This study aimed to determine work-rest schedules for visual tasks of different lengths by evaluating visual fatigue and visually induced motion sickness (VIMS) using an optical head-mounted display (OHMD). Thirty participants were recruited to perform 15 and 30 min visual tasks using an OHMD. After completing each visual task, participants executed six levels of rest time. Critical flicker fusion frequency (CFF) values, relative electroencephalography indices, and Simulator Sickness Questionnaire (SSQ) scores were collected and analyzed. Results indicated that after completing the 15 and 30 min visual tasks, participants experienced visual fatigue and VIMS. There was no significant difference between baseline CFF values, four electroencephalography relative power index values, and SSQ scores when participants completed a 15 min visual task followed by a 20 min rest and a 30 min visual task followed by a 30 min rest. Based on our results, a 20 min rest for visual fatigue and VIMS recovery after a 15 min visual task on an OHMD and a 25 min rest for visual fatigue and VIMS recovery after a 30 min visual task on an OHMD are recommended. This study suggests a work-rest schedule for OHMDs that can be used as a reference for OHMD user guidelines to reduce visual fatigue and visually induced motion sickness. With the rapid development of display technologies, augmented reality (AR) has emerged as a next-generation display technology for human–computer interactions. Optical head-mounted displays (OHMDs) are becoming increasingly popular in daily activities. Most OHMDs use microdisplays, eyepieces, and optical combiners to present AR images on a screen . The eyeVergence–accommodation conflict is a common problem with virtual reality head-mounted displays (VR HMDs) and AR HMDs , which aCritical flicker fusion frequency (CFF or CFFF) is a commonly used method to measure visual fatigue, and it is defined as the lowest frequency at which intermittent light stimulation appears to be steady to the observer . Chi andVIMS is a medical condition in which nausea, disorientation, and oculomotor symptoms occur when the vestibular system senses acceleration that is not perceived (or perceived differently) by the visual system . ExtensiMany studies have been conducted on computers or handheld smart devices to prevent and alleviate the visual fatigue caused by viewing digital screen devices. Reddy et al.  developeTo examine the effects of taking a break, Shieh and Chen  reportedUser experiences when viewing AR information on OHMDs are very different from their experiences when using computers, handheld smart devices, or VR HMDs. In contrast to the virtual environment constructed by VR HMDs, when using OHMDs, users have the visual experience of watching AR objects and the real environment simultaneously. In addition, when viewing or controlling computers or handheld smart devices, users can easily change the viewing distance between themselves and the display, whereas traditional OHMD AR images appear at a fixed distance from the eyes . TherefoWith the increasing interest in OHMDs, they are being widely used in various fields. Performing visual tasks on OHMDs for any duration induces visual fatigue and VIMS. Gao et al.  indicateThis study included 30 healthy male participants. Their ages ranged from 21 to 35 years . All the participants were required to have normal or corrected-to-normal vision. The participants were requested not to use digital devices or watch television (TV) 1 h before data collection to avoid visual discomfort and fatigue.Visual acuity was measured using an OPTEC 2000P Vision Tester . Laboratory illumination was measured using an illuminometer . Epson Moverio BT-200  AR glasses were the OHMD used in this study. The display projector had a 960 × 540 red–green–blue resolution and provided a 23° field of view . All parA full factorial design was employed with two factors: length of the visual task time and length of rest time. Participants were asked to watch an experimental movie on an OHMD. The visual task involved two levels of visual task duration and six levels of rest duration. The experimental movie content in this study included a documentary, comedy, action, romance, science fiction, or drama, chosen by each participant. All participants chose a drama movie as the experiment movie. The sequence of the visual task duration for each participant was randomly arranged. Prior to each visual task, critical flicker fusion frequency (CFF) values, electroencephalogram (EEG) signals, and Simulator Sickness Questionnaire (SSQ) scores were recorded as baseline data. After completing each visual task, the CFF values, EEG signals, and SSQ scores were recorded again. Upon completion of each visual task, a 30 min rest was provided with CFF values, EEG signals, and SSQ scores recorded every 5 min.2 and it remained constant throughout the experiment in all tests. The laboratory temperature was maintained at 25 °C, and illumination in the laboratory environment was maintained at 500–600 lx. A white projection screen was used to provide the standard experimental background. Participants wore an OHMD and faced the projection screen, which was placed 2 m in front of them, as shown in The video was projected onto the front visual field of the participants, and the off-axis viewing angle was not investigated in this study. In this study, the OHMD luminance was 500 cd/mPrevious studies have proposed specific work-rest schedules with VDT or handheld smart devices or VR HMDs to prevent and alleviate visual fatigue and VIMS caused by viewing digital screen devices. However, these recommendations do not account for OHMD effects on visual fatigue and VIMS. It remains necessary to examine appropriate OHMD visual task lengths and rest times considering how to prevent and alleviate visual fatigue and VIMS. Therefore, visual tasks and rest durations were independent variables. Two levels of visual task duration (15 and 30 min) and six levels of rest duration  were specified in this study, following the modified visual task periods and rest periods in the study by Moss and Muth , Shieh aDependent variables included the CFF values, EEG relative power indices, and SSQ scores. In this study, the CFF value and EEG relative power indices were used to measure the severity of visual fatigue and recovery from visual fatigue. CFF values were collected for both eyes, and the mean value of three ascending trials  and three descending trials  was used as the CFF value .After completing the visual tasks and during rest, EEG signals were measured. EEG Ag/AgCl electrodes were attached to specific locations on each participant’s scalp. The exploring electrodes were located over the left occipital lobe (O1) and right occipital lobe (O2); the reference electrodes were located on the prominent bone just behind the left ear (A1) and that just behind the right ear (A2). The electrode locations were based on the international 10–20 system . EEG sigThe SSQ scores, comprising 16 symptoms that reflect the severity of visually induced motion sickness (VIMS), were used in this study to measure VIMS severity and recovery. Participants rated each SSQ item on a 4-point Likert scale  . KennedyThe experimental environment was standardized prior to data collection. The SSQ scores were checked to ensure that each participant had no VIMS symptoms at the outset, indicating a score of 0 in the SSQ total severity score. The purpose and procedures of the experiment were explained to the participants in detail. Participants were requested to complete informed consent forms. Subsequently, each participant’s chair and nose pad were adjusted for comfort, and CFF values, EEG signals, and SSQ scores were then measured as baseline values.Visual task conditions and movie content were arranged randomly. At the beginning of each visual task, a movie was presented on the OHMD and participants were asked to focus their attention on watching the movie. Upon completion of the visual task, a 30 min rest was provided, and CFF values, EEG signals, and SSQ scores were recorded every 5 min. The experimental procedure sequence is shown in t-test and Wilcoxon signed-rank t-test were used to examine the EEG electrode locations’ (O1 and O2) effects on the five EEG relative power indices. Spearman correlation analysis was used to evaluate the relationship between the CFF value and relative EEG power indices. The statistical significance level was set at p = 0.05. All statistical analyses were performed with SPSS Statistics version 22.0  software. Line graphs were made with Excel version 23.0 .Statistical analyses using analysis of variance (ANOVA) and a Wilcoxon signed-rank test were conducted to evaluate how the length of the visual task time and length of the rest period affect the CFF value, EEG relative power indices, and SSQ scores. The Kolmogorov-Smirnov test was performed to examine the normality of the data distribution if the normality assumption was confirmed. If the normality assumption was violated, the Wilcoxon signed-rank test was used for corresponding variables. Duncan’s multiple range tests were conducted as post hoc tests. In addition, the paired p < 0.001; 30 min: Z (240) = 4.551, p < 0.001).The results of the Kolmogorov–Smirnov test indicated that the CFF values, EEG relative power indices, and SSQ scores violated the normality assumption. The post-experiment CFF values were significantly lower than those at baseline in the two visual tasks (15 min: Z (240) = 4.712, p = 0.110). Additionally, after the 30 min visual task and 15 min rest, there were no significant differences in CFF values at baseline and at the end of the 15 min rest period (Z (240) = 1.661, p = 0.100).The results of CFF values are presented in p = 0.83, relative β power index Z (240) = −0.21, p = 0.63, relative θ power index Z (240) = −0.49, p = 0.63, relative β/α power index Z (240) = −0.44, p = 0.66, and relative θ/α power index Z (240) = −0.03, p = 0.98; 30 min visual task: relative α power index Z (240) = −0.66, p = 0.51, relative β power index Z (240) = −0.44, p = 0.66, relative θ power index Z (240) = −0.74, p = 0.46, relative β/α power index Z (240) = −0.95, p = 0.34, and relative θ/α power index Z (240) = −0.11, p = 0.91). Hence, analyzing the EEG relative power index data from one occipital lobe electrode site was sufficient.For EEG relative power indices, the Wilcoxon signed-rank test was conducted to examine the effects of EEG electrode location (O1 and O2) on the five EEG relative power indices, as shown in p = 0.003), relative β index (Z (240) = 2.401, p = 0.017), relative θ index (Z (240) = 3.671, p < 0.001), and relative β/α index (Z (240) = 3.262, p = 0.001)), but no significant effects on θ/α (relative θ/α index (Z (240) = 1.242, p = 0.213)), as shown in The 15 min visual task had significant effects on four EEG power indices (relative α index (Z (240) = 2.951, p = 0.003); the relative β index value had significantly decreased (Z (30) = −2.851, p = 0.003); the relative θ index value had significantly increased (Z (30) = −2.405, p = 0.017); the relative β/α index value had significantly decreased (Z (30) = −3.256, p = 0.001). This shows that after completing the visual task, participants had visual fatigue. The relative β and β/α indices decreased with increasing rest times. After a 10 min rest, there were no significant differences in relative β index value at baseline and at the end of the 10 min rest period (Z (30) = −1.841, p = 0.066); after a 20 min rest, there were no significant differences in relative β/α index value at baseline and at the end of the 20 min rest period (Z (30) = −1.244, p = 0.213). The relative α and θ indices increased with increasing rest times. After a 20 min rest, there were no significant differences in relative α index value at baseline and at the end of the 20 min rest period (Z (30) = −0.782, p = 0.482), and no significant differences in relative θ index value at baseline and at the end of the 20 min rest period (Z (30) = −1.676, p = 0.094). Additionally, after the 15 min visual task and 20 min rest, there were no significant differences in the four EEG power indices at baseline and at the end of the 20 min rest.After a 15 min visual task, the relative α index value significantly increased (Z (30) = −2.915, p < 0.001), relative β index (Z (240) = 2.792, p = 0.005), relative θ index (Z (240) = 3.202, p = 0.001), and relative β/α index (Z (240) = 4.062, p < 0.001)), but had no significant effect on θ/α (relative θ/α index (Z (240) = 1.201, p = 0.229)), as shown in The 30 min visual task had significant effects on four EEG power indices (relative α index (Z (240) = 4.021, p = 0.013); the relative β index value had significantly decreased (Z (30) = −2.854, p = 0.004); the relative θ index value had significantly increased (Z (30) = −1.944, p = 0.041); the relative β/α index value had significantly decreased (Z (30) = −2.232, p = 0.026). This shows that after completing the visual task, participants had visual fatigue. The relative β and β/α indices decreased with increasing rest times. After a 30 min rest, there were no significant differences in relative β index value at baseline and at the end of the 30 min rest period (Z (30) = −0.031, p = 0.975); and no significant differences in relative β/α index value at baseline and at the end of the 25 min rest period (Z (30) = −0.216, p = 0.829). The relative α and θ indices increased with increasing rest times. After a 25 min rest, there were no significant differences in relative α index value at baseline and at the end of the 25 min rest period (Z (30) = −0.031, p = 0.926); after a 30 min rest, there were no significant differences in relative θ index value at baseline and at the end of the 30 min rest period (Z (30) = −0.041, p = 0.494). Additionally, after the 15 min visual task and 30 min rest, there were no significant differences in the four EEG power indices at baseline and at the end of the 30 min rest.After a 30 min visual task, the relative α index value significantly increased (Z (30) = −2.478, p = 0.003) and nausea scores in 30 min (Z (240) = 4.011, p < 0.001); oculomotor scores in 15 min (Z (240) = 4.744, p < 0.001) and oculomotor scores in 30 min (Z (240) = 4.713, p < 0.001); disorientation scores in 15 min (Z (240) = 3.854, p < 0.001) and disorientation scores in 30 min (Z (240) = 4.414, p < 0.001); total severity scores in 15 min (Z (240) = 4.721, p < 0.001) and total severity scores in 30 min (Z (240) = 4.715, p < 0.001)), as presented in p = 0.315) was not significantly different from baseline. In addition, after a 15 min rest, nausea (Z (30) = 3.124, p = 0.101), oculomotor (Z (30) = 2.245, p = 0.211), and total scores (Z (30) = 4.215, p = 0.345) were not significantly different from baseline. After the 30 min visual task followed by a 15 min rest, the disorientation score (Z (30) = 2.245, p = 0.445) was not significantly different from baseline. In addition, after a 20 min rest, nausea (Z (30) = 2.224, p = 0.211), oculomotor (Z (30) = 3.12, p = 0.351), and total scores (Z (30) = 3.152, p = 0.245) were not significantly different from baseline.The 15 and 30 min visual tasks had significant effects on nausea, oculomotor function, disorientation, and total scores (nausea scores in 15 min (Z (240) = 2.952, Spearman correlation was conducted to investigate the relationship between the four relative EEG power indices and the CFF value for the visual task duration and rest duration, as presented in This study aimed to determine work-rest schedules for visual tasks of different lengths by evaluating visual fatigue and VIMS while using an OHMD. The results indicated that decreased CFF values were observed after participants completed 15 and 30 min visual tasks at 1.48 Hz and 2.02 Hz, respectively. Wu et al.  identifiIn this study, the participants’ relative θ index significantly increased after executing the OHMD visual tasks. The relative θ index trend contradicted the results reported by Hsu and Wang . One expFor visual fatigue recovery, after completing a 15 min visual task followed by 10 min of rest and a 30 min visual task followed by 15 min of rest, the participants’ CFF values were not significantly different from baseline. After completing a 15 min visual task followed by 20 min of rest and a 30 min visual task followed by 25 min of rest, the four EEG relative power indices showed no significant difference between the baselines. Hsu and Wang  reportedVIMS severity level increased with increasing visual task time, as shown in Apart from the SSQ for VIMS severity level measurement, Liu et al.  used EEGThe results of this study showed that after completing a 15 min visual task followed by a 20 min rest and a 30 min visual task followed by a 25 min rest, the participants’ CFF values, four EEG relative power index values, and SSQ scores showed no significant difference from baseline. Thus, this study suggests a 20 min rest for visual fatigue and VIMS recovery after a 15 min visual task on an OHMD and a 25 min rest for visual fatigue and VIMS recovery after a 30 min visual task. Yoshimura and Tomoda  recommenWhen using an OHMD, the visual fatigue level and severe VIMS symptoms may increase the risk of injury at the workplace or during daily activities. Hence, this study provides a work-rest schedule for OHMD use: a 15 min visual task and 20 min rest, and 30 min visual task and 30 min rest, to alleviate the users’ visual fatigue and VIMS severity levels, and to enhance the safety and health in the workplace. The current study proposed a work-rest schedule of a 15 min visual task period followed by a 20 min rest break and a 30 min visual task period followed by a 25 min rest period, as the participants’ severity of visual fatigue and VIMS did not significantly differ from baseline levels. However, a visual task time exceeding 30 min and how many rest breaks are required to prevent and alleviate visual fatigue and VIMS still need to be explored.Considering the impact of the movie’s content on participants’ emotional experience, they avoided emotions such as boredom and sleepiness that could affect the experimental results. This study allowed all participants to choose movie content that interested them and all participants chose the drama movie. Depending on the movie’s content, there may be different sound and lighting effects, which may affect the severity of visual fatigue and VIMS; therefore, it is worth further exploring the impact of different movie content on visual fatigue and VIMS in the future.The current study did not examine the gender effect on visual fatigue and VIMS while using the OHMD. Larese Filon et al.  examinedIn this study, an OHMD with a fixed head position was used as the experimental equipment. The results may be different when the AR tasks or the presenting modes of the OHMD (such as the world-fixed mode) are changed. Furthermore, the experiments in this study were conducted in a sitting position. It is reasonable to postulate that other visual tasks and use conditions, for example, watching an operation flow video and performing a task simultaneously, may generate different results. Additionally, the current study did not consider the interaction between the context of use and personal factors, such as the nature of the visual task, using the OHMD, and environmental and personal factors , which might also influence the results and warrant further exploration.This study aimed to determine work-rest schedules for visual tasks of different lengths by evaluating visual fatigue and VIMS while using an OHMD. The results indicated that after completing the 15 and 30 min visual tasks, the participants experienced visual fatigue and VIMS. For the 15 min visual task followed by a 20 min rest and the 30 min visual task followed by a 25 min rest, the participants’ CFF values, four EEG relative power index values, and SSQ scores were not significantly different from baseline values. Therefore, this study suggests a 20 min rest for visual fatigue and VIMS recovery after a 15 min visual task on an OHMD and a 25 min rest for visual fatigue and VIMS recovery after a 30 min visual task. This study provides a work-rest schedule for OHMD use that can be used as a reference for OHMD user guides to reduce visual fatigue and VIMS and to enhance the safety and health in the workplace.


Electrospray ionization and positive ion multiple reaction monitoring were used for detection. The time for of analysis was 13 min. The calibration curves range of tacrolimus, rapamycin, everolimus and pimecrolimus were in the range of 1–100 ng·mL−1, mycophenolate mofetil in the range of 0.1–10 ng·mL−1 and mycophenolic acid at 10–1000 ng·mL−1. All correlation coefficients were >0.993. The coefficients of variation  for inter-day and intra-day precision were less than 10%, while the spiked recoveries were in the range of 92.1% to 116%. Our method was rapid, sensitive, specific, and reproducible for the simultaneous determination of six immunosuppressants in human whole blood. Importantly, our approach can be used to monitor drug concentrations in the blood to facilitate disease treatment.A high-performance liquid chromatography-tandem mass spectrometry method was established for the simultaneous determination of mycophenolic acid, mycophenolate mofetil, tacrolimus, rapamycin, everolimus and pimecrolimus in human whole blood by optimizing the QuEChERS  preparation method. Whole blood was extracted into ethyl acetate, salted out with anhydrous magnesium sulfate, and purified with ethylenediamine-N-propyl silane adsorbent. The supernatant was evaporated under nitrogen until dry and finally reconstituted in methanol. Chromatographic separation was performed on an Agilent Poroshell 120 EC-C18 column in methanol (mobile phase A)-water  at a 0.3 mL·min Allorejection is a significant issue during allotransplantation procedures. Many immunosuppressive drugs can be used to suppress a recipient’s immune response by inhibiting abnormal immune responses in the body . Based oSeveral analytical methods have been developed , includiIn this study, a quick, easy, cheap, rugged, effective and safe (QuEChERS) method was used for sample preparation. Generated by Anastassiades and Lehotay , this meHere, we combined the HPLC-MS/MS technique with the modified QuEChERS method for the extraction and purification of immunosuppressants from whole blood samples. Further, we developed a quantitative method to analyze the concentrations of MPA, MMF, FK-506, RAPA, EVER and pimecrolimus (PIM) in human whole blood. The method yielded a good limit of detection (LOD), LOQ, linear range, precision, accuracy, and matrix effects (ME). Thus, the approach provides a reference method for the detection of immunosuppressant concentrations and provides guidance for the clinical use of drugs.18 column). There was no obvious interference near the selective ion chromatogram. The ME of MMF and FK-506 was <80% . RAPA and EVER showed significant matrix inhibition effects in medium and high concentration QC samples, with a substantial ME of approximately 50%, so the ME was not ignorable (2 > 0.993) for all analytes ; (2) Agilent Eclipse Plus C18 ; (3) Agilent Poroshell 120 EC-C18 , and (4) Agilent ZORBOX SB-C18 . Columns were used to compare the concentrations of target analytes for HPLC-MS/MS analysis  Phenomenex Luna Omega 3 µm PS Canalysis .18 100 Å column showed poor separation performance, low response values, and poor peak shapes -MeOH, 0.1% acetic acid water (plus 10 mM ammonium acetate)-MeOH, 0.1% formic acid water (plus 10 mM ammonium acetate)-MeOH, 0.2% formic acid water (plus 10 mM ammonium acetate)-MeOH, and 0.5% formic acid water (plus 10 mM ammonium acetate)-MeOH. The optimal mobile phase solution was 0.1% acetic acid water (plus 10 mM ammonium acetate)-MeOH.The efficiency of commonly used extraction solvents, such as ACN, MeOH, acetone, and EA was assessed by comparing the peak areas of target analytes in samples. For analysis, 1.5 mL organic extraction solvent (described earlier) was added to target analytes at similar concentrations. The precipitation of organic solvents is to reduce the dielectric constant of water, leading to dehydration, mutual aggregation, and biomolecule precipitation within the surface water layer. Due to the complex matrix in whole blood and associated influencing factors, EA displayed a more effective extraction efficiency, with good peak shape and less interference a. To imp4 as the water removal agent by comparing the detection peak area of the target in the sample. Similar concentrations of the target analyte were treated with 300 mg, 350 mg, 400 mg, 450 mg, and 500 mg of MgSO4 , Florisil, amino-bonded silica gel (NH2), graphitized carbon (GCB) were purchased from Agela Technologies ; anhydrous magnesium sulfate (MgSO4) was purchased from Tianjin Damao Chemical Reagent Factory .Standards: MMF, RAPA, EVER and PIM were purchased from Toronto Research Chemicals ; MPA and FK-506 were purchased from the National Institute for Food and Drug Control . Chromatographically pure acetonitrile (ACN), methanol (MeOH), formic acid and acetic acid were purchased from Dima Technology ; chromatographically pure acetone and ethyl acetate (EA) were purchased from Safran Technology ; octadecyl-bonded silica gel (C18), −1 for each drug was stored in a −20 °C refrigerator. The standard curve working solution and the quality control working solution were diluted proportionally with MeOH to mix the 6 drugs. The concentrations of standard curve solution of FK-506, RAPA, EVER and PIM ranged from 1 ng·mL−1 to 100 ng·mL−1 ; MPA ranged from 10 ng·mL−1 to 1000 ng·mL−1; MMF ranged from 0.1 to 10 ng·mL−1 .The refined weighing MPA, MMF, FK-506, RAPA, EVER and PIM standard drugs were dissolved in MeOH. A stock solution with a mass concentration of 500 μg·mL−1, respectively. Similarly, the three MPA concentrations were 20, 500, and 800 ng·mL−1, respectively, and the three MMF levels were 0.2, 5, and 8 ng·mL−1, respectively.Low-, medium-, and high-concentration quality control (QC) samples were prepared by adding different volumes  of mixed standard solutions to 500 μL of blank whole blood samples. Low, medium, and high FK-506, RAPA, EVER and PIM concentrations were 2, 50, and 80 ng·mL4 and 50 mg PSA adsorbent for purification. The tube was vortexed for 60 s and centrifuged at 12,000 rpm for 10 min. The supernatant was removed, dried under nitrogen, and re-dissolved in MeOH.Peripheral venous blood from volunteers was collected into disposable anticoagulation blood collection tubes. The whole blood samples were pretreated with the optimized QuEChERS method and stored at −20 °C. For analysis, blood was thawed at room temperature and vortexed. Then, 500 μL blood and 2.0 mL EA were added to a centrifuge tube and vortexed for 30 s, followed by the addition of 350 mg MgSO−1, Drying gas temperature: 300 °C, Capillary voltage: 4000 V. The analytes, the monitored ions, the retention time, collision energy and fragmentation voltage are shown in Electrospray ionization (ESI), Positive ion mode, Multiple reaction monitoring (MRM), Drying gas (Drying Gas) flow rate: 11 L·min18 column  from Agilent ; mobile phase A was water containing 0.1% acetic acid and 10 mM ammonium acetate, and mobile phase B was MeOH. The gradient elution procedure was as follows: 0~1.0 min, 5% B; 1.0~1.5 min, 5~45% B; 1.5~3.0 min, 45~90% B; 3.0~10.0 min, 90% B; 10~10.1 min, 90~5% B; 10.1~13 min, 5% B; the flow rate was 0.3 mL·min−1, the column temperature was 40 °C, and the injection volume was 5 μL.The chromatographic column was an Agilent Poroshell 120 EC-C−1, RAPA and EVER at 500 ng·mL−1, MMF at 10 ng·mL−1 and MPA at 1000 ng·mL−1). Both samples were processed and analyzed by HPLC-MS/MS.The following parameters were established: linearity, selectivity, precision, recovery and ME. The selectivity was evaluated by analyzing different samples. Two blank whole blood samples were taken, one of which was spiked with the control mixture solution , and the vertical coordinate indicated the peak area of the substance to be measured (Y). The LOD was calculated at three times the signal-to-noise ratio (S/N), and LOQs were calculated at ten times the S/N.Blank whole blood samples were added to the working solution of the control mixture and processed to generate a standard mixture of whole blood at the following final concentrations: FK-506, RAPA, EVER and PIM at 1, 5, 20, 50, 75, and 100 ng·mLBlank whole blood samples were used to prepare QC low, medium, and high concentration samples, and processed. Three parallel samples were tested for each QC concentration and measurements continued for 3 days. Method precision and recovery were determined by comparing the added mass concentration with the measured mass concentration of samples.An appropriate volume of blank human whole blood was processed to generate a blank whole blood matrix solution (the reconstitution solution was methanol). To this, a working solution of the control mixture of the six drugs was separately added to generate three mass concentration levels: low, medium, and high. The measured peak area (A) of the test substance was compared with the peak area (B) of the test substance obtained by direct injection of the corresponding mass concentration of the standard solution, and the ME (A/B × 100%) of this method was calculated. An ME between 80% and 120% indicated that substrate enhancement/inhibition was acceptable and negligible.We simultaneously detected six immunosuppressive drugs in human whole blood using LC-MS/MS and a modified QuEChERS method. Compared with the conventional QuEChERS method, our method optimized and improved extractant conditions, salting and purifying agents. The validated method can be used for cost-effective immunosuppressant detection in whole blood with good precision, accuracy, and within-range linearity. The method is therefore ideal for the accurate detection of drug concentrations in patients with autoimmune disease, diseases caused by hypersensitivity reactions, and those undergoing transplantation.


Angiogenic tissue engineering is a vital problem waiting to be settled for periodontal regeneration. Erythropoietin, a multieffect cytokine, has been reported as a protective factor for cell fate. According to our previous study, erythropoietin has a significantly angiogenic effect on periodontal ligament stem cells. To further explore its potential effects and mechanism, we studied biological behaviors of periodontal ligament stem cells under inflammatory microenvironment induced by different concentrations  of tumor necrosis factor-in vitro. RNA sequencing was used to detect the downstream signaling pathway. Quantitative real-time polymerase chain reaction was conducted to examine mRNA expression level. Western blot and immunofluorescence were applied to testify the protein expression level.  Cell Counting Kit-8 was used for cell proliferation assay. Annexin V-PI-FITC was used for cell apoptosis through flow cytometry. Matrigel plug was adopted to measure the angiogenic capacity  Periodontal ligament stem cells upregulated apoptosis and suppressed autophagy and angiogenesis under inflammatory microenvironment. Erythropoietin could activate autophagy to rescue apoptosis and angiogenesis levels of periodontal ligament stem cells through the Akt/Erk1/2/BAD signaling pathway under inflammatory microenvironment.  Erythropoietin could protect periodontal ligament stem cells from inflammatory microenvironment, which provided a novel theory for periodontal regeneration. Periodontitis, a commonly infectious oral disease, is deemed as the sixth disease of the globe, which would risk psychological and physical health . Periodoβ-catenin and p38/MAPK signaling pathway to enhance osteogenesis of PDLSCs [β, iNOS, and CD68 expression [Tissue reconstruction also relies on the cytokines. Erythropoietin (EPO), a glycoprotein secreted by the kidney, is reported as a promising candidate cytokine for periodontal tissue engineering. EPO plays a great effect on osteogenic and angiogenic differentiation of mesenchymal stem cells (MSCs) , 12. It f PDLSCs , 17. EPOpression , 19. Colvia upregulating autophagy [Cumulative studies have proved that autophagy is closely related to cell differentiation and apoptosis. Autophagy is considered as a helpful process for cell viability, which could enable cells to adapt to the change and pressure of circumstance . Jiang eutophagy . Researcutophagy . It is eutophagy .The Akt signaling pathway always participates in autophagy, apoptosis, and differentiation. Akt-related autophagy maintains the stemness of mouse embryonic palatal mesenchymal stem cells, which could suppress cleft palate development . It has According to the existing literatures and our previous study, we aimed to investigate the antiapoptosis and angiogenesis of EPO under inflammatory microenvironment. We also explored the involvement of autophagy and Akt signaling pathway in this process, wishing to find a novel approach for periodontal repair.α-MEM , 10% fetal bovine serum (FBS) , and 1% penicillin-streptomycin solution , was added into the flask to obtain periodontal ligament stem cells. The cells usually crawled out about 2 weeks later. The culturing incubator  was set as 37°C and 5% CO2.With fully informed consent, we collected 50 healthy orthodontic teeth from adolescent patients (12-20 years old) under the approval of the Biomedical Ethics Committee of the Affiliated Stomatology Hospital of Southwest Medical University (Lot No. 2020112600). After collecting teeth, the periodontal ligaments were scraped from the tooth root and incubated reversely for 4 hours in a culture flask. The medium, mixture of After digesting and washing, the fourth-generation (p4) PDLSCs were used to detect specific surface markers under flow cytometry instrument . Mesenchymal stem cell surface antibodies   and hematopoietic stem cell surface antibodies   were selected to examine.β-glycerate , 0.1 μmol/L dexamethasone , and 50 mg/L vitamin C . Osteogenic induction solution was changed every 3 days and maintained for 28 days. Then, cells were fixed by paraformaldehyde and stained by alizarin red solution . The lipogenic induction solution was formulated with D-MEM, 10% FBS, 10 μmol/L dexamethasone , 200 μmol/L indomethacin , 0.5 mmol/L 3-isobutyl-1-methylxanthine (IBMX) , and 10 mg/L insulin . Cultured for 28 days, the cells were fixed and stained by oil red O solution . Calcium nodules and lipid droplets were observed under a fluorescent inverted microscope .Osteogenesis and lipogenesis induction were used to detect the multidirectional differentiation ability of PDLSCs. The osteogenic induction solution formulation consisted of D-MEM , 10% FBS, 10 mmol/L sodium μL of CCK8 solution and 90 μL α-MEM were added to each well, incubated for one hour and then detected at 450 nm absorbance in an enzyme microplate reader .Cell Counting Kit-8 (CCK8)  was used for cell proliferation assay. The p4 PDLSC was inoculated in 96-well plates at a density of 2000 cells per well, and the cells were divided into different treated groups. Detection was performed on days 1, 3, 5, and 7 after inoculation, respectively. Overall, 10 μL binding buffer. Then, PDLSCs were stained with 2 μL Annexin V and 1 μL propidium iodide (PI)  and incubated for 30 min.Annexin V-FITC-PI double-staining assay was performed under flow cytometry to detect cell apoptosis level. Cells were collected after incubating for 1 day and diffused in 400 μL amplification system using the SYBR FAST qPCR Master Mix Kit  and then performed amplification in Bio-Rad/CFX96 fluorescence quantitative PCR instrument . The specific conditions of denaturation, annealing, and extension were as follows: 95°C for 3 min, 95°C for 5 sec, 56°C for 10 sec, and 72°C for 25 sec in 40 cycles. The forward and reverse primer  sequences used in the experiment are shown in β-actin, and the changes in gene expression were calculated by the 2-△△CT method.Total RNA was extracted according to the instructions of the Total RNA Extraction Kit , and then, RNA was reverse transcribed to cDNA using the Takara Reverse Transcription Kit  for subsequent experiment. We prepared a 20 Total protein was extracted under the guidance of the kit  instructions. The protein concentration of each group was determined using the BCA protein concentration assay kit . SDS-PAGE gels were configured, and the proteins were electrophoresed vertically and transferred to PVDF membranes . The membranes were blocked for 2 hours in blocking buffer  at room temperature. Then, membranes were incubated with primary antibody at 4°C overnight. Washed in TBST solution for 3 times, membranes were incubated with anti-rabbit IgG, HRP-linked antibody  at room temperature for 1 hour. ECL developer  was added and photographed in a chemiluminescence imaging system . The primary antibodies are listed in μL to the precooled 96-well plate and placed in the incubator for 30 minutes. Pretreated PDLSCs were added to the wells at a density of 2000 cells per well. Tube formation in each well was observed after 6 hours and photographed under fluorescent inverted microscope.Melted matrix gel  was evenly added in the volume of 50 α treatment) and treated groups (TNF-α and EPO treatment) was examined through mRNA sequencing to discover downstream regulatory pathways of EPO. Each group had 3 biological replicates. RNA extraction, specific RNA library preparation, RNA sequencing, and bioinformatics analysis were done by OE biotech Co., Ltd. . The sequence raw data has been submitted to NCBI Sequence Read Archive (Accession number PRJNA824457). The sequence results have been validated by qPCR.The transcriptome expression of PDLSCs in control groups (TNF-μL blocking buffer (0.2% Triton-100 and 5% donkey serum) was added onto each sheet. Then, the cells were incubated with the primary antibody (VEGF-a or LC3B) at 4°C overnight. After rewarming to room temperature, the cells were dealt with the secondary antibody, DyLight680  for 1 hour. DAPI and phalloidin  were used to stain cell nucleus and cytoskeleton. Images of cells were observed and captured under Olympus SpinSR confocal laser microscope .PDLSCs were treated under different conditions on the cell climbing sheets for 1 day. Fixed by paraformaldehyde, 1000 α (treated for 3 days): 0, 10, 20, 50, and 100 ng/mLConcentration gradient of TNF-α) (treated for 3 days): 0, 5, 10, 20, and 50 IU/mLConcentration gradient of EPO (under inflammatory microenvironment induced by 20 ng/mL TNF-α (50 ng/mL), ② TNF-α (50 ng/mL)+LY294002 (10 μM)+EPO (20 IU/mL), and ③ TNF-α (50 ng/mL)+EPO (20 IU/mL)To explore the roles of the Akt/ERK1/2/BAD signaling pathway (treated for 1 day): ① TNF-α (50 ng/mL), ② TNF-α (50 ng/mL)+3-methyladenine (3-MA) (5 mM)+EPO (20 IU/mL), and ③ TNF-α (50 ng/mL)+EPO (20 IU/mL)To explore the roles of autophagy (treated for 1 day): ① TNF-All experiments were all performed under p4 PDLSCs. Experiment groups of this research were mainly divided into 4 parts:t-test was used to compare among two groups. It was considered as statistically significant when P < 0.05.Statistical calculation was completed at GraphPad Prism 9.0 software . Results were presented in the form of mean ± standard deviation (SD). Each experiment has been performed at least three times. One-way ANOVA was used to determine multiple-group comparisons. And Students' β, IL-8) significantly upregulated with the ascent of TNF-α concentration  indicated that TNF- Figures . Additio Figures .β and IL-8 downregulated under the treatment of EPO (Cell viability suggested that EPO could rescue the proliferation of inflammatory PDLSCs on days 3, 5, and 7, especially in 20 IU/mL groups . The mRNt of EPO .α.Under EPO treatment, qPCR and western blot revealed that the Bax/Bcl2 ratio was downregulated, indicating a declining trend of cell apoptosis Figures . It was According to the mRNA/protein expression trend, EPO improved the expression of VEGF-a, IGF-1, and FGF-2 in a concentration-dependent manner Figures .α was also promoted by EPO, based on the results of the mRNA/protein expression trend of Beclin1 and LC3B (Figures Autophagy depressed by TNF- Figures .α+EPO group (α+EPO group (RNA sequencing was conducted to explore the signaling pathway aroused by EPO, recommending significant upregulation of the Akt signaling pathway . To testPO group . And celPO group . Images PO group .α+EPO group (α+EPO group (As an autophagy inhibitor, 3-MA was added to investigate how EPO regulated cell autophagy on apoptosis and angiogenesis. Both qPCR and western blot inferred that 3-MA could downregulate cell autophagy, further changing antiapoptosis and angiogenesis induced by EPO Figures . MatrigePO group . And celPO group . ResultsPO group .α is excessively expressed in the process of periodontitis, which degrades periodontal tissue and fastens cell apoptosis [α is the core inflammatory cytokine during periodontitis, which is suitable for establishment of inflammatory microenvironment [Periodontitis is always triggered by dental bacterial plaque and accelerated by local or wholesome factors. Regarding the pathological process, it involves the invasion of bacteria, activation of immune reaction, recession of junctional epithelium, and depredation of alveolar bone . To defepoptosis , 37. TNFironment –40. Biolironment –44.α to mock the microenvironment of periodontitis, and we established a TNF-α concentration gradient to explore biological behaviors of inflammatory PDLSCs. Coherent with existed literatures [α inhibited cell viability and increased expression of inflammatory genes via the NF-κB signaling pathway. Fang et al. [α could also induce apoptosis and oxidative stress of PDLSCs, which was analogous to our study. In our research, TNF-α agitated the expression of IL-1β and IL-8, suppressed cell proliferation, and enforced Bax/Bcl2 expression ratio, especially in 50 ng/mL and 100 ng/mL groups. And other researchers also used 20 ng/mL or 50 ng/mL TNF-α to mimic the inflammatory microenvironment, which supported results. As the same, those also reckoned that proliferation rate was evidently suppressed and inflammatory cytokines expressed most on 72 hours [Here, we selected TNF-eratures , 45, 46,g et al.  and Mengg et al.  mentione72 hours , 49.α would decrease or increase cellular autophagy. Some researchers pointed that TNF-α contributed to autophagy to protect PDLSCs from apoptosis at an early stage, while attenuating autophagy in a long run [α often downregulated LC3B, Beclin1, and Atg7, resulting the osteogenic decline of PDLSCs. According to the results, the expression level of Beclin1 and LC3B was further diminished with the increasing concentration of TNF-α, which aggravated apoptosis. As the same, the secretions of VEGF-a, FGF-2, and IGF-1 were declined with the increasing concentration of TNF-α, which denoted the decreasing angiogenic level. Taken together, autophagy impaired by TNF-α was considered as a protective factor for PDLSCs in the inflammatory microenvironment. Collectively, 50 ng/mL TNF-α was picked for the continuing experiment.Autophagy is reckoned as a double-edged sword for biological behaviors. In some views, autophagy played a harmful role in the pathogenesis, which indicated that autophagy was positively relevant to inflammatory level and apoptosis , 51, whilong run . Chen etlong run  held tha+/CD90+/CD44+ stem cells—such as PDLSCs, dental pulp stem cells, and bone marrow progenitor cells—which demonstrated that the EPO/EPOR signaling pathway played a crucial role in regulating biological behaviors of these cells. And PDLSCs were also characterized as CD105+, CD90+, and CD44+ mesenchymal stem cells. The expression of EPOR on CD105+/CD90+/CD44+ PDLSCs provided basics for EPO treatment. Therefore, we conducted experiments focusing on the protective effects of EPO for PDLSCs. In the inflammatory microenvironment, proliferation of PDLSCs was enhanced with the treatment of EPO, especially in the 20 IU/mL group. IL-1β and IL-8 were obviously resisted after the treatment of EPO. Likewise, a similar experiment showed that the inflammation level of PDLSCs could be attenuated by ascorbic acid, revealing that inflammation of PDLSCs was significantly related with the NF-κB signaling pathway and DNMT1, which could activate expression of proinflammatory cytokines [EPO has been learned as a multifunctional cytokine/drug for wound healing and bone regeneration, particularly in the realm of periodontology –56. An aytokines . The expα or PI3K/Akt specific inhibitor—LY294002, denoting that the Akt/Erk1/2/BAD signaling pathway was activated through phosphorylation. p-Erk1/2 and p-BAD changed following the change of phosphorylated Akt. VEGF-a, FGF-2, and IGF-1 levels may be regulated by p-Erk1/2 [The Akt signaling pathway always participated in the regulation of apoptosis, autophagy, and angiogenesis , 29. Aktp-Erk1/2 ; Bax/Bclp-Erk1/2 ; Beclin1p-Erk1/2 . Additiop-Erk1/2 . LY29400α+EPO group; Bax/Bcl2 ratio increased compared to the TNF-α+EPO group. Noticeably, 3-MA reduced tube numbers enhanced by EPO and augmented cell apoptosis rescued by EPO. Comprehensively, autophagy lifted by EPO could also moderate apoptosis and angiogenesis of PDLSCs under inflammatory microenvironment.Autophagy also dually moderates apoptosis and angiogenesis , 25. In in vivo experiments would be conducted to testify its in vivo effects. Due to the limitations of fundamental experiment, clinical performance was not certain, requiring further research.However, there still exists drawback, such as lack of animal experiments. A step further, Above all, EPO attenuated inflammation, reduced apoptosis, rescued autophagy, and augmented angiogenesis of PDLSCs under inflammatory microenvironment. And its potential mechanism was also conducted. EPO activated autophagy to moderate apoptosis and angiogenesis via the Akt/Erk1/2/BAD signaling pathway (Supplementary Figure It could be demonstrated that EPO could protect biological behaviors of PDLSCs from inflammatory microenvironment and promote angiogenic tissue regeneration, which brought a brand-new sight for periodontal tissue engineering.


The coronavirus disease (COVID-19) pandemic has already affected more than 400 million people, with increasing numbers of survivors. These data indicate that a myriad of people may be affected by pulmonary sequelae of the infection. The aim of this study was to evaluate pulmonary sequelae in patients with bilateral COVID-19 pneumonia according to severity 1 year after hospital discharge.COVID-FIBROTIC is a multicenter prospective observational cohort study for admitted patients with bilateral COVID-19 pneumonia. Pulmonary functional outcomes and chest computed tomography sequelae were analyzed 12 months after hospital discharge and we classified patients into three groups according to severity. A post hoc analysis model was designed to establish how functional test changed between groups and over time. A multivariable logistic regression model was created to study prognostic factors for lung diffusion impairment and radiological fibrotic-like changes at 12 months.Among 488 hospitalized patients with COVID-19 pneumonia, 284 patients had completed the entire evaluation at 12 months. Median age was 60.5 ± 11.9 and 55.3% were men. We found between-group differences in male sex, length of hospital stay, radiological involvement and inflammatory laboratory parameters. The functional evaluation of pulmonary sequelae showed that severe patients had statistically worse levels of lung diffusion at 2 months but no between group differences were found in subsequent controls. At 12-month follow up, however, we found impaired lung diffusion in 39.8% unrelated to severity. Radiological fibrotic-like changes at 12 months were reported in 22.7% of patients (102/448), only associated with radiological involvement at admission  and LDH .Our data suggest that a significant percentage of individuals would develop pulmonary sequelae after COVID 19 pneumonia, regardless of severity of the acute process.Trial registration clinicaltrials.gov NCT04409275 The online version contains supplementary material available at 10.1186/s12931-022-02166-8. The COVID-19 pandemic has affected more than 400 million people to date , underliIn the acute phase of COVID-19, mechanical ventilation patient autopsies showed more pronounced hyperplastic and metaplastic changes of pneumocytes and interstitial fibrosis than those receiving conventional oxygen . Early hApproximately a third of patients with severe pneumonia presented with fibrotic changes, defined as presence of parenchymal bands, traction bronchiectasis and/or honeycombing within 6 months of discharge. In these patients, need for non-IMV was identified as a risk factor , and 25%A recently published study analyzed functional changes over 1 year in severe COVID-19 pneumonia survivors, excluding mechanically ventilated patients. They report pulmonary function improvement at follow-up, but 33% of patients showed diffusing capacity of the lungs for carbon monoxide (DLCO) abnormalities, while fibrotic changes on HRCT were described in less than 5% .We hypothesized that patients with COVID-19 pneumonia may develop pulmonary sequelae regardless of treatment or not with mechanical ventilation. The aim of our study was to investigate persistent fibrotic-like lesions and changes in lung function in a cohort of patients with bilateral COVID-19 pneumonia 1 year after hospital discharge.2) < 94% in room air at sea level, arterial partial pressure of oxygen to fraction of inspired oxygen (PaO2/FiO2) ratio < 300 mmHg, respiratory frequency > 30 breaths/min, or lung infiltrates > 50% , and spirometry . DLCO was determined by the single breath technique using an infrared analyzer, correcting for hemoglobin values. All procedures were performed according to American Thoracic Society (ATS) and European Respiratory Society (ERS) guidelines –21.The 6-min walk test (6MWT) measures the distance that a patient can walk quickly on a flat, hard surface in a period of 6 min. It evaluates the global and integrated responses of all systems involved during exercise, including pulmonary and cardiovascular systems, systemic circulation, peripheral circulation, blood, neuromuscular units and muscle metabolism , 23.Control chest X-rays (CXRs) were performed in all patients 2 months after discharge using standardized techniques with computed radiography equipment. Pulmonary damage was quantified using the RALE score. HRCT scans  were obtained with subjects in the supine position during breath hold at end-inspiration. Axial reconstructions were performed with a slice thickness of 1 mm, with 1 mm increment, 512 mm × 512 mm (resolution 0.625 mm/10). The same protocol was used in each center, adjusted to the different CT machines. HRCT images were evaluated for presence of ground-glass opacities (GGO), consolidations, bronchiectasis, parenchymal bands and reticulations as defined by the Fleischner society glossary of terms . FibrotiDuring the screening visit , demographic data  and medical history  were collected. Data associated with the acute episode were also collected . All data were retrieved from electronic medical records and de-identified data were entered into an electronic database (Veridata™ EDC). Patients were assessed 2 months after discharge  by collecting residual dyspnea using the modified British Medical Research Council (mMRC) , CXR andGroup 1: hospitalized mild disease : hospitalized patients who required supplemental oxygen by mask or nasal prongs.Group 2: hospitalized severe disease : hospitalized patients who required non-invasive ventilation (non-IMV) or high flow nasal oxygen cannula (HFNC).Group 3: hospitalized critical  disease : hospitalized patients who required respiratory support by intubation and invasive mechanical ventilation (IMV).For further analysis, patients were stratified according to WHO Ordinal Outcomes Scale  into thrWe followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE)  guidelinQualitative variables were described using frequencies and percentages, and quantitative variables by means and standard deviation. Normality for continuous variables was checked using the Shapiro-Wilks test and if their normal distribution was not confirmed, variables were expressed with median and interquartile range.Mean comparison was carried out using the Student t-test in the presence of normality, and if otherwise, using the Mann–Whitney U test. For qualitative variables, comparison of percentages between groups was carried out using Fisher's exact test for dichotomous variables or chi-square test for contingency tables with more than two categories. Patient pulmonary function at follow-up was assessed with mixed linear models for quantitative outcomes, with individual identification key as random effect and time and/or severity status and interaction as independent factors. Cochran-Q test was used in the case of dichotomous outcomes. Factors associated with diffusion impairment and fibrotic pattern at 12 months were studied using multivariable logistic regression. The variables for the multivariable analysis were selected using Akaike’s Information Criterion in a backward-forward stepwise procedure.Effect of time and severity in DLCO, 6MM and FVC was evaluated using linear mixed models with time  and severity  as fixed effects and individuals as random effect; interaction between severity and time was also included. Compound symmetry was used for correlation between time measures of same individual. Similarly dyspnea was dichotomized into 2 or more versus less than 2, model proposed was a generalized linear mixed model with time, severity and interaction between both fixed effects and individuals as random effect. Post-hoc analysis was carried out in the interaction term with p value adjustment according to Bonferroni method for 36 tests.Between May 1 and July 31, 2020, 932 patients were attended for COVID-19 pneumonia in participating hospitals, 481 of whom were initially considered for follow-up. At the end of the study, we retrieved full data in 377, 312 and 284 patients at 2, 6 and 12 months, respectively  years, and 55.3% (157/284) of them were men, which significantly increased as did severity: 50.2% (105/209) vs. 60.9% (14/23) vs. 73.1% (38/52) [p = 0.010]. We also found between-group differences in length of hospital stay [p < 0.001], RALE scores [p < 0.001] and laboratory parameters  [all p < 0.001]. In contrast, there were no significant differences between groups in age, comorbidities, smoking, or body mass index (BMI)  of patients had diffusion impairment (< 80% of predicted DLCO) at 60 days, with an improving trend at 180 and 365 days  [p < 0.001]. The mean (% of predicted) DLCO in V1, V2 and V3 was 78.5 (19.1), 81.6 (16.4) and 84 (16.4), respectively [p < 0.001]  across the whole cohort was present in 14.3% [54/377], 9.3% 29/312] and 6.7% [19/284] of patients at 2, 6 and 12 months, respectively [p = 0.001] and mean FVC (% of predicted) was 99 (17.9), 100.8 16.5) and 104.2 [.5 and 109/312 andWe next analyzed dyspnea across the entire cohort. Dyspnea ≥ 2 at mMRC scale was 21.5% [59/275], 11.3% [31/275] and 9.8% [27/275] at 2, 6 and 12 months  , female sex , BMI  and ferritin levels  . In the remaining 200 patients, the most frequent radiological pattern was GGO, reported in 73.5% [147/200] (32% when we consider the study cohort [147/448]), with between-group differences  and LDH   1 year after acute COVID 19 infection. Furthermore, we identified radiological fibrotic-like changes on chest CT in almost 23% of our study cohort [n = 448]. However, these sequelae were not associated with other markers of severity previously described in ARDS cases , such asAlong with an increasing number of people affected over time, survival has improved since the start of the pandemic , but witAlthough functional recovery from severe COVID-19 pneumonia occurs within 1 year after discharge, approximately one third of patients (39.8%) still have decreased DLCO. These findings are in line with the results from a Chinese cohort , albeit A recent systematic review showed that histopathological findings of diffuse alveolar damage caused by COVID-19 are indistinguishable from those provoked by other causes. At final, chronic/fibrotic phase was identified, showing a honeycomb lung with collagen fibrosis of the alveolar spaces and an interstitium with thickening of the alveolar wall together with squamous metaplasia of the alveoli . These cThe ARDS repair process involves rapid fibroblast proliferation and this leads to extracellular matrix deposition , which iIn our study cohort, any radiological abnormality at 12 months persisted in 27% [123/448] of patients. This percentage is similar to that obtained by Pan et al. (53/209)  and someWe define fibrotic-like changes as the presence of traction bronchiectasis, parenchymal bands and/or reticular pattern , 26, 39.Mechanical ventilation is a recognized factor in fibrosis development , caused Whether these changes involve scarring from the acute process or whether they might progress over time is still unclear . AlthougThis study has several limitations. Firstly, data on functional tests or CT scans prior to admission were not available for assessment of longitudinal changes. Secondly, following the study protocol, chest CT was performed initially only in patients with radiological or functional changes at 2 months, and at 12 months when initial CT was altered. This approach aimed to minimize radiological exposure and overload in radiology services during the pandemic, and follows guidelines recommended by several societies , 45. In Approximately one third of patients who survived a severe COVID-19 pneumonia had impaired lung function and dyspnea 12 months after hospital discharge, and 23% developed fibrotic-like sequelae, in both severe and mild patients. These findings confirm the need for follow-up of patients with severe SARS-CoV-2-induced pneumonia to clarify whether fibrotic changes may progress over time.Additional file 1: Figure S1. Interaction plot severity and time in 6MWT based on linear mixed model post-hoc analysis. V1 (2 months), V2 (6 months) and V3 (12 months). Group 1: mild; group 2: moderate; group 3: severe. No between-group differences were found at any time. 6MWT = 6 min walk test.Additional file 2: Table S1. Chest CT at 2-month follow-up according to severity group. Table S2. Table for reviewer. Characteristics of enrolled patients vs. lost patients.


Pregnant women may use EOs in case of morning sickness, nausea, stress management, etc. Little is known about the potential danger that EOs represent for the placenta and therefore for the pregnancy. Our aim was to explore and compare the placental toxicity and potential endocrine disrupting effects of niaouli, orange, tea tree, wintergreen and ylang-ylang EOs, and their key compounds: 4-terpineol, 1,8-cineol, limonene, methyl salicylate and benzyl salicylate. We studied the release of four hormones and the activation of P2X7 receptor in JEG-Tox human placental cells as key biomarkers for endocrine toxicity. We observed that niaouli, orange, tea tree, wintergreen and ylang-ylang EOs and their key components disrupted at least one of the studied hormones but none of them activated the P2X7 cell death receptor. The tested EOs appear then to be more hormonal modulators rather than EDCs in human placental cells. The hormonal effects observed with the key components were very different from those observed with the EOs. EOs are very complex mixtures, and it is essential to study whole EOs rather than their components individually in safety assessment. Currently, there is a trend in developed countries towards a more natural lifestyle with less attraction to medical treatments and an increasing use of natural products such as essential oils (EOs). Empirical data say that EOs have many beneficial properties for health, for example niaouli and tea tree EOs possess antiseptic, antiviral and antifungal properties; orange and ylang-ylang EOs decrease stress and anxiety; and wintergreen EO reduces inflammation. EOs are also used in a multitude of daily life products including food flavorings, soaps, lotions, shampoos, hair styling products, cologne, laundry detergents and even insect repellents . Routes Pregnancy is a time when various complications can occur. However, it is also a time when many drugs are contraindicated. That is why many pregnant women prefer to use herbs, herbal preparations and EOs to treat pregnancy-related symptoms and minor disorders. EOs are then used in different situations: in diffusion devices to purify the atmosphere of the house, decrease anxiety and stress, and improve the quality of sleep; in inhalers to treat respiratory infections such as cold, sinusitis and bronchitis; and in skin preparations to reduce neuralgia, digestive disorders, pain and to prevent or treat stretch marks or even to prepare childbirth. They may also use EOs in commercially available or homemade household products for cleaning to avoid chemicals contained in traditional cleansing products that they perceive as hazardous for their pregnancy.Many people, including pregnant women, seem to consider EOs as safe alternatives because of their natural origin, but natural does not mean safe. Women often use aromatherapy without medical advice during pregnancy  and randSome studies have reported cases of prepubertal gynecomastia and premature thelarche in children after the use of lavender and tea tree EOs ,13, suggThe hPlacentox assay, selected by the public–private PEPPER platform for the pre-validation of methods for endocrine disruptors characterization, is based on the human placental JEG-Tox cell model and allows the study of not only hormones disruption (both steroids and polypeptides), but also adverse health effects in the same cells , to meetThe aim of the present study was to evaluate the placental toxicity of some of the most used EOs in the world , and their potential endocrine-disrupting effects. To achieve our goal, we first studied the release of human hyperglycosylated chorionic gonadotropin (h-hCG), human placental lactogen (hPL), estradiol and progesterone, and the activation of P2X7 receptor in human placental JEG-Tox cells after incubation with the whole EOs. To better understand EOs effects, we then studied the same biomarkers after incubation of JEG-Tox cells with EOs’ key components: 1,8-cineol (main component of niaouli EO), limonene (main component of orange EO), 4-terpineol (main component of tea tree EO), benzyl salicylate (suspected endocrine-disrupting component of ylang-ylang EO) and methyl salicylate (main component of wintergreen EO).The analysis of EOs revealed that there was 56.34% of 1,8-cineol in niaouli EO, 95.18% of limonene in orange EO, 36.98% of 4-terpineol in tea tree EO, 94.56% of methyl salicylate in wintergreen EO and 2.16% of benzyl salicylate in ylang-ylang EO . To bett−3%, 0.17 × 10−2% and 0.17 × 10−1%, according to the literature [Before studying hormones release and P2X7 receptor activation, we investigated JEG-Tox cell viability after incubation with the EOs. Any concentration inducing a loss of cell viability of at least 30% was considered as cytotoxic  and rejeterature ,14.−3%, 0.17 × 10−2% and 0.17 × 10−1% corresponded to 0.94 × 10−4%, 0.94 × 10−3% and 0.94 × 10−2% of 1,8-cineol, respectively, since the tested batch contained 56.34% of 1,8-cineol (−2% led to an elevated progesterone secretion (×2.21 compared to the negative control) and an elevated h-hCG secretion at 0.17 × 10−1% (×1.57 compared to the negative control), unlike 1,8-cineol (−2% than the control (×1.21) and this increase became significant at 0.17 × 10−1%, (×1.49), while 1,8-cineol led to a lower hPL secretion than the control at 0.94 × 10−3% and 0.94 × 10−2% (×0.89 and ×0.76 at −4% and 0.94 × 10−2% (×0.51), while niaouli EO had no effect on estradiol , while limonene induced a lower secretion than the control at 0.16 × 10−3% and 0.16 × 10−2%  contrary to limonene  and estradiol at 0.6 × 10−2% (×1.71), while tea tree EO had no effect on progesterone and estradiol (−1%. (×1.38) but 4-terpineol did not.Tea tree EO at 0.17 × 10erpineol . Tea trestradiol b,c. ConvThe rises of hormonal secretion induced by tea tree EO and 4-terpineol were not concentration-dependent.−3%, 0.17 × 10−2% and 0.17 × 10−1% corresponded to 0.16 × 10−3%, 0.16 × 10−2% and 0.16 × 10−1% of methyl salicylate, respectively, since the tested batch contained 94.56% of methyl salicylate (−1% (×1.33 compared to the control) contrary to methyl salicylate that reduced progesterone secretion at 0.16 × 10−1% (×0.69). EO also stimulated h-hCG secretion at 0.17 × 10−2% (×1.47) and this stimulation was significant at 0.17 × 10−1% (×1.85), while methyl salicylate had no effect on h-hCG , but not methyl salicylate, which had no effect on hpL secretion  but benzyl salicylate did not (−2% (×1.33 compared to the control). At the same concentration, benzyl salicylate had no effect but it significantly enhanced hPL secretion at 0.36 × 10−3%  while ylang-ylang EO had no effect on this hormone  appear in contradiction with previous in vitro studies that demonstrated estrogenic effects of tea tree EO in MCF-7 human breast cells ,14. The To better understand the effects of the tested EOs on hormone release, we tested their key compounds in the same conditions. 1,8-cineole, limonene, 4-terpineol, methyl salicylate and benzyl salicylate did not disrupt hormone release in the same way as their corresponding EOs. 1,8-cineol, which represents about 55% of niaouli EO, induced lower secretions of estradiol and hPL than control cells whereas niaouli EO raised them. 4-terpineol, composing about 37% of tea tree EO, had progestational and estrogenic effects that the tea tree EO did not have, but it did not raise hPL, unlike tea tree EO. Benzyl salicylate, approximately present at 2% in ylang-ylang EO, induced higher hPL and h-hCG secretions than control cells whereas ylang-ylang EO only altered hPL secretion. Limonene and methyl salicylate constitute about 95% of orange and wintergreen EO, respectively. Limonene induced an antiestrogenic effect, contrary to orange EO that induced an estrogenic effect; methyl salicylate induced an antiprogestational effect, opposite to wintergreen EO, which induced a progestational effect. Our results showed that none of the five compounds activated P2X7 receptor, like the tested EOs, leading to the same conclusion: these compounds seem to be hormonal modulators rather than endocrine disruptors. It is interesting to note that the most abundant compounds  did not have the same effects as the whole oil. This means that the other compounds, even very minor, also participate in the hormonal effects of EOs. Two hypotheses can be raised. First, the additive or synergistic effects, also called the cocktail effects, of all the components are a parameter to be considered when evaluating EOs, particularly in the field of EDCs. Second, the minor compounds could be responsible for the hormonal effects of EOs. Indeed, unlike other toxic substances, EDCs do not necessarily exert their effects in a dose-dependent manner.In 2019, benzyl salicylate was added to the list of the 14 substances to be screened as potential endocrine disruptors by the European Scientific Committee for Consumer Safety (SCCS) and was included in the Community rolling action plan (CoRAP) of the European REACH regulation to be assessed for endocrine disruption. Indeed, in vitro and in vivo published studies have showed that benzyl salicylate alters estrogens. The yeast estrogen screen (recombinant yeast expressing estrogen receptor α) and the E-screen assay (proliferation of MCF-7 cells), in vitro assays showed estrogenic, antiestrogenic or even no effects of benzyl salicylate ,54,55,56Our study highlights the difficulty and complexity of predicting the endocrine disrupting potential of EOs, based only on the safety data related to one of their components, even a major one. Each EO contains many different chemicals that vary in structure and composition, some EOs contain over 150 substances. It is therefore essential to study the entire EO rather than its individual components in order to conclude on a potential effect of EOs. Furthermore, according to our results, tea tree, niaouli, orange, wintergreen and ylang-ylang EOs appear to be more hormonal modulators than endocrine disruptors in placenta, as they did not activate P2X7 receptor. To confirm this statement, it would be necessary to perform further in vitro experiments to study other adverse health effects at the cell level. Mitochondrial alterations, already identified as key elements in understanding placental disorders induced by EDCs , could b® was obtained from ThermoFisher Scientific , and Alamar Blue probes from Alfa aesar . Hyperglycosylated hCG and hPL ELISA kit were purchased from MyBioSource  and estradiol and progesterone from Cisbio . The niaouli EO  viridiflora, which is a synonym for M. quinquenervi), orange EO (Citrus sinensis), tea tree EO , wintergreen EO , the ylang-ylang EO (Cananga odorata) were obtained from Laboratoires Léa nature . EOs components, benzyl salicylate , methyl salicylate , 4-terpineol , limonene  and 1,8-cineol  were purchased from Sigma-Aldrich . Positive controls were used to ensure that the cell model responded correctly in our experimental conditions and were obtained from Sigma-Aldrich.Chemicals and reagents. Minimum essential medium (MEM), fetal bovine serum (FBS), 2 mM glutamine, 10,000 U/mL penicillin and 10,000 µg/mL streptomycin, trypsin-EDTA 0.05% and phosphate buffer saline (PBS) were provided by Gibco  and cell culture plastics such as flasks and microplates by Corning . YO-PRO-1EOs’ composition analysis: the composition of all tested EOs was analyzed by GC–MS analysis according to NF ISO 11024 standard with the SHIMADZU GC-FID 2010 PLUS/MS QP 2010SE, column RxiR 5 SILM 60 m, 0.25 mm ID and 0.25 µM df.m/z).The following analytical conditions were used for both apparatus and columns: the oven temperature was programmed at 50 °C, held for 5 min, then increased to 240 °C at a rate of 3 °C/min; the injector temperature was set at 240 °C; the carrier gas was helium with a flow rate of 1 mL/min; a splitting ratio of 1:50 was applied; the injection volume was set to 1 μL. For the GC–MS mass spectrometry interface, the MS source temperature was set at 220 °C with an ionization energy of 70 eV and an interface temperature of 240 °C. A full scan was recorded (50–700 The identification of constituents was achieved by comparing mass spectra with the Mass Spectra Library (NIST 98) compounds and by comparison of the retention indices (RI) calculated from the injection of a C8–C20 hydrocarbons alkanes mixture with retention indices from the literature on both columns. Some selected pure standards were also used.2 polystyrene flasks. Cell cultures were maintained in a cell culture incubator . When the JEG-3 cells reached subconfluency, they were detached using trypsin-EDTA and counted. The cellular suspension was diluted and seeded in 96-well microplates at a cellular density of 80,000 cells/mL (200 µL/well), then kept at 37 °C for 24 h. Stock solutions of niaouli, orange, tea tree, wintergreen and ylang-ylang EOs were obtained after dilution to 2/3 in absolute ethanol, and then diluted in MEM supplemented with 2.5% FBS to obtain three concentrations: 0.17 × 10−3%, 0.17 × 10−2% and 0.17 × 10−1% (v/v). Cells were incubated for 72 h with the different concentrations of EOs, according to the literature [Human placental cell culture: The JEG-3 human trophoblast cell line was obtained from the American Type Culture Collection (ATCC HTB-36). Cells were cultured in minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine, 0.5% penicillin and streptomycin, in 75 cmterature ,14, in Mterature . EOs’ coex = 535 nm and λem = 600 nm) with a Tecan Spark® microplate reader . Triton® X-100 was used as a positive control for cytotoxicity in the Alamar Blue assay.Cell viability—Alamar Blue assay: The Alamar Blue stock solution (0.1 mg/mL) was prepared in PBS buffer and stored at 4 °C away from light. The working solution used for the test was obtained by diluting the stock solution at 1/11 in culture medium supplemented with 2.5% FBS. After removing the supernatants and rinsing the cells with PBS, the Alamar Blue working solution was distributed to the wells. Then, the microplate was placed in the incubator for 6 h and read (λ® microplate reader was used for both techniques. Substances of very high concern (SVHC) due to their endocrine-disrupting properties were used as positive controls: bisphenol A (BPA) for estradiol release, 4-tert-amylphenol (AP) for progesterone release and diethylstilbestrol for h-hCG and hPL releases.Placental and sexual hormones quantification: After centrifugation of the 96-well microplates, the cell supernatants were collected and the hormones were quantified: human hyperglycosylated chorionic gonadotropin (h-hCG), human placental lactogen (hPL) by ELISA according to the supplier’s instructions (MyBioSource), and estradiol and progesterone by FRET according to the supplier’s instructions (Cisbio). The Spark® assay: P2X7 cell death receptor activation was evaluated using the YO-PRO-1® assay [® probe only enters into cells after pore opening induced by P2X7 receptor activation and binds to DNA, emitting fluorescence. A 1 mM YO-PRO-1 stock solution was diluted at 1/500 in PBS just before use and distributed in the wells of the microplate. After a 10 min incubation time at room temperature, the fluorescence signal was read  using a Spark® microplate reader. BPA was used as a positive control for P2X7 receptor activation in JEG-Tox cells [Cell death P2X7 receptor activation—YO-PRO-11® assay . The YO-ox cells ,20.p-values expressed with the symbol *), and to compare EOs with their components at the same concentration . The significance thresholds were * p < 0.1, ** p < 0.01, *** p < 0.001, # p < 0.1, ## p < 0.01 and #### p < 0.0001.Results exploitation and statistical analysis: Results are expressed in percentage or fold change compared to control cells and presented as means of at least three independent experiments ± standard errors of the mean. A statistical analysis was performed using GraphPad Prism 8 software . A one-way analysis of variance followed by a Dunnett’s test with the risk α set at 5% was performed to compare the EOs’ and components’ incubation with a negative control (Different points have been addressed in this paper. First, we demonstrated that niaouli, orange, tea tree, wintergreen and ylang-ylang EOs appear to be hormonal modulators rather than endocrine disruptors in human placental cells. Second, the key components of EOs did not have the same hormonal effects as whole EOs, proving the complexity of EOs and the need to fully characterize EOs in terms of components and pesticides before launching any safety assessment. In order to conclude on the potential toxic effects of EOs on the placenta, it is thus essential to study whole EOs rather than their components individually.


Fridericia platyphylla, displays a remarkable antitumoral effect against in vitro mPCa cells cultured as bidimensional (2D) monolayers. Considering that three-dimensional (3D) cell cultures provide a more accurate response to chemotherapeutic agents, this study investigated the antiproliferative/antimetastatic effects of BrA and the molecular mechanisms underlying its action in mPCa spheroids (DU145) in vitro. BrA at 60–100 μM was cytotoxic, altered spheroid morphology/volume, and suppressed cell migration and tumor invasiveness. High-content analysis revealed that BrA (60–100 µM) reduced mitochondrial membrane potential and increased apoptosis and necrosis markers, indicating that it triggered cell death mechanisms. Molecular analysis showed that (i) 24-h treatment with BrA (80–100 µM) increased the protein levels of DNA disruption markers (cleaved-PARP and p-γ-H2AX) as well as decreased the protein levels of anti/pro-apoptotic  and cell survival markers (p-AKT1 and p-44/42 MAPK); (ii) 72-h treatment with BrA increased the protein levels of effector caspases  and inflammation markers (NF-kB and TNF-α). Altogether, our results suggest that PARP-mediated cell death (parthanatos) is a potential mechanism of action. In conclusion, BrA confirms its potential as a candidate drug for preclinical studies against mPCa.Metastatic prostate cancer (mPCa) is resistant to several chemotherapeutic agents. Brachydin A (BrA), a glycosylated flavonoid extracted from  Prostate cancer (PCa) is the second most frequent cancer type in men and the fifth leading cause of death worldwide ,2. In 20Fridericia platyphylla (Cham.) L.G. Lohmann (synonym Arrabidaea brachypoda) is a plant native to Brazil that produces unique dimeric glycosylated flavonoids [F. platyphylla, Brachydin A  have promoted therapeutic benefits for cancer treatment, with an important role in mPCa chemotherapy ,7,8. Friavonoids . Brachydavonoids  with antavonoids . The firn A BrA; , exerts Three-dimensional (3D) culture models are multicellular tumor spheroids with a structural and histomorphology organization, cell–cell/cell–matrix interactions, and authentic pathophysiological conditions similar to in vivo models ,16. TheiThe details regarding the collection, extraction, isolation, and identification of Brachydin A (BrA) were previously reported . The Bra® (American Type Culture Collection—Cat. Nº HTB-81TM) and maintained in RPMI 1640 medium  with 10% Fetal Bovine Serum (Gibco), 1% antibiotic/antimycotic mix  and 0.024% NaHCO3 . DU145 cells were kept in a 3110 Series II CO2 water-jacketed incubator  at 37 °C with a 5% CO2 atmosphere and 96% relative humidity. All culture procedures followed the recommendations proposed by Bal-Price and Coecke [The prostate tumor DU145 cell line, isolated from a metastatic brain site, was purchased from ATCCd Coecke . All exp3) formation .DU145 tumor spheroids were obtained using the agarose coated-overlay method, according to Friedrich et al. . Brieflyg for 10 min for adhesion to the well bottom. Then, the spheroids were washed carefully with PBS (pH 7.4) and suspended in acid phosphatase buffer . After incubating the plates for 90 min at 37 ºC, 1 mol/L NaOH was added to the wells, and the absorbance values were recorded in a microplate reader  at λ = 405 nm. The cell viability percentage (%) was obtained by comparing the absorbances of each treatment with the NC, which was considered as 100%. All analyses were performed with six spheroids/replicates (n = 6) and three biological experiments (n = 3).The acid phosphatase assay was performed according to Friedrich et al.  to quant®; Sigma-Aldrich) was performed according to Riss et al. [n = 6) and three biological experiments (n = 3). The resazurin assay  in three biological experiments (n = 3).The volume, morphology, and integrity of DU145 spheroids were analyzed based on the methods reported by Friedrich et al.  and Vincg. After removing the supernatant, the cells were suspended in TrypLE™ Enzyme 1X (Gibco), incubated at 37 °C for 5 min, and manually shaken every 1 min. The cells were diluted with culture medium and centrifuged for 5 min at 300× g. The supernatant was discarded, the resulting pellet was suspended, and 250 cells were seeded in 6-well plates and incubated for seven days. The DU145 colonies were washed with PBS and fixed with methanol: acetic acid: water  for 30 min and stained for 25 min with 5% Giemsa diluted in phosphate buffer . The cells were washed with distilled water, and the colonies were counted using a manual hematological counter. The plating efficiency and the survival fraction were calculated according to Franken et al. [n = 2) in three biological experiments (n = 3).The clonogenicity assay with disaggregated cells from DU145 spheroids was performed according to Mikhail et al. . The DU1n et al. . All ana®  based on the protocol proposed by Vinci et al. [® solution (10 mg/mL) was diluted in incomplete RPMI medium to the working concentration (200 μg/mL), pipetted into a 96-well plate, and incubated at room temperature for 3 h to fix the ECM to the well bottom. Subsequently, the remaining non-adhered ECM was carefully removed, and the wells were washed twice with PBS at room temperature and filled with 1% bovine serum albumin  blocking solution diluted in PBS. After the initiation step, DU145 spheroids suspended in culture medium were transferred to the plates pre-coated with ECM gel for 30 min, and treated with RPMI 1640 (NC), 1% DMSO (SC), 50 µM DTX (PC), or BrA  for a further 30 min. The DU145 spheroid images corresponding to time 0 (t0) at 24 h and 48 h were acquired. For analyzing cell migration, the circumference of the migrated cells around the spheroid was measured in each image using the AxioVision SE64 Rel. 4.9.1 software (Carl Zeiss) with the “measure” tool. The spheroid growth was analyzed by measuring its area comprised of the spheroid and cell migration presented in μm3. All analysis were performed with six spheroids/replicates (n = 6) in three biological experiments (n = 3).The 3D cell migration analysis with DU145 spheroids was performed with ECM Geli et al. . The com2 incubator at 37 °C for 30 min for BSG solidification. An image for each tumor spheroid was recorded at 0, 24, 48, and 72 h intervals using the Zeiss Primo Vert microscope on the 4× objective and the Zen2 software (Zeiss). After capturing the images, the area covered by the spheroids was measured using the “measure” tool in μm3. The spheroid area (invaded area × spheroid area) at each time point was calculated concerning the area at t = 0, and the data were converted into percentage (% t0). All analyses were performed with four spheroids/replicates (n = 4) in three biological experiments (n = 3). The 3D cell invasion assay was performed according to Vinci et al.  and Bereg, the supernatant discarded, and TrypLE enzyme (1X) was added to each tube. The tubes were incubated in a 5% CO2 incubator at 37 °C for 10 min and manually shaken every 2 min. The cells were diluted with complete culture medium and centrifuged for 5 min at 300× g. The cell pellet was suspended in annexin-binding buffer (1X), transferred to 5 mL flow cytometry tubes , and incubated with annexin V-FITC for 15 min in an ice bath. Next, PI (2 μg/mL) was added to the tubes and the samples were immediately analyzed in the BD LSR Fortessa® Flow Cytometer  under an excitation λ = 488 nm and emission λ = 530/575 nm laser . Viable cells were not stained with annexin-V or PI; early apoptotic cells were labeled only with annexin-V; late apoptotic cells were labeled with both annexin-V and PI; necrotic cells were marked only with PI. All analyses were performed with 10,000 events from eight spheroids/replicates (n = 8) in three biological experiments (n = 3).Flow cytometry analysis was performed using the Dead Cell Apoptosis Kit with annexin V-FITC and propidium iodide (PI)  after 72 h of treatment with BrA (40–100 µM), RPMI 1640 (NC), 1% DMSO (SC) or 50 µM DTX (PC). Then, the culture medium (80 μL) was removed, and the remaining medium with the spheroids was transferred to a 2.0 mL microtube. One sample was composed of eight spheroids to obtain a sufficient cell number. Thus, the spheroids were centrifuged for 5 min at 300× ® Chimney Well µClear®; Greiner Bio-One; Monroe, NC, USA) filled with complete medium and maintained for 96 h in a cell culture incubator. After initiation, the spheroids were treated with RPMI 1640 (NC), 1% DMSO (SC), 50 µM DTX (PC), or BrA (40–100 µM). Along with the treatments, CellEvent Caspase 3/7 reagent (2.5 μM in Hank’s solution) and Hoechst 33342 (10 μM in PBS) were added to all the wells. The samples were monitored for 24, 48, and 72 h using the HCS platform. The acquisition parameters were “brightfield” (Channel 1), FITC , and DAPI . The “autofocus” was set to “brightfield”, and the plates were scanned using the acquisition mode “one field of view per well” with the 4× objective. The acquired images were analyzed using the In-Cell Investigator v.1.3 software . Apoptotic cells were quantified using the “average fluorescence intensity” recorded in the FITC-channel. Representative images from DU145 spheroids were acquired by merging Hoechst (Blue/DAPI) and Caspase 3/7 (Green/FITC). All analyses were performed with four spheroids/replicates (n = 4) in three biological experiments (n = 3).All apoptosis analyses were performed using the HCS platform In-Cell Analyzer 2200  with the CellEvent Caspase 3/7 Green reagent , following the manufacturer’s instructions. The DU145 spheroids were started in 96-well black plates with U-shaped transparent bottoms (CellStarn = 4) in three biological experiments (n = 3).Necrotic cells were analyzed using the HCS platform In-Cell Analyzer 2200  with PI (Thermo Fischer Scientific) dye, following the manufacturer’s instructions. The experimental design and treatments were performed as described for the apoptosis assay reported above. Along with the treatments, PI (10 μg/mL in PBS) and Hoechst 33342 (10 μM in PBS) were added to each well, and the samples were analyzed at 24, 48, and 72 h of treatment. The acquisition parameters were “brightfield” (Channel 1), Cy3 , and DAPI . The “autofocus” was set to “brightfield,” and the plates were scanned with acquisition mode “one field of view per well” in the 4× objective. The acquired images were analyzed using the In-Cell Investigator v1.3 software . Necrotic cells were quantified using the “average fluorescence intensity” recorded on the Cy3 channel. Representative images from DU145 spheroids were acquired by merging Hoechst (Blue/DAPI) and PI (Orange/Cy3). All analyses were performed with four spheroids/replicates (n = 4) and three biological experiments (n = 3).All images for the mitochondrial membrane potential (Δψm) analysis were acquired using the HCS platform In-Cell Analyzer 2200  with the MitoStatus Red™ dye , following the manufacturer’s recommendations. The experimental design and treatments were performed as described for the apoptosis assay reported above. Along with treatments, MitoStatus Red (200 nM in DMSO) and Hoechst 33342 (10 μM in PBS) were added to each well and analyzed at 0.5, 4, 24, 48, and 72 h. The acquisition parameters were “brightfield” (Channel 1), Cy5 , and DAPI . The “autofocus” was set to “brightfield,” and the plates were scanned with the acquisition mode “one field of view per well” with 4× objective. The acquired images were analyzed using the In-Cell Investigator v1.3 software . The Δψm potential was quantified using the “average fluorescence intensity” recorded in the Cy5 channel. Representative images of DU145 spheroids were acquired by merging Hoechst (Blue/DAPI) and MitoStatus (Pink/Cy3). All analyses were performed with four spheroids/replicates (n = 4) in three biological experiments (n = 3).All images for mitochondrial redox status analysis were acquired on the HCS platform In-Cell Analyzer 2200  using the MitoSOX™ Red Mitochondrial Superoxide Indicator reagent , following the manufacturer’s instructions. The experimental design and treatments were performed as described for the apoptosis assay reported above. Along with the treatments, MitoSOX™ Red (5 μM in DMSO) and Hoechst 33342 (10 μM in PBS) were added to each well and analyzed at 24, 48, and 72 h. The acquisition parameters were “brightfield” (Channel 1), TexasRed , and DAPI . The “autofocus” was performed in “brightfield,” and the scanned plates with acquisition mode “one field of view per well” with the 4× objective. The images were analyzed using the In-Cell Investigator v1.3 software . Mitochondrial superoxide activity was determined based on the “average fluorescence intensity” recorded on the TexasRed channel. Representative DU145 spheroids images were acquired by merging Hoechst (Blue/DAPI) and MitoSox (Red/TexasRed). All analyses were performed with four spheroids/replicates (NIH.gov accessed on 6 March 2021). All analysis were performed with ninety-six spheroids/replicates (n = 96) in two biological experiments (n = 2).Protein expression was analyzed by western blotting after 24 and 72 h of treatment with BrA (80–100 µM) or 1% DMSO (SC). After 24 h of exposure, whole-cell lysates from DU145 spheroids were obtained with Lysis Buffer  and Protease Inhibitors Cocktail . Next, proteins (20 µg) were separated by standard 10–15% SDS-PAGE gel electrophoresis, transferred to a nitrocellulose membrane, and incubated overnight with primary antibodies against BCL-2, BAD, RIP3K, PARP, p-γ-H2AX, p-AKT, p-ERK1/2 (dilution 1:1000 µL), or α-tubulin (dilution 1:2000 µL), which was used as the loading control. All antibodies were acquired from Cell Signaling Technology . The membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000 µL) and revealed using ECL Western Blotting Detection Reagents . The enhanced chemiluminescence was detected using the ImageQuant LAS 4000 mini documentation system . After 72 h of exposure, whole-cell lysates from DU145 spheroids were obtained with RIPA Lysis and extraction buffer containing Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific). Then, protein samples (10 µg) were treated with Bolt 4X LDS Sample Buffer and 10X Bolt™ Sample Reducing Agent (Thermo Fisher Scientific) and denatured at 70 °C for 10 min. The proteins were separated using the NuPAGE™ 4–12% Bis-Tris Gel and MiniGel Tank system (Thermo Fisher Scientific) and further transferred to PVDF and nitrocellulose (iBlot™ Transfer Stacks) membranes using the iBlot2 Gel Transfer Device (Thermo Fisher Scientific). Immunodetection was performed using primary antibodies (1:500 µL) against NF-kB/p65, CASP8, CASP7, CASP3, BAX, TNF-α , or β-Actin , which was used as the loading control. After this, the membranes were incubated with goat anti-rabbit IgG H&L conjugated with horseradish peroxidase  and revealed using the SuperSignal™ West Pico Substrate (Thermo Fisher Scientific). The chemiluminescence signals were detected using the L-Pix Chemi  system coupled with L-Pix Image software (Loccus Biotechnology). The densitometric values were calculated with the aid of the ImageJ software . The results were initially submitted to data distribution analysis  using the Shapiro–Wilk test. All samples showed parametric distribution, and the data were analyzed using analysis of variance (ANOVA) followed by Dunnett’s post-test, considering The cytotoxic effects of BrA flavonoid in DU145 spheroids were evaluated using the acid phosphatase and resazurin assays. In the former assay, BrA decreased cell viability at exposure times equal to or greater than 48 h and at concentrations ≥ 60 µM . Next, wAfter evaluating the cytotoxic effects of BrA, we analyzed whether it altered the volume, morphology, and integrity of DU145 spheroids . BrA at Besides its antiproliferative potential, BrA flavonoid may prevent tumor cells to generate metastases that are commonly resistant to chemotherapy. The cell migration  and tumoAfter demonstrating the cytotoxicity, phenotype alterations, and antimetastatic properties of BrA flavonoid in DU145 spheroids, we examined how BrA induced cell death using annexin-V and PI by flow cytometry . After 7We performed HCS analysis in DU145 spheroids  to furthConsidering that BrA flavonoid induces mitochondrial membrane depolarization, apoptosis, and necrosis, which affected cell viability, cell migration, and tumor invasion in DU145 spheroids, we examined which molecular mechanisms underlie these effects. We selected the higher BrA concentrations (80 and 100 µM) to analyze several markers of cell death  and cell proliferation pathways . After 2Metastatic prostate cancer (mPCa) has been treated with low molecular weight chemotherapeutics drugs that selectively induce cell death . TaxanesF. platyphylla (Cham.) L.G. Lohmann (synonym Arrabidaea brachypoda), popularly known as “cipó-una”, produces unique dimeric glycosylated flavonoids named brachydins, with antimicrobial, anti-inflammatory, and antiparasitic activities [In Brazil, the native plant tivities ,10,36. Btivities . Similartivities . TherefoNevertheless, mPCa cells cultured in 2D monolayers have no structural/functional properties and may not reproduce in vivo functions  . On the BrA flavonoid affected the volume/morphology of DU145 spheroids after 72 h exposure. The analysis of tumor spheroids is counted among the most relevant tools for evaluating the effectiveness of chemotherapy drug candidates . BesidesCurrently, many candidate molecules have antiproliferative potential, but tumor cells are able to escape the primary tumor and generate metastases that are commonly resistant to chemotherapy. Therefore, the search for new chemotherapeutic agents is based on finding molecules that can delay metastases formation . The celBesides migration and cell invasion, another biological factor that affects chemoresistance in mPCa is the cell death resistance promoted by the high expression of anti-apoptotic proteins . Nowaday2•−) is predominantly produced during oxidative phosphorylation [2•− can oxidize in living cell mitochondria [The HCS analysis performed with MitoStatus Red™, a cationic fluorescent dye that accumulates in the mitochondria of viable cells but not in depolarized mitochondria, revealed that BrA (40–100 µM) decreased the Δψm of DU145 cells in tumor spheroids after 30 min (0.5 h) of treatment. The depolarization of Δψm is one of the first events in the apoptotic cascade; after triggering it, cytochrome c (Cyt C) is released into the cytoplasm, with the formation of Apoptotic peptidase activating factor 1 (Apaf-1), and subsequent caspase-9 activation . The oxirylation . Neverthchondria ) demonstchondria .In the apoptosis cascade, after Δψm depolarization, Cyt c release, and Apaf-1 formation, the activation of “apical” caspases 2, 8, 9, and 10 directly cleaves “effector” caspases; caspases-3 and 7 are the main proteolytic enzymes of intracellular substrates in the final steps of cell death . The CelBesides phenotypic results obtained with the HCS platform in DU145 spheroids, molecular analysis is essential to describe mechanisms of action. BrA downregulates the expression of phospho-AKT and up-regulates cleaved-PARP protein expression in metastatic prostate PC3 cells in 2D monolayers . HoweverAltogether, the results from imaging (HCS) and molecular (western blotting) assays suggest that the parthanatos cell death pathway was the possible mechanism of action with respect to BrA in DU145 spheroids. Certainly, cleaved-PARP is a point that directs death receptor signaling for either apoptosis, necrosis, necroptosis, or parthanatos ,60. FurtParthanatos cell death is associated with (i) initial mitochondrial Δψm permeabilization (after 30 min); (ii) large-scale DNA fragmentation by cleaved-PARP and phospho-γ-H2AX overactivation  ; and  after 24 h exposure and apoptosis/necrosis mixed-effects after 72 h exposure may lead to results that indicate a close connection between parthanatos and other cell death pathways, i.e., the co-targeting of these distinct death pathways, together with cleaved-PARP up-regulation . In our It is essential to highlight that the findings from the present study were obtained with DU145 cells, which can form well-differentiated tumor spheroids in vitro that can point to an even more invasive potential ,67. HoweHere we demonstrated that the phytochemical BrA has cytotoxic, antiproliferative, anti-migratory, and anti-invasive effects that are possibly mediated by PARP overactivation (parthanatos) cell death in DU145 tumor spheroids in vitro. Thus, BrA has promising potential for preclinical studies of mPCa treatment.