 lung cancer patients were downloaded from TCGA database (https://genome-cancer.ucsc.edu/). In addition, we obtained data of 230 samples from the cBioPortal database  to analyze the relationship between PRKCDBP methylation level and mRNA expression.P was obtained by Bonferroni's correction. CpG site methylation of tumor tissues and para-tumor tissues from TCGA were compared using a Wilcoxon signed-rank test. Kaplan-Meier survival analysis was used to assess the prognostic value of the level of PRKCDBP methylation. ROC curve was used to evaluate the diagnostic value of distal PRKCDBP methylation for NSCLC. P < 0.05 was considered statistically significant.We performed statistical analysis using SPSS version 20.0 . Nonparametric Friedman test was used to determine methylation differences between two groups among tumor tissues, 3-cm para-tumor tissues, and distant non-tumor tissues. Adjusted We collected paired tissue samples from 44 patients with NSCLC and classified their clinical pathology information . The avePRKCDBP . This may be due to the close proximity of the para-tumor tissues and tumor tissues.Subgroup comparisons by age, gender, smoking history, lesion location and pathological type showed that the PRKCDBP was significantly higher than that of the pGL3-basic control plasmid, which confirmed that the fragment we analyzed could initiate gene expression . And our results also suggest that the in vitro methylation of the fragment (PRKCDBP_Me) can significantly reduce the promoter activity of the fragment  with DNA demethylation reagent 5'-aza-deoxycytidine and found the mRNA expression of PRKCDBP increased after treatment. These results indicated the expression of gene PRKCDBP was regulated by promoter methylation. We then performed double luciferase assays to determine the promoter activity of the fragment analyzed in this study and confirmed this fragment had transcriptional promoting function.PRKCDBP encoding protein) is a member of the Cavin family, which is involved in the formation and functions of caveolae, including signal transduction, lipid regulation, endocytosis, and tumorigenesis. The Cavin family gene expression is down-regulated in breast cancer. In studies of neuroblastoma and breast cancer, it was found that PRKCDBP methylation can determine its prognosis. Our study found that the promoter of PRKCDBP was hypermethylated in lung cancer tissue as well as in para-tumor tissues, while was hypomethylated in the distant non-tumor tissues. It suggested that the methylation level of PRKCDBP might affect the development of NSCLC and the methylation changes might be detected at the early stage of its onset.Cavin-3 (. It was reported that PRKCDBP could interact with BRCA1 protein and be involved in DNA damage response and participate in the BRCA1-mediated tumor suppressor pathway. Due to the limited sample size, we did not verify the relationship between PRKCDBP and BRCA1. However, the methylation levels of PRKCDBP promoterin female patients is higher in both tumor tissues and para-tumor tissues than in distant non-tumor tissues. It seems that PRKCDBP hypermethylation should be more sensitive in female NSCLC patients than males. This phenomenon might be related to the biologic property of PRKCDBP protein interacting with BRCA1.The incidence of lung cancer in China is higher in male patients than in female patients. In recent years, due to changes in smoking habits, the incidence of lung cancer in women has also shown a growing trendPRKCDBP was a downstream target of TNF α-induced tumor cell apoptosis signaling, and was activated by NF-κB. It was also found that PRKCDBP could enhance the protein stability of P53, and promote apoptosis by enhancing P53 function. The hypermethylation of PRKCDBP causes down-regulation of P53, which promotes the progression of malignant tumors. This also suggests a multifaceted function of PRKCDBP in NSCLC.PRKCDBP methylation is indeed associated with NSCLC. PRKCDBP methylation is a potential and promising candidate biomarker for non-small cell lung cancer. The mechanism of PRKCDBP on NSCLC remains unclear and further experimental verification is needed in the future.In summary, this study showed that the level of


The coronavirus disease (COVID-19) is a respiratory tract infection caused by the new virus SARS-CoV-2. The acute phase of the infection may in certain individuals be followed by another longer phase of disease (long COVID) of unknown etiology probably associated in certain cases with autoimmune activation. It has been shown that COVID-19 can trigger autoantibody production and in genetically predisposed patients may cause the onset or exacerbation of autoimmune diseases. We are reporting a case of mild COVID-19 infection complicated by autoantibody production and cutaneous and gastrointestinal symptoms and subsequently diagnosed with systemic sclerosis (SSc). A 47-year-old man with no history of any autoimmune diseases and in good health became sick together with his family on the 12th of November with mild symptoms: tiredness, fever, cough, and sore throat. Oropharyngeal swab for SARS-CoV-2 tested positive. He was isolated at home and did not require hospitalization. Three weeks later he presented with clinical manifestation compatible with suspicion of SSc. He briefly presented with skin rush, periorbital edema and conjunctivitis, vomiting, dysphagia, burning sensation in the skin, above all in the fingertips and around the mouth, puffy fingers, Raynaud’s phenomenon, pain at the fingertip of the middle finger where a depressed area was noticed without a clear ulceration. ANA showed a strongly positive nucleolar pattern. Anti-PM/Scl 75 and PM/Scl 100 resulted positive. High-resolution computed tomography (HCRT) showed early stage of interstitial lung disease (ILD). The patient was diagnosed with SSc based on the persistence of autoantibodies and the clinical and radiological pictures according to the ACR/EULAR classification . There are several cases described in the medical literature of possible new onset of SLE after COVID-19 infection. This is the first case that describes a possible new onset of SSc. Conclusion: SARS-CoV-2 may trigger systemic sclerosis. Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis in the skin and internal organs associated with vascular injury and immune system dysregulation . SSc is It is known that SSc can be triggered by viral illnesses and interferon signature . A viralCoronavirus disease 19 (COVID-19) is an infection caused by a novel coronavirus SARS-CoV-2, which became a pandemic in 2020 after an initial outbreak in Wuhan, China. It is speculated that SARS-CoV-2 can disrupt self-tolerance and trigger autoimmune responses through cross-reactivity with host cells , 9. The We present a case of mild COVID-19 infection complicated with clinical cutaneous and gastrointestinal manifestation, autoantibody production, and radiological finding compatible with the diagnosis of systemic sclerosis (SSc).H. pylori, HCV, HBV, and HIV were negative. Immunofluorescent antinuclear antibody test using HEp-2 cells (ANA) showed strongly positive nucleolar pattern, and this raised suspicions of SSc   correlate with a positive ANA, with the nucleolar pattern and a positive anti PM/Scl 75 and PM/Scl 100. Moreover, this patient was not critically ill due to COVID-19; he was not hospitalized, he did not experience any dyspnoea or desaturation, and he did not require any oxygen therapy. When the cough, sore throat, and fever disappeared, he experienced something else, something new. This is the second phase; the autoimmunity had replaced the initial viral symptoms.Can a COVID-19-induced inflammation mimic SSc? Or is it a real SSc? The radiological finding of ground glass opacity may occur both in COVID-19 infection and in the early stage of non-specific interstitial pneumonia (NSIP) related to SSc . The patpatients , 11. Thepatients . Interespatients , 11 the It has been described that COVID-19 disease evolves in overlapping phases. The first is the viral phase that may be asymptomatic or mild. The second phase is the inflammatory phase where autoinflammation/autoimmunity can occur. The third phase is the hypercoagulability phase, and the fourth phase is characterized by organ damage . In thisPatient was diagnosed with SSc by a rheumatologist at the Uppsala University Hospital according to the ACR/EULAR classification . It can be argued that PM/Scl 75 and PM/Scl 100 are not the classical SSc antibodies such as anti-centromere, anti-topoisomerase I, or anti-RNA polymerase III, but they are still quite common in SSc especially in the overlap form with polymyositis . DiffereAccurate follow-up is therefore necessary to confirm the SSc diagnosis and to differentiate it both from a transient autoinflammatory response to COVID-19 and from other autoimmune conditions that can overlap SSc. Despite the challenges in diagnostics, there is no doubt that COVID-19 infection has in this patient triggered an autoimmune process that is still active. At the six month follow-up, the clinical manifestation is still the same and the autoantibodies are still present. Long term follow-up is necessary in this mild and non-rapidly progressive case.BANK1, FAM167A-BLK, IL23R, IRF5, STAT4, TBX21, and TNFSF4 as susceptibility genes for the development of SSc (The mechanism of how SARS-CoV-2 triggers autoimmune diseases is under debate. Molecular mimicry due to the immune cross-reaction between epitopes and host antigens may be a possible explanation. Interferon production and cytokine activation can lead to disruption of immune tolerance in genetically predisposed subject. Defect of the function of dendritic cells that operate at the interface between innate and adaptive immunity is another possible mechanism . It is rt of SSc . It coulIn conclusion, we want to emphasize the necessity to accurately investigate and to test autoantibodies in all the patients that present with unclear and new symptoms immediately after a COVID-19 infection. Autoimmune diseases may hide in the “long COVID” patient-group. It is possible that additional cases of SSc induced by COVID-19 will be reported in the near future.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.Written informed consent was obtained from the patient for publication of this case report.The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Subwavelength grating (SWG) metamaterials have garnered a great interest for their singular capability to shape the material properties and the propagation of light, allowing the realization of devices with unprecedented performance. However, practical SWG implementations are limited by fabrication constraints, such as minimum feature size, that restrict the available design space or compromise compatibility with high-volume fabrication technologies. Indeed, most successful SWG realizations so far relied on electron-beam lithographic techniques, compromising the scalability of the approach. Here, we report the experimental demonstration of an SWG metamaterial engineered beam splitter fabricated with deep-ultraviolet immersion lithography in a 300-mm silicon-on-insulator technology. The metamaterial beam splitter exhibits high performance over a measured bandwidth exceeding 186 nm centered at 1550 nm. These results open a new route for the development of scalable silicon photonic circuits exploiting flexible metamaterial engineering. Subwavelength grating (SWG) metamaterials consist of periodic arrangements of dielectric structures with a period substantially smaller than the wavelength of the propagating light. Within this regime, the grating effectively acts as a homogeneous material whose optical properties  are determined by the ensemble of the constituent materials and can be varied by properly designing the geometry of the grating unit cells ,2. This The use of comparatively small grating periods represents the main technological challenge in the realization of high-performing devices based on SWG metamaterials. Structures sometimes include small features near the resolution limit of dry deep-ultraviolet (DUV) lithography tools . SeveralIn order to overcome these limitations, immersion DUV lithography has been increasingly investigated for the fabrication of photonic devices. Immersion DUV lithography is compatible with high-volume production and, compared to dry lithography, allows to achieve a three-fold improvement in device size reproducibility, with one-sigma variations below 1% across the wafer, and an almost two times reduction of line edge roughness ,25. ThesHere, we exploit a fabrication technology based on 300-mm SOI wafers and immersion DUV lithography to experimentally demonstrate a broadband integrated beam splitter based on an SWG-engineered multi-mode interference (MMI) coupler. The device has a silicon thickness of 300 nm and nominal minimum feature size of 75 nm, well below the resolution capabilities of dry DUV lithography. Full three-dimensional finite-difference time-domain (3D FDTD) simulations show excess losses smaller than 1 dB within a broad bandwidth of 230 nm, with negligible power imbalance and phase errors. The fabricated device has a behavior well in line with simulation predictions, exhibiting high performance over a bandwidth exceeding 186 nm.MMI couplers consist of a large waveguide section that can sustain the propagation of multiple guided modes. When light is injected in the device through one of the input ports, it excites a linear combination of these modes, each one propagating with its own propagation constant rinciple ). If outrference ). Lπ is on cores .In ,32, the /Λ = 0.6 . The perThe width of the access waveguides to the multi-mode section is set to W quality . The 0.4nsitions a are useAsymmetric Mach–Zehnder interferometers were fabricated to experimentally characterize the designed device. A range of MMI lengths from LWe found that with this duty cycle, MMIs with LThe fabricated devices have been characterized in the spectral domain using two fiber-coupled tunable laser sources covering together the 1460 nm–1680 nm wavelength range. Before entering the chip, light polarization was set to TE by means of an in-line polarization controller. On the chip, input and output grating couplers were used to couple light between integrated waveguides and standard single mode fibers. Different spectral regions were characterized by adjusting the angle of the optical fibers between 11° and 30°. The fiber collecting light at the output of the chip was finally connected to an external photodetector to measure the wavelength-dependent transmission of the device. Experimental results for LWe have reported on the use of a 300-mm SOI fabrication platform with DUV immersion lithography to implement a high-performing MMI beam splitter based on an SWG metamaterial. The high resolution of immersion lithography allowed designs with nominal feature size of 75 nm. The use of an SWG core instead of a conventional solid silicon core resulted in a broadband and compact MMI, with about a two times reduction in the length of the multi-mode section. The device size could be further scaled down by reducing the length of the input and output tapers, which represent the longest sections in the current design.The experimental characterization of devices showed low excess losses below 1 dB with negligible power imbalance and phase errors over a bandwidth of 186 nm, confirming the high quality of the fabrication and making the device suitable, for example, for coarse wavelength division multiplexing transceivers. These results demonstrate for the first time SWG metamaterials with a fabrication process compatible with high-volume production while simultaneously reaching feature sizes that would commonly require electron-beam lithography. We believe that the use of lithographic techniques routinely available in complementary metal-oxide-semiconductor (CMOS) processes will be of fundamental importance to bring the potentialities of refractive index engineering toward commercial exploitation. This will enable the fabrication of high-performance devices for fiber-to-chip coupling, power splitting, polarization management, and spectral filtering, with promising applications, for example, in coherent communications, sensing, and spectroscopy.


The rapid outbreak of COVID-19 around the world has adversely affected the mental health of the public. The prevalence of anxiety among the public has increased dramatically during the COVID-19 pandemic. However, there are few studies evaluating the effects of positive psychological responses and information-seeking behaviors on anxiety experienced among social media users during the COVID-19 pandemic.This study evaluated the prevalence of anxiety and its associated factors among WeChat users in mainland China during the early stages of the COVID-19 pandemic.From February 10 to February 24, 2020, a nationwide, web-based cross-sectional survey study was carried out using convenience sampling. Participants’ levels of anxiety, positive psychological responses, and information-seeking behaviors were assessed. The survey was distributed among WeChat users via the WeChat smartphone platform. Chi-square tests and multivariable logistic regression analyses were performed to examine the factors associated with anxiety.This study found that the prevalence of anxiety  among WeChat users in China was 17.96% (446/2483) during the early stages of the COVID-19 pandemic. Results of multivariable logistic regression analysis showed that information-seeking behaviors such as cannot stop searching for information on COVID-19, being concerned about the COVID-19 pandemic, and spending more than 1 hour per day consuming information about the pandemic were found to be associated with increased levels of anxiety. Additionally, participants who chose social media and commercial media as the primary sources to obtain information about the COVID-19 pandemic were found more likely to report anxiety. Conversely, participants who were confident or rational about the COVID-19 pandemic were less likely to report anxiety.This study found that positive psychological responses and information-seeking behaviors were closely associated with anxiety among WeChat users during the COVID-19 pandemic in China. It might be paramount to enhance mental well-being by helping people respond to the COVID-19 pandemic more rationally and positively in order to decrease symptoms of anxiety. In January 2020, the World Health Organization classified COVID-19 as a public health emergency of international concern ,4. The sThe COVID-19 pandemic generated a psychological crisis among the public as the prevalence of mental disorders, including anxiety and depression, increased -18. VariPositive psychological responses to the COVID-19 pandemic among the public play a crucial part in reducing anxiety. Positive thoughts and attitudes could help individuals cope with stressors . A recenResearchers have studied the pathogenic and epidemiological characteristics, epidemiological trends, as well as the prevention and treatment of COVID-19 -43. HoweIn order to examine the prevalence of anxiety among WeChat users during the COVID-19 pandemic, a cross-sectional study design using convenience sampling was employed via a web-based WeChat platform developed by the Department of Environmental Health of China Medical University in China. Due to the urgency of the COVID-19 pandemic and the necessity of timely acquisition, the questionnaire was first uploaded to this web-based WeChat platform. Next, the survey was distributed via all the open WeChat groups of the research assistants, to render the survey easily available to their close contacts. The survey was distributed between February 10 and February 24, 2020, during which we achieved a sample size that was adequate in accordance with previous related studies -47. The The inclusion criteria for participation in this study were as follows: at least 18 years of age, ability to read and write Chinese, ability to complete the web-based questionnaire by themselves using a smartphone, ability to offer electronic signed informed consent, and voluntary participation. The exclusion criteria were as follows: undergoing any therapy for psychological illness, history of any drug dependence, and diagnoses of any diseases or impairments that would prevent them from completing the questionnaire independently. The questionnaire had been preset for submission only after all the questions were answered within the range of the selected choices. Only data from complete questionnaires were analyzed.This study was carried out in conformance with the Declaration of Helsinki (1989). The study protocols were approved by the Ethics Committee of China Medical University.The survey collected participants’ demographic characteristics, including gender , age, marital status (married or other), occupation, education, and monthly income. Occupation was further classified into government worker, health care worker, professional staff , employee of enterprises, commercial personnel, soldier, student, and other. Education was further classified into junior college or below, bachelor’s degree, and master’s degree or above. Monthly income was categorized as follows: ≤¥5000 (≤US $725.19), ¥5001-10,000 (US $725.34-1450.39), and >¥10,000 (>US $1450.39).P<.001]. The indicators of reliability and validity were good.The validated Chinese version of the GAD-7, one of the most reliable tools to measure generalized anxiety disorder, was used to assess anxiety among the study participants during the early stages of the COVID-19 pandemic . PsycholP<.001). The indicators of reliability and validity were good.Positive psychological responses included “being confident,” “being hopeful,” and “being rational” in the past 2 weeks. They were measured by “yes” or “no” questions (Cronbach α=.788). The results of the factor analysis indicated that the three items of the positive psychological responses tested were extracted as one component, which contributed to 70.932% of the variance  cannot stop searching for information about the COVID-19 pandemic, (2) being concerned about the COVID-19 pandemic, (3) time spent consuming information about the COVID-19 pandemic , and (4) sources of information about the COVID-19 pandemic . Responses to the item “cannot stop searching for information about the COVID-19 pandemic” included “agree,” “not sure,” and “disagree.” Whether participants were concerned about the COVID-19 pandemic and their sources of information about the pandemic were assessed by “yes” or “no” questions.anxiety or no anxiety, as measured by the GAD-7 scale). Multivariable logistic regression analysis showed that the factors associated with anxiety were assessed while controlling for confounding variables. The variables included in the multivariable logistic regression analysis included age as a continuous variable and other variables as categorized variables. The responses were not included in the analysis when more than 95% of individuals had the same response to the categorical independent variables. Findings were considered statistically significant when a two-tailed P value was <.05.SPSS software  was used to perform all statistical analyses. Chi-square tests were applied to assess bivariate associations with anxiety and the variables of interest. Anxiety was assessed using the binary variable  Chinese WeChat users who participated in our survey experienced anxiety. A total of 2501 adults participated in this survey and 2483 of them provided complete and logical answers, resulting in a valid response rate of 99.28%. The demographic characteristics of the participants and results from the bivariate analysis of anxiety are presented in P<.001).The prevalence of anxiety according to the participants' positive psychological responses are presented in P<.001) than other participants. The prevalence of anxiety was also observed to be higher for participants who spent more than 1 hour a day on searching for information about the COVID-19 pandemic (P<.001).The distribution and prevalence of anxiety with different information-seeking behaviors during the COVID-19 pandemic is shown in P=.001).Our study findings also showed that participants who sought information about COVID-19 via social media and commercial media  had a higher prevalence of anxiety  than those who did not seek information through social and commercial media  ,53. HoweOur study findings showed that anxiety was closely associated with positive psychological responses. Among the positive psychological responses, being confident and being rational were observed to be associated with lower prevalence of anxiety, which suggests that being confident and rational might prompt individuals to cope better with adverse events and difficult challenges during public health emergencies. Being rational also has been found to influence anxiety in previous studies, wherein people who were more positive coped better with life stressors and had low levels of anxiety ,60. WeChPeople could cope with stressors better and improve their mental health by means of rational-emotive behavioral interventions and cognitive behavioral therapies to reduce anxiety and improve psychological abilities, including emotion regulation and stress management ,29,63,64This study also found that excessive consumption of information about COVID-19 might be closely linked with anxiety. Consuming information about the pandemic for more than 1 hour a day was observed to be associated with anxiety. Our results are consistent with other studies showing that anxiety is associated with increased use of smart phones, leading to excessive exposure to COVID-19 news ,67,68. TMoreover, sources of information about COVID-19 were found to be significantly associated with anxiety, especially information obtained via social media was observed to have a positive correlation with anxiety. Excessively searching for health information on social media during traumatic events  can result in sleep disorders, distress such as anxiety and depression, and posttraumatic stress disorder -89. SociSeveral limitations should be acknowledged. First, the data were collected using a self-administered web-based questionnaire via smartphones, which may have resulted in information bias and misclassification bias. It is possible that participants did not provide accurate information in the study in order to either be included in the study or to move quickly through the survey. To minimize the bias as much as possible, a pilot study was conducted to acquire participants’ perception of the questionnaire. Therefore, the collected questionnaire had been filtered with data cleaning, checking for consistency and logicality of the answers, adjusting invalid and missing values. Due to the severity of the COVID-19 pandemic and the necessity of timely acquisition of data, the survey was distributed via WeChat rather than conducted using a face-to-face approach. Additionally, this survey among WeChat users used a convenience sampling method during the first 2 months of the COVID-19 pandemic. Therefore, generalizability of the results may be limited to the whole population of China. As the survey was shared within academic WeChat groups, it is possible the results reported in our study are from a population that has a higher education level than the general population, and this may have affected anxiety levels either positively or negatively, because the participants likely had a stronger understanding of the pandemic. Future studies should be carried out with random sampling. Finally, the results may be limited by residual confounding from unmeasured confounders such as regional economic disparity, allocation of health resources, and an individual’s social status. To minimize the potential errors induced by unmeasured confounders, the survey was conducted across 3 municipalities and 22 provinces via WeChat, one of the most popular social platforms in China.The COVID-19 pandemic has adversely affected the mental health of the public and resulted in a high prevalence of anxiety among them, especially during the early stages of the pandemic. Certain behaviors such as cannot stop searching for information on COVID-19, being concerned about the COVID-19 pandemic, spending more than 1 hour a day consuming information about the COVID-19 pandemic, and using social media and commercial media as the primary source of information about the COVID-19 pandemic were found to be associated with anxiety. In contrast, positive psychological responses such as being confident and being rational were found to be negatively correlated with anxiety. Prompt measures and guidance from public health authorities on choosing reliable and trusted sources of information might decrease the negative effects of overconsumption of COVID-19–related information and improve the overall mental health of the public.


Screening for colorectal cancer (CRC) using the faecal immunochemical test (FIT) is widely advocated. Few studies have compared the rate of detecting colonoscopic pathologies in single compared to double FIT-positive follow-up colonoscopy-compliant individuals in a two-sample national FIT screening program.To compare CRC incidence in double FIT-positive versus single FIT-positive individuals using a retrospective cohort of patients from a tertiary hospital in Singapore.Retrospective cohort study.Data was extracted from one public tertiary hospital in Singapore.1,422 FIT-positive individuals from the national FIT screening program who were referred to the hospital from 1st January 2017 to 31st March 2020 for follow-up consultation and diagnostic colonoscopy.The exposure of interest was a positive result on both FIT kits. The main outcome was a follow-up diagnostic colonoscopy finding of CRC. The secondary outcome was a diagnostic colonoscopy finding of a colorectal polyp.Incidence density of CRC was 1.15 and 13.10 per 100,000 person-months, in the single and double FIT-positive group, respectively. This resulted in an incidence rate ratio of 11.40 . Colorectal polyp detection was significantly higher (p < 0.01) in the double  compared to the single  FIT-positive group.The key limitation of this study was the relatively small cohort derived from a single tertiary hospital, as this had the effect of limiting the number of incident cases, resulting in comparatively imprecise CIs.Double FIT-positive individuals are significantly more likely to have a colonoscopy finding of incident CRC or premalignant polyp than single FIT-positive individuals. Clinicians and policymakers should consider updating their CRC screening protocols accordingly. Colorectal cancer (CRC) screening has been shown to improve oncological outcomes by earlier detection and treatment of premalignant adenomatous polyps or CRC itself . CRC scrSingapore has adopted two-sample FIT in its national CRC screening program as this has been shown to increase sensitivity by potentially detecting neoplasms that may have been missed in one-sample FIT , 4. The Individuals with two positive FIT samples (“double FIT-positive”)–which would have been sampled over two days–should intuitively be more likely to have colonic pathologies compared to individuals with only one positive FIT sample (“single FIT-positive”). However, few studies have examined these outcomes , 7. ThusThis study was ethically approved by the National Healthcare Group’s Domain Specific Review Board  in accordance with the Declaration of Helsinki. All data was analysed anonymously.This research was done without patient involvement. Patients were not invited to comment on the study design and were not consulted to develop patient relevant outcomes or interpret the results. Patients were not invited to contribute to the writing or editing of this document for readability or accuracy. Informed consent was provided as part of enrolment to the national CRC screening program for both data analysis and publication.st January 2017 to 31st March 2020 for follow-up consultation and diagnostic colonoscopy. The index date was the date of referral to the hospital. These individuals are referred directly to NUH by the program coordinator due to their residential address and not by personal choice. The socio-demographic distribution by residential address is representative of the national demographics.The study population  was a retrospective cohort enrolled through convenience sampling comprising all individuals who (I) had completed and submitted two FIT kits under the national CRC screening program, (II) received at least one positive FIT-result, and (III) were referred to a single tertiary institution ) between 1st December 2020). This resulted in a final sample of 1,422 participants, or 15.0% loss to follow up.Individuals were excluded from the study if they (I) had missing or incomplete results for either FIT kit, or (II) had transferred to another hospital at the point of data extraction (31The exposure of interest was defined as a positive result on both FIT kits. The main outcome (incident CRC cases) was defined as a follow-up diagnostic colonoscopy finding of a CRC, regardless of staging. The secondary outcome was defined as a follow-up diagnostic colonoscopy finding of a colorectal polyp. Colonoscopy reports were verified by using the hospital’s electronic medical records system. Colonoscopy findings were grouped into normal, pre-malignant polyp and CRC. Pre-malignant polyp was defined as a tubular adenoma ≥ 10 mm in size, an adenoma of any size with villous features or high grade dysplasia, or a dysplastic serrated lesion of any size.Apart from the main exposure and outcome of interest, our data included participants’ ethnicity , age, date of two-sample FIT kit collection, date of referral to NUH, and date of follow-up medical consultation and colonoscopy.For demographics, frequencies and proportions were used to report ethnicity, colonoscopy findings, predictive values and compliance to follow-up colonoscopy. Median and interquartile range (IQR) were used to summarise participant age. For colonoscopy-compliant participants, median and IQR was used to report follow-up time (in days) between date of referral to the hospital and date of colonoscopy. Incident CRC cases were summarised using frequencies and proportions.To examine possible effects of confounding, chi-square tests were used to compare ethnicity and colonoscopy compliance, and Mann-Whitney U tests for age and follow-up time, between single and double FIT-positive groups.A chi-square test was used to compare incident CRC cases and polyp detection between the groups. Incidence density (ID) of CRC cases was derived using person-months at risk, calculated from month of birth to the month of colonoscopy. Positive predictive value was tabulated as a proportion of positive colonoscopic findings over total colonoscopy performed in each group. Positive colonoscopic findings are defined as pre-malignant and cancer lesions. Incidence rate ratio (IRR) was used to compare relative risk of CRC between the single FIT-positive and double FIT-positive groups. Confidence intervals (95% CIs) were reported for IRR.The median age of the sample was 66 years (IQR = 59–72 years). Overall compliance to follow-up colonoscopy was 51.7%. The distribution of sample demographics, colonoscopy compliance, and outcomes of interest between single and double FIT-positive patients can be found in n = 670; median = 53 days, IQR = 32–81 days) and double FIT-positive  groups; U = 53,222.5, p = 0.12.Median follow-up time between date of referral to the hospital and date of colonoscopy was calculated for colonoscopy-compliant patients between the single  refer to .In colonoscopy-compliant patients, the double FIT-positive group was approximately 11 times more likely to have CRC and a significantly higher incidence of colorectal polyps than the single FIT-positive group. This correlated with the higher PPV in the group with double FIT-positive tests. These findings have significant implications to several stakeholders.There is the need to expedite colonoscopy for those who are double FIT-positive to ensure prompt detection of CRC and premalignant polyps. With increasing advocacy on CRC screening and being current to screening recommendations, a higher number of patients who are at least single FIT-positive is expected if screening uptake increases. This could put strains on healthcare systems with finite facilities for colonoscopy, resulting in a need to prioritise which patients should more urgently undergo the procedure.At the national level, countries that are still advocating CRC screening using only one-sample FIT should reconsider their approach. Double FIT-positive is likely to have increased specificity, resulting in fewer false positive findings during follow-up , 7. ReceMore work is also required to reinforce the importance of prompt follow-up after a positive FIT result. As observed in our study and the literature, colonoscopy compliance rate after a positive FIT is approximately 50% . Using sThe key strengths of this study were (I) the representativeness of the sample to the average-risk study population recommended to our hospital via the national screening programme, with low loss to follow up (15.0%), and (II) the ability to verify outcomes of interest via the hospital’s clinical database. The key limitation of this study was the relatively small cohort derived from referrals of the national CRC screening program to a single tertiary hospital, as this had the effect of limiting the number of incident cases, resulting in comparatively imprecise CIs. The various ethnic groups in Singapore could also be a potential confounder given possible difference in knowledge, beliefs and attitude towards CRC screening. Nonetheless, our findings observed similar socio-demographic characteristics between both groups. This should be the case and suggests that our data is representative of national demographics as FIT-positive hospital referrals are performed geographically on a national level, and population sociodemographic characteristics are similarly distributed across Singapore . AlthougS1 File(XLSX)Click here for additional data file.