Increasing childhood obesity is a global issue requiring potentially local solutions to ensure it does not continue into adulthood. We systematically identified potentially modifiable targets of obesity at the onset and end of puberty in Hong Kong, the most economically developed major Chinese city.n ≤ 7119, WHR n = 5691) and ~17.6 years (n = 3618) at Bonferroni-corrected significance, and multivariable regression to adjust for potential confounders followed by replicated multivariable regression (n = 308) and CpG by CpG analysis (n = 286) at ~23 years. Findings were compared with evidence from published randomized controlled trials (RCTs) and Mendelian randomization (MR) studies.We conducted an environment-wide association study (EWAS) and an epigenome-wide association study of obesity to systematically assess associations with body mass index (BMI) and waist–hip ratio (WHR) in Hong Kong’s population-representative ‘Children of 1997’ birth cohort. Univariable linear regression was used to select exposures related to obesity at ~11.5 years , physical activity, snoring, binge eating, and earlier puberty were positively associated with BMI at ~17.6 years, while eating before sleep was inversely associated with BMI at ~17.6 years. Findings for birth weight, dairy intake, and binge eating are consistent with available evidence from RCTs or MR studies. We found 17 CpGs related to BMI and 17 to WHR.These novel insights into potentially modifiable factors associated with obesity at the outset and the end of puberty could, if causal, inform future interventions to improve population health in Hong Kong and similar Chinese settings.This study including the follow-up survey and epigenetics testing was supported by the Health and Medical Research Fund Research Fellowship, Food and Health Bureau, Hong Kong SAR Government (#04180097). The DNA extraction of the samples used for epigenetic testing was supported by CFS-HKU1. With improving living standards and socioeconomic development, non-communicable chronic diseases pose a heavy burden on society in both developed and developing countries . ObesityEnvironment-wide association studies (EWAS) enable us to assess a variety of exposures across the human environmental exposome in a high-throughput manner , similarIn addition to the environmental factors, it is increasingly realized that epigenetic factors, which are also modifiable, may play an important role in obesity . DNA metIn this situation, Hong Kong, a non-western developed setting, can provide unique insights into health determinants. Most Chinese people in Hong Kong are first-, second-, or third-generation migrants from the neighbouring province of Guangdong in southern China. Dietary habits of people in Hong Kong are similar to those in southern China, although also influenced by western culture . Lifestyn = 8327) population-representative Chinese cohort in Hong Kong  was calculated based on waist and hip circumference collected in Survey I conducted in 2008–2009. At ~17.6 years, in both phases of the Biobank Clinical follow-up, BMI was assessed by bio-electrical impedance analysis (BIA) with a Tanita segmental body composition monitor . Waist and hip circumference measurements were made using a tape twice following a standard protocol by trained technicians and nurses. Given BMI has a more accepted cut-off value than WHR for children, we classified obesity as BMI ≥20.89 kg/mage 17.6 . In the DNA methylation was conducted in 286 participants randomly selected from the 308 participants in the follow-up survey. DNA were extracted from buffy coat samples previously stored at −80° using EZI DNA blood kit (QIAGEN) with magnetic particle technology. DNA methylation was assessed using the Illumina Methylation EPIC Beadchip, which interrogated the methylation status of over 850,000 CpG sites. We conducted quality control using the ‘ewastools’ package , which i−4 for obesity at ~17.6 years) to account for multiple testing (n = 308) at age ~23 years and compared the direction of associations with those at earlier age groups (~11.5 and ~17.6 years). Associations with consistent directions of associations in earlier age groups (~11.5 or ~17.6 years) with those at ~23 years suggest a consistent association by age. Additionally, to account for the time lag between the age at which physical measurements were taken and age at exposure collection, we included a time difference variable in the adjusted models for BMI. Finally, exposures that remained after controlling for confounders, were compared with the evidence from existing RCTs and Mendelian randomization (MR) studies, a study design which uses genetic variants as instrument and provides less confounded associations  was not used given the relatively small sample size in the epigenome-wide association study. To estimate genomic inflation, we used a Bayesian method that estimates inflation more accurately in epigenome-wide association studies based on the empirical null distributions  consent for the original survey and subsequent record linkage was obtained from the parents, next of kin, caretakers, or guardians (informants) on behalf of the participants by the informant agreeing and subsequently completing the questionnaire at enrollment, this manner of obtaining consent was approved by The University of Hong Kong Medical Faculty Ethics Committee over 20 years ago. Informed written consent for subsequent Surveys and in-person follow-up was obtained from a parent or guardian, or at ages 18+ years from the participant. Ethical approval for this study, including the follow-up survey at ~23 years and comprehensive health-related analyses, was obtained from the University of Hong Kong-Hospital Authority Hong Kong West Cluster, Joint Institutional Review Board, Hong Kong Special Administrative Region, China .Specifically, for obesity at ~11.5 years, we found sex , higher birth weight, maternal second-hand smoking, higher parental weight, family history of diabetes, gestational diabetes, and more water consumption were associated with higher BMI, while being small for gestational age and spending more time having meals were associated with lower BMI at ~11.5 years . Except For obesity at ~17.6 years, in addition to some shared factors, including sex, birth weight, parental weight, and maternal second-hand smoking, we found some aspects of diet , physical activity, health status , earlier puberty and binge eating associated with higher BMI at ~17.6 years . Being aRegarding the comparison with RCTs and MR studies, we found RCTs on dark chocolate consumption , water cRBM16, SCN2B, SLC24A4, TECPR2, KSR1, RPTOR, GTF3C3, ZNF827, TXNDC15, C2, and RPS6KA2 and 17 for WHR in the genes LANCL2, C6orf195, MIR4535, CTRL, LYRM9, DCDC2, DIRC3, RPS6KA2, LPP, NFIC, MIR7641-2, ZNF141, RNF213, and OPA3 (RPS6KA2 was a shared CpG for both BMI and WHR. The genetic inflation factors (lambda) were 1.006 and 1.005, respectively.In the epigenome-wide association study of 286 participants we identified 17 CpGs for BMI at ~23 years in the genes and OPA3  and 4. cIn this environment- and epigenome-wide association study, we systematically examined associations of over 400 exposures with obesity in a unique Chinese birth cohort, as well as the association of DNA methylation with obesity. Building on the previous studies in this birth cohort , we not Our study found that maternal second-hand smoking was consistently associated with obesity at different ages, which is consistent with the concerns repeatedly raised in previous studies , and addRegarding dietary factors, as the dietary assessments were more comprehensively conducted in the Biobank Clinical follow-up, the identified dietary factors were mainly for obesity at ~17.6 years. Interestingly, we found that children who consume more ASB have higher BMI, which is consistent with meta-analyses of cohort studies . ConsistAnother interesting finding is that milk consumption was not related to obesity at ~11.5 years, while reduced-fat/skim milk consumption was associated with higher BMI at ~17.6 years, with a consistent direction of associations for BMI at ~23 years. Our findings are consistent with a previous study in this cohort, which showed milk consumption frequency was not associated with BMI at 13 years , howeverWe also found tea or coffee consumption were associated with higher BMI at ~17.6 years. RCTs of coffee or tea consumption are scarce among children and adolescents because they require long-term adherence. MR studies do not suggest that coffee consumption affects obesity , so the Echoing the increasing attention to the role of mood and emotion in obesity control , we founAs regards lifestyle, consistent with previous observational studies , we founConsistent with previous studies , we founRPS6KA2 was associated with both BMI and WHR, consistent with the previous epigenome-wide association studies of obesity in different populations  feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Mone Zaidi as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Environment-wide and epigenome-wide association study of adiposity in "Children of 1997" birth cohort" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:1. The authors analyzed the association between exposures and obesity using linear regression. It is recommended to additionally perform logistic regression by converting BMI and waist circumference into binary dependent variables. Appropriate scientific evidence for cut-off values of BMI and waist circumference in adolescence and adulthood, respectively, should be provided. Regarding waist circumference, it is necessary to consider the cut-off value of each male and female.2. The authors used a false discovery rate (FDR)<0.05 as the significance threshold for the epigenome-wide association study. However, considering the previous studies including 'Rakyan VK, et al. Epigenome-wide association studies for common human diseases. Nat Rev Genet 2011', it is appropriate to use a significance threshold between E-08 to E-06 for epigenome-wide association studies. If the sample size is not sufficient to proceed with the analysis using these significance thresholds, it is appropriate to delete the epigenome-wide association study from the manuscript.3. In the limitations of this study, the authors used the following expression: "… despite the minimal level of confounding in this cohort…"In presenting the limitations of this study, the authors used the following expression: "despite the minimal level of confounding in this cohort"Considering the following points, this expression is wrong:This is an observational study, and a large amount of confounding effect should be considered on the association between obesity and exposures analyzed in this study including diet, physical activity, mood and feelings, family history, and socioeconomic status. Since the confounders considered in the multivariable analysis are very limited, the possibility of residual confounding remains large. These limitations should be detailed in the discussion.4. Since the outcome variables were measured by body mass index and waist-hip ratio, it is more appropriate to use the term "obesity" rather than "adiposity". It is recommended that the term "adiposity" used in the manuscript be changed to "obesity".5. Only part of the analysis code is presented, not the entire code. The whole analysis code used for the actual analysis needs to be provided.6. Provide information regarding definition of obesity according to age.7. Provide general population characteristics of each age group 8. Provide the time difference between the age at the time of physical measurement and age at the exposure (survey period) and consider to include as variable.9. Provide the basis for expressing onset and at end of puberty.10. Change the term 'health' to another term as it is inappropriate to list  etc.11. Modify the way the table is organized and correct the order of variable description in table. For example, make two tables 4 and 5 into one, and check the order of enumeration of variables Reviewer #1 (Recommendations for the authors):1. The authors analyzed the association between exposures and obesity using linear regression. It is recommended to additionally perform logistic regression by converting BMI and waist circumference into binary dependent variables. Appropriate scientific evidence for cut-off values of BMI and waist circumference in adolescence and adulthood, respectively, should be provided. Regarding waist circumference, it is necessary to consider the cut-off value of each male and female.2. The authors used a false discovery rate (FDR)<0.05 as the significance threshold for the epigenome-wide association study. However, considering the previous studies including 'Rakyan VK, et al. Epigenome-wide association studies for common human diseases. Nat Rev Genet 2011', it is appropriate to use a significance threshold between E-08 to E-06 for epigenome-wide association studies. If the sample size is not sufficient to proceed with the analysis using these significance thresholds, it is appropriate to delete the epigenome-wide association study from the manuscript.3. In the limitations of this study, the authors used the following expression: "… despite the minimal level of confounding in this cohort…"In presenting the limitations of this study, the authors used the following expression: "despite the minimal level of confounding in this cohort"Considering the following points, this expression is wrong:This is an observational study, and a large amount of confounding effect should be considered on the association between obesity and exposures analyzed in this study including diet, physical activity, mood and feelings, family history, and socioeconomic status. Since the confounders considered in the multivariable analysis are very limited, the possibility of residual confounding remains large. These limitations should be detailed in the discussion.4. Since the outcome variables were measured by body mass index and waist-hip ratio, it is more appropriate to use the term "obesity" rather than "adiposity". It is recommended that the term "adiposity" used in the manuscript be changed to "obesity".5. Only part of the analysis code is presented, not the entire code. The whole analysis code used for the actual analysis needs to be provided.Reviewer #2 (Recommendations for the authors):Please provide information regarding definition of adiposity according to age.Please provide general population characteristics of each age group Please provide the time difference between the age at the time of physical measurement and age at the exposure (survey period) and consider to include as variable.Please provide the basis for expressing onset and at end of puberty.Please changed the term 'health' to another term as it is inappropriate to list  etc.Please modify the way the table is organized and correct the order of variable description in table. For example, make two tables 4 and 5 into one, and check the order of enumeration of variables  Reviewer #1 (Recommendations for the authors):1. The authors analyzed the association between exposures and obesity using linear regression. It is recommended to additionally perform logistic regression by converting BMI and waist circumference into binary dependent variables. Appropriate scientific evidence for cut-off values of BMI and waist circumference in adolescence and adulthood, respectively, should be provided. Regarding waist circumference, it is necessary to consider the cut-off value of each male and female.Thank you very much for your helpful comments. In the environment-wide association study, we used BMI, a continuous outcome, to maximize power in the selection of exposures passing Bonferroni-corrected significance. Then we examined the associations of these exposures with BMI after controlling for confounders in multivariable regression. Following your suggestions, in the multivariable regression, we converted BMI into a binary outcome  and performed logistic regression adjusted for potential confounders. We adopted the obesity cut-off values of BMI for different age groups with sex-specific cut-offs (1):– BMI ≥ 20.89 kg/m2 for boys and BMI ≥ 21.20 kg/m2 for girls at age 11.5– BMI ≥ 24.73 kg/m2 for men and BMI ≥ 24.85 kg/m2 for women at age 17.6The directions of associations are consistent with those using BMI as continuous outcome, the comparisons are shown in Table 2 and Table 4.It would be interesting to explore the associations with central obesity as a binary outcome as well, however, given the lack of high-quality evidence about the cut-off values for waist circumference and waist-to-hip ratio in Asian children and adolescents, we did not perform logistic regression on central obesity risk. We have revised the methods and results:We added “Given BMI has a more accepted cut-off value than WHR for children, we classified obesity as BMI ≥ 20.89 kg/m2 for boys and BMI ≥ 21.20 kg/m2 for girls at age 11.5; and BMI ≥ 24.73 kg/m2 for men and BMI ≥ 24.85 kg/m2 for women at age 17.6 (1).”We updated Tables 2 and Table 4:“Except for paternal diabetes which was not significant, the rest of exposures all showed consistent associations with obesity risk (Table 2).”“Regarding the association with obesity risk at 17.6 years, birth weight, being a twin, maternal second-hand smoking, physical activity and energy drinks intake were not significant but the directions of associations are consistent (Table 4).”We added in the discussion:“Third, we obtained similar results for BMI and obesity risk, however, given the lack of high-quality evidence about the cut-off values for waist circumference and waist-to-hip ratio in Asian children and adolescents, we did not perform logistic regression on central obesity risk.”2. The authors used a false discovery rate (FDR)<0.05 as the significance threshold for the epigenome-wide association study. However, considering the previous studies including 'Rakyan VK, et al. Epigenome-wide association studies for common human diseases. Nat Rev Genet 2011', it is appropriate to use a significance threshold between E-08 to E-06 for epigenome-wide association studies. If the sample size is not sufficient to proceed with the analysis using these significance thresholds, it is appropriate to delete the epigenome-wide association study from the manuscript.Thank you for your helpful suggestion. We have changed the threshold to 1×10-6 in the epigenome-wide association study, and updated the methods and results accordingly.We revised to “The significance was considered as p<1×10-6, genome-wide significance (5×10-8) was not used given the relatively small sample size in the epigenome-wide association study.”We revised to “In the epigenome-wide association study of 286 participants we identified 17 CpGs for BMI at ~23 years in the genes RBM16, SCN2B, SLC24A4, TECPR2, KSR1, RPTOR, GTF3C3, ZNF827, TXNDC15, C2, and RPS6KA2 and 17 for WHR in the genes LANCL2, C6orf195, MIR4535, CTRL, LYRM9, DCDC2, DIRC3, RPS6KA2, LPP, NFIC, MIR7641-2, ZNF141, RNF213, and OPA3 .”3. In the limitations of this study, the authors used the following expression: "… despite the minimal level of confounding in this cohort…"In presenting the limitations of this study, the authors used the following expression: "despite the minimal level of confounding in this cohort"Considering the following points, this expression is wrong:This is an observational study, and a large amount of confounding effect should be considered on the association between obesity and exposures analyzed in this study including diet, physical activity, mood and feelings, family history, and socioeconomic status. Since the confounders considered in the multivariable analysis are very limited, the possibility of residual confounding remains large. These limitations should be detailed in the discussion.Thank you very much for your very helpful suggestion, we totally agree that our study is an observational study and inevitably open to residual confounding, although we have controlled for sex, housing type at birth, household income at birth, maternal second-hand smoking during pregnancy, maternal age at birth, maternal education, maternal birthplace, and the interaction of maternal education with maternal birthplace. So, we also compared the associations from multivariable regression with available evidence from Mendelian randomization studies, which uses genetic variants as instrument to minimize confounding. We have added more in the discussion.We revised from: “Finally, despite the minimal level of confounding in this cohort for some key exposures, such as breastfeeding, residual confounding likely exists, so the associations are not definitive.”To: “Finally, although we controlled for several confounders in the multivariable analysis, residual confounding may still exist given our study is observational. Comparing our study with evidence from MR studies which are less likely to be confounded (2-5), we found a consistent direction of association for dairy intake (39) and binge eating (40) being associated with higher BMI. Evidence for some exposures, such as tea and chocolate consumption, is still lacking; further MR studies are needed to assess causality.”4. Since the outcome variables were measured by body mass index and waist-hip ratio, it is more appropriate to use the term "obesity" rather than "adiposity". It is recommended that the term "adiposity" used in the manuscript be changed to "obesity".Thank you very much for your very helpful suggestion. We have changed the term "adiposity" to "obesity" in the manuscript throughout.5. Only part of the analysis code is presented, not the entire code. The whole analysis code used for the actual analysis needs to be provided.Thank you very much for your comments. We previously showed part of the analysis code in the environment-wide association study because the analysis code is similar for BMI and WHR, and for different ages. As you suggested, we have uploaded the whole analysis code along with the revised manuscript.Reviewer #2 (Recommendations for the authors):Please provide information regarding definition of adiposity according to age.Thank you very much for your helpful suggestion. We added the definition of obesity by the World Health Organization (WHO), and also addressed the cut-off values specific to Asian population by age and by sex.“Obesity is a well-established risk factor for multiple chronic diseases, including cardiovascular disease, diabetes, and cancer (6). According to the World Health Organization (WHO), obesity is defined as “abnormal or excessive fat accumulation that presents a risk to health” (7). Body mass index (BMI) is the most appropriate measure for overweight and obesity because the cut-offs account for age, sex, and ethnicity (7). Obesity has increased substantially in many settings, including Hong Kong…”We clarified the obesity cut-off by adding the following sentence:2 for boys and BMI ≥ 21.20 kg/m2 for girls at age 11.5; and BMI ≥ 24.73 kg/m2 for men and BMI ≥ 24.85 kg/m2 for women at age 17.6 (1).”“Given BMI has a more accepted cut-off value than WHR for children, we classified obesity as BMI ≥ 20.89 kg/mPlease provide general population characteristics of each age group Thank you very much for your helpful suggestion. We have added information of each age group  in Table 1.Please provide the time difference between the age at the time of physical measurement and age at the exposure (survey period) and consider to include as variable.Thank you very much for your suggestions. We understand that the time difference between the age at the time of physical measurement and age at the exposure (time of survey period) is an important aspect and sometimes may lead to imprecision in measurement in large cohort studies. The Biobank Clinical follow-up at 17.6 years involved collecting questionnaires from children and parents, and anthropometric measurements from participants in one visit, so the time difference is minimal. However, for exposures collected at baseline and the Survey I, there may be a difference between the age at which physical measurements were taken and age at the exposure. To address this issue, we included a variable for the time differences associated with these exposures, and results were similar after including the time difference variable (Supplementary file 2). We added the following sentences in the Methods and Results:“Additionally, to account for the time lag between the age at which physical measurements were taken and age at exposure collection, we included a time difference variable in the adjusted models for BMI.”“The associations of selected exposures with BMI at ~11.5 years and at 17.6 years were similar after adjusting for the time difference between age at anthropometric measurements and age of exposure collection (Supplementary file 2).”We added: “After accounting for the time difference between age at anthropometric measurements and age of exposure collection, we also obtained similar results.”Please provide the basis for expressing onset and at end of puberty.Thank you very much for raising this question. We considered puberty because puberty is an important stage which involves a re-orientation from childhood priorities to adulthood (24). Exposures associated with adiposity at puberty may be important for health in later life (25). We considered onset and at the end of puberty separately, because due to the changes in hormones during puberty, exposures related to obesity at the onset and at the end of puberty may be different, the same exposure may also have different effects on obesity. We added more explanation in the Introduction section:“We focused on puberty because it is an important stage involving a re-orientation from childhood priorities to adulthood (24). Exposures associated with obesity at puberty may be important for health in later life (25). Considering that exposures related to obesity at the outset and at the end of puberty may be different, and the associations of the same exposure with obesity may vary by age, we conducted the EWAS at different ages.”Please changed the term 'health' to another term as it is inappropriate to list  etc.Thank you very much for pointing out the inappropriate term, we have incorporated your suggestion throughout the manuscript by changing ‘health’ into ‘health status’, with a note that the term here refers to physical health condition. Please refer to the tracked changes in the revised manuscript (Exposures and categorization of Methods) and revised Supplementary file 1, Figures 1 and 2.We revised to “The exposures considered … child’s health status , parents’ health status,…”Please modify the way the table is organized and correct the order of variable description in table. For example, make two tables 4 and 5 into one, and check the order of enumeration of variables Thank you very much for this suggestion, we have incorporated your suggestion and reorganized the tables by combining Tables 2 and 3, Tables 4 and 5, Tables 6 and 7, Tables 8 and 9.References1. Cole TJ. Establishing a standard definition for child overweight and obesity worldwide: international survey. BMJ. 2000;320(7244):1240-.2. Reed ZE, Micali N, Bulik CM, Davey Smith G, Wade KH. Assessing the causal role of adiposity on disordered eating in childhood, adolescence, and adulthood: a Mendelian randomization analysis. Am J Clin Nutr. 2017:ajcn154104.3. Gill D, Brewer CF, Del Greco MF, Sivakumaran P, Bowden J, Sheehan NA, et al. Age at menarche and adult body mass index: a Mendelian randomization study. Int J Obes. 2018;42(9):1574-81.4. Bell JA, Carslake D, Wade KH, Richmond RC, Langdon RJ, Vincent EE, et al. Influence of puberty timing on adiposity and cardiometabolic traits: A Mendelian randomisation study. PLoS Med. 2018;15(8):e1002641.5. Chen YC, Fan HY, Yang C, Hsieh RH, Pan WH, Lee YL. Assessing causality between childhood adiposity and early puberty: A bidirectional Mendelian randomization and longitudinal study. Metabolism. 2019;100:153961.6. Gallagher EJ, LeRoith D. Obesity and Diabetes: The Increased Risk of Cancer and Cancer-Related Mortality. Physiol Rev. 2015;95(3):727-48.7. World Health Organization. Regional Office for the Western P. The Asia-Pacific perspective : redefining obesity and its treatment: Sydney : Health Communications Australia; 2000 2000.


Oncomelania hupensis, has not changed much in recent years. Different environmental types have different impacts on snail breeding, and understanding these differences is conducive to improving the efficiency of snail monitoring and control and to saving resources. Based on previous epidemiological data, we selected 199 villages in 2020 and 269 villages in 2021 from transmission control, transmission interruption, and elimination areas of snail breeding. Snail surveys were conducted in selected villages using systematic sampling and/or environmental sampling methods in six types of snail-breeding environments . All live snails collected from the field were evaluated for Schistosoma japonicum infection using the microscopic dissection method, and a subsample of snails was subjected to loop-mediated isothermal amplification (LAMP) to assess the presence of S. japonicum infection. Snail distribution data and infection rate and nucleic acid positive rate of schistosomes in snails were calculated and analyzed. The 2-year survey covered 29,493 ha of the environment, in which 12,313 ha of snail habitats were detected. In total, 51.16 ha of new snail habitats and 107.76 ha of re-emergent snail habitats were identified during the survey. The occurrence rate of snails in canals  and undefined environments  was relatively high in 2020, and the density of snails in bottomlands  and undefined environments  was relatively high in 2021. Of the 227,355 live snails collected in this study, none were S. japonicum-positive as determined by microscopy. Of the 20,131 pooled samples, however, 5 were S. japonicum-positive based on LAMP analysis, and they were distributed in three environmental types: 3 in bottomland, 1 in dry land, and 1 in a canal. The bottomland environment has a high risk of schistosomiasis transmission because it contains a large area of newly emerging and re-emerging snail habitats, and it also had the most breeding snails infected with S. japonicum. Thus, this habitat type should be the key target for snail monitoring and early warning and for the prevention and control of schistosomiasis.The goal of schistosomiasis prevention and control in China is shifting from transmission interruption to elimination. However, the area inhabited by the intermediate host, the snail  Schistosoma japonicum, which is transmitted through various means. In China, this disease poses a serious public health threat, and its transmission is largely dependent on the freshwater snail Oncomelania hupensis, which serves as the sole intermediate host for S. japonicum [S. japonicum infections [Schistosomiasis is a zoonotic disease caused by the parasitic trematode worm aponicum ,2. Despiaponicum  has led fections . Currentfections .O. hupensis has remained at about 3.5 billion m2 since 2004, and newly emerging and re-emerging snail habitats have been reported in many areas [S. japonicum to complete its life cycle in the wild [S. japonicum infection in snails can miss its presence, which increases the risk of schistosomiasis transmission [However, the area inhabited by ny areas ,6. Thus,ny areas  has a withe wild . Existinsmission . Researcsmission .O. hupensis, detect S. japonicum infection of snails in different environments in the field, and map newly emerging and re-emerging snail habitats. We studied O. hupensis for two consecutive years from 2020 to 2021 in schistosomiasis-endemic areas, including transmission control, transmission interruption, and elimination areas where snails breed.Due to the wide distribution of snails and the variety of snail-breeding environments, it is increasingly important to identify the risks in each environment, implement targeted prevention and control measures, and improve the level of monitoring and early warning. This approach will lay a solid foundation for shifting schistosomiasis control in China from transmission interruption to elimination. The goals of this study were to evaluate the distribution of Based on epidemiological data from previous years, we selected one administrative village from each endemic county with expected potential risk of schistosomiasis transmission. In 2020 and 2021, 199 and 269 villages, respectively, were selected. The villages mentioned above shared at least one of the following characteristics: (1) previously had a high prevalence of schistosomiasis, and the snail habitats in their environment remained essentially unchanged; (2) exhibited an increase in breeding areas or density of living snails, detected infected snails or the emergence of new snail habitats, and had a relatively high rate of infection or positivity for antibodies in humans or livestock according to the latest survey; (3) experienced significant changes in their snail-breeding habitats due to natural disasters, such as floods or earthquakes, or the construction of large-scale water conservancy, transportation, and other projects; or (4) underwent large-scale population migration or movement, which increased the likelihood of schistosomiasis occurrence.S. japonicum in all snails collected , and loop-mediated isothermal amplification (LAMP) was used to detect at least 500 live snails . In order to improve the monitoring efficiency of the risk environment, the number of live snails detected by LAMP in each snail environment was not to be less than 100  and/or suspicious environments, and the total number of snail inspection frames in each risk monitoring village was not to be less than 200 frames, and all snails in the frame were to be captured. In the spring of each year, systematic sampling and/or environmental sampling methods were used to investigate snails in the environment with snails and/or suspicious environment, and the longitude and latitude of each environment were recorded. Crushing microscopy was used to detect We conducted the snail survey in each village in the spring of 2020 and 2021 using systematic sampling and/or environmental sampling methods.2 (31.7 cm × 31.7 cm). The interval distance between checkpoints was determined based on the size and length of the environment at that time. We adopted the method of inspecting every 5 m and 10 m for river ditches and ponds. To conduct snail surveys on rivers and lakes, beaches, fields, and other areas, we adopted a vertical and horizontal systematic sampling  survey method. We set several parallel inspection lines on the beaches and fields, then set inspection points (boxes) along each inspection line at equal distances. For the distance between lines and the distance between points on each line, we used a space of 5 to 20 m based on the size of the beaches and fields. We increased the distance length for environments for large beaches, but none exceeded 50 m. We divided it into several blocks in an environment with an extensive beach area. Then, we conducted a vertical and horizontal systematic sampling survey within each block so that the distribution of checkpoints evenly penetrated the entire survey area. We captured all the snails at the inspection point and registered the location, environment type, number of snails, and other relevant information. Then, in the laboratory, we observed the survival rate, detected the positive rate of snails, and recorded the occurrence rate of positive snail frames.For environments where snails are abundant and scattered, we adopted a systematic sampling survey method. The specific process was to set up a checkpoint at a certain distance and check one frame at each point, with a frame size of 0.1 m2. Then, within each block, we used a vertical and horizontal system to select points (frames), generally 100 to 200 points (frames) per area; if we did not detect snails at four adjacent points (frames) in the vertical and horizontal system sampling, we searched for four points (frames) in the area between the four points where snails are prone to breeding.During the survey, we set up a systematic sampling survey with a frame every 5 m or 10 m equidistant for snail-breeding environments, such as rivers, ditches, ponds, and ridges. If we did not detect snails at two adjacent points (frames), we selected an environment where snails are easy to breed in the interval between the two points (frames) and randomly inspected two points (frames) simultaneously. We used systematic sampling combined with environmental sampling to calculate the area with snails, which extended 20 m from the left to the right. For a large beach, we first divided it into several blocks, each with an area of about 133,340 m2 frame. Based on the previous survey, we employed a systematic sampling approach in environments containing breeding snails. In neighboring habitats, we conducted environmental sampling first and then systematic sampling when we found living snails. We determined the distance between any two frames and the number of frames used based on the ecological characteristics of the surveyed environments. Subsequently, we tallied and collected all snails within the frames and brought them to the laboratory for examination.We surveyed various environments, including canals, ponds, paddy fields, dry lands, bottomlands, and undefined areas. To count snails, we used a 0.1 mO. hupensis snails based on their shell morphology, we placed them in a dish filled with dechlorinated water at room temperature for 2–3 h. Any snail showing signs of movement, extension of soft tissue, or contraction after being probed was considered alive. The living snails were gently crushed using another thick slide and examined under a microscope after adding a drop of dechlorinated water. To determine if a snail was infected with S. japonicum, we examined its soft tissue under a microscope and looked for sporocysts and/or cercariae. If we observed sporocysts and/or cercariae of S. japonicum in the snail’s soft tissue, we considered it infected (After identifying and washing the infected .We used the TIANamp Genomic DNA Kit (TIANGEN BIOTECH (BEIJING) CO., Ltd., Beijing, China) and followed the manufacturer’s protocol and obtain the required DNA from snails using this DNA extraction kit. Groups of five tubes of extracted DNA from the same environment were pooled into one sample for loop-mediated isothermal amplification (LAMP) detection.S. japonicum was detected by using genomic DNA extracted from pooled snail samples as a template for the LAMP assay. The primers targeting the mitochondrial 28S rRNA gene of S. japonicum were used in the reaction, which was carried out in a final volume of 25 µL. The primer sequences were: F3 (5′-GCTTTGTCCTTCGGGCATTA-3′), B3 (5′-GGTTTCGTAACGCCCAATGA-3′), FIP (5′-ACGCAACTGCCAACGTGACATACTGGTCGGCTTGTTACTAGC-3′), and BIP (5′-TGGTAGACGATCCACCTGACCCCTCGCGCACATGTTAAACTC-3′).The DNA of The volume of each component was 2.0 µL (genomic DNA template), 12.5 µL (2 × LAMP reaction buffer), 1.0 µL (amplification primer mixture), 7.5 µL (sterilized deionized water), 1.0 µL (Bst DNA polymerase), and 1.0 µL (chromogenic reagent (New England Biolabs).S. japonicum and sterilized deionized water were used to replace the extracted DNA template, respectively. After incubating the reaction tubes at 65 °C for 60–90 min, we observed visually without the need for any additional instrumentation or analysis. Tubes with green liquid indicated the presence of S. japonicum DNA and were considered positive, while the remaining tubes with yellow-brown liquid were negative.Positive and negative controls were included in each amplification reaction, where genomic DNA extracted from adult All selected villages were uniformly numbered and located in longitude and latitude. We collected the area of new and recurrent snails corresponding to each geographical location and built a database from 2020 to 2021. We then imported the database in to ArcGIS 10.1 software to generate a map of the distribution of snails in traditional and newly developed or re-emerging snail habitatsWe recorded annual data and organized it in a Microsoft Excel spreadsheet. We calculated the snail distribution area by multiplying the length (m) and width (m) of the surveyed environments. The dimensions were based on the furthest distance between two frames where either live or infected snails were found. To determine statistical significance, we calculated confidence intervals (CIs) using standard formulas based on the binomial distribution.We first selected all the transmission control counties and at least 20% of the counties with snails (including transmission interruption counties and elimination counties). We then selected some villages in these selected counties to survey snail breeding in the field in areas with different environmental types. Ultimately, we surveyed 199 villages in 2020 and 269 villages in 2021.We conducted this investigation in an area covering 29,492.81 ha. Among the 12,312.92 ha confirmed to contain breeding snails, 51.16 ha were newly developed snail habitats, including canals (2623 ha), ponds (247.23 ha), paddy fields (2416.77 ha), dry lands (1073.31 ha), bottomlands , and undefined environments (723.06 ha). Additionally, 1072.76 ha were re-emergent snail habitats, including canals (952.26 ha), ponds (79.64 ha), paddy fields (300.98 ha), dry lands (150.12 ha), bottomlands , and undefined environments (299.22 ha).During the snail investigation in 2020–2021, we investigated 931,735 frames and discovered 227,355 living snails in 67,798 frames. The percentage of frames containing living snails varied between 6.88%  and 7.69% . Furthermore, the average live snail density ranged from 0.19  in 2020 to 0.3  .The percentages of frames containing living snails in the undefined and canal environments were high  in 2020, while the values in pond and dry land environments were low  .The average density of snails varied among the different environments. The mean densities of snails in bottomland and undefined environments were relatively high  in 2021, whereas densities in pond and dry land environments were low  in 2020 .S. japonicum-positive snails were detected by microscopy. We randomly selected soft tissue from 134,302 dissected snails, which accounted for 59.07% of the total snail population. Then, we extracted DNA from these samples to obtain 20,131 mixed nucleic acid samples. Among those mixed samples, only five were positive for S. japonicum in the LAMP analysis. Three of the positive nucleic acid-mixed samples were found in a bottomland environment , one was found on a dry land environment , and one was found in a canal  environment. The mean positive rate of mixed nucleic acid samples was 0.018% (95% CI: 0.00–0.039%) in 2020 and 0.059% (95% CI: 0.00–0.13%) in 2021  in 2020, and in the canal environment, with the second-highest average positive rate .It is crucial to comprehend the distribution of its vector, somiasis ,19. SnaiO. hupensis is the only intermediate host of S. japonicum. The effective control of snails, mainly through environmental management, has played a significant role in successfully controlling this parasite in China [O. hupensis control is one of the important means to control schistosomiasis and is also one of the important measures in the medium- and long-term planning of schistosomiasis control in China. At the same time, China has been actively exploring methods, such as biological and drug snail control, to combat schistosomiasis. However, due to the highly complex breeding environment of schistosomiasis, the large size of the existing snail population, and restricted access to some snail-breeding areas, it has been challenging to implement effective snail control measures. As a result, the snail-breeding area remains extensive making it difficult to achieve the goal of eradicating snails in the future. In addition, the close communication between people in schistosomiasis-endemic areas, the frequent circulation of goods, and the frequent scattering of livestock in endemic areas make the transmission risk of schistosomiasis japonica still existent. Therefore, studies of the breeding environments of O. hupensis are needed for the rational use of resources, control of snails, reduction in the risk of schistosomiasis transmission, and provision of data support for accurate prevention and control of snails as well as for a reference for the elimination of schistosomiasis in the future.Newly developed and re-emergent snail habitats are still a big challenge in endemic areas  because in China . O. hupeThis study has some limitations that should be acknowledged. Firstly, the selection of villages at potential risk of schistosomiasis was not randomized, and due to cost constraints, only one third of the dissected snails were tested using the LAMP method. This may have resulted in a biased estimation of the snail infection status. Secondly, while the LAMP results could identify areas with transmission risk, the pooling strategy used could not provide an accurate estimation of the snail infection rate. Future research should explore modeling methods to estimate infection rates and transmission intensity accurately. Third, the snail-breeding environment is very complex, which makes it difficult to define certain areas as being a single environment type. To address this issue, we used an undefined environment category, which makes the snail survey more comprehensive and accurate.S. japonicum, as shown by nucleic acid detection. In the future, the monitoring and early warning of snails in bottomland areas should be a top priority for the prevention and control of schistosomiasis.The areas of newly developed and recurrent snail habitats are basically consistent with the results predicted by our previous research. Among the six kinds of snail-breeding environments, bottomland areas have a high risk of schistosomiasis transmission because they contain large areas of newly emerging and re-emerging snail habitats and they have the highest proportion of breeding snails infected with


BackgroundA dental implant is one of the most commonly used treatments to replace missing teeth. A reasonable number of implant cases necessitate using a bone graft before or at the time of implant placement. This study aims to evaluate the quality and readability of online patient-centered information about implant bone grafts.MethodologyThis cross-sectional study used Google, Yahoo, and Bing search engines. The keywords were entered to screen 900 websites. The DISCERN, Journal of the American Medical Association (JAMA), and Health on the Net (HON) code tools evaluated the included websites for quality. The Flesch reading-ease score (FRES), Flesch-Kincaid grade level, and simple measure of gobbledygook tests measured readability. Statistical analysis was done using SPSS version 25 .ResultsA total of 161 websites were included; 65 (40.4%) of the included websites belonged to a university or medical center. Only five (3.1%) websites were exclusively related to dental implant treatments. DISCERN showed moderate quality for 82 (50.9%) websites. There was a statistical difference between commercial and non-profit organization websites. In the JAMA evaluation, currency was the most commonly achieved in 67 (41.6%) websites. For the HON code, four (2.5%) websites were certified. Based on FRES, the most common readability category was “fair difficult,” accounting for 64 (39.8%), followed by “standard” in 56 (34.8%) websites.ConclusionsThe study findings suggest that English-language patient-centered information about implant bone grafts is challenging to comprehend and of low quality. Hence, there is a need to establish websites that provide trustworthy, high-quality information on implant bone grafts. Tooth loss can be a serious issue affecting millions of people worldwide. In fact, in 2015, a staggering 276 million people experienced complete tooth loss. Poor oral hygiene and careless behavior about oral health can lead to periodontal problems and teeth loss. Recent studies investigating the reasons for teeth extraction found that the most common reason was dental caries, followed by periodontal diseases .For replacing missing teeth, people opt for different treatments. The survival rate differs according to the type of treatment. For example, the single crown supported by implant survival rate was 96.363%. On the other hand, it was 94.525% for fixed partial dentures and 91.27% for implant tooth-supported prostheses after five years [After tooth extraction, biological and physiological bone remodeling occurs. There are some debates about bone resorption and the preservation of alveolar bone with the use of an implant because of the percentage of bone loss around the implant, even if it is low. Until now, there is not enough evidence to conclude whether the implant can preserve the alveolar bone height ,7. The aImplant placement requires good bone quality and morphology, which includes bone height and width. In cases where bone is deficient, bone graft or ridge augmentation is indicated. There are several types of bone graft materials, each with their specific use and properties . Based oThe utilization of websitesThe technique used for finding websites involved searching and choosing from a common English search engine, such as Google, Yahoo, and Bing. The searches were accomplished in incognito mode to guarantee impartial outcomes, and cookies and browser data were cleared preceding browsing. The search was based on the following keywords: “Dental bone graft” and “Implant bone graft” in the search algorithm. Despite only a small fraction of users typically clicking on results beyond the first page of Google ,17, we iWe implemented exclusion criteria to eliminate irrelevant websites. Websites with non-English content, those with a minimal amount of information about bone grafts, sites primarily featuring auditory or visual content, scientific publications or books, websites involving banner advertisements or promotional products, sponsored links, or discussion forums, websites restricting direct access, and websites lacking information about ridge augmentation were excluded. Finally, duplication was checked and removed.The remaining incorporated websites were classified following the framework proposed by Ni Riordain and McCreary in 2009 . They weQuality assessmentThe evaluation of the websites was performed using well-established assessment tools, which included the Journal of the American Medical Association (JAMA)  criteriaThe DISCERN questionnaire offers a reliable approach to assess the quality of written information concerning various therapies for specific health conditions. This questionnaire comprises 16 questions categorized into three distinct sections. The first part, encompassing questions 1 to 8, primarily evaluates the publication’s credibility, aiming to find its reliability as a source of information about a particular therapy. The second part consists of questions from 9 to 15, focusing specifically on the quality of provided content about treatment options. Lastly, question 16 corresponds to the overall quality score of the evaluation. The sum of scores was calculated as the total score. Hence, 80 was the maximum, and 16 was the minimum. Reliability and treatment quality scores reflected the sum of questions 1 to 8 and 9 to 15, respectively. A five-point Likert scale was utilized to assign scores to each question, with a rating of one indicating no or poor quality and five signifying good quality . To estaThe Journal of American Medical Association has published the JAMA benchmarks, which encompass the following four criteria: authorship , attribution , currency , and disclosure (ownership and disclosure of any conflicts of interest). Each item is evaluated as achieved or not .Each website was additionally assessed for the transparency and quality of online health information using the HON  Foundation. It is worth mentioning that while HON primarily concentrates on evaluating transparency and quality, it does not evaluate the accuracy of online health information. HON includes the following eight criteria: attribution, authority, complementarity, confidentiality, justifiability, financial disclosure, openness, and advertising policy . The evaReadability assessmentRegarding the readability assessment, three tools were utilized, namely, the Flesch-Kincaid grade level (FKGL), the simplified measure of gobbledygook (SMOG), and the Flesch reading ease (FRE) scale. FKGL ranges from 0 to 18, with 18 being the most difficult text to read. The SMOG readability test gauges the education level required to comprehend a text; the higher the score, the more difficult the text to comprehend, with each score corresponding to education level (7-8 means the text is readable for the 7th to 8th-grade level). However, the FRE score can range from 0 to 100; the higher the score, the easier the text to read. An FRE score of 90-100 means the text is very easy to understand, 80-89 is easy, 70-79 is fairly easy, 60-69 is standard and ideal for most general audience material, 50-59 is fairly difficult to read, and below 50 is difficult to read (graduate level). All readability tests were conducted using the automated formulas for the intended formulas.Ethical considerationsAs this study relies solely on public data, ethical approval and consent were not necessary.Data analysisThe statistical data was analyzed using the statistical software SPSS version 25 . The results were presented in tables, with each mean value accompanied by its standard deviation. A p-value of less than 0.05 was considered statistically significant for comparative tests.Available websites and categorizationSearching the two terms “Dental bone graft” and “Implant bone graft” in Google, Bing, and Yahoo resulted in 28,200,000, 757,000, and 861,000 for the term “dental bone graft” per engine, respectively. For “Implant bone graft,” it resulted in 23,000,000 for Google, 821,000 for Bing, and 1,330,000 for Yahoo. The first 150 websites per search term from each search engine were screened across the eligibility criteria. Among the 900 websites, 161 were included. Figure Quality assessmentQuality was assessed using DISCERN, JAMA benchmarks, and HON. Total DISCERN had a mean score of 47.97 ±17.62), and the overall quality (question 16) mean was 2.94 (±1.16). The reliability and treatment quality sections had a mean of 23.88 (±8.427) and 21.16 (±8.623), respectively. Table , and theRegarding JAMA benchmarks, none of the websites achieved the four items, while three items were achieved in 21 13%) websites; nine of these websites belonged to non-profit organizations. No significant difference was observed between the number of achieved items among different affiliations (p = 0.62). JAMA evaluation includes four items. Currency was the most commonly achieved in 67 (41.6%), followed by authorship in 61 (37.9%) websites. Attribution was met in 28 (17.4%) websites only, with 13 (8.1%) belonging to non-profit organizations with statistically significant differences (p = 0.02). None of the websites achieved the disclosure item. The summary of the JAMA evaluation among affiliations is shown in Table % websiteReadabilityFRE scale ranged from 19.7 to 78.5 with a mean of 56.76 (±9.1), and the most common category was “fair difficult,” accounting for 64 (39.8%), followed by “standard” in 56 (34.8%) websites. Only eight (5%) websites were “fair easy”. Figure Word and sentence count varied among websites, ranging from 171 to 3,365 with a mean of 1,078.2 (±590.8) and 7 to 227 with a mean of 59.3 ±34.9), respectively. Different means according to affiliation are shown in Table .9, respeThe internet has improved many aspects of life, including access to healthcare through telemedicine. While it can be helpful in reducing health illiteracy, there are risks of inaccurate information and incomplete knowledge delivery . With thA dental implant is the first and most reliable choice for replacing missing teeth, and more than half of the implant cases require bone graft . The assContent types and presentationAfter filtering the 900 screened websites for Google, Bing, and Yahoo, only 161 websites were included, with the most common reason for exclusion being that the website is not related. The studies about dental implants and peri-implantitis included 32 and 27 websites, respectively ,15. ThisBased on website affiliations, most websites belonged to universities or medical centers, followed by commercial websites. This is more or less consistent with the other studies, as the study about implants had non-profit organizations and university or medical centers as the most common. However, in the peri-implantitis study, it was commercial and non-profit organizations. Noteworthy, governmental websites were the least in both and in this study ,15. GoveQuality assessmentDISCERN evaluates two aspects, namely, the reliability of the information and the quality of content about the treatment. Among the 16 questions, the poorest scores were related to the references. The mean score for question four was 1.68 (±1.4), lower than the studies in different fields ,15,28. TJAMA evaluation showed similar findings to other studies in terms of the number of achieved items per website, with most websites not achieving any item. Our data showed a statistically significant difference in attribution among different affiliations, with non-profit organizations being higher in including sources of information.Websites that adhere to the HON code are expected to provide accurate, transparent, and up-to-date information; protect user privacy; and clearly disclose the source and qualifications of the content creators . SimilarReadability assessmentLow health literacy has been found to have a negative impact on health and quality of life. Research has shown that older adults with low health literacy are more likely to experience chronic health conditions and have a higher risk of mortality . PopulatLimitationsIt is important to acknowledge the limitations of our study. We only searched three search engines. Additionally, we only used six assessment tools for quality and readability. Although they are commonly used, we encourage further research that addresses these limitations. In addition, our study evaluated the content in the English language only.The study findings suggest that English-language patient-centered information about implant bone grafts is challenging to comprehend and lacks good quality. This can potentially contribute to the spread of misinformation and the persistence of unrealistic patient expectations. As a result, there is a need to establish websites that provide trustworthy, high-quality information on implant bone grafts. Such resources should be free from commercial biases and presented in an easily understandable way for the average patient.


Cells create physical connections with the extracellular environment through adhesions. Nascent adhesions form at the leading edge of migrating cells and either undergo cycles of disassembly and reassembly, or elongate and stabilize at the end of actin fibers. How adhesions assemble has been addressed in several studies, but the exact role of actin fibers in the elongation and stabilization of nascent adhesions remains largely elusive. To address this question, here we extended our computational model of adhesion assembly by incorporating an actin fiber that locally promotes integrin activation. The model revealed that an actin fiber promotes adhesion stabilization and elongation. Actomyosin contractility from the fiber also promotes adhesion stabilization and elongation, by strengthening integrin-ligand interactions, but only up to a force threshold. Above this force threshold, most integrin-ligand bonds fail, and the adhesion disassembles. In the absence of contraction, actin fibers still support adhesions stabilization. Collectively, our results provide a picture in which myosin activity is dispensable for adhesion stabilization and elongation under an actin fiber, offering a framework for interpreting several previous experimental observations. The formation of cell adhesions is important for numerous biological processes, including cell migration, differentiation, survival, tissue morphogenesis, and wound healing. However, to fully understand how adhesions control biological processes, we need to unravel the biophysical principles underlying their assembly and maintenance. Experimentally, assessing how adhesion proteins form and maintain adhesions is difficult. The mechanisms by which adhesion proteins regulate adhesions are not well understood, and controversy exists on the roll of cell contractility. Here, we developed a model of adhesion assembly and characterized how adhesions elongate in the presence and absence of contraction. We found that force is not needed for the transition of adhesions into elongated morphologies. The ability of cells to form adhesions with the extracellular environment is critical to many physiological and pathological processes, including wound healing, tissue morphogenesis, embryonic development, and cancer metastasis –3. Adhesμm of the light microscope [Nascent adhesions appear in the lamellipodium as clusters containing an average of 20–50 integrins, with a diameter of ~100 nm, smaller than the ~0.25 croscope , 22. Thecroscope . When thcroscope , 22–24.Actomyosin contractility from the actin fibers stabilizes nascent adhesions and supports integrin connections with the ECM through reinforcement of their bonds with ECM ligands. Experiments with myosin inhibitors and dominant negative mutants have shown that actomyosin force also promotes the recruitment of intracellular accessory proteins and the maturation of adhesions , 25–28. In this study, we sought to understand the mechanisms of adhesion elongation and stabilization at the end of an actin fiber by testing the contributions of actomyosin force, inside-out activation, and substrate stiffness to these mechanisms. Because the transient nature of nascent adhesions makes it difficult to distinguish adhesion assembly from elongation and stabilization, here we applied computational modeling  of the integrin-ligand bonds followed catch-slip kinetics, in which an initial strengthening of the bond occurred with increasing force, followed by weakening at higher forces (Fmyo) was applied to all ligated integrins. Depending on the spring constant of the harmonic interaction between integrin and ligand, which mimicked substrate stiffness and contributed to the substrate force, Fsub, and depending upon the magnitude of Fmyo in the fiber region, the total force on the integrin-ligand bonds was calculated and the probability of rupture determined. Activation, ligand binding and unbinding resulted in cycles of integrin diffusion, activation, and de-activation, which mimicked nascent adhesions. The main objective of the model was to test the effect of an actin fiber on the orientation and stability of the adhesion. The effects that fiber contractility had on several adhesion properties were evaluated by running simulations in the presence and absence of contraction, and using varying Fmyo. The effect of substrate stiffness, which further contributed to the total force on the integrin-ligand bonds, was also assessed.We extended our computational model of adhesion assembly based on overdamped Langevin dynamics , 32 by ir forces . The effr forces . The fibWe confirmed that integrin adhesions are elongated under actin fibers using COS7 kidney epithelial cells. Cells were co-transfected with mScarlet-paxillin to label adhesions and EGFP-F-tractin to label actin. Imaging by TIRF showed adhesions assembling at the advancing leading edge , and larFmyo, up to 40 pN, and substrate stiffness, k, from 0.1 to 0.7 pN/nm. The average percentage of integrin-ligand bonds first increased with increasing Fmyo, then it decreased, and this effect was more marked at high substrate rigidities , a four-fold increase in Fmyo, from 5 pN to 20 pN, increased the average percentage of ligated integrins from ~25% to ~35%, a total increase of about 40%  stochastic force from thermal effects, FT; (ii) actomyosin contractility in the region of the actin fiber, Fmyo = 5–40 pN, a range comparable to the contractility of actomyosin bundles in cells [iii) force from the substrate limiting integrin motion away from the ligand, Fsub, proportional to the substrate stiffness k . Coolecules . In the olecules , 54.Fmyo between 5–40 pN, adhesions initially stabilized, reaching an optimum at Fmyo = 25 pN, but then destabilized for Fmyo > 30 pN , it can used in , and dt k, proportional to the substrate stiffness, and equilibrium distance L = 0.01 nm. The integrin-ligand bond lifetime, τ, depends on the total force on integrin, Fi, and is the inverse of the rupture rate: τ = 1/koff. The rupture rate follows a catch-slip bond kinetics, in which the lifetime of the bond initially increases with force Fi, then decreases, as shown in koff, is computed from a double exponential function, including a strengthening pathway represented by a first exponential term with a negative exponent; and weakening pathway, represented by a second exponential term with a positive exponent [The bond between integrin and the ligand has spring constant, exponent , 69, as:FT, a specific τ exists for each bond. The rupture rate, koff, determines the probability of unbinding as: Since the force on each bond is unique, depending on whether integrin is in the fiber region, and the magnitude and direction of In the model, the location where the actin fiber overlaps with the adhesion is incorporated as a 300 nm wide strip in the middle of the domain . In thisPbundling, is the probability that free integrins in the fiber area activate faster. Outside the fiber, integrins activate at a rate kon = 1 s-1, as in our previous study [kon = 3 s-1 and the remaining 50% present kon = 1 s-1. When the probability of actin bundling is 1, all integrins under the fiber present kon = 3 s-1. The increase in rate of integrin activation under the fiber mimics talin-mediated activation of integrin [ζi. We found that reducing diffusion increases adhesion alignment in the direction of the fiber, but to a smaller extent than increasing the activation rate  and assigns all simulation parameters to specific variables, including integrin diffusion constant, integrin and ligand concentrations and substrate stiffness; then, it sets the initial random positions of integrins and the positions of the ligands in the lattice. The step function runs iteratively, and each iteration corresponds to one timestep, until the total time of 300–500 s is reached. The main objective of the step function is to evaluate the total force on each integrin and update its position and bound state.The algorithm consists of two parts: an initialization function and a step function. The initialization function sets the domain geometry and the boundary conditions , and number of ligands (441). Integrins are initially distributed on the 1 x 1 μm domain, by assigning lization , 38.Fi, acting on each integrin; based on Fi, it updates integrin positions. Transitions between unbound and bound states of integrin are determined by kinetic rates.The step function is divided into three parts: it first evaluates integrin positions; it computes the total force, dt = 0.0001 s to dt = 0.00001 s or dt = 0.000001 s does not change the results, both in the presence and absence of an actin fiber, and using substates with different rigidities  glass bottom dishes, at 15,000 cells per dish in Gibco DMEM  with 5% FBS (VWR Scientific). Cells were co-transfected with 500 ng pcDNA3.1-mScarlet-paxillin and 300 ng pEGFP-F-tractin  complexed in Gibco OptiMEM with Mirus TransIT LT1  following the manufacturer’s recommended DNA:transfection reagent ratio. Two-channel time-lapse image series were acquired at 2 s intervals (per channel) using a Nikon 100x/1.49NA CFI Apo objective, Nikon TiE Inverted Microscope with Perfect Focus System 3 and motorized TIRF, and a Photometrics Prime 95B camera. mScarlet-paxillin was imaged with 561 nm laser excitation with a 620/60 nm emission filter. EGFP-F-tractin was imaged with 488 nm laser excitation and a 535/45 nm emission filter. Image processing was performed with Fiji software.S1 FigA. Distribution of the percentage of ligated integrins in different conditions of bundling and Fmyo (between 0–30 pN). B. Distribution of final tension on ligated integrins in different conditions of bundling and Fmyo (between 0–30 pN). All data are computed between 100–500 s of simulations, extracting values every 1 s. C. Average angle of the adhesion using Fmyo = 10 pN (black), 20 pN (dark gray), 30 pN (medium gray), 40 pN (light gray) and two fiber widths: 0.2 and 0.8 nm. Errorbars indicate standard deviation from the mean. The angle is calculated from the direction of the first principal component considering the 2D positions of ligated integrins. All data are computed between 100–500 s of simulations, using k = 0.6 pN/nm, and extracting values every 1 s.(TIFF)Click here for additional data file.S2 FigA-B. Distribution of the total time spent by integrins in the ligated state, in different conditions of Fmyo (between 0–30 pN), without (A) and with (B) actin bundling. C. Average angle of the adhesion relative to the actin fiber. Three conditions are tested: absence of bundling; bundling with reduced diffusion of integrin in the fiber region (using 5-fold higher ζi than outside the fiber); and bundling with increased activation rate (using 3-fold higher kon relative to kon outside the fiber). The angle is calculated from the direction of the first principal component considering the 2D positions of ligated integrins. All data are computed between 100–500 s of simulations, using k = 0.6 pN/nm, and recording every 1 s.(TIFF)Click here for additional data file.S3 FigA. Average percentage of ligated integrins varying the width of the actin fiber and using Pbundling = 1. Errorbars indicate standard deviation from the mean. B. Number of binding events per second varying the width of the actin fiber and using Pbundling = 1. C. Distribution of total time in the ligated state, calculated as sum of the ligand-bound lifetimes of for each integrin over the course of 500 s of simulations, using Pbundling = 1 and increasing the width of the fiber. D. Average angle of integrin adhesion, relative to the direction of the actin fiber, using Pbundling = 1 and varying the width of the fiber. The angle is calculated from the direction of the first principal component of the 2D positions of ligated integrins. Errorbars indicate standard deviation from the mean. All data are computed in the absence of actomyosin contractility and are evaluated between 100–500 s of simulations, from 3 independent runs using k = 0.6 pN/nm and considering the total number of ligated integrins every 1 s of simulations.(TIFF)Click here for additional data file.S4 FigA. Distribution of force on ligated integrins using actin flow velocity of 0, 10, 30, and 100 nm/s, in different conditions of Fmyo (between 0–30 pN) and using FT = 0. B. Distribution of force on ligated integrins using actin flow velocity of 0, 10, 30, and 100 nm/s, in different conditions of Fmyo (between 0–30 pN) and including thermal fluctuations. All data are computed between 100–500 s, from 3 independent runs using k = 0.6 pN/nm. The distributions are normalized by their maximum value in each plot.(TIFF)Click here for additional data file.S5 FigA. Average percentage of ligated integrins varying timestep, in the presence (fiber) or absence (no fiber) of actin bundling. When present, a fiber of 300 nm width is used. B. Average percentage of ligated integrins varying timestep, and using k = 0.2 pN/nm and k = 0.4 pN/nm. An actomyosin force of 20 pN is applied to ligated integrins in the fiber area, considering a fiber width of 300 nm. All data are computed between 100–500 s, from 3 independent runs. Errorbars indicate standard error from the mean.(TIFF)Click here for additional data file.


The number of Japan’s international students has rapidly increased in the last decade. This study examines the relationship between menstrual abnormalities in cycle and symptoms, stress level, and lifestyle of female international students in Japan across three time periods, namely pre-arrival, difficult, and current time periods. A cross-sectional design was employed, and data were collected through a self-administered questionnaire, including the menstrual distress questionnaire (MDQ), between December 2022 and February 2023. The questionnaire was distributed to 56 female international students from two universities in Japan, and a total of 29 valid responses were collected. We found varying menstrual cycle abnormalities and severity of menstrual symptoms across three time periods, with the difficult period after arrival in Japan displaying the highest symptom severity. Higher stress levels were significantly associated with more severe menstrual symptoms. Lifestyle habits such as alcohol consumption were also linked to menstrual symptoms. The current study emphasizes the importance of providing menstrual education, support, and resources to address international students’ unique challenges in managing their menstrual health while studying abroad. By promoting awareness, education, and access to healthcare services, universities may contribute to international students’ well-being and academic success.  Menstruation is a physiological phenomenon, and it serves as an indicator of the status of female hormones, acting as a barometer of health. On the other hand, menstrual disorders encompass abnormalities in the menstrual cycle, volume, duration, onset or cessation, and accompanying symptoms such as premenstrual syndrome (PMS) and dysmenorrhea. Lifestyle, socio-demographic, genetic, and psychological factors , as wellDuring the last decade, the number of international students has rapidly increased in Japan . InternaMoreover, a survey conducted among 153 Chinese female international students revealed that 61.4% of respondents expressed a desire to obtain information and seek advice regarding women’s sexual and reproductive health, with 85.1% of them explicitly mentioning requests related to menstruation . This inRegarding the temporal changes in menstrual symptoms, a study comparing PMS in Japanese female university students at admission and 3 months later  reportedPrevious research suggests that menstrual symptoms may worsen during periods of high stress ,9,10, anWe conducted a cross-sectional study using a self-administered questionnaire, and the data collection period was from 5 December 2022 to 28 February 2023. The study targeted all 90 female international students from two campuses at two universities in Tokushima Prefecture. The targeted universities are major academic institutions the region, known for their ability to attract international students. We excluded individuals who did not receive an explanation regarding the research and did not provide consent to participate; those who were unable to respond to the questionnaire in Japanese, English, or Vietnamese; and those who were not residing in Japan during the distribution of the questionnaire or were experiencing menstrual cessation  due to pregnancy or other reasons.The questionnaire was anonymous and available in both paper and web formats. It was prepared in two versions with identical content: (a) Japanese (with furigana) and English and (b) Vietnamese. To mitigate bias between the two response formats, we implemented several measures, including providing clear instructions to all participants, ensuring consistent content across both formats, and carefully reviewing the data for any anomalous patterns or inconsistencies. The questionnaire consisted of the following sections:This part of the questionnaire includes questions on demographic factors, including prior study enrollment in Japan, current course and year of enrollment, nationality, age, age at first menstruation, marital and childbirth status, date of arrival in Japan, history of menstrual education, and present influence on academic performance.Menstrual Status (12 items): Menstrual cycle length, regularity, duration, perceived amount of bleeding, medical history, individuals consulted regarding menstruation, level of knowledge;Symptoms during premenstrual and menstrual phases (49 items) using a 46-item Menstrual Distress Questionnaire (MDQ) ,25 groupLifestyle (7 items): Frequency of breakfast consumption , sleep duration , exercise frequency , alcohol consumption , smoking , living status ;Stress (10 items): overall, menstrual-related, health-related, academic-related, financial situation, interpersonal relationships, food, culture shock, homesickness, and language barrier. Respondents were asked to rate each item on a scale of 0  to 4 (severely felt).This part of the questionnaire includes questions during the three critical time periods: (1) the pre-arrival period (PP); (2) the most difficult period (DP); and (3) the current period (CP). The respondent was asked to specify the time of arrival in Japan and the time of DP, and the following items were queried:https://jp.surveymonkey.com/ accessed on 5 December 2022) and checked the “consent” checkbox on the first page before proceeding with the questionnaire. It took about 20 min to complete the questionnaire. The explanatory documents were prepared in Japanese, English, and Vietnamese, and the participants were informed that participation was voluntary and that refusing to participate would not affect their academic achievements. The research was approved by the Tokushima University Hospital Research Ethics Committee for Life Sciences and Medicine (No. 4253), and permission for implementation was obtained from the respective institution’s authorities.Research cooperation was obtained from each university. Researchers gave face-to-face explanations to the international students using explanatory documents, and questionnaires were distributed to the international students in their preferred language. The explanatory documents included a URL and QR code for the web survey, allowing respondents to choose between paper-based or web-based responses. For paper-based responses, the participants indicated their consent by checking a consent confirmation section on the questionnaire and then answering the questions. The collected questionnaires were retrieved using collection boxes or similar methods. For the web survey, participants accessed the Survey Monkey site ; Sleep duration less than seven hours (Yes/No); Daily exercise frequency (Yes/No); Alcohol consumption (Yes/No); Smoking (Yes/No); Living alone (Yes/No).Statistical tests were conducted using SPSS Statistics version 28.0 for Windows . Descriptive statistics were performed on the obtained data for each variable. Regarding lifestyle, stress, menstrual conditions , and individuals consulted regarding menstruation, the following were analyzed at each of the three time periods. For variables with corresponding data, Cochran’s Q test was used to compare proportions for knowledge levels of normal and abnormal menstrual conditions, knowledge levels of menstrual disorders, and PMS. Friedman’s test was used for MDQ scores, and Friedman’s or Wilcoxon signed-rank test was employed for stress fluctuations. If statistically significant differences were found, the Bonferroni test was conducted. MannThe questionnaire was distributed to 56 individuals, who could be mobilized during the time, and 44 responses were collected, with 25 respondents completing the survey online and 19 on paper. In order to minimize potential bias in the results, we excluded seven women who had ever used hormonal medication for contraception or gynecological treatment at any of the three time periods, as the medications may influence hormone levels in the body, leading to disruptions in menstrual regularity and menstrual pain. After excluding another eight participants who did not complete the MDQ questionnaire, the total number of valid responses was 29.Five respondents were on government scholarships, twenty-three on private scholarships, and one on an exchange program. Of these, 14 were from junior colleges, 3 from undergraduate programs, 1 from a master’s program, and 11 from doctoral programs. Regarding nationality, there were 15 from Vietnam, 5 from Mongolia, 3 from Indonesia, 2 from China, and 4 from other Asian countries. The mean (±standard deviation: SD) age was 27.3 (±5.2) years, with a mean age of 23.9 (±2.4) years for individuals from Vietnam and 31.0 (±5.0) years for individuals from other countries. When considering program level, the mean age was 24.1 (±2.6) years for junior college students, 26.0 (±3.5) years for undergraduate students, and 31.5 (±5.1) years for graduate students. The mean length of stay in Japan was 36.6 (±18.2) months, ranging from 7 months to 5 years and 9 months. The mean length of stay for individuals from Vietnam was 44.3 (±10.3) months, while for individuals from other countries, it was 28.4 (±21.5) months. When considering program level, the mean length of stay was 44.2 (±11.7) months for junior college students, 41.3 (±27.1) months for undergraduate students, and 26.6 (±19.0) months for graduate students. The most challenging mental and physical health period occurred from the beginning of their stay in Japan until 4 years and 2 months later, with an average duration of 15.1 (±14.9) months since their arrival. For individuals from Vietnam, the average duration was 18.8 (±13.2) months, while for individuals from other countries, it was 11.3 (±16.1) months. When considering program level, the average duration was 18.9 (±14.4) months for junior college students, 22.7 (±22.0) months for undergraduate students, and 8.9 (±12.5) months for graduate students.p = 0.013) in the percentage of students with regular menstruation among the three time periods. There was also a significant change in the perception of normal menstrual flow (p = 0.022).The mean (±SD) age of first menstruation was 13.4 (±2.2) years. Total scores on the MDQ in premenstrual and menstrual distress were highest during difficult times, but there was no significant difference among the three time periods. Regarding menstrual education, 69.0% of the respondents received it at school, while 65.5% received it at home before coming to Japan. 86.2% of the respondents were aware of dysmenorrhea to some extent, and 51.7% were aware of PMS before coming to Japan. Knowledge about dysmenorrhea increased after coming to Japan. There was a significant change in the level of knowledge about PMS during the three time periods, with more people having knowledge during difficult times or at present compared to before coming to Japan. The person most commonly consulted about menstruation was the mother. When staying in Japan, fellow international students seem to play a consultant role on par with the mother regarding menstruation issues.Common premenstrual symptoms include fatigue, irritability, mood swings, and backache before coming to Japan; backache and fatigue and mood swings during difficult times; and irritability and backache or painful or tender breasts (breast tenderness) at the current period A. CommonDuring the premenstrual and menstrual phases, there was a strong desire for snack foods during three time periods .Regarding the impact of premenstrual symptoms on academic performance, during the current period, a considerable impact was reported by 2 respondents (6.9%) and a slight impact by 13 respondents (44.8%). Regarding symptoms during the menstrual phase, it had a considerable impact on 5 respondents (17.2%) and a slight impact on 17 respondents (58.6%). The reasons for not seeking medical consultation despite the impact of symptoms (multiple answers) were being busy for 10 respondents (34.5%), economic reasons for 6 respondents (20.7%), language issues or embarrassment for 5 respondents each (17.2%), and not knowing where to seek consultation for 1 respondent (3.4%).p < 0.001), with significant differences between before coming to Japan and the difficult period (p = 0.014) and with significant differences between the difficult and current periods (p = 0.038). There were also significant differences in stress levels related to the economic situation (p = 0.002) and interpersonal relationships (p = 0.024). Levels of language barrier stress (p = 0.001), cultural adaptation stress (p = 0.001), and homesickness stress (p = 0.038) after coming to Japan in the current period were significantly lower than those in the difficult period.When comparing the time periods with high total MDQ scores, three patterns emerged: the pattern with the highest scores before coming to Japan (pattern 1), the pattern with the highest scores during the difficult period (pattern 2), and the pattern with the highest scores at the current period (pattern 3). The means of MDQ total scores for the three patterns are shown in There were associations of changes in total MDQ score patterns with the current enrollment program and nationality. Respondents in junior colleges and undergraduate programs tended to have higher MDQ scores before coming to Japan. However, graduate program students tended to have higher MDQ scores during the difficult period. In addition, Vietnamese respondents tended to have higher scores before coming to Japan .p = 0.002). Individuals with irregular menstruation also experienced higher levels of cultural adaptation stress. In the current period, individuals who slept <7 h had a higher prevalence of abnormal menstrual bleeding (p = 0.033), and those who experienced language-related stress or homesickness-related stress had a higher prevalence of abnormal menstrual cycle length  (data not shown).There were no smokers. Regarding menstrual abnormalities, during the difficult period, there was a significant association between the regularity of menstruation and the severity of cultural adaptation stress , and the current period. We confirmed that international students who experienced difficulties during a specific period were more likely to report menstrual abnormalities. In addition, we revealed associations of stressors specific to international students, such as language and cultural stress, with the menstrual cycle length and regularity. Our results are consistent with some previous studies, which have found stress caused disturbances in the menstrual cycle at the hormonal level  and is aThere were no significant differences in the MDQ scores during the three time periods. Nevertheless, the severity of menstrual symptoms was associated with overall stress during the difficult period after arrival. In the premenstrual phase, the severity of symptoms was related to menstrual, interpersonal, food, and cultural stress. The severity of symptoms during the menstrual phase was associated with academic, economic, interpersonal, and cultural stress. In the current phase, the severity of menstrual symptoms was related to overall, menstrual, academic, and homesick stress. Previous studies have reported that menstrual symptoms were associated with daily life stress  and percIn this study, it became evident that sleep duration is associated with the amount of bleeding during menstruation. No significant correlations existed between menstrual symptoms’ severity and lifestyle factors such as diet, sleep, and exercise. However, among students who consumed alcohol after coming to Japan, the symptoms were significantly higher during the difficult premenstrual and current menstrual phases. The association between alcohol consumption and menstrual symptoms was consistent with previous studies ,8. StudeWe found that there are three patterns of the changes in MDQ scores: higher scores during the pre-arrival period (pattern 1), higher scores during the difficult period (pattern 2), and higher scores during the current period (pattern 3). The total ratio of individuals with either pattern 2 or pattern 3 was 60.7% for premenstrual symptoms and 62.1% for menstrual symptoms, which indicates that more than half experienced a worsening of menstrual-related symptoms after coming to Japan. These results are consistent with previous studies which have reported that among Chinese international students, the proportion of individuals who reported changes in menstruation was 18.5% after staying for half a year  and 49% In addition, individuals with stronger symptoms before coming to Japan were mostly short-term college or undergraduate students. However, those with stronger symptoms during the difficult period were predominantly graduate students. This difference can be attributed to the lower average age of short-term college and undergraduate students compared with graduate students and differences in knowledge and experience regarding menstruation, menstrual symptoms, and their management. Similarly, the stronger symptoms before coming to Japan among Vietnamese individuals can be explained by their younger average age and longer duration of stay. Universities should offer menstrual education to international students upon admission and consistently after that, regardless of their duration of stay in Japan. This will enhance students’ awareness and knowledge of the subject.There were more pain symptoms in the most common symptoms experienced before and during menstruation, according to the MDQ, followed by negative emotions. In the present study, among the symptoms experienced before menstruation, many international students reported experiencing backache, while only about half of them experienced such pain in a previous study targeting Japanese individuals ,30. MoreOur results are consistent with the previous literature ,34, whicRegardless of the duration of stay in Japan, inappropriate lifestyle habits such as lack of sleep and alcohol consumption, as well as increased stress levels, are related to the regularity of menstruation, the amount of bleeding, and the severity of accompanying menstrual symptoms. Various factors, including the academic program and nationality, influence these abnormalities. The awareness of PMS is low upon arrival in Japan, highlighting the importance of incorporating knowledge about menstruation, menstrual irregularities, appropriate lifestyle habits, and stress management during orientation sessions. Providing information that fosters understanding and connects to appropriate healthcare-seeking behaviors is necessary. In addition, establishing on-campus consultation services for menstruation concerns is crucial.The current study is the first study conducted in Japan targeting menstrual changes among international students. The identification of three distinct patterns of menstrual changes offers valuable insights for further investigation into the factors influencing these patterns, particularly pattern 2 and pattern 3. Additionally, an intriguing finding from the study is that the perceived most difficult time period does not always align with the highest intensity on the MDQ, suggesting potential associations with age and study program. The diverse stressors experienced by international students, along with their native culture, likely contribute to the complexity of these findings.The study has limitations that need to be considered. First, being a cross-sectional study, it does not establish cause-and-effect relationships. Secondly, recall bias is possible as respondents rely on their memory to recollect past events. The longer the duration since the events occurred, the greater the potential for bias. The study’s narrow focus on university students from two sites also led to a small sample size. Furthermore, most participants were from Asia, making applying the findings to a wider population difficult. Within the framework of this paper, we did not specifically focus on analyzing the impact of age as well as potential influence of cultural differences and overall wellness prior to coming to Japan on menstrual issues. Future research should involve longitudinal studies that include more dimensions and diverse international students, allowing for more robust and comprehensive insights.This study sheds light on the experiences of international students with menstrual symptoms and their impact on daily life. It emphasizes the importance of providing menstrual education, support, and resources to address international students’ unique challenges in managing their menstrual health while studying abroad. By promoting awareness, education, and access to healthcare services, universities may contribute to international students’ well-being and academic success.