Patients with immune-mediated rheumatic diseases (IMRD) are at increased risk of infections, including significant morbidity and high mortality. Considering the potential for unfavorable outcomes of SARS-CoV-2 infection in patients with IMRD, several questions were raised regarding the impact of COVID-19 at the start of the pandemic.This paper presents the protocol of a study that aims to prospectively evaluate patients with IMRD and a confirmed COVID-19 diagnosis .The study comprised a prospective, observational cohort (patients with IMRD and COVID-19) and a comparison group (patients with only IMRD), with a follow-up time of 6 months to evaluate differences in health outcomes. The primary outcomes will be changes in IMRD disease activity after SARS-CoV-2 infection at 4 time points: (1) at baseline, (2) within 4-6 weeks after infection, (3) at 3 months after the second assessment (±15 days), and (4) at 6 months (±15 days). The secondary outcomes will be the progression rate to moderate or severe forms of COVID-19, need for intensive care unit admission and mechanical ventilation, death, and therapeutic changes related to IMRD. Two outcomes—pulmonary and thromboembolic events in patients with both IMRD and SARS-CoV-2 infection—are of particular interest and will be monitored with close attention .Recruitment opened in May 2020, with 1300 participants recruited from 43 sites as of November 2020. Patient recruitment will conclude by the end of December 2020, with follow-up occurring until April 2021. Data analysis is scheduled to start after all inclusion data have been collected, with an aim to publish a peer-reviewed paper in December 2020.We believe this study will provide clinically relevant data on the general impact of COVID-19 on patients with IMRD.Brazilian Registry of Clinical Trials RBR-33YTQC; http://www.ensaiosclinicos.gov.br/rg/RBR-33ytqc/DERR1-10.2196/24357 COVID-19 was declared a pandemic on March 11, 2020, by the World Health Organization .Patients with immune-mediated rheumatic diseases (IMRD) are at increased risk of infections, including significant morbidity and high mortality . It is wSome Italian, American, French, and Chinese databases have started to demonstrate that the risk of poor outcomes is quite similar to the general population and could be linked more closely with comorbidities and aging than IMRD itself -11. HoweConsidering Brazil as a continental country and with relevant regional and socioeconomic differences, as well as discrepancies concerning basic sanitation and access to public and private health care systems, it is important to generate data related to disease activity, treatment management, and survival curves in patients with both IMRD and COVID-19.This paper presents the protocol for the ReumaCoV-Brasil Registry (Brazilian Registry of Patients with Immuno-mediated Chronic Inflammatory Diseases Infected by SARS-CoV-2). This trial was registered with the Brazilian Register of Clinical Trials (RBR-33YTQC) on June 1, 2020.The primary objective is to prospectively evaluate patients with IMRD who had COVID-19, according to the Brazilian Ministry of Health criteria  Multime, and to The inclusion phase started on May 20, 2020, with the recruitment of patients from 43 centers in 5 regions of the country , and wilHospital das Clínicas, Universidade Federal de PernambucoArtrocenter Clínica Médica – Unidade Taubaté, São PauloCentro de Pesquisas Clínicas, São PauloClínica Jozélio Freire de Carvalho, BahiaClínica Médica do Hospital Baia Sul, Santa CatarinaClínica Omura Medicina Diagnóstica, São PauloFaculdade de Ciências da Saúde de Barretos, São PauloFundação Hospitalar do Estado do AcreFundação Universidade Estadual do CearáHospital da Secretária da Saúde do Distrito Federal – Instituto Hospital de Base do Distrito FederalHospital das Clínicas da Faculdade de Medicina da USP, São PauloHospital das Clínicas da Faculdade de Medicina de Marilia, São PauloHospital das Clínicas da Universidade Estadual de Campinas, São PauloHospital das Clínicas da Universidade Federal de GoiásHospital das Clínicas da Universidade Federal de Uberlândia, Minas GeraisHospital das Clínicas, Faculdade de Medicina de Ribeirão Preto – USP, São PauloHospital das Clínicas, Universidade Federal de Minas GeraisHospital de Base, Fundação Faculdade Regional de Medicina São José Do Rio Preto, São PauloHospital de Clínicas de Porto Alegre, Universidade Federal do Rio Grande do SulHospital de Clínicas, Universidade Federal do ParanáHospital dos Servidor Público Estadual de São PauloHospital Geral de Fortaleza, CearáHospital Getúlio Vargas, PernambucoHospital Governador Celso Ramos, Santa CatarinaHospital Moinhos de Vento, Porto AlegreHospital Nossa Senhora da Conceição, Rio Grande do SulHospital Regional da Unimed de Fortaleza, CearáHospital Santa Casa de Misericórdia de Vitória, Espírito SantoHospital São Lucas da PUCRS, Pontifica Universidade Católica do Rio Grande do SulHospital São Paulo, Universidade Federal de São PauloHospital Universitário Clementino Fraga Filho (1), Universidade Federal do Rio de JaneiroHospital Universitário Clementino Fraga Filho (2), Universidade Federal do Rio de JaneiroHospital Universitário de Londrina, Universidade Estadual de Londrina, ParanáHospital Universitário Lauro Wanderley, Universidade Federal da ParaíbaHospital Universitário, Universidade Federal de Juiz de Fora, Minas GeraisInstituto de Medicina Integral Professor Fernando Figueira, PernambucoLaboratório de Imunopatologia Keizo Asamy, PernambucoSanta Casa de Misericórdia de Belo Horizonte, Minas GeraisUniversidade de Brasília, Distrito FederalUniversidade de Santo Amaro, São PauloUniversidade Estadual do Rio de JaneiroUniversidade Federal de Ciências da Saúde de Porto Alegre, Rio Grande do SulUniversidade Federal do AmazonasOver the course of 6 months, participants will be questioned regarding disease activity, clinical manifestations of COVID-19 , concomitant medications management, social distancing, flu vaccination, survival curves, and mortality rates.The secondary objectives will include assessing the autoantibodies profile , as well as immunoglobulins  and the serologic conversion rate for SARS-CoV2 (IgG and IgM).ReumaCoV-Brasil is a multicenter, observational, prospective registry implemented to monitor adult IMRD patients with a confirmed diagnosis of COVID-19, according to the Brazilian Ministry of Health criteria .Using a nationwide sampling strategy, it is a 2-phase study: (1) cross-sectional evaluation (inclusion) with information about previous or current symptoms of COVID-19 and clinical characteristics at baseline, which can be performed via telephone call  or by a face to face visit, if possible; and (2) prospective follow-up concerning the IMRD characteristics with 2 face to face visits, every 3 months (3-month and 6-month assessments), after viral infection.Regardless COVID-19 diagnosis, eligible patients include those aged 18 years or over with a prior diagnosis of IMRD, according to the American College of Rheumatology or European League against Rheumatism criteria, including rheumatoid arthritis ,17, systThe exclusion criteria included the presence of other primary immunodeficiency diseases, past organ or bone marrow transplantation, neoplasms within the last 5 years, current chemotherapy, HIV diagnosis, and thymus diseases. The controls will be patients with IMRD but without a diagnosis of COVID-19, matched for sex, age, and IMRD type. The exclusion criteria stated above will apply to the control group as well.Considering the moderate to severe forms of COVID-19 as the dependent variable and an estimated rate at 20%, according to the current literature, as well as the proportion of 1 case for 1 control and an error α=5% and β=20%, the sample calculation was approximately 576 IMRD patients with positive COVID-19 and 576 IMRD patients without COVID-19 .When an IMRD case is identified, the patient will be invited to participate in the study and will be enrolled after reading and signing the informed consent form . The cliThe primary outcomes will be changes in IMRD activity after SARS-CoV-2 infection at 4 time points: (1) baseline; (2) within 4-6 weeks after SARS-CoV-2 infection; (3) 3 months after inclusion in the study (±15 days); (4) 6 months after inclusion (±15 days) . If the The disease activity assessment will be performed using a global patient assessment and physician assessment, using a numerical visual scale (0-10), as well as specific and validated disease activity measurements or prognostic factors related to systemic vasculitis . In addiRheumatoid arthritisClinical Disease Activity Index Axial spondyloarthritisAnkylosing Spondylitis Disease Activity Score-C-Reactive Protein Ankylosing Spondylitis Disease Activity Score-Erythrocyte Sedimentation Rate Bath Ankylosing Spondylitis Disease Activity Index Psoriatic arthritisPsoriasis Area Severity Index and nail disease Disease Activity in Psoriatic Arthritis Minimal Disease Activity Systemic erythematous lupusModified Systemic Erythematous Lupus Disease Activity Index ,47Sjögren syndromeEuropean League Against Rheumatism Sjögren's Syndrome Disease Activity Index Systemic sclerosisRodnan score Raynaud’s phenomenon and digital ulcersBehçet disease (BD)Behçet’s Disease Current Activity Form Inflammatory myopathiesManual Muscle Testing in 8 muscles Health Assessment Questionnaire Systemic vasculitisBirmingham Vasculitis Activity Score, version 3 Five-Factor Score Details about previous lab exams will also be recorded, such as erythrocyte sedimentation rate, C-reactive protein, rheumatoid factor, anti-CCP, antinuclear antibody, anti-ENA (extractable nuclear antigen), anti–double-stranded DNA, human leukocyte antigen B27, complement, anticardiolipin IgG and IgM, ANCA, and cryoglobulins.The secondary outcomes will be related to COVID-19 itself, such as hospitalization, need of intensive care unit, mechanical ventilation, time from onset of symptoms or hospitalization to development of severe acute respiratory syndrome (SARS), outcome after SARS (improvement or death), laboratory changes , and pharmacological , experimental , and supportive treatments. Moreover, information about changes in IMRD treatment  and new autoimmune manifestations will be also recorded for all patients.Two outcomes—pulmonary and thromboembolic events in patients with both IMRD and SARS-CoV-2 infection—are of particular interest and will be monitored with close attention and entail the collection of more data  because they have been reported as an important aspect of moderate and severe forms of COVID-19, and heparin and direct oral anticoagulants are used to manage them. Considering that some researchers have shown antiphospholipid autoantibodies in patients from the general population without IMRD, another secondary endpoint is to evaluate these aspects as well as to verify if patients with IMRD could have more thromboembolic events compared to the third group (patients without IMRD) ,56.The explanatory variables will be age, sex, geographic localization, race, education level, profession, employment situation, comorbidities, IMRD details , concomitant medications, and COVID-19 symptoms , time of infection, municipality, and vaccination against the flu.If IMRD patients without symptoms of COVID-19 enrolled in the control group test positive for anti–SARS-CoV-2 antibodies (IgM and/or IgG) after scheduled lab exams at baseline, they will be considered as having COVID-19 and will be moved from the control group to the intervention group (asymptomatic group).A total of 14 mL of blood will be collected for further lab testing, according to the protocol described in The total blood tube and another tube with RNAlater added will be shipped to the Keizo Asami Immunopathology Laboratory, located at the Federal University of Pernambuco and maintained at –80 °C .The data will be collected by appropriate and trained health care staff, particularly rheumatologists from public and private backgrounds. In addition, 4 study coordinators will provide regular assistance and training regarding data quality. Regular data inspection and quality control will be performed throughout the lifetime of the study. Research sites will retain identifiable study data securely. The data custodians will work together to establish a suitable trial data repository for the anonymized study data set following the conclusion of primary and secondary data analysis. Anonymization will ensure patients’ privacy and adherence to the previously mentioned guidelines.The study’s endpoints are as follows:Domain 1: changes in disease activity and modifications in lab exam resultsDomain 2: outcomes concerning moderate and severe forms such as hospitalization rate, need for intensive care unit and mechanical ventilation, total hospitalization time, and death; date of a major serious event Domain 3: humoral response regarding anti–SARS-CoV-2 antibodies and autoantibodies related to IMRD.The data will be analyzed in a descriptive way using absolute and relative frequencies for categorical variables and quantitative measures  for numerical variables. The normality of the data will be verified using the Kolmogorov-Smirnov test.The chi-square association test will be used to assess any differences among the endpoints, or the Fischer exact test for small samples. The linear associations among variables will be evaluated using the Pearson correlation or Spearman, when appropriate.For the evaluation of the behavior of clinical variables between 2 or more points in time by group, analysis of variance (ANOVA) will be used, with repeated measures to be made. In case of nonnormality of the data, the averages of the groups at each time point will be compared using the Kruskal-Wallis nonparametric test. To compare the means between the groups, the Wilcoxon nonparametric test will be used.t test. In case of violation of the assumption of normality, the Mann-Whitney nonparametric test will be used.The comparison between the means of numerical variables with normal distribution will be verified through the Student Adjusted multiple linear regression models will be used to assess the simultaneous effects of sex, age, time of disease, disease activity, comorbidities, concomitant medications, and other confounding variables, according to the group and predefined outcomes. For dichotomous dependent variables, a logistic regression model will be preferred. Survival analysis models, including log rank and Kaplan-Meier tests, adjusted for confounding variables, will be developed to assess the main outcomes over time. The time defined as the end date will be the date of a major event, such as illness with confirmation or suspicion of infection, hospitalization, or death.P value <.05 will be defined as significant.SPSS, version 20 (IBM Corp), will be used in all analyses, and a The results of this research will be presented in an aggregated form, guaranteeing confidentiality and ensuring that there are no risks to patients’ well-being and care. This protocol was approved by the Brazilian Committee of Ethics in Human Research on April 5, 2020 , and registered on the Brazilian Registry of Clinical Trials (RBR-33YTQC) on June 1, 2020. The project is in the data collection phase, which is expected to end in May 2021.The data are owned and held by the Brazilian Society of Rheumatology (Sociedade Brasileira de Reumatologia). Data can be obtained upon request.The authors will ensure safe and proper conduct of the study, in agreement with the International Conference on Harmonization Guideline for Good Clinical Practice and the Declaration of Helsinki , and resThe ReumaCoV-Brasil is a Brazilian registry of rheumatic patients with COVID-19, supported by the Brazilian Society of Rheumatology. This study was designed to provide more information on epidemiological data and specific outcomes in Brazilian patients with IMRD across the country. In addition, the immunophenotyping may help us to understand the different outcomes according to genetic background and host response ,59. With


Due to the lack of earthquake-resistant rural houses, small and medium earthquakes caused massive casualties in rural China. In 2004, China began implementing the Earthquake Rural Housing Safety Project Policy (ERHSPP) to reduce earthquake losses, mainly promoting the adoption of earthquake-resistant structures in rural residents’ self-built houses through subsidies, training of construction craftsmen, and provision of earthquake-resistant housing drawings. We conducted a field survey, collecting 1169 rural households in Pingliang, Gansu Province, and 1501 rural households in Yuxi, Yunnan Province, China. We studied Earthquake-Resistant Construction Behaviors (E-RCB) by the logistic and the ordered logistic regression models. Results show that government housing subsidy promotes E-RCB of rural households; E-RCB was affected by ERHSPP, positively correlated with economic status and housing earthquake damage; E-RCB was negatively correlated with structure danger, house age, and earthquake experience; and housing earthquake damage, not earthquake experience, strikingly promoted E-RCB in rural China. The results could provide suggestions in communication risk strategies for the government. We suggest the local government should promote local acceptable disaster propaganda, provide hierarchical housing subsidies, pay attention to housing seismic supervision, publish earthquake-resistant housing design drawings, and conduct more earthquake-resistant technical training for rural craftsmen. However, the lack of supervision and guidance during rural houses’ construction caused huge losses after the earthquake. From 2001 to 2005, 60 disastrous earthquakes occurred in mainland China, affecting approximately 7684 million people, 144,300 kmTo enhance the seismic performance of rural self-built houses, ERHSPP was implemented in Xinjiang in 2004 as a pilot, and it was officially promoted throughout China in 2007 in the Opinions on the Implementation of Earthquake Safety Project for Rural Residential Buildings . AccordiERHSPP has been successful in improving the earthquake resistance of rural houses. None of the newly built rural earthquake-resistant houses in Xinjiang province after 2004 were damaged after more than 60 earthquakes of magnitude ≥5 . TherefoPrior research on household disaster preparedness discussed psychological aspects and adaptive behaviors. We would like to explore the factors affecting actual behaviors. The Government housing subsidy in this article refers specifically to the special housing subsidies in ERHSPP, The Earthquake-Resistant Construction Behaviors (E-RCB) refer specifically to used seismic drawing, made seismic patands, and chosen qualified contractors when building the house. This paper adopts logistic and ordered logistic regression to quantitatively analyze E-RCB, ERHSPP effect, and other aspects to investigate:Did government housing subsidy promote rural household E-RCB or not?Are government project’s effect, house risk, economic level, and earthquake experience related to E-RCB or not?E-RCB in Yuxi and Pingliang, China, are conducted as the case study through questionnaire surveys. This article includes five parts: Existing research on household disaster preparedness discussed psychological aspects and adjustment behavior. Psychological research on household disaster preparedness discussed the factors that influence individual risk perceptions. Differences in individual perceptions of disaster risk are related to gender, age, education, socio-economic status, household income, housing economic value, the risk level of the residence, the number of children, individual responsibility for taking preventive measures, and psychosocial factors ,6,7,8. OIn terms of adjustment behaviors, studies discussed household and individual disaster preparedness and adjustment behaviors, mainly including the apportionment of economic losses such as earthquake insurance ,13,14,15Another possible research direction is to analyze the relationship between government project effect and household disaster preparedness. According to the practice of ERHSPP, this article defines “Earthquake-Resistant Construction Behaviors (E-RCB)” as the use of seismic drawing, seismic patands, and qualified contractors when rural households build their own houses. The hypotheses of E-RCB are as follows.Scholars have studied the impact on the government engineering projects, and whether the impact on residents’ behavior is positive or negative. There are different opinions.The government’s dissemination of disaster preparedness knowledge to encourage people to understand local disaster risk information is considered to be related to residents’ disaster preparedness behavior. The government’s disaster mitigation plan can promote residents to understand their location’s disaster risk ,24. In eResidents’ government trust is considered to be related to residents’ disaster preparedness. Han took Yushu earthquake survivors as samples, and pointed out that the two self-evaluation indicators of individual government trust and household disaster preparedness were significantly negatively correlated . AdditioSpecial government housing subsidies and similar economic subsidies are considered related to residents’ disaster preparedness behavior. Taylan found linking house maintenance credits with adoption of risk mitigation measures, motivating homeowners for taking earthquake preparedness measures in Turkey . On the Additionally, households may also passively rely on government actions. Wu’s research on rural communities in Kaohsiung City found that after the implementation of the disaster reduction plan, 50% of the communities passively rely on the assistance of the government and experts, and only about 1/6 of the communities have mobilized more materials for disaster reduction preparations with government policy support . Tian usTherefore, previous studies have different views on the impact of government engineering measures on residents’ behavior. This article provides the following hypotheses:Hypothesis 1a (H1a). Government project effect and E-RCB are positively correlated.Hypothesis 1b (H1b). Government project effect and E-RCB are negatively correlated.Hypothesis 1c (H1c). Government housing subsidy and E-RCB are positively correlated.Hypothesis 1d (H1d). Government housing subsidy and E-RCB are negatively correlated.The comparison between the new and old built rural houses can reflect the effect of ERHSPP. We considered house age to be related to the seismic performance of the house. According to the Second National Agricultural Census in 2006 , in ruraBy the Standard for Seismic Appraisal of Buildings (GB 50023–2009) , the expHypothesis 2 (H2). Structural danger and house age are negatively related to E-RCB.We considered the economic level represented by income directly related to the household earthquake preparedness behavior . EconomiHypothesis 3 (H3). Economic level and E-RCB are positively correlated.Considering that a catastrophe is a small probability event, it may be challenging to motivate households to build strong houses for earthquake preparedness, which is time-consuming and labor-intensive. Previous studies generally believed that the individual’s past disaster experiences such as directly experiencing large-scale destructive earthquakes and experiencing earthquake losses will help them decide how to respond to earthquakes. Compared with individuals who have not personally experienced earthquakes, individuals who have experienced earthquakes have taken more disaster preparedness measures ,45,46. BHypothesis 4 (H4). Earthquake experience and E-RCB are positively correlated.Wu has conducted field surveys on the affordability costs of the seismic fortification of rural houses in Pingliang, Gansu Province, in 2010, and Yuxi, Yunnan Province, in 2011 .The sample questionnaires collected valid sample information of 2670 rural households, among 1169 samples from Pingliang, Gansu Province, and 1501 samples from Yuxi, Yunnan Province. The sample questionnaires are the data source of this study. We used multi-stage stratified sampling, selecting villages with Earthquake Intensity Degree ≥ 7. Each family chose one person to form a list and stratified according to local population information sampling. Interviewers conducted face-to-face interviews due to the education level of the interviewees. This study uses Stata 15 for data analysis. Two survey sites are shown in 2 area. Yuxi and Pingliang City both belong to the earthquake-affected area.Both places have experienced major earthquakes in 1970 and 2008. The 7.7 Ms Tonghai earthquake on 15 January 1970, caused 15,621 deaths, and 338,456 houses collapsed. The entire area of Yuxi City caused heavy losses due to the 1970 Tonghai earthquake. The 8.0 Ms Wenchuan Earthquake on 12 May 2008 caused 69,227 deaths and severely damaged more than a 100,000 kmThis research mainly analyzes whether government project effect, house risk, economic level, and earthquake experience are related to E-RCB. The descriptive statistics of the variables are shown in The first dependent variable is Policy Subsidy, which reflects received the government housing subsidy when building this house with seismic structure. The questionnaire asked, “Have you received government subsidies for building/purchasing this house?”, 1 for “Yes” and 0 for “No”. 6.03% sample households received special housing subsidies. We only raised this question in Yuxi, and the total sample size for this variable is 1501.The second dependent variable is Earthquake-Resistant Construction Behaviors (E-RCB), which consists of three measures: used seismic drawing, made seismic patands, and chosen qualified contractors when building the house. 16.93% sample households completed all three measures, 9.78% sample households completed two measures, and 5.54% sample households completed one measure.Seismic Drawing: Used seismic drawing when building this house. The questionnaire asked “Is the construction drawings used when building this house?”, “Does the construction drawings consider seismic resistance?”, 1 for “Used” and “Considered”, 0 for “Used” but “Not considered”, and 0 for “Not used”. 15.88% sample households used seismic drawing.Seismic Patands: Made seismic patands when building this house. The questionnaire asked “Has the foundation of this house been treated?”, 1 for “Treated, there are ground ring beams” and 0 for “Simple treatment” and “No treatment”. 27.75% sample households made seismic patands.Qualified Contractors: Chosen qualified contractors with seismic construction skills when building this house. The questionnaire asked, “Who built this house?”, 1 for “Qualified construction team” and 0 for “Unqualified construction team” and “Friends and relatives I find myself”. 9.48% sample households chose qualified contractors.Policy Information: Knew the seismic fortification intensity of the area (or the seismic fortification standard to be achieved when building houses). The questionnaire from Pingliang asked “Do you know the seismic fortification intensity in this area?” and the questionnaire from Yuxi asked “Do you know the seismic fortification standards to be met when building houses in this area?”, 1 for “Known” and 0 for “Never heard of it”. 10.15% sample households knew the seismic fortification intensity.Government Trust: Recognition degree of “the local government’s earthquake countermeasures are adequate”. The questionnaire asked “How would you rate ‘the local government’s countermeasures to the earthquake are adequate’”; the options “Inconsistent”, “Not very consistent”, “Relatively consistent”, and “Consistent” are assigned a value of 0–3 in turn. 37.42% sample households are relatively consistent with this opinion.Structural Danger: The corresponding risk of the building structure can be divided into “Ring beam structure or steel frame”, “Brick–concrete or brick–wood”, and “Adobe” concerning Seismic Field Work Part 3: Survey Specifications (GB/T 18208.3-2011) . The hazHouse Age: The questionnaire asked, “When was this house built?” House Age is equal to the survey year minus the house construction year, and the result is in logarithm. The mean of the sample logarithm house age was 2.400.Annual Income: The questionnaire asked, “How much income did your family have in the past year?” The annual family income is RMB. 5000 yuan, 5000–8000 yuan, 8000–10,000 yuan, 10,000–20,000 yuan, 20,000–30,000 yuan, 30,000–50,000 yuan, 50,000–80,000 yuan, 80,000–100,000 yuan, 100,000–200,000 yuan, 200,000–500,000 yuan, and more than 500,000 yuan are assigned 1–11 points in turn. The mean of sample households’ annual income was within the range of 8000–20,000 yuan.Earthquake Experience: Experienced 1970 Tonghai or 2008 Wenchuan earthquake. Questionnaire of Pingliang asked “Have you encountered an earthquake disaster so far?”: 1 for “Have experienced an earthquake disaster” and 0 for “Have experienced other disaster “ and “Have not suffered a disaster”. Questionnaire of Yuxi asked “Have you heard of the 1970 Tonghai earthquake?”: 1 for “Experienced personally” and 0 for “Heard of” and “Never heard of”. Out of the sample households, 55.06% experienced the 2008 Wenchuan or 1970 Tonghai disastrous earthquake.Housing Damage: The house has previously collapsed due to an earthquake. The questionnaire asked “What degree of earthquake disaster did you suffer?”: 1 for “My house completely collapsed” and “My house partially collapsed” and 0 for “No house damage”. Out of the sample households, 30.19% experienced self-housing collapse due to an earthquake.To conduct hypothesis testing, this article regards the explanatory variable “Earthquake-Resistant Construction Behaviors (E-RCB) “ as an ordinal variable, and establishes an ordered logistic regression model with the expression:We aimed to explore whether the policy subsidy has an incentive effect on E-RCB and whether the household received the policy subsidy as a dependent variable. The use of seismic drawings that reflect technical services, the qualified construction team that reflects the training of construction craftsmen, and the seismic construction of self-built houses are used as independent variables. We only raised this question in Yuxi, and the total sample size for this variable is 1501. In This paper uses E-RCB as the dependent variable. We tested the interaction effects of the independent variables, and the results showed that there was no significant interaction effect between the independent variables, so we chose to use the following model. Model 1 is the basic model to examine the impact of government project effect on E-RCB. Model 2 adds house variables based on Model 1 to investigate the influence of house risk on E-RCB. Model 3 adds economic variables to Model 2 to examine the impact of the economic level on E-RCB. Model 4 adds experience variables based on model 3 to investigate the impact of earthquake experience on E-RCB. The results of Model 1–4 are shown in The results of Model 1 show that after controlling government trust, compared with households that did not know the local seismic fortification standards, households that know the standards enacted more E-RCB; the increase is about 94.3%  translated into the local language that is easy to understand .The Chinese government can provide policy subsidies, which are graded based on family income and provide more subsidies to poor families for housing special subsidies in rural earthquake-hazardous areas, helping families with financial difficulties build affordable earthquake-resistant houses. This discovery is similar to Wu’s conclusion, when policy subsidies are not graded based on family income and provide more subsidies to poor families. The result is often that families with higher economic affordability are more likely to receive policy subsidies, and poor families cannot use the policy, because they cannot afford out-of-pocket expenses after policy subsidy reduction .Timing of housing seismic supervision: The Chinese government may pay attention to the seismic fortification supervision in the process of building new houses in rural areas, and in the restoration and reconstruction after natural disasters, so that newly built rural houses meet the local seismic fortification standards.The local government should continue to publish unified, earthquake-resistant housing design drawings, which are suitable for local architectural styles in the local disaster reduction plan.The local government can conduct more earthquake-resistant technical training for rural craftsmen and encourage welcoming NGOs and community residents to participate in craftsman training and disaster education.


Cyclodextrins (CDs) are a group of cyclic oligosaccharides produced from starch or starch derivatives. They contain six (αCD), seven (βCD), eight (γCD), or more glucopyranose monomers linked via α-1,4-glycosidic bonds. CDs have a truncated cone shape with a hydrophilic outer wall and a less hydrophilic inner wall, the latter forming a more apolar internal cavity. Because of this special architecture, CDs are soluble in water and can simultaneously host lipophilic guest molecules. The major advantage of inclusion into CDs is increased aqueous solubility of such lipophilic substances. Accordingly, we present studies where the complexation of natural compounds such as propolis and dietary plant bioactives  with γCD resulted in improved stability, bioavailability, and bioactivity in various laboratory model organisms and in humans. We also address safety aspects that may arise from increased bioavailability of plant extracts or natural compounds owing to CD complexation. When orally administered, α- and βCD—which are inert to intestinal digestion—are fermented by the human intestinal flora, while γCD is almost completely degraded to glucose units by α-amylase. Hence, recent reports indicate that empty γCD supplementation exhibits metabolic activity on its own, which may provide opportunities for new applications. Cyclodextrins (CDs), also known as cyclomaltoses, cycloamyloses, and Schardinger dextrins, are a group of cyclic oligosaccharides that are produced from starch or starch derivatives by the bacterial enzyme cyclodextrin glycosyltransferase (CGTase) ,2. CDs cThis review aims to provide a short historical overview as well as a summary of the synthesis, properties, pharmacokinetics, and safety of CDs. Furthermore, the differences among the three main CD types are addressed with respect to their complex formation and route of administration. The present state of knowledge regarding the stability, bioavailability, and bioactivity of γCD complexes with selected natural compounds and dietary plant bioactives is described. Finally, the biological activity of empty γCD is also reviewed.Bacillus amylobacter -transferase) is a unique extracellular enzyme capable of catalyzing the cyclization reaction to form nonreducing CDs from starch, amylose, and other polysaccharides. In addition, CGTase catalyzes two other main transglycosylation reactions  and exhibits a weak hydrolyzing activity. A common feature of the reactions catalyzed by CGTase is the cleavage of an α-glycosidic bond. However, coupling is the reverse reaction of cyclization, where CD is the substrate and the generated linear malto-oligosaccharide is transferred to an acceptor substrate. During disproportionation, a linear malto-oligosaccharide is cleaved, and the shorter product is transferred to an acceptor substrate ,13. The Bacillus species are the most extensively studied enzymes , for, for2], The CD molecules have a hydrophilic outer wall because of the free hydroxyl groups on the outside of the molecules and a less hydrophilic or hydrophobic inner wall imparted by the ether oxygen atoms in the glycosidic hemiacetals and the carbon-hydrogen atoms . AccordiIn addition to the central cavity, an essential feature of CDs is the location of the hydroxyl groups . There aThe capacity to form inclusion complexes with lipophilic substances resulted in a broad range of applications for CDs, especially in analytical chemistry, agriculture, the pharmaceutical field, food, and hygienic articles . When deBased on pharmacokinetic studies of CDs, orally administered CDs have low bioavailability, and systemically absorbed CDs disappear rapidly from the body, mostly unmetabolized in urine ,30,31,32It has been shown that orally administered αCD at a dose of 200 mg/kg body weight (BW) in rats exhibited an absorption rate of 1%. Excretion of intact αCD after oral gavage was carried out via the kidneys. In this context, it is interesting that no intact αCD was found in the feces, which indicates that αCD is completely fermented by the intestinal microbiota, such as resistant starch or other types of fermentable dietary fiber . IntraveMale Wistar rats employed by Kubota et al. (1996) received βCD orally at a dose of 500 mg/kg. Here, βCD reached the maximum plasma concentration within 40 min with an absorption rate of 0.6% . IntraveThe enzyme β-amylase hydrolyses starch from the nonreducing end. Although γCD is resistant to degradation by β-amylase, it is a substrate of salivary and pancreatic α-amylase, whereas α- and βCD are not. The α-amylase hydrolyses α-bonds from large polysaccharides such as starch. γCD is almost completely degraded to malto-triose, maltose, and glucose, similar to the digestion of starch and linear dextrins ,33,34. TA high parenteral dose of βCD (200 mg/kg) in rats led to a decreased elimination rate, suggesting that this high dose may be nephrotoxic . The renNo signs of toxicity from γCD have been shown in acute toxicity studies in mice and rats after oral, subcutaneous, intravenous, and intraperitoneal administration . A dietaOverall, all three parent CDs are “generally recognized as safe” (GRAS) by the U.S. FDA for use as a food additive ,46,47. FDuring complexation, guest molecules and CD molecules come into contact with each other to unite and form a complex . In mostIn aqueous solutions, the internal cavity of CDs is occupied by water molecules. These water molecules can be substituted by less polar guest molecules. The driving force is the polar–apolar interaction between water and CD, which results in an energetically unfavored environment. Complex formation takes place through substitution with an appropriate guest molecule or, more commonly, some lipophilic moiety of the molecule, which is less polar than water molecules ,8. This Various methods have been applied to prepare CD complexes: The solution method, coprecipitation method, neutralization method, slurry method, kneading method, and grinding method ,50. FactThe host-guest interaction of OA and UA with βCD was studied by Huang et al. . They foAnother studied complex is caffeic acid phenethyl ester (CAPE)-γCD. The structure of CAPE, a key anticancer phenolic compound in New Zealand propolis, as well as the complex structure of CAPE-γCD, is shown Escherichia coli and Staphylococcus aureus was higher than that of zein-THY-NF without γCD. Therefore, the webs are of interest for application as antibacterial food packaging materials [In 2017, approximately 70% of global CD production consisted of βCD, while the shares of αCD and γCD were approximately 15% and 5%, respectively. However, due to its favorable toxicological profile, γCD, which was initially less produced, is becoming increasingly attractive as a pharmaceutical excipient . CDs havaterials .As reported by Fenyvesi, Vikmon, and Szente , the fooSince CDs are poorly absorbed through biological membranes due to their relatively high molecular weight and large number of hydrogen donors and acceptors , it is nRegarding the use of CDs in pharmaceutical products to form complexes with lipophilic drugs, parenteral application also matters. It should be noted that parenteral administration is mainly suitable for βCD derivatives such as HPβCD. αCD and βCD are not recommended because of their reported nephrotoxicity. The low aqueous solubility of βCD is also not an advantage for its use as a parenteral drug . SimilarCaenorhabditis elegans compared to worms receiving pure γCD or pure tocotrienols. Initially, it was observed that the nematodes absorbed a fluorescent dye, which was encapsulated in γCD for this assay, from the gastrointestinal tract and accumulated the dye in the cytoplasm of the intestinal cells. After testing the ingestion of γCD inclusion compounds, the oral administration of tocotrienols in γCDs was analyzed. Accordingly, the authors suggested that γCD is an excellent vehicle for the oral gavage of hydrophobic substances such as tocotrienol [A study in male Wistar rats examined the effects of different tocotrienol emulsions (13.9 mg tocotrienol) with or without γCD on γ-tocotrienol  and betulinic acid (BA)  were encHowever, in a study that used dietary Kuding tea extract as a UA source  encapsulated in 12.88% γCD (comprising 150 mg UA/kg BW), young male C57BL/6 mice had increased liver weight and hepatic fat accumulation compared to control mice, which received only a high-fat, high-fructose, Western-type diet for 6 weeks. In addition, the mice from the complex group showed increased hepatic peroxisome proliferator activated receptor gamma (Pparγ) and CD36 molecule (Cd36) mRNA levels as well as elevated plasma cholesterol levels and increased cytochrome P450, family 7, subfamily a, polypeptide 1 (CYP7A1) mRNA, and protein levels. Analyzing the enzymes of hepatic xenobiotic metabolism showed that there was a substantial elevation of cytochrome P450, family 3, subfamily a (CYP3A) and glutathione S-transferase, alpha 1 (GSTA1) mRNA, and protein levels in KTE-γCD mice. Pure UA-treated mice exhibited a moderate elevation of CYP3A and GSTA1. In line with the assumption that CDs can mask the taste of guest molecules . This stBrazilian green propolis supercritical extract (GPSE) has been reported to be rich in artepillin C  ,86,87,88Increasing the stability, bioavailability, and bioactivity of natural compounds and plant bioactives is an important issue in food science and pharmaceutics. As reviewed here, this goal can be achieved by encapsulating these often lipophilic molecules in γCD. A growing number of studies indicate that such CD complexes often lead to enhanced biological/pharmacological efficiency, increased stability, and better taste and odor.However, one should consider that improving the bioavailability of dietary plant bioactives may be accompanied by some risks, and a critical evaluation as well as a risk assessment is needed. Encapsulation enables the intake of higher dosages of natural compounds and plant bioactives. As shown for KTE encapsulated in γCD, high dietary supplementation can lead to an increased hepatic phase I and phase II response . Hence, Similarly, the complexation of natural products such as the bee product propolis could also increase the bioavailability of potential allergens. Propolis allergies per se were more of an occupational disease affecting beekeepers in the past. However, since propolis is frequently included in dietary supplements, more and more, an increasing number of people are affected by this allergy .Moreover, by complexing herbal extracts into CDs, one may increase the bioavailability of undesirable substances such as pesticides or contaminants. The selection and quality of the raw material is therefore of particular importance. It should also be considered that CDs mask the bitter taste and odor of their complexation agents, as reported for KTE. A bitter taste often results in food aversion by animals, thereby protecting them against the consumption of toxic compounds, although not all bitter compounds are toxic .CDs have a long history as host molecules in inclusion complexes and were long presumed to be inert molecules. However, only recently has it become evident that CDs are not just carrier molecules, but also undergo digestion and fermentation in the digestive tract. In particular, the first studies on γCD supplementation revealed promising effects on energy metabolism. Therefore, the impact of empty CDs on metabolism and their possible nutritional or pharmaceutical applications need additional investigation.


GJB1) gene, which encodes the connexin32 protein. A small number of patients with GJB1 mutations present with episodic neurological dysfunction and reversible white matter lesions, which has not been adequately reported. Here, we aim to enable clinicians to further understand this particular situation through systematically reviewing all published relevant cases.X‐linked Charcot‐Marie‐Tooth type 1 (CMTX1) is an inherited peripheral neuropathy caused by mutations in the gap junction beta 1 , cerebrospinal fluid (CSF) analysis, and nerve conduction study (NCS).We identified 47 cases of CMTX1 associated with episodic central nervous system (CNS) dysfunction from 38 publications. CMTX1 patients experienced episodic CNS deficits at a young age, ranging from infancy to 26 years, and 45 (95.7%) of them were male. The CNS symptoms manifested as facial, lingual, or limb weakness in 44 (93.6%), dysarthria or dysphagia in 39 (83.0%), facial or limb numbness in 15 (31.9%), and ataxia in 10 (21.3%) patients. The duration of episodic symptoms ranged from 3 minutes to 6 months. Thirty (63.8%) CMTX1 cases have reported obvious predisposing factors, among which the most common factors were infection or fever (27.7%), travel to high altitude (12.8%), and intensive exercise (8.5%). As for brain MRI, most abnormal signals were found in bilateral deep white matter (88.9%) and corpus callosum (80.0%). In addition, most of the NCS results were abnormal, including prolonged latency, reduced amplitude, and slowed conduction velocity. The motor nerve conduction velocity (MNCV) of median nerve was the most detectable and valuable, ranging from 25 to 45 m/s.We have reported the most comprehensive summary of the demographic and clinical profile from 47 CMTX1 patients with episodic CNS deficits and provided new insight into the phenotype spectrum of CMTX1. We hope that our study can help clinicians make early diagnosis and implement the best prevention and treatment strategies for CMTX1 patients with episodic CNS deficits. GJB1) gene on chromosome Xq13.1, which is responsible for encoding the gap junction protein called connexin32 (Cx32).Charcot‐Marie‐Tooth (CMT) is widely known as a hereditary neurological disease that mainly affects the motor and sensory fibers of the peripheral nervous system (PNS).GJB1 mutation .Although only peripheral neuropathy is present in most CMTX1 patients, episodic neurological dysfunction has been found in a few CMTX1 patients.GJB1 and white matter/leukoencephalopathy /central nervous system/episodic/recurrent/transient/attack/stroke. The screening process of the articles is shown in Figure Through a comprehensive literature search on the PubMed electronic database, the articles related to CMTX1 patients with episodic neurological dysfunction were retrieved, and the case reports were systematically collected. The search terms were: CMTX/X‐linked Charcot‐Marie‐Tooth/CMTX1/connexin32/Two researchers independently searched the literature. The inclusion criteria for selected studies were as follows: (1) the case reports of CMTX1 patients with episodic neurological dysfunction; (2) the reports published in English; (3) extractable information in each article. The exclusion criteria met one of the following items: (1) duplicated publication of the smaller dataset; (2) without extractable data; (3) review or abstract. The divergent parts were discussed and agreed.For each eligible case report, the following information is extracted and recorded, including the first author's name, publication year, region, demographic characteristics, family history, genetic mutation, clinical manifestations, the results of MRI, CSF analysis, and NCS. In order to classify the clinical factors of CMTX1 patients, some of the above information was further broken down as follows:The region of the patients was drawn from “Asia” (including China/Japan/Korea/India/Turkey), “North America” (including the USA), “Europe” , and “Oceania” .The CNS manifestations were classified as “facial, lingual, or limb weakness”, “dysarthria or dysphagia”, “facial or limb numbness”, “ataxia”, “aphasia”, “pyramidal sign”, and “others”.The PNS examinations were sorted into “areflexia or hyporeflexia”, “pes cavus or hammer toes”, “muscle atrophy or weakness in distal limb”, and “absent or diminished sense”.Predisposing factors were typed into “infection or fever”, “travel to high altitude”, “intensive exercise”, “trauma”, “poor sleep”, “puerperium”, “hyperventilation”, and “prolonged sun exposure”.The MRI lesions can be divided into “bilateral deep white matter”, “corpus callosum”, “splenium of corpus callosum”, “parieto‐occipital regions”, “posterior limbs of internal capsule”, “cerebellar peduncles”, and “others”.Cerebrospinal fluid results were classified into “normal”, “elevated protein levels”, and “elevated white blood cells”.The parameters from NCS including “distal latency”, “compound muscle action potential (CAMP) amplitude”, “motor nerve conduction velocity (MNCV)”, “sensory nerve action potential (SNAP)”, and “sensory nerve conduction velocity (SNCV)” were extracted from median, ulnar, peroneal, tibial, and sural nerves.Treatment was broken down into “corticosteroid and/or intravenous immunoglobulin (IVIg) therapy”, “symptomatic and supportive treatment”, and “without any treatment”.t‐test was used for comparison of continuous variables, while categorical variables were compared by χ2 test. All statistical analyses were performed with SPSS Statistics 25 . A P value of 0.05 was considered statistically significant.Continuous variables are expressed as mean ± standard deviation (SD), and categorical variables as percentages. The A total of 189 articles were retrieved, and 79 clearly not relevant articles were excluded by reading titles and abstracts. By reading the full text of the remaining 110 articles, 22 were not related to episodic neurological dysfunction, 15 were cellular or animal studies, 15 were reviews, 11 reported other types of CMT patients, four had incomplete data, three were not published in English, and two were duplicated publications with the smaller dataset. Finally, we identified 47 cases from 38 articles that met the criteria of our systematic review. A total of 47 cases were selected in the present study. Tables GJB1. Moreover, we re‐analyzed the pathogenicity of all the mutations in our systematic review by using gnomAD database, REVEL scores, and ClinVar database, and included case reports and other relevant studies. Of the 47 patients we systematically reviewed, 37 (78.7%) had a family history consistent with X‐linked inheritance. All patients presented the data of genetic mutations, including 39 (83.0%) missense mutations, four (8.5%) deletion mutations, three (6.4%) nonsense mutations, one (2.1%) insertion mutation, and one (2.1%) regulatory mutation in the noncoding region of The clinical manifestations in CMTX1 patients with episodic neurological deficits are presented in Tables The MRI and CSF findings of CMTX1 patients with episodic CNS deficits are shown in Tables NCS was mentioned in 40 CMTX1 patients with episodic CNS deficits, of which 38 were abnormal and 17 had specific data for extraction Tables . The proP = 0.199).The data on the treatment were available in 27 patients, of which 14 (51.9%) received corticosteroid and/or IVIg therapy, nine (33.3%) received symptomatic and supportive treatment, and four (14.8%) patients did not receive any treatment .In our systematic review, we found that the CNS manifestations did not appear to be correlated with the course and severity of peripheral neuropathy, which posed a challenge for making a clear diagnosis. Furthermore, clinicians may have a harder time making a correct diagnosis for patients with episodic CNS deficits as the first symptom. In these cases, CMTX1 should be distinguished from other episodic neurological diseases, including transient ischemic attack (TIA), mitochondrial encephalopathy with lactic acidosis and stroke‐like episodes (MELAS), acute disseminated encephalomyelitis (ADEM), and adrenoleukodystrophy (ALD).GJB1 gene. To date, more than 400 GJB1 mutations have been identified.GJB1 mutation.The final diagnosis of CMTX1 relies on the sequencing of the In summary, we have reported the most comprehensive summary of the demographic and clinical profile from 47 CMTX1 patients with episodic CNS dysfunction and provided new insight into the phenotype spectrum of CMTX1. In clinical practice, CMTX1 should be considered in patients presenting with episodic CNS dysfunction and abnormal white matter signals on MRI. Moreover, detailed neurological examination and NCS will provide critical clues to a correct diagnosis of CMTX1.The authors declare no financial or other conflict of interests.Table S1. Re‐analysis of the pathogenicity for all the mutations in CMTX1 patients with episodic neurological dysfunctionClick here for additional data file.Table S2. The clinical manifestation in CMTX1 patients with episodic neurological dysfunctionClick here for additional data file.Table S3. The results of MRI and CSF analysis in CMTX1 patients with episodic neurological dysfunctionClick here for additional data file.Table S4. The NCS results in CMTX1 patients with episodic neurological dysfunctionClick here for additional data file.


This study aimed to confirm the associations between stress and physical activity (PA) in Korean adolescents with atopic dermatitis (AD) based on data from the 2018–2019 Korea Youth Risk Behavior Web-Based Survey. The AD groups were divided into adolescents who were not diagnosed with AD, adolescents who were diagnosed with AD more than one year ago, and adolescents who were diagnosed with AD within one year. We defined the regular PA group and the non-PA group using the American College of Sports Medicine guidelines for children and adolescents: moderate to vigorous PA ≥5 times per week, including vigorous PA ≥3 days per week and muscle strengthening exercises ≥3 times per week. We performed logistic regression analysis to calculate the stress odds ratio (OR) and 95% confidence interval (CI) by group using model 1, adjusted for age, sex, and body mass index, and model 2, additionally adjusted for drinking, smoking, economic statuses, academic achievement, asthma, and rhinitis. In the group diagnosed with AD within one year, stress was 41% and 32% higher according to models 1 (1.41 (1.31–1.52)) and 2 (1.34 (1.20–1.50)), respectively. However, the stress OR was 30% lower in adolescents who completed regular PA than in the non-PA group (model 1: 0.71 (0.58–0.87); model 2: 0.68 (0.57–0.84)), even if diagnosed with AD within one year. In conclusion, the stress of adolescents with AD was significantly higher than that of adolescents without AD. The stress was significantly lower in the group with regular PA, and it was more robust in adolescents diagnosed with AD within one year. Atopic dermatitis (AD) is a disease characterized by repeated cycles of exacerbation and improvement of itchy eczema . Among aAD degrades quality of life by affecting an individual’s sleep, emotional and mental health, physical activity (PA), and social functioning . In partAmong various measures to manage mental health, regular PA is known to contribute to improving physical function and mental stress ,16. An aN = 117,343) were used in the final analysis.Raw data from the 2018 (14th) and 2019 (15th) KYRBWS, a government-approved statistical survey examining the health behaviors of middle and high school students in Korea, were analyzed in this study. The raw data were gathered from the Korea Centers for Disease Control and Prevention website after submitting an application outlining the purpose and plan of use and obtaining approval. The KYRBWS was conducted on 7th to 12th graders in 800 middle and high schools in Korea, selected by stratifying the population, allocating the sample, and extracting the sample. A total of 60,040 out of 62,823 (95.6%) participants completed the KYRBWS-14, and 57,303 out of 60,100 (95.3%) participants completed the KYRBWS-15. Data from these participants  by a physician?” were included in the non-AD group. Those who answered “Yes” to the same question were additionally asked, “Have you been diagnosed with atopic dermatitis  by a physician in the past 12 months?” to further divide AD participants into the AD ≥1 year and AD <1 year groups.For stress, those who chose “extremely high,” “high,” or “moderate” to the question, “How is your day-to-day stress level?” were considered to have stress, whereas those who chose “low” or “none” were considered to have no stress.To define regular PA, participants were asked, “How many days in the past 7 days have you engaged in PA  that increases your heart rate above the normal rate or makes you short of breath for at least 60 min a day?” and were divided into the “≥5 days” or “<5 days” categories. Participants were also asked, “How many days in the past 7 days have you engaged in vigorous PA that makes you extremely short of breath or sweat for at least 20 min a day?” and were divided into the “≥3 days” or “<3 days” categories. Participants were also asked, “How many days in the past 7 days have you engaged in muscle strengthening exercises, such as push-ups, sit-ups, lifting weights, using dumbbells, pull up bar, and parallel bar?” and were divided into the “≥3 days” or “<3 days” categories .2) were examined. Smoking status was determined using the yes-or-no question, “Have you ever smoked at least one cigarette?” and drinking status was determined using the yes-or-no question, “Have you ever had at least one shot of alcohol?” Economic status was determined using the question, “What is your household’s economic status?” The responses were grouped into “high” and “middle-high,” or into “middle,” “middle-low,” and “low.” Academic achievement was determined using the question, “How were your school grades in the past 12 months?” The responses were grouped into “high” and “middle-high,” or into “middle,” “middle-low,” and “low.”For physical characteristics, participants’ age, sex, and body mass index  compared to the non-AD group using a complex samples regression. Differences in smoking status, drinking status, economic status, academic achievement, moderate and vigorous physical activity (MVPA), and muscle strengthening exercises among the AD groups were analyzed with chi-square tests. The stress odds ratio (OR) according to the AD group was compared using multiple logistic regression. The ORs and 95% confidence intervals (CI) for the AD groups were presented in models 1 and 2 based on adjustment. Model 1 was adjusted for age, sex, and obesity , and model 2 was additionally adjusted for drinking status, smoking status, economic status, academic achievement, asthma, and rhinitis. Furthermore, the stress OR according to AD and PA was analyzed using the same method. Statistical significance was set at α = 0.05 for all analyses.All statistical analyses were performed using STATA version 15.0 . A complex sample design was used to compute representative values for the Korean adolescent population. The physical characteristics according to the AD group  were compared using the mean, standard error, and percentage within each group (%). The participants’ characteristics according to AD groups were examined by the p < 0.001). The AD <1 year group had a higher percentage of respondents who reported smoking and drinking but lower MVPA and muscle strengthening exercises compared to the non-AD group . However, the AD ≥1 year group only had a significantly higher OR in model 1, at 1.17 (1.11–1.23), but not in model 2, at 1.06 (0.98–1.14)  in model 1 and 1.34 (1.20–1.50) in model 2 (98–1.14) .p < 0.001). The OR for stress was also significantly lower in the AD ≥1 year group, with 0.83 (0.72–0.97) in model 1 and 0.80 (0.70–0.93) in model 2 (p < 0.05). Finally, the OR for stress was also significantly lower in the AD <1 year group, with 0.71 (0.58–0.87) in model 1 and 0.68 (0.57–0.84) in model 2 (p < 0.001)  in model 1 and 0.76 (0.72–0.81) in model 2 (< 0.001) .This study analyzed data from the KYRBWS obtained from the Korean adolescent population. The risks of stress in adolescents diagnosed with AD within one year were 41% and 34% higher than in non-AD adolescents in the two adjusted models, respectively. However, among those diagnosed with AD, adolescents who engaged in regular PA had a significantly lower stress risk compared to the non-PA adolescent group, and these results were more robust among adolescents diagnosed with AD within one year.In this study, we showed that stress was significantly higher in the AD group than in the non-AD group. These results are consistent with those of prior cross-sectional ,22 and lβ = −0.24; 95% CI, −0.27 to −0.20). As shown in the systematic review by Mücke et al., high PA reduces stress-related cortisol and heart rate responsiveness, even when participants were exposed to high stress levels [2max) by 8.5% and reduced anxiety symptoms by 13.7% [In this study, regular PA was associated with a lower risk of stress compared to the non-PA group. The results from previous studies confirmed that regular PA can help improve physical and psychological health among children and adolescents ,26. In as levels . They res levels ,30,31. Aby 13.7% . In geneby 13.7% . In factby 13.7% . TherefoIn this study, we could not examine the exact effects of regular PA on adolescents’ allergic immune responses. However, we observed that regular PA was positively associated with psychological stress in the Korean adolescent population. Some previous studies have reported that PA is associated with AD symptoms, and that patients with severe AD participate in approximately 50% less PA  . In geneThis study has a few limitations. First, this study used nationally representative data obtained via an online survey, and thus, we could not obtain information about AD treatment history and severity. However, one strength of this study is that the study data were extracted from a sample of more than 100,000 individuals by a government agency. Second, this was a cross-sectional study; therefore, the causality between stress and PA in adolescents with AD could not be determined. However, we specified the time of diagnosis to within one year, and the survey asked about PA within the past week, which enabled an understanding of the association between the current level of stress and PA. Third, the KYRBWS used multiple-choice questions, so the exact amount of PA could not be determined. However, the survey contained separate questions for moderate PA, vigorous PA, and muscle strengthening exercises, so we were able to distinguish intensity and form of exercise.In this study, adolescents with AD had a higher stress OR than adolescents without; in particular, those who had been diagnosed with AD within one year had a 30% higher risk. However, adolescents with AD who regularly engaged in MVPA and muscle strengthening exercises had significantly lower stress. Thus, it would be beneficial to emphasize the importance of PA in managing mental health for adolescents with AD who have a high risk of stress.


Osteological analyses presented evidence of residual rickets, a healed trauma, dental pathological conditions, and pipe notches. The combined isotope analyses yielded results for 43 individuals which suggested a diverse range of geological origins, including at least 16% of the population being non-local. The inclusion of δ13CVPDB had intriguing implications for three individuals who likely did not have origins in the Cape Town region nor in Europe. Peptide analysis on the dental enamel of 25 tested individuals confirmed they were all biologically male. We suggest that isolated enamel may provide crucial information about individuals’ pathological conditions, geographical origins, diet, and biological sex. These data further demonstrated that a combined approach using multiple osteological and biochemical methods is advantageous for human remains which are poorly preserved and can contextualise a site with little direct evidence.Skeletal remains discovered in Simon’s Town, South Africa, were hypothesised as being associated with a former Dutch East India Company (VOC) hospital. We report a novel combined osteological and biochemical approach to these poorly-preserved remains. A combined strontium ( In the context of human remains, many macroscopic osteological techniques are only informative if the skeletal elements are complete enough to allow for accurate measurements. As a result, the introduction of biochemical techniques, such as isotope and proteomics analysis, has expanded possibilities to obtain information from poorly-preserved remains on an individual and population level, and are being conducted more frequently in recent research3. In Simon’s Town, South Africa , oxygen (δ18OVPDB), and carbon (δ13CVPDB) isotope analyses using tooth enamel informs about childhood geographical origins and dietary preferences by comparing the data to expected local values from the Cape Town region of South Africa21. As isotope analyses can only confirm non-local origins through deviations of expected ranges, any individuals which fall within expected ranges are assumed local, despite the possibility of alternative interpretations given the wide range of strontium ratios for the Cape region.Addressing questions of the individuals’ origins contributed to overarching investigations of the site by utilising a combined funerary archaeological, osteological, proteomics, and isotopic approach. Osteological examination and the increasingly popular technique of proteomics to identify sex-specific tooth enamel proteins provide context into population demographics for the buried individuals. Funerary archaeology focusing on burial styles and associated artefacts provide further context to potential differing local In this paper, the results of the osteoarchaeological, funerary, multi-isotope, and proteomics research are presented. These data uniquely contribute to a larger research project that investigates the context of the Simon’s Town site by providing empirical data to support the limited historical sources in ongoing research. Further, this research contributes to the low recovery rate of VOC-related burial grounds, while illustrating the importance and future potential of biochemical techniques when used on poorly-preserved skeletal remains. Finally, we demonstrate the first known large scale multimethod approach in bioarchaeological literature that combines data from historical archives, archaeological contexts, macroscopic osteology, multi-isotope, and peptide analysis to interpret both the individual and population level context for poorly-preserved skeletal remains.All information from the funerary, osteological, and biomolecular analyses of the 43 individuals analysed for this research is presented in Table 123/184) (Table 10/184), but there remain unknown burial styles due to partially or fully disarticulated burials (51/184). There was also considerable evidence of grouped (mass) burials (n = 20) containing two or more individuals . Additionally, disorganised placement of graves resulting in frequent secondary deposition . Seven coffin burials were identified through remnants of preserved wood, coffin silhouettes, and coffin nails. A few burials had associated artefacts (27/184), which included buttons, button hooks, pins, small textile fragments , a glass trade bead necklace , as well as numerous fragmentary pipe stems and pipe bowls, of which one had a maker’s mark from Gouda, The Netherlands. Two European coins were associated with burial 37C, one of which was identifiable as a seventeenth-century VOC 6 stuiver. An eighteenth-century French silver écu was associated with burial 74.The predominant burial style for the 184 individuals was supine, with the arms positioned either along the sides of the body or crossed over the pelvis or torso 13/184  of non-adults ranged from 11 to 20 years of age, while the youngest individual was approximately 3 to 6 years old.Osteological age-at-death estimations were limited to broad group classifications of non-adults (≤ 20 years) or adults (> 20 years)25 Table . A mandi26 and evidence of a healed ankle fracture. Dental assessments for 22 individuals consistently revealed typical dental pathologies (21/22). These affected individuals exhibited evidence of caries , calculus , enamel hypoplasia , and antemortem tooth loss . Although not pathological, pipe notches were also observed in four individuals' maxillary and mandibular anterior and premolar teeth. There were no observations of intentional dental modifications in the form of decorative filing or tooth evulsion as prevalent amongst numerous African28 and Indian Ocean basin29 populations.In most cases, macroscopic assessment of the skeletal remains was not possible. The only identifiable pathological conditions were residual ricketsMass-spectrometry based peptide analysis was performed on the tooth enamel of 25 randomly selected individuals to identify sex chromosome-linked protein isoforms. Peptides specific to the X-chromosome amelogenin isoform (AMELX peptide sequence = SIRPPYPSY) and the Y-chromosome isoform (AMELY peptide sequence = SM(ox)IRPPY) were consistently detected in all individuals. Thus, in this research with poorly-preserved remains, the detection of both AMELX and AMELY peptide sequences strongly demonstrated that all 25 sampled individuals were chromosomally male 33. The analysed individuals for this research produced a range of 0.7074 to 0.7158. Consequently, most analysed individuals (39/43) exhibited ratios congruent with local Cape Town ranges. In contrast, the 87Sr/86Sr of three individuals  were lower than the expected Cape Town range, ranging from 0.7074 to 0.7088, and were thus considered non-local.Published strontium isotope ratios from relevant archaeological faunal and human data were assessed to identify baseline strontium ranges for the region. The entire Cape Town region is geologically represented by Quaternary sand, Precambrian Malmesbury Precambrian Cape granite suite, and Cambrian Table Mountain groups Fig. . Publish3 plant consumption, with some inclusion of C4 plants. A δ13C range of analysed dentine from contemporary sites suggested a local dietary range of − 18.8‰ to − 13.5‰34, corresponding with a significantly higher contribution of C3 foods than C413. Further, human enamel carbonate data from pre-contact and VOC contemporary Cape Town populations are consistent with that of δ13C from dentine, with a δ13CVPDB range of − 13.4‰ to − 6.1‰, representing a C3 to C3–C4 mixed diet19. Notably, enamel and collagen isotope analyses in prior studies have used δ13C values suggestive of C4 diets as a proxy for non-local origins, alongside dental modifications and 87Sr/86Sr13.Dietary expectations for the Cape Town region during the seventeenth to eighteenth centuries suggested a predominance of Cδ13CVPDB data range from − 13.9‰ to − 1.3‰. The δ13CVPDB of most individuals (36/43) fell within a range of − 12.3‰ to − 7.9‰, indicating a C3 dominant to mixed C3–C4 diet, which is consistent with comparable enamel data from the region19, Three burials  had low δ13C values, indicating C3 dominant diets just outside the established local range (− 13.9‰ to − 13.6‰). However, C3 values may be influenced by numerous factors including canopy type, water levels36. Therefore, burials 18B, 43B, and 97B were considered local, whereas four individuals  who consumed C4 dominant diets (− 5.3‰ to − 1.3‰), were suggested to be of non-local origins.The δ18O values from tooth enamel represent the oxygen isotopic composition of ingested and imbibed water. While this includes plant and animal foods, and respiration, the highest proportional contribution to the δ18O values comes from imbibed water37. Therefore, precipitation in the Cape region was reviewed to assess the expected δ18O ranges for the Cape region using the Global Network of Isotopes in Precipitation (GNIP) database, combined with other published precipitation, ground and tap water δ18O values, to create an expected range of δ18O values for the region39 47. Additionally, recent studies have suggested that extra care should be taken when using precipitation values as proxies for imbibed water, as large variations can be observed in δ18Oenamel-carbonate depending on the region and environment39. There is little known about the intra-dental elemental variation of δ18O values from this region and period, which may influence the interpretation of δ18OVPDB values35. Consequently, δ18O values are presented but were evaluated carefully, and only values which were considerably lower than might be expected for the region have been suspected as non-local given regional climate and precipitation δ18O values.The n39 Fig. . Precipiδ18OVPDB data from the enamel of a contemporary population was made to better understand expectations for local populations. Therefore, we compared our results to previously unpublished human enamel δ18OVPDB data of 35 individuals from the contemporary colonial Cobern Street burial ground in Cape Town , and this research ; t(79) = 10.874, p = < 0.001. Such dissimilarity in the oxygen isotope ranges between sites can tentatively suggest the Simon’s Town population of this research came from significantly different climactic origins than those of the Cobern Street population. In particular, burial 48 with a δ18OVPDB value of − 10.0‰, seems highly unlikely to be local given the coastal proximity, altitude, climate and precipitation levels in the Cape region. However, it is noted that small differences in the data could be the result of differing pretreatments, as the Cobern Street enamel was not pretreated44. Therefore, it must be stressed that if local human enamel δ18OVPDB data are published, or the relationship between precipitation and δ18Ocarbonate from the Cape region is investigated, this evaluation of Simon’s Town δ18OVPDB values should be revised39.Results from an independent two-tailed 87Sr/86Sr baseline range, three individuals were identified as non-local . The addition of δ18O and δ13C isotopes aided in identifying further non-locals, which for δ18O included burial 48 (− 10.0‰), and for δ13C included burials 42B, 72, 105, and 138 with a C4 dominant diet (− 5.3‰ to − 1.3‰). These results suggest that at least 16% (7/43) of the analysed individuals were non-local to the Cape Town region. The statistically significant difference between the oxygen data from Cobern Street and this research may suggest an underestimation of the extent of non-local origins in this population.While the geologically diverse Cape Town region resulted in a broad n = 63 adults)48. Additionally, a predominance of the male sex was observed at the Marina Residence hospital burial site 11. We stress, however, that the complete absence of identified females in this research is distinct. Further, while the majority were adults, six non-adult individuals were identified, with one young child (aged 3 to 6 years) and the remaining non-adults presenting ages of 11 to 20 years. While the employment of adolescents is consistent with VOC personnel archival data, the employment of young children (< 10 years) was unlikely9. The 87Sr/86Sr and δ13C isotope data from the permanent first molar of the 3- to 6-year-old child was consistent with local expectations ; therefore, it is possible that the child had local origins. However, as this combination of isotope data could also be reflective of alternative origins, it is also possible they may have been the child of a passenger transiting through Simon’s Town aboard a VOC, or other company, ship.Demographic assessments in the form of sex, age, and associated material culture revealed two distinct demographic aspects that suggested a relation to the VOC hospital. The 25 individuals analysed for sex-specific peptides all had isoforms derived from both X and Y chromosomes. Identifying the AMELY peptide, SM(ox)IRPPY, in all tested individuals strongly suggested that all assessed individuals were biologically male, supporting the hypothesis that the site likely represented VOC personnel. To further investigate the significance of the sex distribution, we compared our results to the demography of previously excavated contemporary burial sites from Cape Town. For example, the Cobern Street burial site reported a slight skew towards male presence over females ; therefore, we expect a general or residential population to have a higher representation of non-adults, and in particular young children (< 12 years old). In contemporary Cape Town sites, the total non-adult population varies depending on burial site context. For example, non-adult representation at the Cobern Street burial site was approximately 30% (36/119), and at the Marina Residence hospital burial site in Cape Town, approximately 8% (6/78)11. Comparatively, the 3% non-adult representation in the Simon’s Town burial site more closely resembled the Marina Residence demographic. Reviewing available VOC archival data9 demonstrated that while non-adult individuals were employed, they were not aboard VOC ships in large numbers. Only 2% of all listed personnel and passengers aboard VOC ships were categorised as “Jongen”, which referred to "boys" aged between 10 to 17 years, employed as assistants for various specialist roles on board the ship. Overall, the age and biological sex results from this research represented the expected demographic of a VOC hospital burial site.In addition to this data, the expectation in a typical cemetery populace would be to find higher inclusions of infant burials considering this period was reported as having a relatively high infant mortality rate, with up to 27% of infants not surviving their first yearThe predominant burial style was supine with arms alongside or crossed over the body. Regarding associated material culture, there was evidence in some burials of small fragments of textile, rope, and small pins, suggesting enshrouding prior to interment. Few individuals were found with clothing buttons on or near the torso, implying some individuals were fully clothed at time of burial. Some associated artefacts supported a link between the burial ground and the Dutch colonial era (> 1652 CE). Finds such as European coins  dating to the seventeenth and eighteenth centuries, a trade bead necklace, and numerous fragmentary pipe stems and bowls (with a Dutch maker’s mark) were found throughout the site.28 and Indian Ocean basin29 populations. It must be noted that Cape Town would have already had a high degree of cultural admixture by the 1700s CE49. Dental modification increased in frequency across South Africa between the eighteenth and nineteenth centuries, particularly in the West, as associated with the increased population of enslaved people from other African and Indian Ocean nations. Therefore, if the buried population included the burials of enslaved people, it was expected to find intentional dental modification in the form of chipping or evulsion as was found at other VOC Cape region sites50. The only modifications to dentition observed were distinctly notched grooves from pipe smoking in four individuals, occurring from the persistent clenching of clay pipes. Notched grooves were supported by the presence of clay pipe fragments, some directly associated with the burials. As clay pipe use was most prevalent between the sixteenth and nineteenth centuries51, this observation was consistent with the period during which the VOC hospital was in use.There were no instances of intentional dental modifications in the form of decorative filing or tooth evulsion as prevalent amongst numerous African87Sr/86Sr ranges for multiple countries of interest, including South African, European, and some young volcanic Indian Ocean basin regions. Of note was that of all 43 87Sr/86Sr, no individuals exhibited extremely radiogenic ratios (> 0.7280), as have been identified in other analysed Cape Town sites, which has been suggested to indicate enslaved persons from regions such as Mozambique52. When considering the multi-isotope data distributions, six individuals  presented distinctly higher or lower ratios or values from the general sampled individuals and thus merit a more detailed discussion.The strontium isotope data of all 43 individuals analysed are compatible with known 87Sr/86Sr of 0.715812 . The 87Sr/86Sr of burial 48 (0.710458) was within the expected local Cape Town range, with a δ13CVPDB value of − 10.6‰ indicated a mixed C3–C4 diet. Yet, burial 48 exhibited the lowest δ18OVPDB of − 10.0‰. Low δ18O values may correlate with regions far from the equator, in higher altitudes, further distance from the sea, or areas with heavy precipitation. Therefore, it is possible that burial 48 was non-local considering Cape precipitation data.Burial 66 exhibited the highest 812 Fig. , a δ18OV87Sr/86Sr of 0.707589, but high δ18O–δ13C values. The oxygen isotope value for this individual was the highest in the range at δ18OVPDB = − 3.6‰. Similarly, their δ13CVPDB value was second highest at − 1.9‰, representing a C4 dominant diet. Further, burial 43D had the lowest 87Sr/86Sr of 0.707409, with a δ18OVPDB value of − 5.9‰, and a δ13CVPDB value representative of a mixed C3–C4 diet (− 9.4‰). Burial 43A also exhibited a lower 87Sr/86Sr of 0.708839, with a δ13CVPDB value of − 11.9‰ (mixed C3–C4 diet) and a δ18OVPDB value of − 6.7‰. Such low 87Sr/86Sr are suggestive of regions with a younger geology53 or with increased influences from marine derived strontium, and are not considered characteristic of the Cape Town region.Burial 42B had a low δ13C values indicative of dominant C4 diets. Burial 105’s 87Sr/86Sr of 0.709102 was within the local range, with a δ18OVPDB value of − 6.5‰, yet δ13CVPDB value of − 5.3‰ is suggestive of a mixed diet with significantly higher C4 inclusion than is expected for the Cape region. Burial 72 also had an 87Sr/86Sr within the expected local Cape Town range (0.710528), and a δ18OVPDB of − 6.6‰, yet also indicates a high δ13CVPDB value of − 3.8‰, suggesting a diet more dominant in C4 plants. Finally, burial 138 exhibited a local 87Sr/86Sr (0.712174), with a δ18OVPDB of − 5.0‰. The δ13CVPDB value of burial 138 was the highest amongst the analysed sample (δ13CVPDB = − 1.3‰), and like burial 42B, suggests a C4 dominant diet.Three further individuals exhibited high 4 plants. The VOC occupation of the Cape region introduced C4 plants to the predominantly C3 diet. Yet, most research on the area has found that local diets were C3 dominant to mixed C3–C4. A pure C4 diet has been considered indicative of migration from elsewhere into the Cape in other contemporary sites54, therefore, we can conclude that these individuals did not originate from the Cape region.While exact origins cannot be identified, burials 42B, 72, 105, and 138 originated from regions where diets were dominant in C87Sr/86Sr, δ18OVPDB and δ13CVPDB data between Simon’s Town (this research) and the contemporary Cobern Street site. Differences included more confined 87Sr/86Sr and δ13C values, and minimal overlap in the δ18O data. However, the δ18O was assessed with discretion due to intra-dental variations or variations introduced by pretreatments44. This is of note as the Simon’s Town samples were pretreated, whereas the Cobern Street samples were not. When comparing the data of all three isotopes between the sites, independent two-tailed t-tests highlighted statistical differences between the two populations for 87Sr/86Sr ; t(40) = 4.241, p = < 0.001) and δ13O  = 10.874, p = < 0.001), while the δ13C values were not statistically different ; t(71) = 1.369, p = 0.175). The higher 87Sr/86Sr and δ13CVPDB values from Cobern Street were combined with dental modifications to identify enslaved individuals who were displaced from outside of the Cape region34. However, considering the sites are contemporary, there are notable and significant population differences between those buried at Simon’s Town versus those at Cobern Street. While further comparable data from the region would be required to make stronger inferences between sites, the statistically significant differences in the multi-isotope data between these two contemporary VOC-related sites suggested that the populations were dissimilar in geographical origins.Finally, it is worth briefly highlighting that a clear difference may be observed between the boxplots comparing the 7/43) of geographically diverse non-local individuals in the Cape Town region. Directly comparable isotope data from a contemporary VOC related burial ground highlighted statistically significant differences between the 87Sr/86Sr and δ18O values of Simon’s Town and Cobern Street populations. The few associated artefacts found with burials were congruent with the Dutch colonial era. However, the age and sex demographics more closely resembled the contemporary Marina Residence hospital burial ground in Cape Town. As only males were identified through peptide analysis (25/25), this strongly supported an atypical burial population, such as a location catering to VOC personnel.Taking all available data into account, the overall combined isotope results suggested that the 43 analysed individuals exhibited a combination of possible local individuals, and 16%  with six non-adults. Moreover, 25 individuals assessed for enamel peptide analysis presented with both AMELX and AMELY sequences which determined that all 25 individuals were biologically male. The most prevalent burial style was supine with arms down either side of the body or flexed over the chest or torso. The material finds from the site included fragments of clay pipes, European trade coins, trade beads, buttons, and textile remnants.87Sr/86Sr), as well as oxygen (δ18OVPDB) and carbon (δ13CVPDB) from enamel carbonate was applied. Our findings demonstrated that a combined isotope approach was valuable in identifying a geographically diverse population, with the addition of δ18O and δ13C being of use in identifying non-locals. At least 16% of the analysed population (7/43) were non-local to the Cape Town region, supporting the link between this site and the former VOC hospital’s burial ground.As isotope analyses could only be applied to tooth enamel; a multi-isotope approach using strontium  due to poor preservation affecting visibility. Sampling involved the extraction of two teeth per individual, with one tooth specifically for aDNA analysis to avoid contamination, using stainless steel tools. A total of 84 individuals were sampled. Permits were obtained for destructive sampling/analysis, and for the export of human remains outside of South Africa , completed the excavation of the site. A forthcoming site report detailing the excavation will be publicly available. The skeletal remains were poorly preserved; therefore, osteological observations were limited to dental modifications or gross pathological conditions. Non-adults were aged by assessing dental eruption, formation and epiphyseal fusion14C dating, carbon and nitrogen isotopes in collagen, and aDNA, but all tests revealed that neither collagen nor aDNA had preserved.Several preliminary tests on dentine and tooth roots were conducted for 55.Peptide analysis uses tooth enamel to assess the sex-chromosome encoded enamel-forming proteins, amelogenins, which are laid down during the development of our teeth, and therefore inform us of biological sex. Amelogenin proteins are sexually dimorphic markers of X and Y chromosomes and can therefore indicate whether an individual is chromosomally male or female56. The enamel surface was abraded to remove surface contaminants and washed in Milli-Q water. Peptide extraction and analysis was performed55 with enamel etching in 60 μL of 5% (v/v) HCl extended to 60 min. Peptides were purified using C18 resin-loaded ZipTips with the final 60% acetonitrile/0.1% formic acid elution lyophilised. Each sample was redissolved in 0.1% aqueous TFA (10 µL), centrifuged  and 8 µL of the supernatant transferred into a tapered LC vial. Samples were analysed with a Dionex U3000 nanoLC coupled to a ThermoFisher LTQ FT Ultra Mass Spectrometer containing a nano-ESI source. An injection volume of 2 µL was loaded onto a C18 Pepmap300 loading column  using 0.1% aqueous TFA as a loading solvent. Sample separation was performed using a C18 Pepmap300 column  with a gradient of two mobile phases: mobile phase A  and mobile phase B . Peptides were eluted using a 30 min linear gradient of mobile phase B from 0 to 55% at a flow rate of 0.4 µL/min followed an increase to 90% B over 0.1 min, which was held for 5 min before returning to 0% B over 0.1 min for a column equilibration period of 25 min. The nano-ESI source was operated a voltage of 1.7 kV in the positive ion mode. The (entrance) capillary temperature was set at 275 °C, with inner capillary voltage value set on 37 V and tube lens value of 145 V. Three data channels were acquired. Full scan FTMS spectra were acquired over a 400–1200 m/z range at a nominal resolution of 100,000 (at m/z = 400). MS/MS channels were set to acquire precursor ions at m/z 440.2 and m/z 540.3, with a window of m/z 8, corresponding to the 2+ ions of the AMELY and AMELX peptides, respectively. Precursor ions were fragmented in the ion trap of the spectrometer using He collision gas at a nominal collision energy of 35. Product ions were detected in the ion trap. The instrument was controlled, and data visualised by Xcalibur software (Thermo Fisher). The presence of AMELX and AMELY peptides was determined by plotting chromatograms for the transition m/z 540.3 to 714.4 (characteristic for AMELX), and m/z 440.2 to 645.4 (characteristic for AMELY).A total of 25 samples were processed for targeted proteomic (LC–MS) analysis, where sample protocols required uncleaned enamel chips or complete tooth crowns87Sr/86Sr), oxygen (δ18OVPDB) and carbon (δ13CVPDB) isotopes are established methods in archaeology to investigate geographic origins, mobility, and dietary habits of past organisms61. Oxygen and carbon, classified as light isotopes, are influenced by the diet and fractionation processes occurring within the body. Conversely, strontium isotope ratios which are classified as heavy isotopes, are indicative of the underlying geology without undergoing any fractionation processes within the body62. Due to the predominant mineral content in human dental enamel, it is particularly beneficial in archaeological analysis due to its resilience to diagenetic changes64. As the interest of this research focuses local vs. non-local origins, the first (M1) and second (M2) molars were selected for sampling. During amelogenesis of these teeth, the individual's dietary intake containing strontium, oxygen and carbon isotopes representative of their geographical environment and diet, are incorporated into the enamel’s calcium hydroxyapatite 6(OH)2)69. Strontium can be incorporated into HAP through substitution at the calcium (Ca) sites, while oxygen and carbon can be incorporated via structural carbonate (–CO3) substitution at both –PO4 and –OH sites70. As HAP does not remodel, the Sr and carbonate substitution sites in HAP represent the isotopic ratios and values incorporated into the enamel during early childhood (during the first 0–3 and 3–8 years of life for M1 and M2 respectively)63. Consequently, strontium ratios and oxygen isotopic values from human teeth are representative of the underlying geology and water which are incorporated into the body through diet and imbibed water.Analysis of strontium , C4  and CAM  photosynthetic pathways73. CAM plants have not been explicitly discussed in this research due to the complexity of their mechanisms, as they can produce similar dietary δ13C values to both C3 and C4 pathway plants. The inorganic (apatite) components of human tissue are analysed in this research and represent the total diet, inclusive of the protein component that is typically represented by collagenous dietary isotope analysis75.Carbon isotope values can broadly infer diet due to isotopic differences between plants utilising C87Sr/86Sr, δ18O, and δ13C isotope analysis following preparation protocols outlined by Kootker et al.52. Enamel powder was sampled at the Faculty of Archaeology chemistry laboratory at Leiden University, The Netherlands. The surfaces of all sampled cusps were abraded with a Dremel rotary tool to remove external organics. Then, approximately 10 mg of enamel powder was removed in a band across the height of the crown  using a Dremel rotary tool with diamond burr accessories. Initial indicators of enamel preservation were considered at various stages of sampling by assessing hardness of tooth enamel, response of enamel to drilling, and colour of raw enamel powder69. As all drilled teeth had well-preserved enamel, and the duration of interment was expected to be no more than 300 years, no further preservation checks  were deemed necessary. Each sample was treated with 1 mL of a 3% NaOCl solution at room temperature for 24 h, followed by four Milli-Q® water rinses. Then, 1 mL of acetic acid–Ca acetate buffer (pH = 4.75) solution was added for at room temperature for 24 h, followed by the same four Milli-Q® wash rinsing procedure. The samples were transferred to the department of Earth Sciences at the Vrije Universiteit Amsterdam, The Netherlands, where approximately 2 ± 1 mg of enamel powder was subsampled for 87Sr/86Sr and transferred to the USA class 100 clean laboratory at the Department of Earth Sciences for Sr purification. For combined δ18O and δ13C, 0.3 mg ± 10% was subsampled in clean Exetainer® vials and transferred to the stable isotope facility. Strontium column extraction and sample loading were performed following previously published protocols52.A total of 43 individual samples were taken for 86Sr/88Sr of 0.119476. The NIST® SRM® 987 standard averaged 0.710258 ± 0.000008 throughout this study . The intermediate precision over the period 2017–2021 using the same method was 0.710254 ± 0.000018 . All measurements were normalised to an accepted value of 0.710240. The total procedural blanks (n = 3) contained less than 56 pg strontium. The 87Sr/86Sr are reported plus ± 2 standard error (2SE), representing the analytical uncertainty calculated from 240 cycles of 8.1 s integration time (12 blocks of 20 cycles) within each run.The Sr isotope compositions were measured using a Thermo Scientific™ Triton Plus™ thermal ionisation mass spectrometer (TIMS) housed at the Vrije Universiteit Amsterdam. The strontium ratios were determined using a static routine and were corrected for mass fractionation to an δ18O and δ13C, the enamel samples were flushed with helium gas, then reacted with orthophosphoric acid (H3PO4) (100%) at 45 °C for 24 h. The isolated carbon dioxide (CO2) was analysed using a Thermo Finnigan GasBench II preparation device interfaced with a Thermo Finnigan Delta + mass spectrometer at the Earth Sciences Stable Isotope laboratory, Vrije Universiteit Amsterdam, The Netherlands. The data were normalised to the Vienna Peedee Belemnite (VPDB) scale using an in-house carbonate reference material (VICS) calibrated against NBS19 and LSVEC-certified reference materials. The international control standard IAEA-CO1 was used to check instrument performance , mean − 2.46‰, n = 14 for the Cobern Street data (2013)). The reproducibility of IAEA-CO1 during the analytical session was ± 0.12‰ (1σ) for both sessions. Both δ18O and δ13C are expressed relative to Vienna Pee Dee Belemnite (VPDB) standard in per mil.For Permits were obtained for the destructive sampling, analysis, and export of human skeletal remains outside of South Africa. This was approved by the South African Heritage Resources Agency  of the National Heritage Resources act, 1999 (Act 25 of 1999) and Regulation 3(3) of PN 298  under CASE# 18061907SB0704E, and export permit under Section 32(19) of the National Heritage Resources Act (Act 25 of 1999), under CaseID: 14380), with the permit holder being Tim Hart.Supplementary Table S1.