Tourette syndrome (TS) is a neurodevelopmental disorder characterized by motor and phonic tics.To assess the safety and efficacy of deutetrabenazine , a vesicular monoamine transporter 2 inhibitor, in children and adolescents with TS.Alternatives for Reducing Tics in TS (ARTISTS) open‐label extension (OLE) (NCT03567291) was a 54‐week, global, phase 3, open‐label extension study of deutetrabenazine (6–48 mg daily) conducted May 28, 2018 to April 3, 2020 with a 2‐week randomized withdrawal period. Participants (6–16 years of age) had TS and active tics causing distress or impairment. Safety (primary outcome) was assessed by treatment‐emergent adverse events (TEAEs) and clinical laboratory testing. Efficacy was measured by the Yale Global Tic Severity Scale‐Total Tic Score (YGTSS‐TTS).P = 0.78). Several exploratory measures showed sustained improvement throughout the treatment periods.The intent‐to‐treat population  had a median (range) duration of exposure of 28.4 (0.3–52.9) weeks. Of 227 participants in the safety analysis, 161 (70.9%) reported ≥1 TEAE , of which 95 (41.9%) were treatment related. The most frequently reported TEAEs were headaches, somnolence, nasopharyngitis, weight increases, and anxiety. No additional safety signals were observed. Worsening of YGTSS‐TTS after the 2‐week randomized withdrawal was not statistically significant (least squares mean difference, −0.4; In this long‐term, open‐label trial, deutetrabenazine was well tolerated with low frequency of TEAEs. There was no significant difference in tics between treatment arms during the 2‐week randomized withdrawal period, however, descriptive statistics and comparison with baseline showed a numeric improvement in tics, quality of life, and other measures. Tourette syndrome (TS) is a childhood‐onset neurodevelopmental disorder characterized by motor and phonic tics. Behavioral and emotional disorders, such as attention deficit hyperactivity disorder (ADHD) and obsessive‐compulsive disorder, frequently co‐occur with tics and together increase the likelihood of academic challenges, stigmatization, and poor self‐perception.Comprehensive behavioral intervention for tics or pharmacotherapy are usually the first treatment options for TS.P = 0.69, Cohen d = −0.07; ARTISTS 2: LSD = −0.8 [95% CI = −3.9 to 2.3], P = 0.60, Cohen d = −0.11) compared with placebo. Of note, because ARITSTS 1 and ARTISTS 2 were 12 weeks or less, these trials were not designed to investigate efficacy and safety of long‐term deutetrabenazine treatment.Deutetrabenazine  is a VMAT2 inhibitor approved by the USFDA in adults for treatment of TD and chorea associated with HD.Here, we report a long‐term study, ARTISTS OLE , designed to evaluate the safety and tolerability of long‐term deutetrabenazine therapy for tics associated with TS in children and adolescents.off medication. During the follow‐up period, assessments were performed at week 55 (1 week after treatment completion) and adverse events were collected via telephone 2 weeks after the last dose of deutetrabenazine.ARTISTS OLE was a 56‐week phase 3, open‐label, single‐arm, 3‐part extension study for participants who completed 1 of 2 parent studies (ARTISTS 1 or ARTISTS 2).Participants were eligible for ARTISTS OLE after a 1‐week washout following completion of 1 of the double‐blind, placebo‐controlled parent studies. The key inclusion criteria for the ARTISTS 1 and ARTISTS 2 parent studies were 6 to 16 years of age (inclusive), at least 20 kg body weight at baseline, diagnosis of TS per Diagnostic and Statistical Manual of Mental Disorders, 5th Edition criteria, active tics causing distress or impairment, and an YGTSS‐TTS of at least 20. Key exclusion criteria included stereotypy associated with autism spectrum disorder or diagnosis of bipolar disorder, schizophrenia, or another psychotic disorder.Participants received deutetrabenazine twice daily  for up to 54 weeks (participants on the 6‐mg dose received once‐daily dosing), with an initial 7 week titration period. During the titration period, dose adjustments could be made no more than once every 5 days and only in 6‐mg increments; dose adjustments were not allowed during the maintenance periods.Safety was assessed by evaluating treatment‐emergent adverse events (TEAEs), clinical laboratory test results, vital sign measurements, electrocardiogram (ECG) findings, physical examination findings (including body weight and height measurements), concomitant medication usage, neurological examination, Columbia‐Suicide Severity Rating Scale, and Children's Depression Inventory 2nd Edition (CDI 2). The key secondary endpoint was the change in the YGTSS‐TTS from week 28 to week 30. The YGTSS is a semi‐structured clinician rating instrument that evaluates the number, frequency, intensity, complexity, and interference of motor and phonic tics.The intent‐to‐treat (ITT) analysis set includes all participants enrolled in the study, regardless of whether they received study medication. The safety analysis set includes all participants who received at least one dose of deutetrabenazine. The responder‐randomized, withdrawal‐modified ITT population includes all participants who completed the initial 28‐week open‐label period, were randomized, received study medication during the randomized withdrawal period, had YGTSS‐TTS results at both the week‐28 and week‐30 visits, and had a ≥25% reduction in the YGTSS‐TTS from baseline in the parent study to week 28.The protocol was submitted to the appropriate Independent Ethics Committee or Institutional Review Board according to local regulations. Signed and dated informed consent from the parent/legally acceptable representative and a signed and dated assent, if necessary, was obtained for each participant.Of the 228 participants who were enrolled, all were included in the ITT population with a mean age of 12 years   participants reported at least 1 TEAE, equating to an exposure‐adjusted incidence rate (EAIR) of 2.77/patient‐year Table . TEAEs wP = 0.78). A ≥20% increase in YGTSS‐TTS during the randomized withdrawal period occurred in 17 (32.1%) participants in the deutetrabenazine group and 10 (40%) in the placebo group.During the 2‐week randomized withdrawal period (weeks 28–30), a numeric worsening was observed in both groups, as follows: at week 28 , mean (SD) YGTSS‐TTS was 14.8 (6.57) and 14.3 (7.52) for the deutetrabenazine and placebo treatment groups, respectively; at week 30, there were mean (standard error [SE]) increases (worsening) in the YGTSS‐TTS from the start of the randomized withdrawal period (week 28) of 1.5 (0.78) and 1.9 (1.44) for the deutetrabenazine and placebo treatment groups, respectively. The results were not clinically meaningful or statistically significant  YGTSS‐TTS was 27.5 9.24), and a sustained decrease (indicating improvement) in the mean was observed at weeks 8, 15, 28, 41, and 54, respectively, with a slight increase (indicating worsening) at follow‐up week 55 after a 1‐week washout from active treatment. Mean (SE) reductions in YGTSS‐TTS at each on‐treatment visit were similar, with a range of −6.2 (0.83) to −8.3 (0.97) Fig. . At week, and a sQuality of life related to tics was assessed and showed improvement that was sustained through the treatment period but worsened 1 week after treatment ended. At baseline, the mean (SD) TS‐CGI was 4.0 (0.85) and through the initial 28‐week open‐label period and the re‐titration, open‐label maintenance and follow‐up period there was a sustained decrease at weeks 8, 15, 28, 41, and 54 with a slight increase at the week 55 follow‐up. At week 8, the mean (SE) change from baseline was −0.7 (0.06), which was sustained through week 54, and at week 55 (follow‐up visit) mean TS‐CGI scores were worse than at any on‐treatment visit Fig. .Descriptive statistics of several exploratory measures showed similar patterns of sustained improvement throughout the treatment periods. Impact of tics on TS‐PGII was rated as “somewhat” to “a lot”  at baseline, and at week 8 there was a mean (SE) change of −0.5 (0.07), indicating improvement, which was sustained through week 54. At baseline, mean (SD) of the C&A‐GTS‐QoL activities of daily living subscale score was 25.0 (19.52) and there was a mean (SE) change of −4.4 (1.48) at week 28 and −2.7 (2.10) at week 54, suggesting benefit of treatment. Obsessive‐compulsive behavior was measured by CY‐BOCS; mean (SD) total score was 3.0 (6.16) and improvements were indicated by mean (SE) changes from baseline of −0.7 (0.38) at week 28 and −1.7 (0.59) at week 54. Impact of tics on TS‐PGIS score, YGTSS impairment score, YGTSS GSS, and tic‐free interval improved by weeks 2 to 4, and improvements were sustained throughout the treatment period. The visual analogue scale in the C&A‐GTS‐QoL indicated a nominal improvement by week 28, which was sustained throughout the treatment period.This long‐term, open‐label study demonstrates that use of deutetrabenazine, a VMAT2 inhibitor, in children and adolescents with TS has a low frequency of TEAEs, and suggests that in this pediatric population, deutetrabenazine does not produce any new safety signals beyond the known safety profile.This open‐label trial was terminated early, because of the parent studies, ARTISTS 1 and ARTISTS 2,In this study of children and adolescents with tics associated with TS, open‐label treatment with deutetrabenazine produced no significant difference between the active and placebo arms during the randomized withdrawal period lasting 2 weeks. It is possible, however, that the 2‐week withdrawal period was too short to observe any statistical difference or clinically meaningful increase in tics. However, descriptive statistics and comparison with baseline showed a numeric reduction in tics and improvements in quality of life and other secondary measures through the end of treatment, followed by a numeric increase (worsening) at follow‐up 1 week after treatment was withdrawn compared with the last on‐treatment visit. At follow‐up 1 week after treatment was withdrawn, no placebo‐controlled statistical analysis was performed, and the likelihood of a placebo effect is high. Therefore, worsening during the washout period may be biased.The parent studies (ARTISTS 1 and ARTISTS 2) did not meet their primary endpoints, possibly because of marked heterogeneity in the patient population and subtherapeutic dosing.Limitations of the study include the open‐label design and the early termination. As an extension study that invited participants who had already received deutetrabenazine, there may be bias toward participants with a favorable deutetrabenazine response. Higher variability in results was observed by week 54 for the majority of endpoints, which can be attributed to the smaller number of participants who completed the end of treatment visit before study termination. The randomized withdrawal period was 2 weeks, and this duration may not have been sufficient to evaluate persistence of effect. Additionally, effects from the withdrawal period may have been masked by a strong placebo effect, which has been documented for YGTSS‐TTS assessments in patients with TS.In this long‐term, open‐label extension study in pediatric patients with TS, deutetrabenazine was generally well tolerated with a low frequency of adverse events, which is consistent with the known safety profile of deutetrabenazine in studies conducted in adults. Although there was no significant difference in tics between the active and placebo arms during the 2‐week randomized withdrawal period, descriptive statistics and comparison with baseline showed a numeric reduction in tics and improvements in quality of life and other outcome measures, followed by a worsening at follow‐up 1 week after treatment was withdrawn.(1) Research project: A. Conception, B. Organization, C. Execution; (2) Statistical Analysis: A. Design, B. Execution, C. Review and Critique; (3) Manuscript Preparation: A. Writing of the First Draft, B. Review and Critique.J.J.: 1A, 1C, 2A, 2C, 3BB.C.: 1A, 1C, 2A, 2C, 3BD.O.C.: 1A, 2A, 2C, 3AJ.J.S.: 2B, 2C, 3BB.J.G.: 2C, 3BE.A.G.: 1A, 1C, 2A, 2C, 3BD.A.S.: 1A, 1C, 2A, 3A, 3BM.W.: 1A, 2A, 3A, 3BJ.M.S.: 1A, 2A, 3A, 3BE.H.: 1C, 2C, 3BJ.A.: 1A, 2A, 2C, 3A, 3BH.B.: 2B, 2C, 3A, 3BM.F.G.: 1B, 2B, 2C, 3A, 3BEthical Compliance Statement: Protocols were approved by the appropriate national and local authorities and independent ethics committees or institutional review boards at each study site before study initiation. Protocol amendments were also submitted. The study was conducted in full accordance with the International Council for Harmonization Good Clinical Practice Consolidated Guideline E6 and any applicable national and local laws and regulations. A personally signed and dated informed consent form was obtained from the parent/legally acceptable representative, and a signed and dated assent, depending on the child's age, as appropriate, was obtained from each patient (if the patient was able) before any study‐specific procedures or assessments were performed, and after the aims, methods, anticipated benefits, and potential hazards were explained. We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this work is consistent with those guidelines.Funding Sources and Conflicts of Interest: This study was funded by Teva Branded Pharmaceutical Products R&D, Inc. The funders were involved in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication. D.A.S., M.W., and J.A. were employees of the funder at the time of this research. M.F.G. is an employee of the funder. J.M.S. was an employee of Teva Pharmaceuticals International GmbH at the time of this research, and E.H. and H.B. are employees of Teva Pharmaceutical Industries Ltd.Financial Disclosures for Previous 12 Months: J.J. reported receiving grants or contracts from AbbVie, CHDI Foundation, Dystonia Coalition, Emalex Biosciences, Medtronic Neuromodulation, The Michael J. Fox Foundation for Parkinson Research, Parkinson's Foundation, Revance Therapeutics, and Teva Pharmaceutical Industries, royalties or licenses from Cambridge, Elsevier, Medlink: Neurology, Lippincott Williams and Wilkins, UpToDate, and Wiley‐Blackwell; and consulting fees from AbbVie, Eon BioPharma, Neurocrine, Revance Therapeutics, and Teva Pharmaceutical Industries. B.C. reported receiving grants, honoraria or consulting fees from the American Academy of Child and Adolescent Psychiatry; Emalex; Harvard Medical School /Psychiatry Academy; The National Institute of Mental Health; Partners Healthcare; Skyland Trail; Neurocrine; Teva/Nuvelution; and Tourette Association of America‐CDC Partnership. D.O.C. reported receiving grants or contracts from AbbVie, Acadia Pharmaceuticals, Alerity Therapeutics, Annexon, Neurocrine, Genentech‐Roche, Novartis, Prilenia, Spark Therapeutics, and uniQure; consulting fees from Annexon, Spark Therapeutics, Alterity Therapeutics, Novartis, and Teva Neuroscience; data safety monitoring board or advisory board duties from Photopharmics; and leadership or fiduciary role in MSA Coalition and Huntington Study Group. J.J.S. reported receiving consulting fees from Teva Branded Pharmaceutical Products R&D, Nuvelution, Bracket/Signant Health, St. Jude Medical, Revance Therapeutics, Amneal, Impel Pharmaceuticals, Medtronic, and AbbVie. She has received research fees from Amneal. She serves on the data safety monitoring board for BlueRock Therapeutics, and has advisory board duties for Photopharmics. B.J.G. was an employee of Nuvelution and Blackstone Life Sciences. E.A.G. reported receiving consulting fees from Nuvelution and TS Pharma Inc.


Exposure to air pollution is one of the greatest environmental risks for human health. Air pollution level is significantly driven by anthropogenic emissions and meteorological conditions. To protect people from air pollutants, China has implemented clean air actions to reduce anthropogenic emissions, which has led to rapid improvement in air quality over China. Here, we evaluated the impact of anthropogenic emissions and meteorological conditions on trends in air pollutants in a coastal city (Lianyungang) in eastern China from 2015 to 2022 based on a random forest model. The annual mean concentration of observed air pollutants, including fine particles, inhalable particles, sulfur dioxide, nitrogen dioxide, and carbon monoxide, presented significant decreasing trends during 2015–2022, with dominant contributions (55–75%) by anthropogenic emission reduction. An increasing trend in ozone was observed with an important contribution (28%) by anthropogenic emissions. The impact of meteorological conditions on air pollution showed significant seasonality. For instance, the negative impact on aerosol pollution occurred during cold months, while the positive impact was in warm months. Health-risk-based air quality decreased by approximately 40% in 8 years, for which anthropogenic emission made a major contribution (93%). Air polioxide SO, and volr PM10) . It was 0 . It watter PM2. and inhar (PM10) ,7 sourcer (PM10) ,9,10,11.3), which showed a stable but even a slight increase from 2013 to 2017 [2 showed the most significant decrease over China due to efficient emission reduction controls for the power plant sector [Relatively serious air pollution is mainly distributed in developing and populous countries, such as China. Air pollution is generally characterized by complicated mixtures along with complex chemical reactions ,13,14,15t sector ,21,22,232.5 concentrations decreased from 61.8 to 42.0 μg m−3 from 2013 to 2017 over China, with dominant contributions (approximately 91%) from anthropogenic emission abatements. Furthermore, they also found that the improvement in PM2.5 air pollution was driven by three measures, including strengthening industrial emission standards, upgrades on industrial boilers, phasing out outdated industrial capacities, and promoting clean fuels in the residential sector [2.5 concentrations that could slow down the aerosol sink of hydroperoxyl (HO2) radicals [Recently, many studies applied an air quality model to assess the contribution of anthropogenic emissions and meteorology to changes in air quality during the recent years in China ,24,25,26l sector . Howeverl sector  and a raradicals ,29. Thosradicals ,30,31,32radicals ,34, and radicals ,38,39,40radicals ,41,42.In addition to emissions, meteorological conditions also play an important role in affecting air quality, especially in the typical coastal regions. However, the impact of meteorological conditions on air quality at a typical coastal region was not well quantified. In this study, we applied a machine learning approach to evaluate the impact of anthropogenic emissions and meteorology on trends in air quality during 2015–2022 in a coastal city of eastern China . Additionally, drivers of health-risk air quality index (HAQI) and the corresponding premature mortality from 2015 to 2022 were also investigated.2.5, PM10, CO, NO2, O3, and SO2, in Lianyungang, which is a typical coastal city in eastern China, was obtained from the China National Environmental Monitoring Center network . The meteorological parameters were taken from ERA5 reanalysis data of the European Centre for Medium-Range Numerical Prediction (ECMWF), which is accessible at https://cds.climate.copernicus.eu/ (last access 10 January 2023). The meteorological data include zonal wind speed (U10), meridional wind speed (V10), temperature (T), boundary layer height (BLH), solar radiation (SR), sea level pressure (SP), total cloud coverage (TCC), total precipitation (TP), relative humidity (RH), zonal wind speed of 500 hpa (U500), meridional wind speed of 500 hpa (V500), 500 hpa vertical wind speed (W500), 850 hpa zonal wind speed (U_850), 850 hpa meridional wind speed (V850), and 850 hpa vertical wind speed (W850). The horizontal and temporal resolutions of these meteorological data were 0.25 degree and 1 h, respectively. The geographic location of Lianyungang is shown in In the present study, the hourly mass concentration of air pollutants, including PM2.5, PM10, CO, NO2, O3, and SO2, respectively. A more detailed description of such meteorological normalization approach can be found elsewhere [2.5, PM10, CO, NO2, O3, or SO2) was first established. The 70% and 30% of the input data were divided as training and testing data sets, respectively. In this processing, the RF model’s built-in importance indexes could be obtained. In addition, a fivefold cross validation was applied to evaluate the model performance. The normalized mean square error (NMSE), root mean square error (RMSE), and correlation coefficient between observation and prediction were calculated for evaluation metrics.In the present study, a meteorological normalization approach based on the RF algorithm  was appllsewhere ,36,37,42lsewhere . BrieflyTo obtain weather-normalized  prediction, the total 1000-time prediction was achieved. To do so, the model input data for daily meteorological variable features were randomly resampled from the historical weather data during 2001–2022, while time variables were resampled. This process was repeated in the corresponding 1000 predictions. For each processing, a specific model prediction result was then taken. The final deweathered concentration for the specific air pollutant was obtained by aggregating the 1000 predictions using arithmetic mean.i refers to individual month. The model configuration used in this study is consistent with previous studies [In addition, we applied a meteorological impact index method to evaluate the extent of meteorological influence on the concentrations of air pollution in different years and/or months. This method has been described in a previous study . Briefly studies . In this studies .(1)MET 2.5, PM10, NO2, SO2, CO, and O3) by using Equation (2). The RR based on the equivalent concentration of the pollutants 0 is the threshold concentration, below which the pollutant proves no obvious adverse health effects:ith criteria pollutant  . MeanwhiFinally, Equation (7) shows that the overall HAQI is determined by the maximum of all 2.5 and O3 can reflect health effects of atmospheric pollutants. A detailed calculation method can be found in Apte et al., (2015) [3 and stroke, ischemic heart disease (IHD), chronic obstructive pulmonary disease (COPD), and lung cancer (LC) linked to PM2.5.The premature mortality (M) attributable to PM, (2015) . Briefly2.5, PM10, NO2, SO2, CO, and O3). Overall, the predicted concentrations of all air pollutants were correlated well (r = 0.71–0.88) within the observed concentrations. The NMSE values of the six air pollutants were in the range of 0.04–0.16. The corresponding RMSE values were in the range of 0.22–34.43. These model validation results could prove the good performance for the RF models.2.5, PM10, NO2, SO2, and CO), the Unix time—as a long-term anthropogenic emission proxy—shows highly relative importance. This reveals the important impact of anthropogenic emission on trends in air pollutants  during 2015–2022. However, the Unix time shows a relatively low contribution to the relative importance, suggesting that the variability of atmospheric ozone in Lianyungang was not mainly driven by anthropogenic emissions.2 concentration showed the largest difference in its concentrations from 2015 to 2022 among the six air pollutants, which decreased from 25.24 to 6.87 µg m−3. The observed PM2.5 and PM10 concentrations decreased from 53.70 and 93.31 to 30.52 and 55.84 µg m−3 in 8 years, respectively. The observed NO2 showed a slight decrease during 2015–2022. The observed SO2 showed the largest decreasing trends (−2.75 µg m−3 a−1), followed by PM2.5 (−3.12 µg m−3 a−1), PM10 (−5.27 µg m−3 a−1), CO (−0.04 mg m−3 a−1), and NO2 (−1.15 µg m−3 a−1). The corresponding deweathered concentrations for these air pollutants showed similar trends, i.e., SO2 (−1.94 µg m−3 a−1), PM2.5 (−1.89 µg m−3 a−1), PM10 (−3.27 µg m−3 a−1), CO (−0.03 mg m−3 a−1), and NO2 (−0.64 µg m−3 a−1). These results reflect a large contribution (55–75%) of anthropogenic emissions to the changes in air quality trend during 2015–2022. However, the deweathered O3 presented an increasing trend during 2015–2022, highlighting the continuous aggravation of O3 air pollution in this region. This is overall consistent with some previous observation and modeling studies [ studies ,46,47.2.5 concentrations were −1.89 µg m−3 a−1, −1.54 µg m−3 a−1, and −2.15 µg m−3 a−1 during 2015–2022, 2015–2018, and 2019–2022, respectively. The deweathered NO2 trend showed a similar trend with a more rapid reduction during the latter period (−1.04 µg m−3 a−1) rather than the former period (−0.06 µg m−3 a−1). These results could be attributed to more rapid reductions in PM2.5 and NO2 from 2020 to 2022. The deweathered SO2 showed a larger reduction during 2015–2018 than that during 2019–2022, highlighting a more effective reduction in SO2 during the former period. This is also consistent with emission inventory studies [2 over eastern China. The deweathered PM10 also presented a larger annual reduction ratio (−5.35 µg m−3 a−1) during 2015–2018 than that during 2019–2022, which could be partly explained by mankind dust emission control (such as urban road dust emissions) [3 presented a comparable trend with a range of 0.21–0.37 µg m−3 a−1, which reflects the continuity of the O3 pollution trend during the different periods in this region. It should be noted that an unexpected short-term emission reduction due to the COVID-19 pandemic lockdown in the spring of 2020 in eastern China has been widely reported [−3 a−1, −5.03 and −3.24 µg m−3 a−1, −1.07 and −0.63 µg m−3 a−1, −2.71 and −1.93 µg m−3 a−1, 0.91 and −0.26 µg m−3 a−1, and −0.04 and −0.03 mg m−3 a−1 for PM2.5, PM10, NO2, SO2, O3, and CO, respectively. The difference between the original trends  ,49. Durireported ,52,53,54ends see  and the 3, the magnitudes of such values for most air pollutants were larger during 2019–2022 than that during 2015–2018, suggesting that meteorological conditions (such as atmospheric diffusion conditions and/or air temperature) during the latter period were more favorable for reducing concentrations of air pollutant than during the former period. Except for O3, the meteorological impact index of the five air pollutants presents relatively high values during warm months, while it presents relatively low values and even positive values during cold months in this coastal city region. These results could provide direct evidence to prove that meteorological conditions during cold months could promote aerosol  and gas  pollution. The meteorological conditions during warm months play an important role in reducing the concentrations of these air pollutants. The meteorological impact index of O3 shows an overall opposite variation compared with other air pollutants, which present relatively low and high values during cold months and warm months, respectively. This is in line with the fact that the most frequent O3 pollution occurs in warm seasons due to unfavorable meteorological conditions, such as strong solar radiation and high air temperature. This is consistent with the RF built-in importance results with the most important prediction feature for solar radiation and U10  to the HAQI in both 2015 and 2022. Anthropogenic emission contributed to approximately 93% of changes in value from 2015 to 2022, where PM2.5 and PM10 contributed approximately 50% to this emission driver. This suggests that the decline in HAQI value from 2015 to 2022 was driven by anthropogenic emission reduction. As reported by a previous study, approximately 0.894 million premature deaths in 2017 were estimated due to PM2.5 and O3 pollution across China [2.5 and O3 pollution in the Lianyungang region. 2.5 and O3 from 2015 to 2022, respectively. Among the five specific causes, PM2.5-IHD and PM2.5-stroke accounted for a major fraction (approximately 71–78%) of the total number of deaths, due to the fact that stroke and IHD dominate the total mortality [2.5 and O3 in 2015, which would cause a total of 11,208 deaths. The specific causes were PM2.5-IHD (3931), PM2.5-stroke (4764), PM2.5-LC (770), PM2.5-COPD (1446), and O3-respiratory (297). Overall, the observed mortality from PM2.5 pollution declined during 2015–2022. The results indicated by the present study were comparable to a previous study, which showed a significant reduction in premature deaths attributable to long-term PM2.5 exposures from 2013 to 2016 in China due to emission control [2.5-related mortality was decreased from 10,088 deaths to 6501 deaths in 8 years, while the deweathered O3-related mortality was increased from 804 deaths to 1192 deaths. The deweathered PM2.5-related mortality presented a decreasing trend (−531 deaths per year). The O3-related mortality presented an increasing trend (38 deaths per year). These results suggest that reduction in exposure risk to ambient PM2.5 was driven by anthropogenic emission control in this region. This is consistent with the results obtained from chemical transport modeling, which showed that air pollution control avoided 0.39 million deaths caused by PM2.5 pollution in China from 2012 to 2017 [3 pollution was increased. The difference of the total deweathered deaths from 2015 to 2022 was 3199, suggesting that China’s clean air actions might avoid air-pollution-induced deaths in the Lianyungang region. The relative change in observed and deweathered values reflected the magnitude of the meteorological impact (see Equation (1)). The highest meteorology-driven impact could lead to 2050 deaths, which occurred in 2021. This suggests that changes in meteorological conditions could significantly affect air pollution deaths in this coastal region. Although we have estimated cause-specific premature mortality in this study, a further comparison between estimation and observation in the real world would be more useful to understand the uncertainty of our estimates and more helpful to further understand the health risk of air pollution in a future study.ction 51–% to the ortality . The hig control . The dew to 2017 . However2.5, PM10, NO2, SO2, and CO decreased from 53.70 µg m−3, 93.31 µg m−3, 29.45 µg m−3, 25.24 µg m−3, and 0.90 mg m−3 to 30.52 µg m−3, 55.84 µg m−3, 21.85 µg m−3, 6.87 µg m−3, and 0.61 mg m−3, during 8 years, respectively. The annual mean concentrations of deweathered PM2.5, PM10, NO2, SO2, and CO presented decreasing trends with decreasing rates of −1.89 µg m−3 a−1, −3.27 µg m−3 a−1, −1.89 µg m−3 a−1, −0.64 µg m−3 a−1, −1.94 µg m−3 a−1, and −0.03 mg m−3 a−1, which contributed 61%, 62%, 56%, 71%, and 75% to the observed trends, respectively. The observed and deweathered O3 presented an increasing trend with increasing rates of 0.92 µg m−3 a−1 and 0.26 µg m−3 a−1, respectively. These results demonstrated the dominant contribution of anthropogenic emission to trends of air pollutants during 2015–2022. The HAQI value decreased from 445 to 262 in 8 years. The contribution of anthropogenic emissions and meteorological conditions to the changes in HAQI were 93% and 7%, respectively. These results suggest that the substantial improvement in overall air quality was driven by anthropogenic emission reduction, which proved the effectiveness of clean air actions on air pollution control in the coastal city. Moreover, the clean air action could avoid 3199 deaths from 2015 to 2022 in the Lianyungang region, highlighting the health benefit of air pollution control policies.In this study, the impact of anthropogenic emissions and meteorology on trends in air quality during 2015–2022 in a coastal city was investigated using an RF modeling approach. The annual mean PM


Anthropometric and body composition variables  were measured. The following fitness tests were also conducted: 30 − 15 Intermittent Fitness Test (VO2max and HRmax), COD (5-0-5 agility test), and speed (10-30msprint test. HRmax and the training load were also measured and monitored using the Rate of Perceived Exertion during the 26 weeks.Twenty elite young football players (age: 13.26 ± 0.19 years; height: 165.8 ± 11.67 cm; body mass: 50.70 ± 7.56 kg; VO2max, between 2D and 4D lengths and Left and Right hand ratios. Also, in AW with Right and Left 4D. The CW and de ACWR with the Right 4D. There were other associations between physical test variables and workload variables.There were associations between HRmax and VO2max, COD, or sprint ability. However, it cannot be ruled out that the absence of statistically significant results may be related to the small sample size and the maturational heterogeneity of the participants.Under-14 soccer players with low right and left-hand 2D:4D ratios did not perform better in the selected fitness tests to assess VO Before the test, all competitors performed warm-ups under a strength and conditioning coach’s guidance. The ICC for this test was 0.92 [30-15IFT was utilized to calculate VOA CR-10 Borg’s scale  was used to record the RPE , 32. BefAdditional calculations were made with RPE. The total daily training load throughout the week was deemed the weekly AW. It was monitored the team’s average weekly AW throughout all 26 weeks of the season and documented (acWL). The formula shown below was used to estimate the CW. The following equation was used to determine each player’s CW , 36:CW = (AWn − 1 + AWn − 2 + AWn − 3) × 0.333 where n = week number.The variable ACWR was used to determine and record the mean ratio of the team’s accumulated workload (AW) members to their CW members. The following formula was utilized to determine the ratio of the individual AW to the individual CW , where n2max, BF%, HRmax, COD, sprint, AW and CW. The following ranges were considered for the correlation coefficient sizes: <0.1 = trivial; 0.1–0.3 = small; > 0.3–0.5 = moderate; > 0.5–0.7 = large; > 0.7–0.9 = very large; and > 0.9 = nearly perfect correlation [2max, VIFT, COD right and left foot, HRmax, BF%, 10 m, and 30 m were set as predictor variables and LF-2D:4D and RF-2D:4D ratios as dependent variables. The significance level was set at p < 0.05.Statistical analyses were performed using GraphPad Prism 8.0.1 . Data are presented as mean and standard deviation (SD). Shapiro–Wilk was applied to check the normality of the data. After its confirmation, Pearson correlation analysis was performed between 2D:4D, VOrelation . To modeTable 2max, HRmax, BF%, COD right and left foot, 10 m, 30 m, TD-top and TD-average.As for the association between variables, as shown in Table 2max, VIFT, COD right and left foot, 10 m, and 30 m) with RF-2D:4D ratio. However, the coefficients found were not statistically significant .Figure 2max, VIFT, COD right and left foot, 10 m, and 30 m) with LF-2D:4D ratio are present. However, the coefficients obtained were not statistically significant .In Fig. 2max, BF%, HRmax, COD, AW, and CW variables; and verify if 2D:4D ratio could explain fitness variables and AW. According to the results, there were no significant associations between physical performance markers and the 2D:4D ratio. In addition, this ratio could not explain fitness variables and AW.The aims of this study were: (i) determine the correlation between 2D:4D, VOThe predictive validity of physical parameters on game performance has been demonstrated at university and professional levels , 38, 39.Several studies –42 have The digit ratios found in the present study  were higher than those of Baker et al.  with 240The 2D:4D ratio effects may be ‘’strong’’ enough to explain general differences in performance/attainment between extreme groups  , but ins2max than other groups . In contrast, another study conducted with basketball athletes aged between 14–15 showed that the post-pubertal subjects performed better than the pubertal and pre-pubertal individuals in Yo-Yo IR1 (770.7 > 613.1 > 430.9 m) [As for the body mass analysis, the standard deviation was about 15% of the sample body mass. This may largely determine upper body strength and power and justify the lack of association between variables. According to previous research , more ma430.9 m) .Considering the present and previous research findings, there is a disparity of results regarding the relationship between the results obtained in different physical fitness tests and athletic performance markers, such as 2D:4D ratio. Although it is thought to be related to diverse gender, phenotypes, and hormone-related traits, including sports ability, results have often been conflicting and based on small sample sizes . For thiThis study possesses both notable strengths and limitations. In terms of resilience, the findings offer coaches valuable insights into the assessment and monitoring of soccer players, particularly considering the absence of associations and predictive capabilities of 2D:4D with other variables. However, it is important to acknowledge certain limitations. Firstly, the participants were exclusively drawn from one team, potentially limiting the generalizability of the results. Additionally, the sample size employed in this study was relatively small, and future investigations should incorporate larger sample sizes to enhance the reliability of the findings. Furthermore, as suggested by prior research , longitu2max, COD, or sprinting ability. However, it cannot be ruled out that the absence of statistically significant results may be related to the small sample size and the study participants’ maturational heterogeneity.Contrary to existing evidence indicating that 2D:4D ratios are good performance markers in individual and team sports, the present study verified that U14 soccer players with low right and left-hand 2D:4D ratios do not show higher performance in selected fitness tests. Similarly, the 2D:4D ratio did not help predict motor performance in tests commonly used by soccer players to estimate their VO


As secondary parameters leukotriene 4 (LTB4), and cytokine release  were quantified.As prostaglandin E2 (PGEverum) or no BITC (control) twice a day for 2 weeks each. Nasturtium intervention resulted in an increase in mean PGE2 levels in serum samples , and ex vivo stimulated peripheral blood mononuclear cells (PBMC) . Using a pre-to-post responder analysis approach, 18 of 34 subjects showed a > 25% PGE2 increase in serum, while it was >25% decreased for 9 subjects . Under the selected conditions, the BITC content of nasturtium did not affect the observed changes in PGE2. Verum intervention also increased mean LTB4 serum level , but not in LPS stimulated PBMC, and significantly increased TNF-α release in stimulated PBMC after 3 h . No change was seen in the anti-inflammatory cytokine IL-10, or the pro-inflammatory cytokines IL-1β, and IL-12.Thirty-four healthy female participants consumed 1.5 g nasturtium containing BITC, (in vitro results, on average, LPS activated PBMC and serum from both groups showed increased PGE2 levels. Further analyses suggest that PGE2 release after intervention could possibly depend on the baseline PGE2 level. Identification of phenotypes that respond differently to the nasturtium intervention could be useful to establish personalized approaches for dosing phytopharmaceuticals medicines.In contrast to the previously reported  The plant is also a component in phytopharmaceuticals for the prevention and treatment of urinary tract infections, and against acute sinusitis or bronchitis (in vitro studies (2 (PGE2) signaling pathway in bacterial lipopolysaccharide (LPS) stimulated peripheral blood mononuclear cells (PBMC) from healthy human volunteers upon treatment with a water extract of nasturtium  and not after drinking the preparation devoid of AITC  levels, and also cytokine release  from ex vivo LPS stimulated PBMC from healthy human volunteers.In our recent eriments . In cont of AITC . Based o2.2.1.Escherichia coli O11:B4, standard BSA solution (10 mg/mL), ammonium persulfate, methanol (>99%) and Tween 20 were from Sigma Aldrich , LymphoPrepTM gradient was purchased from Progen Biotechnik GmbH . Colistin sulfate salt was purchased from Thermo Fisher Scientific Inc. . Trifluoracetic acid  was from AppliChem GmbH . Pronase E (from Streptomyces griseus) was obtained from Merck KGaA . Sodium hydroxide, formic acid , acetonitrile (LC–MS grade), methylene chloride (GC Ultra Grade), and water Rotisolv HPLC were purchased from Carl Roth GmbH+Co. KG . C18ec solid phase extraction cartridges  were obtained from Machery-Nagel GmbH & Co. KG . Benzyl cyanide (≥98%), benzonitrile (≥99.9%), and BITC (≥98.5%) were obtained from Sigma-Aldrich Chemie GmbH  and Na2SO4 anhydrous (≥99%) was from VWR International GmbH . β-Mercaptoethanol was obtained from Merck KGaA (Darmstadt Germany). Ethylene diamine tetraacetic acid  and sodium dodecyl sulfate  was from Serva GmbH .Fetal calf serum (FCS), L-glutamine and phosphate buffered saline , penicillin–streptomycin (P/S) solution, L-glutamine solution, RPMI-1640 and penicillin/streptomycin solution  were purchased from Gibco™, Life Technologies GmbH . LPS from 2.2.via posted flyers, social media, and personal communication in Freiburg, Germany. Subjects were screened for eligibility using a health questionnaire. As the immune system differs between men and women . This intervention trial was registered on German Clinical Trials Register (DRKS) with the ID: DRKS00016548. All participants provided written informed consent for study participation and received financial compensation approved by the ethics committee. A total of 46 healthy female subjects were recruited nd women , the stu2.3.T. majus flowers and leaves was provided by Repha GmbH . A detailed phytochemical analysis of the plant powder was reported by us earlier (verum preparation), and (2) containing heat-inactivated myrosinase, which did not produce ITC (control). In the verum 18.87 ± 4.5 μmol BITC and 2.33 ± 0.05 μmol benzyl nitrile (BCN) were detected, whereas the control contained 0.07 ± 0.06 μmol BITC and 4.22 ± 0.63 μmol BCN per serving. Prior to the intervention, aliquots of the plant powders were analyzed for the absence of relevant microbiological contamination following the guidelines of the German Society for Hygiene and Microbiology  for dried products (herbs and spices) by Eurofins BioTesting Services Nord GmbH .Standardized dried powder of  earlier . For con2.4.Brassicales. A list of foods to avoid was provided. During each of the interventions the subjects consumed one serving of verum or control every morning and afternoon , optionally together with some white bread. The order of consumption (begin with verum or control) was assigned randomly using Microsoft® Excel using the RANDBETWEEN function 1;2. “1” was defined as “control” und “2” as “verum.” No certain block size was used and the randomization was done for each subject during enrollment by one unblinded investigator. The full intervention protocol can be viewed at the DRKS (DRKS00016548).A monocentric, randomized, double-blind, controlled, crossover trial was conducted at the Medical Center of the University of Freiburg, Germany . Vacutainers for serum samples were placed on ice; Li-Heparin vacutainers for PBMC isolation were kept at room temperature. All samples were processed within 2 h after sampling. On day 14, 4 h after the last procedure in the morning, spot urine and blood were collected as on the first day. On day 14, no further intake took place in the evening. This schedule was chosen because the concentration of ITC metabolites in human serum reaches a maximum between 1 and 5 h after consumption .g for 10 min to obtain serum, aliquoted in 200 or 300 μL portions, and stored at −80°C. One 300 μL aliquot was mixed with 100 μL TFA and stored at −80°C until chemical analysis.Urine samples were stored in urine monovette  and reaction tubes at −80°C until analysis. Two 200 μL urine aliquots were mixed with 50 μL TFA for chemical analysis and stored at −80°C. Blood with coagulant was centrifuged at 4°C and 2000× 2.6.6cells/mL were prepared with RPMI, supplemented with 10% FCS, 1% Pen/Strep and glutamine. 1.5 mL of cell suspension was stimulated with 100 ng/mL LPS and incubated at 37°C in a humidified incubator with 5% CO2/95% air atmosphere for 3, 24, or 48 h. Subsequently, the cells were centrifuged at 500× g for 5 min at 4°C. Cell free supernatants were collected, and stored at −80°C. Cell pellets were washed twice with ice cold PBS and stored at −80°C.PBMC were isolated from Li-heparinized blood by centrifugation on a LymphoPrep™ gradient using SepMate™ centrifugation tubes . Cells were washed twice with PBS, and cell viability as well as cell concentration were determined using the trypan blue exclusion test. Cell suspensions of 1 × 102.7.2 and LTB4 using the ELISA kits from Cayman  according to the manufacturer’s instructions.Cell free supernatants and serum samples were used for quantification of PGE2.8.The quantification of cytokines was done using the human IL-10, IL-1β, IL12, and TNF-α uncoated ELISA kits from Invitrogen  following the manufacturer’s protocol.2.9.n = 3) for GLS and hydrolysis product formation.Nasturtium plant powder was analyzed for GLS and the potential to release GLS hydrolysis products. For analysis of the GLS hydrolysis products, 1.5 g of the plant powder was prepared as described in 2.3. After 20 s, 500 μL of the mixture were placed into a solvent resistant extraction vessel, which was placed on an ice-water bath. Then, samples were extracted at 0°C and analyzed by GC–MS as described previously , except 2.10.g, 4°C for 10 min. The supernatant was transferred to SPE cartridges. Prior to transferring, the SPE cartridges were conditioned with 3 mL acetonitrile (ACN) and equilibrated with 3 mL water. After the application of the samples, cartridges were washed with 3 mL formic acid  and the analytes were eluted with 3 mL of FA . Afterwards, the eluates were evaporated to dryness with a steady stream of nitrogen. The urine and serum samples were reconstituted in 200 μL and 100 μL FA , respectively. Aliquots of the samples (5 μL) were then injected into the LC-ESI-MS/MS system.Urine and serum samples were prepared for analysis as described before . Frozen 2.11.The mercapturic acid metabolites  were analysed as described before  with min2.12.g, 4°C for 10 min, the supernatant was applied to prepared SPE cartridges. Preparation of the SPE cartridges was done by conditioning with 3 mL methanol and equilibrating with 3 mL water. After sample application, the cartridges were washed with 3 mL FA (0.1% in water) and the analytes were eluted with 3 mL FA . Subsequently, the serum samples were evaporated to dryness under a gentle stream of nitrogen and redissolved in 100 μL FA . Sample aliquotes of (4 μL) were injected into the LC-ESI-MS/MS system.The sample preparation of serum samples was conducted as described before with minor modifications . The sam2.13.For the LC-ESI-MS/MS analysis the protocol performed by Kühn and colleagues was used . The 4002.14.Creatinine was used as a normalization factor to estimate mercapturic acid metabolites in urine. Quantification of creatinine in urine samples was done using the ELISA kit from Invitrogen AG  following the manufacturer’s protocol.2.15.2 serum level upon nasturtium intervention. According to this, detection of a 25% difference between groups in PGE2 level, measured by ELISA, with 80% power, using a 2-tailed t-test at the 1% significance level and 30% standard deviation, requires 34 participants. The calculation was based on the assumption of a log-normal distribution of the values. Based on an estimated drop-out-rate of 20%, 41 subjects were recruited for the trial. For both intervention phases, the between-subject difference in outcome parameters was calculated. The two-sided Student’s paired t-test or Wilcoxon signed-rank test (due to parameter distribution) was carried out. Normal distribution was assessed using the Kolmogorov–Smirnov test and Shapiro–Wilk test. To analyze for a potential carry-over effect between the verum and control intervention phase, a pre-test was performed by calculating the sum of the measured values in the intervention trial for each participant and compared across the two groups by an unpaired t-test  or GraphPad Prism 6.0 software .The sample size calculation was based on the primary outcome, namely change in PGEd t-test . This wa3.3.1.n = 6) and conflicting time schedules (n = 1). Individuals were aged 23.32 ± 2.56 years, with a body weight of 60.98 ± 5.19 kg, height of 1.69 ± 0.05 m and a BMI of 21.42 ± 1.79 kg/m2. Individuals starting with verum were aged 23.48 ± 2.62 years, with a body weight of 60.98 ± 6.13 kg, height of 1.69 ± 0.05 m and a BMI of 21.33 ± 1.97 kg/m2. Individuals starting with control were aged 23.08 ± 2.37 years, with a body weight of 61.12 ± 3.39 kg, height of 1.68 ± 0.05 m and a BMI of 21.57 ± 1.57 kg/m2. Participants reported mainly mild side effects, which are given in A total of 34 healthy female participants completed the intervention trial. Seven individuals dropped out before allocation . Reasons3.2.verum group at a mean level of 28.36 ± 14.12 nmol/mg creatinine, and from the control group at 5.36 ± 4.53 nmol/mg creatinine , this was the case in 17 subjects from the control group (mean 7.76 nmol/L ± 2.18), verum intervention. We thus re-evaluated these data according to the sequence of the start of the intervention, i.e., verum or control . This was comparable to the control , which indicated an independence of the effect from the BITC content of the plant. Next, we investigated the effect of the intervention on PGE2 release in LPS stimulated PBMC in both phases to simulate an acute bacterial infection. Bacterial LPS is commonly used in such experiments to mimic inflammatory responses in cell and animal studies. After intervention, PGE2 release from 24 h stimulated PBMC increased to a mean of 1.71-fold , and 1.43-fold (control), see 2 release was still upregulated upon intervention . As seen for the serum PGE2 analysis, no significant superior treatment effect of verum compared to control was observed.First, we analyzed the magnitude of difference in outcome in the analysis . Intervep-value of 0.7498 indicated that this was not the case.We additionally analyzed the data for a potential carryover effect between the two intervention phases, but a 2 production, it can also lead to LTB4 formation through 5-lipooxygenase (5-LOX)  stimulated PBMC. We observed that the mean LTB4 level was significantly increased in serum upon verum intervention  . Thus, w2 to the intervention was quite heterogeneous, although the present study population was similar in many baseline parameters such as age, sex, weight, or height. To determine the effect of the intervention at the individual level, we next categorized subjects with a > 25% decrease in PGE2 serum level as “responders,” and those with an increase in PGE2 > 25% as “adverse responders.” Following this, 26% of the verum group could be classified as “responders,” 52% as “adverse responders,” and 22% showed no relevant change. For the control group, 24% of subjects could be classified as “responders,” 58% as “adverse responders,” and 18% showed no relevant change.The response in PGE2 levels from each volunteer and correlated these to the corresponding PGE2 baseline levels. The results are presented in 2 serum levels, B: PGE2 release from 24 h stimulated PBMC). An inverse correlation could be seen between the baseline PGE2 levels and the magnitude of change upon verum intervention in serum samples , and in stimulated PBMC from both intervention groups . However, there are some statistical pitfalls in correlating the baseline value with the change in a factor, such as the artifact of regression to the mean or random variation within subjects, as described by Atkinson and Batterham , the change in the verum group was 241.9 pg/mL , and the change in the control group was 244.3 pg/mL . Since the mean SD in the baseline change was larger than the mean values in the intervention groups, a regression artifact cannot be excluded. In 24 h stimulated PBMC the mean change from baseline was 5496 pg/mL , the change in the verum group was 7862 pg/mL  and the change in the control group was 4725 pg/mL . In this case, a pseudo effect is unlikely. However, having a relatively small sample size and no comparator arm without any treatment, the validity of this analysis is limited.Previous studies addressed the question of whether an individual’s outcome could depend on the baseline level of the parameter of interest, e.g., . Followiatterham . We thus3.4.2 was seen in intervention groups, we also studied the release of cytokines from LPS-activated cells. As given in verum significantly increased TNF-α release in LPS stimulated PBMC after 3 h . Intervention with control preparation had no effect on LPS-stimulated PBMC . After 24 h, the TNF-α level was back to baseline. No change was seen in the anti-inflammatory cytokine IL-10, or the pro-inflammatory cytokines IL-1β, and IL-12  with single intake of fresh watercress  reported a mild pro-inflammatory reaction in LPS-stimulated whole blood cultures from healthy subjects upon consumption of watercress as compared to control group, though. This was shown by significantly increased mean levels of TNF-α, IL-1β, and IL-6  in serum after the verum intervention. Compared to a recent in vitro study, BITC concentration achieved in serum was very low. This could also be an explanation for the different effect on PGE2. However, we observed considerable variation in response between individuals, with some subjects showing an increase in PGE2 response while in another part it was decreased upon intervention. This concurs with other studies, in which usually only up to 40% of subjects responded to intervention, e.g., . This study was conducted in accordance with the Declaration of Helsinki. This intervention trial was registered on German Clinical Trials Register (DRKS) with the ID: DRKS00016548. The patients/participants provided their written informed consent to participate in this study.EL: conceptualization. LF, HT, JS, MK, and FH: methodology. CH, LF, HT, SC, JS, MK, FH, MA, NB, and EL: formal analysis. CH, LF, HT, SC, JS, MK, and FH: investigation. RS, MS, and EL: resources. CH, LF, and EL: writing—original draft preparation. FH, SR, and MS: writing—review and editing. All authors have read and agreed to the published version of the manuscript. The listing order of the authors is without weighting.The intervention trial was partly supported by a grant from Repha GmbH, Langenhagen, Germany. Repha GmbH was not involved in the design, conduction, interpretation, or publishing of the results. FH was funded by the Leibniz Association (J16/2017). The manuscript processing fee was partly funded by the University of Freiburg in the funding program Open Access Publishing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The assessment of factors that can promote the transmission of antibiotic resistance genes (ARGs) across bacteria in the gastrointestinal tract is in great demand to understand the occurrence of infections related to antibiotic-resistant bacteria (ARB) in humans. However, whether acid-resistant enteric bacteria can promote ARG transmission in gastric fluid under high-pH conditions remains unknown. This study assessed the effects of simulated gastric fluid (SGF) at different pH levels on the RP4 plasmid-mediated conjugative transfer of ARGs. Moreover, transcriptomic analysis, measurement of reactive oxygen species (ROS) levels, assessment of cell membrane permeability, and real-time quantitative assessment of the expression of key genes were performed to identify the underlying mechanisms. The frequency of conjugative transfer was the highest in SGF at pH 4.5. Antidepressant consumption and certain dietary factors further negatively impacted this situation, with 5.66-fold and 4.26-fold increases in the conjugative transfer frequency being noted upon the addition of sertraline and 10% glucose, respectively, compared with that in the control group without any additives. The induction of ROS generation, the activation of cellular antioxidant systems, increases in cell membrane permeability, and the promotion of adhesive pilus formation were factors potentially contributing to the increased transfer frequency. These findings indicate that conjugative transfer could be enhanced under certain circumstances in SGF at elevated pH levels, thereby facilitating ARG transmission in the gastrointestinal tract.IMPORTANCE The low pH of gastric acid kills unwanted microorganisms, in turn affecting their inhabitation in the intestine. Hence, studies on the factors that influence antibiotic resistance gene (ARG) propagation in the gastrointestinal tract and on the underlying mechanisms are limited. In this study, we constructed a conjugative transfer model in the presence of simulated gastric fluid (SGF) and found that SGF could promote the dissemination of ARGs under high-pH conditions. Furthermore, antidepressant consumption and certain dietary factors could negatively impact this situation. Transcriptomic analysis and a reactive oxygen species assay revealed the overproduction of reactive oxygen species as a potential mechanism by which SGF could promote conjugative transfer. This finding can help provide a comprehensive understanding of the bloom of antibiotic-resistant bacteria in the body and create awareness regarding the risk of ARG transmission due to certain diseases or an improper diet and the subsequent decrease in gastric acid levels. Antibiotic resistance has been considered a public health threat that prevents the treatment of bacterial infections and results in a continual increase in mortality . HundredBacteria acquire antibiotic resistance mainly through vertical gene transfer (VGT) and horizontal gene transfer (HGT), including conjugation, transformation, and transduction , 5. ARG Escherichia coli and Shigella flexneri are more acid resistant than other bacteria and therefore can survive after exposure to pH 2.0 ± 2.5. Moreover, Salmonella enterica serovar Typhimurium cells are killed following exposure to pH 3.0  levels, assessment of cell membrane permeability, and reverse transcription-quantitative PCR (RT-qPCR) analysis were performed to explore key channels. To the best of our knowledge, this is the first study to reveal the enhancement of conjugative transfer under certain circumstances in SGF, which can facilitate the transmission of ARGs in the gastrointestinal tract. Our findings can help provide a comprehensive understanding of ARB blooms in the body.In this study, simulated gastric fluid (SGF) was prepared E. coli HB101 and E. coli K-12 were exposed to SGF together at pH 3.5 and 4.5, transconjugants began to grow on the plates, and the conjugative transfer frequencies reached (3.79 ± 0.11) × 10−5 and (1.05 ± 0.17) × 10−4, 1.68-fold and 4.66-fold higher than that in the control group at pH 7.0, respectively (P < 0.01). In contrast, when E. coli and Salmonella enterica serovar Aberdeen were exposed to SGF together at pH 3.5, no evident difference was noted between the conjugative frequencies at pH 3.5 and pH 7.0 (control). Moreover, when E. coli and Salmonella Aberdeen were exposed to SGF together at pH 4.5, the conjugative transfer frequency was significantly increased to (2.91 ± 0.34) × 10−6, 2.36-fold higher than that in the control group at pH 7.0 (P < 0.01). This may be due to the higher rate of mortality of the recipient Salmonella Aberdeen cells at pH 3.5 (64.54%) than that of the recipient E. coli K-12 cells at pH 3.5 (38.92%). On the other hand, no significant difference was noted between the mortality of the recipient Salmonella Aberdeen cells (14.38%) and that of the recipient E. coli K-12 cells (17.56%) at pH 4.5 . Thus, SGF can significantly promote the horizontal transfer of ARGs within and across bacterial genera when its pH is maintained at 3.5 or 4.5.To assess the effects of SGF at different pH levels on the horizontal transfer of ARGs within and across bacterial genera, the conjugative transfer of the RP4 plasmid exposed to SGF at various pH levels  or phosphate-buffered saline (PBS) (pH 7.0) was performed with an exposure time of 4 h. As shown in E. coli HB101 and E. coli K-12 at exposure times of 2 and 4 h when the pH was 3.5. The conjugative transfer frequency at 4 h was 3.34-fold higher than that in the control group at 2 h (P < 0.01). In addition, when the pH was 3.5, the frequency of conjugative transfer between E. coli and Salmonella Aberdeen was significantly increased to (1.50 ± 0.43) × 10−6 at 4 h, 1.75-fold higher than that in the control group at 2 h (P < 0.05). Thus, the horizontal transfer of ARGs across bacteria may be improved by increasing the time of exposure to gastric fluid.To determine the effects of the time of exposure to SGF on the horizontal transfer of ARGs within and across bacterial genera, the conjugative transfer of the RP4 plasmid was performed after exposure to SGF for various times. As shown in E. coli through mutagenesis  upon exposure to 50 mg/L or 100 mg/L fluoxetine, amitriptyline, sertraline, or duloxetine compared with that in the control group without drug exposure. Importantly, sertraline exposure had the maximum effect, resulting in a 5.66-fold increase in the conjugative transfer frequency compared with that in the control group. According to clinical recommendations, the prescribed daily doses of fluoxetine, duloxetine, sertraline, and amitriptyline are 20 to 120 mg  cells and recipient Salmonella Aberdeen cells exposed to SGF at pH 4.5. We then validated our findings by measuring ROS levels, assessing cell membrane permeability, and performing RT-qPCR analysis. Compared with the control group at pH 7.0, totals of 1,647 genes were differentially upregulated and 1,660 genes were differentially downregulated in the donor E. coli HB101 cells, while 1,436 genes were differentially upregulated and 1,515 genes were differentially downregulated in the recipient Salmonella Aberdeen cells in the SGF group at pH 4.5 . Moreover, the expression level of sodB, the primary scavenger of O2− (P < 0.01). The expression level of grxA, which encodes glutaredoxins (P < 0.01). The expression levels of other oxidative stress response genes, such as the Fe-S metabolism- and Fe-S cluster scaffold complex subunit-related genes sufC and sufD, were also upregulated by 2.35- to 3.34-fold in the donor cells. Similarly, the expression levels of ahpF, grxA, katG, and sodB were upregulated by 2.60- to 24.93-fold in the recipient cells (P < 0.01). Genes encoding other antioxidant reductases, such as gor, trxC, and fpr, were also upregulated by 2.06- to 4.17-fold (P < 0.01).To study oxidative stress in bacteria, the expression levels of cellular antioxidant-encoding genes were assessed after exposure to SGF . Furthermore, ROS scavenging was performed to validate whether ROS production could enhance conjugative transfer upon exposure to SGF at pH 3.5 and 4.5. The frequency of the transfer of the RP4 plasmid across genera was significantly decreased by 1.50- to 3.98-fold upon exposure to SGF at pH 3.5 and 4.5, and there was no remarkable difference compared with the findings for the control group at pH 7.0 (P < 0.01)  . These rompA, ompC, and ompF , and those of ompA and ompC were upregulated by 3.27- to 6.45-fold in the recipient Salmonella Aberdeen cells (P < 0.01). Moreover, in the donor bacteria, the expression levels of outer membrane-related genes such as bamA, bamB, and bamC, which encode outer membrane protein assembly factors, were upregulated by 2.95- to 4.41-fold (P < 0.01). Furthermore, 2.04- to 47.18-fold increases in the expression levels of osmB, encoding an outer membrane lipoprotein, and tolA and tolR, encoding proteins that maintain outer membrane stability, were noted in the donor and recipient cells (P < 0.01) (nt cells . Further < 0.01) , indicatfim-like genes have been characterized to contribute to cell adhesion in E. coli  . As shown in fimA and fimH in both bacteria were upregulated by 2.34-fold and 1.98-fold, respectively, upon exposure to SGF at pH 4.5. Thus, adhesive pili may play a crucial role in promoting conjugative transfer.Direct cell-to-cell contact is necessary for plasmid transfer , and som E. coli . Transcrtrfap and the mating pair formation system (traF) , and those of atpC, atpD, atpL, atpG, and atpA in Salmonella Aberdeen were upregulated by 2.50- to 4.32-fold (P < 0.01)  system initiated by promoter m (traF) , were mom (traF) . In addim (traF) . Differein vitro, e.g., disinfectants, nanoparticles, and heavy metals  were upregulated, suggesting that membrane permeability could be increased and contribute to the increased conjugative transfer of ARGs in SGF at pH 4.5. To enable the transfer of plasmid DNA from donor to recipient bacteria, physical cell-to-cell contact is necessary during the conjugation process. Type 1 pili have been shown to increase the adhesion of donor E. coli K-12 cells to neighboring cells (fim) were expressed in the donor and recipient cells at pH 4.5, indicating that adhesive pili may enhance plasmid transfer by facilitating cell-to-cell contact between donors and recipients. Thus, SGF at pH 4.5 could increase the conjugative transfer of the RP4 plasmid through ROS overproduction and could enhance cell membrane permeability and adhesive pilus formation.To assess why SGF could promote the conjugative transfer of ARGs at pH 4.5, we performed a transcriptomic analysis. Previously, increased ROS production or membrane permeability due to CuO nanoparticles and Cu plasmid . According cells . In our In summary, we found that SGF at pH 3.5 and 4.5 could enhance the conjugative transfer of ARGs within and across bacterial genera. Furthermore, the consumption of antidepressants, including amitriptyline, fluoxetine, duloxetine, and sertraline, and certain dietary factors, including very high salt, high and medium sugar, and high capsaicin concentrations, could facilitate conjugative transfer. The induction of ROS production, the activation of cellular antioxidant systems, increases in cell membrane permeability, and the promotion of adhesive pilus formation were factors potentially contributing to an increase in the transfer efficiency in SGF at elevated pH levels. These results broaden our understanding of ARG transmission in the gastrointestinal tract and reveal that some diseases or improper dietary intake may decrease gastric acid levels and increase the risk of ARG dissemination.E. coli HB101 containing the RP4 plasmid  and exhibiting Amp, Kan, and Tet resistance was chosen as the donor. E. coli K-12 exhibiting Chl resistance, obtained from our previous mutation study , were chosen as the recipients. E. coli HB101 was cultured in Luria-Bertani (LB) medium  containing 100 mg/L Amp, 60 mg/L Kan, and 40 mg/L Tet. E. coli K-12 was cultured in LB medium containing 25 mg/L Chl, and Salmonella Aberdeen was cultured in LB medium containing 25 mg/L Str. All of the strains were incubated at 37°C for 16 h.Antibiotics  were purchased from Sigma-Aldrich. on study , and Sald-glucose, 2.05 g of NaCl, 0.6 g of KH2PO4, 0.37 g of KCl, and 0.11 g of CaCl2. The SGF solution was autoclaved at 121°C for 15 min. After the pH was adjusted using 5.0 N HCl, 5 mL of a solution of 2.66 mg/mL of pepsin, 20 mg/mL of lysozyme, and 10 mg/mL of ox bile was sterilized using a filter with a pore size of 0.22 μm and added to 1 L of SGF.SGF was prepared according to a previously described method . In brieE. coli HB101(RP4) and recipient E. coli K-12 and Salmonella Aberdeen cells were freshly grown in LB medium at 37°C for 14 to 16 h. Next, the cells were centrifuged at 8,000 × g for 5 min. After the removal of the supernatants, the pellets were washed three times with phosphate-buffered saline (PBS) (pH 7.0) and resuspended in PBS to obtain a cell count of ~109 CFU/mL. Following this, 100 μL of the donor cells and 100 μL of the recipient cells were added to 8 mL of the SGF solution at different pH levels  or PBS (pH 7.0) for 2 and 4 h to achieve a final concentration of ~108 CFU/mL. Subsequently, 500 μL of the bacterial mixtures was plated onto LB agar plates and incubated at 37°C for 36 h. For transfer within genera, transconjugants were selected on LB agar plates containing 100 mg/L Amp, 40 mg/L Tet, 60 mg/L Kan, and 25 mg/L Chl. The recipient bacterial count was determined by plating diluted mating mixtures onto LB agar plates containing 25 mg/L Chl. For transfer across genera, transconjugants were selected on LB agar plates containing 100 mg/L Amp, 40 mg/L Tet, 60 mg/L Kan, and 25 mg/L Str. The recipient bacterial count was determined by pouring the diluted mating mixtures onto LB agar plates containing 25 mg/L Str. The conjugation frequency was calculated by dividing the number of verified transconjugant colonies by the total number of recipients. All of the experiments were performed in at least biological triplicates.Donor E. coli HB101 and Salmonella Aberdeen was assessed.To assess the effects of drug consumption, dietary factors, and ROS scavenging on the conjugative transfer of the RP4 plasmid, four antidepressants , three dietary factors , and 100 μmol/L of thiourea were added to SGF at pH 4.5, and the frequency of conjugation between E. coli K-12 and Salmonella Aberdeen cells were randomly selected and cultured in LB broth containing 100 mg/L Amp, 40 mg/L Tet, 60 mg/L Kan, and 25 mg/L Chl or 25 mg/L Str at 37°C for 14 to 16 h. The plasmid was extracted using a plasmid extraction kit  according to the manufacturer’s instructions and assessed using 1% agarose gel electrophoresis. The unique gene traG in the RP4 plasmid was selected as the target gene and verified by PCR. The RP4-specific primers used for PCR were RP4-f (5′-AAAGCGGACAGCATCAGTAACGAA-3′) and RP4-r (5′-GAGCTTGGTGGCCGCATAGTGTAG-3′). The reaction was performed using a 20-μL reaction mixture containing 10 μL of 2× Rapid Taq master , 1 μL of each primer (10 μmol/L), 2 μL of the extracted plasmids, and 6 μL of nuclease-free water. The thermal cycling conditions were as follows: an initial denaturation step at 95°C for 5 min followed by 30 cycles at 95°C for 30 s, 55°C for 30 s, and 72°C for 1 min and a final extension step at 72°C for 5 min. The PCR product was 104 bp and was assessed using 1% agarose gel electrophoresis .To verify the formation of transconjugants carrying the RP4 plasmid, transconjugant 6 CFU/mL of donor and recipient cells in PBS were incubated with DCF-DA for 30 min at 37°C. Next, the DCF-DA-loaded cells were treated with SGF at different pH levels (3.5 or 4.5) or PBS (pH 7.0) for 30 min. All of the samples were scanned by flow cytometry at an excitation wavelength of 488 nm and an emission wavelength of 526 nm. All of the tests were performed in biological triplicates.ROS production was assessed by flow cytometry  using the 2′,7′-dichlorofluorescein diacetate (DCF-DA) cellular ROS detection assay kit , according to the manufacturer’s instructions. In brief, suspensions of ~10Cell membrane permeability was assessed by flow cytometry, as described previously . In brieE. coli HB101(RP4) and recipient Salmonella Aberdeen cells were prepared as described above. Next, 100 μL of the donor cells and 100 μL of the recipient cells were added to an 8-mL SGF solution at pH 4.5. After mating for 4 h across genera, the bacterial cells were collected by centrifugation at 8,000 × g for 10 min. Total RNA was extracted using the RNeasy minikit , according to the manufacturer’s instructions. Sequencing was performed by Novogene . In the control group, 100 μL of the donor cells and 100 μL of the recipient cells were added to 8 mL of PBS at pH 7.0. Global transcriptional analysis was performed according to methods described in a previous study, with a few modifications (E. coli HB101 (GenBank accession number NC_000913.3) and Salmonella Aberdeen (accession number NC_003197.2). Cufflinks (version 2.2.1) was used to analyze differential expression in triplicate cultures under the same conditions. The fragments per kilobase of transcript per million mapped reads (FPKM) method of gene expression analysis was used to identify differentially expressed genes (DEGs) between the SGF group at pH 4.5 and the control group at pH 7.0. The resultant P values were adjusted using the Benjamini-Hochberg approach , and 7 μL of RNase-free water. The PCR conditions were as follows: an initial step at 95°C for 10 min, 40 cycles at 95°C for 15 s and 60°C for 1 min, and a final step at 95°C for 15 s and 60°C for 1 min. All qPCRs were performed in triplicates using diethyl pyrocarbonate (DEPC)-treated water as the negative control. qPCR was performed using the ABIViiA 7 real-time PCR system . All of the primers are listed in Table S5.The extracted RNA was transcribed to cDNA by RT-PCR using a reverse transcription kit . Next, qPCRs were performed using 20 μL of a reaction mixture consisting of 2 μL of cDNA, 10 μL of SYBR green TC) values for both the reference (16S rRNA) and target genes, the relative expression levels of target mRNAs were calculated by normalization against 16S rRNA gene expression levels. The control group at pH 7.0 was used for calibration. A comparative ΔΔTC method (2−ΔΔCT) was used to assess gene expression differences from triplicate measurements  and the TBtools (version 1.108) data visualization tool were used for graphic drawing. The SPSS Statistics 22.0 software package  was used for all statistical analyses. Significant differences were assessed using the independent-sample PRJNA930988.RNA sequencing data are accessible through the Sequence Read Archive (SRA) under BioProject accession number


H. pylori. The aim of the present study was to prepare a phillygenin derivative (PHI-Der) through demethylation and hydroxylation. The minimum inhibitory concentration of 18 strains of H. pylori from different sources was 8–32 μg/mL in vitro, and the activity increased 2–8 times than that of phillygenin. PHI-Der could significantly inhibit the colonization of H. pylori in vivo, reduce the inflammatory response, and promote the repair of inflammatory damage. Further, we used SwissTargetPrediction to predict that its main targets are ALOX5, MCL1, and SLC6A4, and find that it can inhibit bacterial biofilm formation and reduce bacterial infection of cells. It can enhance the intracellular oxidative capacity of H. pylori to inhibit H. pylori growth. Further, it could prevent the oxidation of H. pylori-infected cells and reduce the inflammatory response, which plays a role in protection. In conclusion, compared to phillygenin, PHI-Der had better antibacterial activity and was more effective in treating H. pylori infection. It has characteristics of high safety, specificity, resistance to drug resistance and better antibacterial activity than phillygenin, it’s a good antioxidant for host cells.Modifying and transforming natural antibacterial products is a novel idea for developing new efficacious compounds. Phillygenin has an inhibitory effect on  H. pylori biofilm; C is PHI-Der that enhance the oxidation of H. pylori; D is PHI-Der reduce the oxidative effect and the expression of inflammatory factors in H. pylori-infected cells.The figure shows the hypothesis of the mechanism of phillygenin derivatives (PHI-Der). A is PHI-Der in the treatment of gastritis mice; B is the inhibitory effect of PHI-Der on  Bacterial species other than H. pylori were cultured on nutrient agar or Luria–Bertani plates at 37°C for 1–2 days. Staphylococcus aureus and other information.We extracted 2.2.First, we dissolved phillygenin (7 mg) in dry dichloromethane (5 mL). Second, we slowly added boron tribromide in dichloromethane dropwise into phillygenin at −50°C  and placed it under ice bath for 1 h. Third, we quenched the reaction with 1 mL methanol and concentrated the reaction solution. Fourth, we performed high-performance liquid chromatography for purification  to obtain PHI-Der at a yield of 45%. After Fourier-transform infrared analysis, mass spectrometry and hydrogen and carbon nuclear magnetic resonance (NMR), the structure of PHI-Der was determined. The 3D structure of PHI-Der was visualized using ChemDraw3D. The SMILES structure was uploaded to PaddleHelix2.3.600) of H. pylori was adjusted to 1.0 × 107 colony forming unit (CFU)/mL. OD600 of the remaining bacteria and the fungus were adjusted to 1 × 106 and 5 × 103 CFU/mL, respectively, and 10 μL was added to each well. Cultivation was performed for 24–72 h before further analyses  and diluted them sequentially. Negative  and positive  wells were used as controls. Bacteria growing in the logarithmic phase on the solid plate were used to create a bacterial suspension with the corresponding medium. Optical density at 600 nm , we diluted  the bacterial liquid, spread it on Columbia agar plates, and performed culture in a three-gas incubator for 4–5 days. We calculated the number of bacteria growing on the agar plate. The drug concentration at which the number of bacteria was inhibited by 99.9% was considered to be MBC.We added 173.6 μL of the culture medium and 6.4 μL of PHI-Der (4 mg/mL) to the first well of the 96-well plate. The wells were sequentially folded and diluted. The wells of phosphate-buffered saline  were used as positive controls. 2.5.H. pylori G27 strain. MICs of metronidazole and PHI-Der were 2 and 16 μg/mL, respectively. We used one-fourth of MIC to induce the strain, which was detected every 3 days over 24 days of induction. The induction concentration was adjusted with changes in MIC. For example, when MIC of metronidazole changed to 16 μg/mL, the induction concentration was adjusted to 4 μg/mL.Long-term contact of bacteria with low doses of drugs causes changes in the medicinal chemical processes of the cells themselves, so that bacteria gradually tolerate drugs. The drug resistance of PHI-Der was detected with the 2.6.5. We inoculated 100 μL/well into 96-well plates and replicated three same samples. Incubation was performed at 37°C for 24 h. The final concentrations of PHI-Der were 200, 150, 100, 50, and 0 μg/mL. Subsequently, incubation was performed at 37°C for 24 h. We added 10 μL of Cell Counting Kit-8  to each well, tapped to mix, and incubated for 4 h. Finally, we measured the absorbance at 450 nm.GES-1 and BGC823  cell suspensions were adjusted to 1 × 102.7.We purchased 6–8-week-old specific-pathogen-free (SPF) C57BL/6 mice from Changsha Tianqin Biotechnology Co., Ltd. . Twenty animals were randomly divided into administration and negative control groups, with ten animals in each group (Not infected with Hp), and raised in SPF environment. The administration group was administered with 10 times the therapeutic dose daily for 3 consecutive days, while the negative control group was administered with PBS solution. The mice were weighed consecutively for 7 days, starting 1 day before administration. Three days after drug withdrawal, the mice in each group were weighed, and the average body weight was calculated. Blood was collected from the eyeball, and the mice were sacrificed through dislocation and neck dislocation. Stomach, kidney, liver, and spleen tissues were obtained for pathological sectioning and hematoxylin and eosin (H&E) staining.2.8.H. pylori. Drugs were administered daily for 3 consecutive days. Two days after drug withdrawal, blood was collected from the eyes of the mice in the infected group, and the mice were sacrificed through cervical dislocation. Gastric tissues were collected and crushed. A portion of gastric tissues was paraffin-sectioned and stained with H&E. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling immunohistochemistry and immunofluorescence histochemistry were performed, and ImageJ was used to quantify the fluorescent signal of the immunohistochemical staining done on the tissue samples.PHI-Der, omeprazole , amoxicillin , and clarithromycin  were dissolved and diluted to 10 mg/mL. We successfully modeled (HPBS001) 6–8-week-old SPF C57BL/6 mice and divided them into four groups: the omeprazole + amoxicillin + clarithromycin group , the omeprazole + PHI-Der (28 mg/kg) group, the omeprazole + PHI-Der (7 mg/kg) group, and the PBS/negative control group. Each group comprised of ten mice. The negative control group comprised of ten mice not infected with 2.9.Helicobacter pylori infection” were screened from the GeneCards database as potential targets of PHI-Der against H. pylori infection. The target interaction relationship was obtained from the STRING database.Homo sapiens.”PHI-Der targets were obtained from the SwissTargetPrediction database.2.10.The three-dimensional structure of PHI-Der was imported into Discovery Studio. We used the module for preparing ligands in molecules to process small molecules. The module mainly minimizes the energy of small molecules and yields CHARMM force fields. The crystal structure of the target protein corresponding to the core target was downloaded from the Protein Data Bank website.2.11.H. pylori G27 bacterial suspension was adjusted to 0.1, and the biofilm formed under microaerophilic conditions for 3 days. PHI-Der was added at concentrations of 128, 64, 32, and 16 μg/mL for 24 h. Its anti-biofilm activity was evaluated using crystal violet  and Alamar blue  staining. The biofilm protein content was determined using the bicinchoninic protein concentration assay kit .The OD of the 2.12.Helicobacter pylori G27 was cultured to the logarithmic phase. The bacterial suspension was adjusted to 1 × 107 CFU/mL. We added 10 μM of 2′,7′-dichlorofluorescein diacetate  to the bacterial suspension, and cultivate in a three-gas incubator for 1 h. The cocultured bacterial solution was washed twice with PBS buffer, in order to remove excess DCFH-DA. Phillygenin and PHI-Der were added at concentrations of 32 μg/mL each. PBS and Rosup were used as negative and positive controls, respectively. After 2 h of action, 15 μL of the solution was added dropwise onto the slides and observed under a fluorescence microscope.2.13.H. pylori suspension was collected and labeled with SYTO9 for 15 min, and then treated with different drug concentrations and co-cultured with the cells for 2 h. The plate is placed under a fluorescence microscope for observation.Bacterial adhesion cell experiment GES-1(Rhodamine staining) cells were cultured in RPMI1640 medium containing 10% FBS without antibiotics, the 4 cells/well) and grown in 96-well plates until 70% confluence. PHI-Der was incubated with cells for 2 h at 37°C. The cells were washed with PBS, incubated with 20 μM of DCFH-DA for 30 min at 37°C, and washed twice with PBS to remove the unabsorbed probe. Suspensions in the serum and antibiotic free medium were infected with H. pylori (1 × 108 CFU/mL). Reactive oxygen species (ROS) levels were measured for 180 min using a fluorescence microplate Synergy HT reader with λex of 485 nm and λem of 530 nm.GES-1 cells were seeded  and reverse-transcribed into complementary DNA using a reverse transcription kit . RT-qPCR was performed using LightCycler according to the kit , with pre-denaturation at 95°C for 30 s, denaturation at 95°C for 10 s, and annealing and extension at 60°C for 30 s for 40 cycles. –∆∆CT method) approach, with 16S ribosomal RNA amplicons used as an internal control for data normalization.2.15.p < 0.05 was considered statistically significant.Data are expressed as mean ± standard deviation. Statistical analyses were performed using SPSS 25.0. One-way analysis of variance was performed, and 3.3.1.18H18O6, and the structural formula of PHI-Der SMILES is OC1 = CC=C(C=C1O)[C@H]1OCC2C1CO[C@H]2C1 = CC=C(O)C(O) = C1. 18H18O6 ideal characteristic peak was [M]+ = 330.33. However, the evident characteristic peak was [M −17]+ = 313.32. One hydroxyl group had been removed because of the unstable connection. 1H NMR  δ 6.64 , 6.54 , 6.36 , 4.03.97 , 3.77 , and 2.65–2.50  and deuterium with chloroform–deuterium with the methanol solvent. Through the preparation route , PHI-Der3.2.H. pylori strains  . Crystal protein , a prote protein .SpoT is a bifunctional enzyme with the properties of guanosine tetraphosphate (p-ppGpp) synthase and hydrolase (Hp1174 is a gene of the major facilitator superfamily (MFS) efflux pump family  indirectly regulated the formation of extracellular polymeric substances (EPSs) that induce functional gastrointestinal diseases , a key enzyme that mediates lipid peroxidation by producing lipid peroxides, plays a central regulatory role in inflammation  and modify and transform them into derivatives  is associated with functional dyspepsia in H. pylori infection .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


F(1) = 4.435, p = 0.041, η2p = 0.086) and improvement in the domains of physical health (F(1) = 4.881, p = 0.032, η2p = 0.094), memory (F(1) = 4.192, p = 0.046, η2p = 0.082) and money (F(1) = 4.862, p = 0.032, η2p = 0.094) in the CQOL-AD. We detected a significantly higher improvement of G1 in memory (F(1) = 4.192, p = 0.046 η2p = 0.082), money (F(1) = 7.147, p = 0.010, η2p = 0.132) and friends (F(1) = 4.828, p = 0.033, η2p = 0.093). The G1 when compared to the G2 did not demonstrate significant effects of the BI-Zarit, in the total scores CQOL-AD, MAAS and DASS-21. The study showed that both psychoeducation alone and integrated with yoga reduced the burden of family caregivers of people with Alzheimer's disease, and that the integration of online yoga practice with psychoeducation potentiated the improvement only in some aspects of the quality of life and subjective perception of burden reduction.We evaluate and compare the effectiveness of two psychoeducation programs on the burden and quality of life of family caregivers of people with Alzheimer's disease: one of psychoeducation alone and the other integrated with yoga. Forty-nine participants were randomly allocated to two groups, 25 to the psychoeducation integrated with yoga group (G1) and 24 to the psychoeducation group (G2). The programs took place online through Google Meet and consisted of eight weekly meetings, lasting 30 min for psychoeducation and 30 min for yoga. Participants were evaluated by the Burden Interview Scale (BI-Zarit), Quality of Life Scale in Alzheimer's Disease-Caregiver Version (CQOL-AD), Depression, Anxiety and Stress Scale (DASS-21), Mindfulness and Awareness Scale (MAAS) and a satisfaction survey. Both groups showed reduced overload according to the BI-Zarit scale (Clinical trial registrationhttps://ensaiosclinicos.gov.br/, identifier RBR-794593r, retrospectively registered.The online version contains supplementary material available at 10.1007/s10433-023-00792-9. Population aging is a worldwide phenomenon and, associated with this process, an increase in chronic degenerative diseases such as dementia is expected ALZINT . It is cAlzheimer's disease (AD) dementia is the most prevalent in the world, ranging from 53 to 75% of cases  0 and 0.5 (no dementia and questionable dementia).p > 0.05)  and psychoeducation group (G2). Eight participants, four in each group, were excluded because they had not participated in at least 75% of the meetings, and one participant did not attend any of the meetings. The final sample consisted of 49 participants, 25 from G1 (mean age = 54.96 ± 9.36) and 24 from the G2 (mean age = 53.54 ± 8.48). The two groups did not differ significantly from each other regarding age; years of schooling; marital status; relationship; hours dedicated to care; family income; and severity of dementia  to 4 , and the higher the score, the greater the overload Scazufca .The Brazilian version consists of 13 items that can score four points each, ranging from poor (1 point) to excellent (4 points). The higher the total score, the better the quality of life. It addresses the domains health, mood, living situation, memory, family, marriage, friends, self, ability to do chores, ability to do things for fun, money and life as a whole. We asked the participants to evaluate how important each of those domains is for their quality of life and then how they see their present situation regarding each domain. The final score is the addition of points of all the items of their present situation. The Alpha Cronbach coefficient of this version is 0.86 Novelli .It consists of 21 Likert-type questions with four response options (0–3). Each subscale assesses the depression, anxiety and stress domains. The total for each domain ranges from 0 to 21, and the final score is multiplied by 2. The final score of each subscale suggests classification as normal, mild, moderate, severe or extremely severe. Items 3, 5, 10, 13, 16, 17 and 21 correspond to the depression domain and scores 0–9 suggest absence  of depression, 10–13 mild depression, 14–20 moderate depression, 21–27 severe depression and above 28 extremely severe depression. Anxiety corresponds to items 2, 4, 7, 9, 15, 19 and 20, and scores 0–7 suggest absence of anxiety , 8 and 9 mild anxiety, 10–14 moderate anxiety, 15–19 severe anxiety and above 20 extremely severe anxiety. Finally, stress corresponds to items 1, 6, 8, 11, 12, 14 and 18 and scores from 0 to 14 suggest normal result (absence of stress), from 15 to 18 mild stress, 19–25 moderate, 26–33 severe and above 34 extremely severe stress. The Brazilian version has an Alpha Cronbach coefficient of 0.92 for depression, 0.86 for anxiety and 0.90 for stress  to 6 (seldom). The minimum score is 15 points, and the maximum is 90, indicating the minimum and maximum level of mindfulness and awareness, respectively . Regarding the criteria, it presented 91.2% sensitivity, 100% specificity, positive predictive value of 100%, negative predictive value of 97.6% and accuracy of 98.1%  to 4 (maximum). The higher the score, the greater the perception of improvement.All assessments at T0 and T1 were carried out in person by the researcher. There was no blinding of the researchers.The psychoeducation program was carried out in eight 30-min virtual weekly meetings through Google Meet. For G1 we applied the same psychoeducation program and 30 min of yoga. Two WhatsApp groups managed by the researcher were created, aiming to encourage participation in the meetings. In addition, we sent videos to G1 encouraging the practice of yoga at least twice a week and a calendar for them to register the days they practiced.Our psychoeducation program provided information about the disease, non-pharmacological strategies to deal with NSD, guidelines on how to recognize the signs of overload, as well as strategies to reduce this overload and improve quality of life.Hatha Yoga and among the techniques known in this system we highlighted the Asanas for our work.The yoga exercises chosen for this work originated from the system known as The postures chosen were adapted to the specific conditions of an audience with little affinity for yoga and, in many cases, for physical activity in general. Although we had to adjust the exercises, we did not compromise the principles that guide a traditional yoga practice. This was done to facilitate adherence and involvement of practitioners.Asanas—20 min of exercises done in a sitting position (on chairs); relaxation—5 min (on chairs); and meditation—5 min (on chairs).Classes 1 and 2: Asanas—20 min divided into exercises in standing and sitting positions (standing and on chairs); relaxation—5 min (on chairs); and meditation—5 min (on chairs).Classes 3, 4 and 5: Asanas—20 min (lying down); relaxation—10 min (lying down).Classes 6, 7 and 8: The classes were conducted as follows:The yoga program classes are detailed in Supplementary Information.In addition to the synchronous online classes, we recorded videos that were sent to the participants and encouraged repetition of the practice during the week.The intervention design is shown in Fig. Group differences were evaluated by the chi-squared and Student's t tests, and the generalized linear mixed model (GLMM) was used to compare scores between and within groups at baseline (T0) and 8 weeks after the intervention (T1)Cohen . All anap > 0.05). Detailed sociodemographic characteristics are presented in Table All participants were female, most of them being daughters, 80% in G1 and 75% in G2. In G1, the mean age was 54.96 (± 9.36) years and in G2, 53.54 (± 8.48) years. The two groups did not differ significantly in terms of age; years of schooling; marital status, degree of kinship with the demented person; hours devoted to care; family income; and severity of CDR dementia (F(1) = 4.435, p = 0.041, η2p = 0.086). Additionally, there was no difference between them (F(1) = 0.316, p = 0.577, η2p = 0.007). In the CQOL-AD scale, there were no differences in the total scores; however, improvement was detected in the domains of physical health (F(1) = 4.881, p = 0.032, η2p = 0.094), memory (F(1) = 4.192, p = 0.046, η2p = 0.082) and money (F(1) = 4.862, p = 0.032, η2p = 0.094) in both groups. Comparing the groups, we detected a significantly higher improvement of G1 in memory (F(1) = 4.192, p = 0.046 η2p = 0.082) and money (F(1) = 7.147, p = 0.010, η2p = 0.132). In the friends domain, there was a difference between the groups (F(1) = 4.828, p = 0.033, η2p = 0.093), but the statistical difference must be related to the low valuation of G2 at T1. The G1 when compared to the G2 did not demonstrate significant effects on most of the primary outcomes, including BI-Zarit, MAAS and DASS-21 , G2 = 95.83% (n = 23). As for specific aspects, they had the perception of being able to deal better or much better with the disease and the symptoms of the family member G1 = 82.6% (n = 20); G2 = 75% (n = 18); felt their burden was reduced or greatly reduced G1 = 82.6% (n = 20), G2 = 50% (n = 11); and observed an improvement or a lot of improvement in their quality of life G1 = 91.3% (n = 22), G2 = 66.7% (n = 15). In their perception of burden reduction, there was a significant difference between the groups, which was stronger in the G1 (χ2(3) = 9.074, p = 0.028)  to excellent (4), and the higher the score, the better the quality of life. We did not detect improvement in the global assessment of quality of life, but in the evaluation by domains there was significant improvement in physical health, memory and money in both groups. In the group that practiced yoga, there was higher improvement in the memory and money domains, in addition to the To assess the subjective perception of burden reduction in our satisfaction survey, we used the question: “After the program, do you believe that your burden has been reduced?” There were four response options, ranging from “There was no reduction in burden” to “It greatly reduced burden” and the higher the score, the greater the reduction. We noticed a more significant reduction in the group that, in addition to psychoeducation, had online yoga classes, even though this result was not confirmed by the BI-Zarit scale. This scale also includes the objective assessment of the burden on the caregiver’s personal life resulting from providing care, and the implications for the relationship between the elderly and the caregiver. The scale has 22 questions, the last question being about how much he/she feels overwhelmed. Responses range from never (0) to always (4) and the higher the score, the more severe the overload. The difference in the result between the subjective satisfaction survey and the BI-Zarit scale may have occurred since the practice of yoga provides physical and mental well-being and, consequently, a feeling of relief from the burden of care. The BI-Zarit scale, on the other hand, may have yielded higher scores (greater burden) because it also assesses aspects of objective burden, such as the financial situation, for example.With the social isolation recently imposed by the COVID-19 pandemic, online support alternatives were implemented to assist the caregiver Below is the link to the electronic supplementary material.