The aim of this study is to describe the characteristics, survival and prognostic factors of a cohort of patients with bone metastases (BMs) from differentiated thyroid carcinoma (DTC).This was a multicenter retrospective observational study including patients diagnosed with BMs from DTC between 1980 and 2021. A Cox regression was performed to study prognostic factors for 5- and 10-year survival. Kaplan–Meier and log-rank tests were performed for the survival analysis and comparison between groups. P = 0.047) and treatment with I131 (HR 0.214 (95% CI 0.069–0.665); P = 0.008) at 5 years, the presence of other metastases  and treatment with I131 (HR 0.23 (95% CI 0.058–0.913); P = 0.037) at 10 years.Sixty-three patients were evaluated. Median follow-up from BM diagnosis was 35 (15–68) months. About 30 (48.4%) patients presented with synchronous BMs. Regarding histology, 38 (60.3%) had the papillary variant. BMs were multiple in 32 (50.8%) patients. The most frequent location was the spine (60.3%). Other metastases were present in 77.8%, mainly pulmonary (69.8%). Concerning treatment, 54 (85.9%) patients received I131, with BM uptake in 31 (49.2%) and 25 (39.7%) received treatment with multikinase inhibitors. Regarding complications, 34 (54%) patients had skeletal-related events, 34 (54%) died and 5- and 10-year overall survival was 42.4% and 20.4%, respectively. Significant prognostic factors in the multivariate analysis were the presence of lymph node involvement (hazard ratio (HR): 2.916; 95% confidence interval (CI): 1.013–8.391;Our study reflects the management of patients with bone metastases from differentiated thyroid carcinoma in real clinical practice in several centers in southern Spain. Overall survival at 5 and 10 years was lower in patients who were not treated with I131, had nodal involvement and/or had other metastases. Bone metastases (BMs) are infrequent in differentiated thyroid carcinoma (DTC), occurring in 2–13% of the cases. It has been estimated that BMs occur in 7–28% of follicular carcinomas and 1.4–7% of papillary carcinomas, although data vary considerably in published studies , treatmeBMs are usually clinically silent at onset. Their most frequent clinical presentation is pain followed by fractures . SkeletaTreatment may be aimed at palliating the symptoms, preserving or restoring the anatomy and/or modifying the disease . The maiThe main objective of our study was to describe the characteristics, management and outcomes in real clinical practice and overall survival in a cohort of patients with BMs from DTC. The secondary objective was to identify prognostic factors associated with overall mortality in this group of patients.This was a retrospective observational multicenter study including adult patients with BMs from DTC treated in six hospitals in Andalusia (Spain) between 1980 and 2021. The study was approved by the ethics committee of Virgen del Rocío University Hospital.The variables collected were sex, age (at DTC and BM diagnosis), follow-up time (using BM diagnosis as time zero), BM diagnosis , histologic variant , ultrasensitive thyroglobulin level at DTC diagnosis, stage according to the eighth edition of the TNM , charactn (%)). The Chi-square test was used for the comparison of proportions. To study risk factors for mortality at 5 and 10 years, a univariate analysis was performed using Cox regression and subsequently a multivariate analysis with the variables that obtained a P < 0.1. The overall survival analysis was performed using the Kaplan–Meier method, and the log-rank test was used to compare survival between groups. A P-value less than 0.05 was considered statistically significant.The statistical analysis was conducted using Statistical Package for Social Sciences (SPSS®) version 25 and R tool for Windows. The descriptive analysis was performed by obtaining the median and the quartiles for quantitative variables (expressed as P50(P25–P75)) and frequency for qualitative variables (expressed as The sample comprised 63 patients.The baseline characteristics of the patients are shown in P = 0.01), had higher I131 uptake (P < 0.001), received higher doses of I131 after BM diagnosis (400 (250–570) vs 0 (0-112) mCi; P < 0.001) – due to their increased uptake of I131 – and received TKI treatment less frequently (P = 0.034) than those with metachronous BMs. There were no other differences between the two groups.The patients with synchronous BMs at DTC diagnosis had fewer metastases in other locations (The treatments performed and the results are shown in P = 0.423), presence of multiple bone metastases (P = 0.304), presence of metastases in other locations (P = 1) or presence of pulmonary metastases (0.823) between patients who received treatment with I131 and those who did not (X2 test).There were no statistically significant differences when comparing the presence of lymph node metastasis (N1), more advanced tumor (T) stages (P = 0.304), antiresorptives (P = 0.212), corticosteroids (P = 0.135), surgery (P = 0.49), radiotherapy (P = 0.707) or TKIs (P = 0.719) than patients with a single BM. The percentage of SRE in patients with a single BM was 65.6% vs 41.9% in patients with multiple BMs, with a result close to significance (P = 0.059).Patients with multiple BM did not receive more treatment with I131 (P = 0.061). Of the eight patients treated with TKIs and antiresorptive agents, 2 (25%) presented mandibular osteonecrosis in the context of treatment with zoledronic acid and TKIs (sorafenib and axitinib in each case). One of them required surgery.Of the 21 patients who received antiresorptive therapy, in 18 (85.7%) it was prescribed after the patient experienced an SRE. Of the three who received these drugs preventively, two had not had an SRE. There were no significant differences between the patients who received this treatment and those who did not, but the results were close to significance in terms of spinal cord compression , metastases in other locations ; P = 0.019, lung metastases , corticosteroid treatment ; P = 0.035, I131 treatment , I131 dose after BM diagnosis ; P = 0.003 and I131 uptake by BMs .The results of the univariate analysis of risk factors for 5-year mortality are shown in When studying combinations of treatments  and their association with mortality, the groups obtained were of insufficient size to perform Cox regression. Therefore, a chi-square test was performed. None of the results reached statistical significance.P = 0.047) and treatment with I131 . The presence of pulmonary metastases yielded a result close to significance .The variables that were statistically significant in the 5-year multivariate analysis were the presence of lymphatic involvement (N1) , corticosteroid treatment , I131 dose after BM diagnosis  and I131 uptake by BMs .The results of the univariate analysis of risk factors for 10-year mortality are shown in P = 0.048) and treatment with I131 . The presence of lymphatic involvement (N1) scored close to significance .The variables that were statistically significant in the 10-year multivariate analysis were the presence of metastases in other locations  and with and without pulmonary metastases (P = 0.012), and differences approached the significance between the groups that received treatment with I131 and those that did not (P = 0.071). When analyzing 10-year survival, there were significant differences between the group with other metastases and the group without metastases (P = 0.013).The overall survival plots according to the most relevant factors that reached statistical significance in the multivariate analysis are shown in et al. . The overall survival at 5 and 10 years was 42.4% and 20.4%, respectively. Furthermore, our study supported I131 treatment as a prognostic factor for survival, as described in other studies  presented osteonecrosis of the jaw. In December 2010, the European Medicines Agency issued safety warnings about ONJ risk in patients receiving sunitinib or bevacizumab. To date, there is little published data on the combination of these two treatments and none in patients with DTC. Moreover, studies on this subject are usually small and retrospective. They are also limited by the fact that most of the patients studied have a short survival and therefore a short treatment with bisphosphonates. In a study conducted in 76 patients with renal carcinoma and bone metastases, the combination of both treatments not only had an impact on patient survival but also increased the risk of osteonecrosis of the jaw  than in our study, although they reported similar rates of fracture (24.5% vs 25.4%) and spinal cord compression (11.9% vs 12.7%). Their percentage of deaths was also lower than ours (27.3% vs 54%), despite a longer median follow-up time in months (48 vs 35). This may be explained by their lower percentage of patients with N1 and pulmonary metastases, which was associated with lower survival in our study. It was not possible to study whether treatment with antiresorptive drugs was protective for SRE, since only three patients received these treatments preventively and not after having had an SRE.Finally, SRE were less frequent in the study by Mazziotti et al.  and 10 years (P = 0.062). This was the only factor related to mortality in the study published in 1999 by Lin et al.  and close to significance at 10 years (P = 0.095), similar to that published by other authors , and the presence of metastases other than BMs was a factor related to lower 10-year survival (P = 0.048). These have previously been described as factors associated with mortality in other studies (P = 0.076). This is congruent with the results of other studies  and in the long follow-up time, with a median of 35 months and a maximum of 515 months.Our study reflects the management of patients with BMs from DTC in real clinical practice in several centers in southern Spain. The use of antiresorptive drugs was lower than recommended based on current evidence on their preventive role in SRE, which were present in 54% of our patients. Overall survival at 5 and 10 years was lower in patients who were not treated with I131, had nodal involvement and/or had other metastases.Given the low frequency of BMs from DTC, multicenter studies involving a larger number of patients will help to improve our understanding of this disease and the treatment of these patients. The implementation of unified management protocols would also be beneficial both to improve the quality of the studies performed and to provide patients with better care.The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.All authors collected the data. A P-G analyzed the data and wrote the first draft of the manuscript. E G-N conceived the study. All authors reviewed and corrected the manuscript.


In recent years, numerous dosing studies have been conducted to optimize therapeutic antibiotic exposures in patients with serious infections. These studies have led to the inclusion of dose optimization recommendations in international clinical practice guidelines. The last international survey describing dosing, administration and monitoring of commonly prescribed antibiotics for critically ill patients was published in 2015 (ADMIN-ICU 2015). This study aimed to describe the evolution of practice since this time.A cross-sectional international survey distributed through professional societies and networks was used to obtain information on practices used in the dosing, administration and monitoring of vancomycin, piperacillin/tazobactam, meropenem and aminoglycosides.A total of 538 respondents (71% physicians and 29% pharmacists) from 409 hospitals in 45 countries completed the survey. Vancomycin was mostly administered as an intermittent infusion, and loading doses were used by 74% of respondents with 25 mg/kg and 20 mg/kg the most favoured doses for intermittent and continuous infusions, respectively. Piperacillin/tazobactam and meropenem were most frequently administered as an extended infusion . Therapeutic drug monitoring was undertaken by 90%, 82%, 43%, and 39% of respondents for vancomycin, aminoglycosides, piperacillin/tazobactam, and meropenem, respectively, and was more frequently performed in high-income countries. Respondents rarely used dosing software to guide therapy in clinical practice and was most frequently used with vancomycin (11%).We observed numerous changes in practice since the ADMIN-ICU 2015 survey was conducted. Beta-lactams are more commonly administered as extended infusions, and therapeutic drug monitoring use has increased, which align with emerging evidence.The online version contains supplementary material available at 10.1186/s13054-023-04527-1. Treating serious infections in the intensive care unit (ICU) can be complex and challenging . CurrentProduct information-derived dosing has limited application in critically ill patients given the altered PKs and challenges in pathogen susceptibility to antibiotics, relative to other population groups in the hospital . Dosing International surveys have been conducted to measure the translation of antibiotic optimization research into clinical practice –15. The The aim of this international cross-sectional study was to survey clinicians working in ICUs worldwide to describe contemporary practices in dosing, administration and monitoring of commonly prescribed antibiotics including glycopeptides, beta-lactams, and aminoglycosides.A panel of international experts formed the writing committee and developed the survey, building upon the foundations of the ADMIN-ICU 2015 survey . The surThe survey was constructed to describe professional characteristics of the respondent. Additionally, the survey collected details of the respondent’s work location and access to resources associated with dosing, administration and monitoring of commonly prescribed antibiotics in the ICU. The country in which respondents worked was categorized by region and economy according to World Bank criteria .The investigators developed clinical vignettes to ascertain information regarding an individual’s practices used for dosing, administering and monitoring of vancomycin, beta-lactams, and the respondent's choice of aminoglycoside. Clinical vignette responses were based on a septic 35-year-old patient, weighing 80 kg with a height of 1.78 m and normal renal function (creatinine clearance = 90 mL/min), with an additional scenario of a 200 kg patient selected for vancomycin. The use of a loading dose (LD), maintenance dose (MD) and frequency, infusion duration, TDM utilization, PK/PD target and dose adjustment method used in clinical practice was surveyed.A range of infusion durations were defined as presented in Additional file From August to December 2021, an open invitation to answer the online survey hosted on the Research Electronic Data Capture (REDCap©) platform was accessible to respondents. Invitations to participate were distributed to members of professional societies as listed in the acknowledgements. Additionally, national coordinators for this study distributed the survey via local networks. Distribution strategies were chosen to maximize the breadth of respondents, to provide an adequate international respondent sample. A reminder email was sent after one and three months. A cross-sectional global representation of clinicians involved in the treatment of critically ill infections were the target of this survey. No incentive was offered to respondents to complete the survey.The data were extracted from REDCap© into Microsoft Excel®. Members from the writing committee  conducted a consensus review of all data. Data were excluded from the final analysis as described in Additional file Data were expressed as median values with inter-quartile range for continuous variables, and as numbers and/or percentages for categorical variables. Data derived from ADMIN-ICU 2015 were also expressed in this way. A sub-group analysis was performed to determine any difference in TDM utilization or beta-lactam prolonged infusion (continuous or extended infusion) administration between academic hospitals (university or university affiliated) and general hospitals. A Chi-square test was used to make this determination. Descriptive summary statistics were produced (using IBM SPSS Statistics v27) to present survey findings.A total of 538 respondents from 409 hospitals in 292 cities in 45 countries completed the survey. Respondents from a variety of regions were represented, including Europe and Central Asia 30%, 163/538), Middle East and North Africa , and East Asia and Pacific . Most respondents were from a high-income country (HIC) , followed by upper-middle-income country (UMIC) , with 23% (125/538) from either a low-income country (LIC) or lower-middle-income country (LMIC) , with 22% preferencing continuous infusion (CI) see Fig. . Most rep = 0.049),  as compared to LMICs and LICs . Furthermore, vancomycin II MDs were larger in HICs as compared to LMICs and LICs  to minimum inhibitory concentration (MIC) ratio of 400–600 mg·h/L , by dosing guidelines 33%) and by linear adjustment post-TDM (31%) (see Table 3% and byp = 0.001),  (36%), followed by 100% fT > 4xMIC (23%). When assessing EI and CI only, the most common PK/PD target remained 100% fT > MIC.Piperacillin/tazobactam was most often administered as an extended infusion (EI) (42%) followed by II (36%) and CI 14%) ,  followed by 100% fT > 4xMIC (25%) (see Table fT > MIC.Most respondents preferred to administer meropenem as an EI 51%), followed by II (38%) and CI (7%) , followed by product information 31%) and pharmacist recommendation (31%)  over 30–60 min (II) . Gentamicin was the preferred aminoglycoside prescribed , followed by amikacin , and tobramycin . Once daily dosing of aminoglycosides was based on actual body weight by most respondents in our 80 kg patient scenario, with dosing strategies presented in Fig. TDM was performed by 82% of respondents see Fig. . TDM wasThe median peak and trough concentration targets are presented in Additional file Dosing software was used by 11%, 3%, and 8% of respondents to adjust vancomycin, beta-lactam, and aminoglycoside doses, respectively  to guide vancomycin dosing in our study. This aligns with the Consensus Guideline , and is Interestingly, only 11% of respondents used dosing software, even though it is the recommended approach in the Consensus Guideline and has been shown to improve target attainment and reduce nephrotoxicity compared to a trough-guided approach , 26. AdoS. aureus infections is established as an AUC/MIC of 400–600 mg·h/L [The vancomycin PK/PD target for 0 mg·h/L . However0 mg·h/L , 27. Thi0 mg·h/L . As with0 mg·h/L .Beta-lactam LDs were mostly used for EI and CIs, which aligns with the 2021 surviving sepsis campaign recommendation . HoweverUptake of beta-lactam TDM varied markedly depending on region, with large variability observed even within HICs. However, TDM utilization was more prevalent in academic hospitals. Future exploration of contributing factors to such variability should be undertaken. The use of TDM has increased from approximately 10% in ADMIN-ICU 2015 to around 40% in our study  was 100% ic shock , 16.Aminoglycosides were mostly administered as a once daily II, which aligns with contemporary guidelines , 18, 35.TDM-guided aminoglycoside dosing is recommended in contemporary guidelines , 18. In Our results showed that a Cmax/MIC target for aminoglycosides was most frequently used, with aminoglycoside peak and trough targets comparable with the ADMIN-ICU 2015 findings , this influence is unlikely meaningful.Fourthly, it is plausible, although unlikely, that changes in practice observed in this study as compared to ADMIN-ICU 2015 were due to sampling a different cohort of respondents, as opposed to representing practice change over time. All data collected were de-identified as required during our ethics approval process. Therefore, it was not possible to report respondents who completed both ADMIN-ICU surveys. However, the survey samples are large enough to infer that they are likely representative of true practice.Fifthly, the use of a simple clinical vignette may have limited the generalizability of our findings. For example, if a patient exhibited augmented renal clearance or developed renal failure, respondents may have been more likely to recommend TDM or prolonged infusions. The simple clinical vignette was selected to be consistent with ADMIN-ICU 2015 in order to compare findings.Finally, the survey responses may not align with those made in clinical practice based on an actual patient and their inevitable complexities. However, a key objective of this study was to characterize changes in practice since the ADMIN-ICU 2015, which was subject to the same limitations and hence comparable. Although, differences in respondent demographics were apparent in the present study, with less experienced respondents and more pharmacists included.Despite these limitations, importantly, we present contemporary dosing, administration and monitoring of a selection of antibiotics used for serious infections and highlighted changes in practice over the last 7-years and how current practice aligns with contemporary evidence and guidelines. Although large variation in dosing and monitoring practices were observed, there have been significant clinical practice changes aligning with contemporary evidence and guidelines. Most notably, beta-lactams are now most commonly administered as an EI, with TDM use increasing in clinical practice. The authors believe that observational methods could be used to better understand current antibiotic dosing practices in this patient cohort on a global scale. This would increase confidence in the findings of this study. Additionally, future research is needed to assess the impact of antibiotic dosing strategies on important patient-centred outcomes.Additional file 1. Supplementary Material, Description of data: Table S1. Definition of infusion types and duration; Table S2. Online survey questions; Table S3. Exclusions; Table S4. Respondents as per Region and Economy; Table S5. Results according to Region and Economy; Table S6. TDM utilization and beta-lactam prolonged infusion administration according to hospital type.


Euphorbia resinifera is a melliferous, medicinal, and endemic plant to Morocco. Nevertheless, its ecological and genetic diversity still unknown. The objective of this study is to analyze the diversity and genetic structure of Moroccan wild populations of E. resinifera using ISSR markers. Twelve natural populations collected from its geographical range in Morocco were analyzed using 14 ISSR primers.r = 0.507; P < 0.0001).A total of 125 bands were obtained, with polymorphism of 74.81%. The polymorphic information content (PIC), resolving power (Rp), Shannon’s information index (I), and total genetic diversity (Ht) were 0.33, 2.8, 0.35, and 0.21, respectively. The analysis of molecular variance showed that 75.56% of the total variability is present within populations and that 24.44% exists among populations. Also, the analysis showed a very low genetic differentiation between groups of mountain range type (FCT = 0.066), mountain versant type groups (FCT =  −0.024), and altitude groups (FCT =  −0.022). Moreover, the geographical distances between populations are correlated with their corresponding genetic distances according to the Mantel test (These results suggest that the population structuring follows a model of isolation by geographical distance. Indeed, the genetic structuring of populations into two groups obtained from PCoA and structure analysis revealed a dependence on the geographical origin of the populations. By contrast, the genetic distances are not correlated with the altitude. Euphorbia is the largest of the Euphorbiaceae family and the second largest genus of flowering plants in the world [Euphorbia resinifera O. berg is one among them, growing naturally in the Atlas Mountains, particularly in Beni Mellal and Azilal provinces [E. resinifera employs both sexual and vegetative means of reproduction to ensure the survival and expansion of its population [E. resinifera populations during the flowering season. Thus, with an annual honey production of about 300 tons, it is an important plant of the solidary agriculture in the region. It is well known as a melliferous plant for its high therapeutic and nutritional quality honey [With nearly 2000 species, the genus he world , 2. Desphe world , the genhe world . In Morohe world . Euphorbrovinces . The plarovinces . It prespulation . The yelty honey , 9, whicty honey .E. resinifera was recognized as having a vast therapeutic properties and benefits. In folk medicine, it is used to treat some types of complicated dermatoses, cancer treatment and control glycaemia in type II diabetics [E. resinifera was identified [Additionally, iabetics –13. In 1entified . This moentified . Moreoveentified , 17, antentified , 19, an entified , 21.E. resinifera populations may help to identify different genotypes and rationalize conservative treatments. Therefore, it has become imperative to establish a research program aiming at the evaluation of the genetic diversity of this species. Our first study on morphological characters showed high phenotypic diversity in twelve Moroccan populations [Nevertheless, the genetic resources of this species underwent important genetic erosion caused by various factors including the overgrazing, the deforestation provoked by a demographic pressure, voluntary or natural fires, and some fungal diseases attacking the plant. Consequently, emergency measures should be taken to safeguard this wild species. Thus, the safeguard and valorization of these local plant genetic resources constitute not only an imperative but also a major component of the stability of natural ecosystems and their valorization in agro-economic, therapeutic, and environmental perspectives. For that, the description of Moroccan ulations . HoweverSeveral types of molecular markers are employed for assessment of genetic diversity and relationships in plant species, including RFLPs (restriction fragment length polymorphisms) , RAPDs . Firstly, global analysis of AMOVA was done to apportion the total genetic variation into two hierarchical levels: among populations (FST) and within populations. Secondly, hierarchical AMOVA analysis was used to partition the variation among:Two mountain range groups of populations: Middle Atlas  and High Atlas Three mountain versant groups of populations: North versant , Southwest versant , and South versant Two altitude groups of populations: very low  and low  Table The band profiles of each gel were scored visually and recorded as presence (1) or absence (0) of bands leading to the construction of data binary matrix . For each primer, the percentage of polymorphic band (PPB), the polymorphic information content (PIC), and resolving power (Rp) were determined. Also, the POPGENE software was used to measure the following parameters: numbers of alleles (Na), effective number of alleles (Ne), genetic diversity within populations (Hs), total gene diversity (Ht), coefficient of gene differentiation (Gst), and Shannon’s information index (I). Partition of the observed genetic variation and calculation of the corresponding em) was approximated estimated through Wright’s island model: Nem = 0.25 (1/FST - 1) [Population’s specific FST indices were also calculated to investigate which population is more divergent from the remaining. The average gene diversity over loci was calculated to estimate intra-population variability. These evaluations were performed using the package ARLEQUIN version 3.01 . The paiFST - 1) . A ManteFST - 1) . To idenFST - 1) , which iFST - 1) . FurtherFST - 1) .E. resinifera populations, of which 101 band were polymorphic. The number of bands ranged from 4  to 13 (UBC840), with an average of 7.21  varied from 0.12 (UBC834) to 0.44 (UBC836) with an average of 0.32, indicating that 32% of total genetic variability was distributed among populations and the remaining (68%) accounted for within populations. These results were congruent with that revealed by FST value, accounting for 0. 244 (Table em = 0.77) which provides information on amount of number of exchanged individuals between populations studied.Estimates of genetic diversity of studied populations are summarized in Table .12 UBC83 to 0.44 When AMOVA was performed, at three hierarchical levels, with two mountain range type, very low genetic differentiation was observed between groups (FCT = 0.066) even though that it is slightly significant Table . This inST values and geographic distances between the 12 populations are presented in Table r = 0.507; P < 0.0001), the geographical distances between populations are correlated with their corresponding genetic distances. These results suggest that the populations’ structure follows a model of isolation by geographical distance. By contrast, no significant association was obtained between the genetic distance and difference of altitude between populations . Thus, the populations “AAS” and “FMJ” with a low difference of altitude (2 m) have a high value of genetic distance (0.376), and the populations “BZO” and “WAW” with a high difference of altitude (743 m) have obtained the low value of genetic distance (0.195). Between 66 pairwise FST values, 54 values are significant, meaning that the populations are widely different from each other. The significant values varied from 0.084  to 0.466 , which mean that populations FMJ/BZO are the most genetically similar and OUZ/KSB are the most divergent. The result indicated that the Bin El Ouidane (BIN) and Bzou (BZO) populations have the lowest value of specific FST index (respectively 0.209 and 0.212), and Elksiba (KSB) population has the highest value of this index (FST = 0.289), implying that this latter population is the most divergent from the others studied. Regarding the intrapopulation variability assessed by gene diversity over loci (data not shown) which oscillated from 0.148 for Elksiba (KSB) to 0.261 for Bin El Ouidane (BIN) and 0.257 for Bzou (BZO), reflecting consequently for any in situ and/or ex situ conservation strategy should aim to include those later populations.The pairwise FK = 2 (H′ = 0.947), indicating the stability of the result for this model. Considering the genotypes as pure when the membership coefficient was greater than 0.80 and as a hybrid or admixture when the membership coefficient was lower than 0.80, 41 individuals among the 60 analyzed (68.33%) were assigned to one of the model’s defined groups. The first group (red) was formed by the individuals from High Atlas Mountain populations, namely Bzou (BZO4), Ouled Ayyad , Bin El Ouidane (BO3 and BO4), all individuals from populations of Ouzoud , Imi n’Ifri (IMI5), and four from Wawla , with a membership coefficient oscillated between 0.815 to 0.990, while the rest of this group were originating from Middle Atlas Mountain populations: Afourer (AFO2 and AFO4), Modj (MOD1), with a membership coefficient ranging from 0.953 to 0.980. Nevertheless, the other seven individuals, namely  from Tagzirt population,  coming from Afourer population, MOD3 from Modj population, IMI1 belonging to Imi n’Ifri population, and WAW3 from Wawla population could be considered as admixed (coefficients ranged from 0.507 to 0.644). The second group (green) contained individuals coming from High Atlas Mountain populations:  of North versant, with a membership coefficient ranging from 0.954 to 0.975, all individuals of Foum Jemaa population , and one individual (IMI2) from Imi n’Ifri population belonging to Southwest versant (coefficients between 0.840 and 0.959) and  collected from South versant having a membership coefficient between 0.922 and 0.986, while the rest of group include bushes coming from Middle Atlas Mountain populations: (AAS3 and AAS4) of North versant with an assignment coefficient of 0.888 and 0.86 respectively,  of Southwest versant with a membership coefficient from 0.932 to 0.969, and all bushes of Elksiba population  from South versant mountain, having a coefficient of assignment oscillating between 0.929 and 0.976. Finally, 12 remaining bushes which could be considered as admixture belonged to the OAY, AAS, AFO, MOD, IMI, and BZO populations (coefficients between 0.508 and 0.775).The structure analysis based on the ΔK method showed that the best number of genetic clusters (K) was 2, suggesting that all individuals fell into two clusters  in Iran by Pahlevani et al.  [Euphorbia prostrata Aiton, Euphorbia peplus L., and Euphorbia terracina L.)  [Euphorbia species by El-Hawary et al.  [Euphorbiaceae (Croton urucurana Baill) (89%) based on ISSR markers [E. telephioides (80.7%) revealed by allozyme markers [E. resinifera. These values are comparable with these obtained by Reginaldo et al. for Brazilian Euphorbiaceae (Croton urucurana Baill) using ISSRs markers (PIC = 0.29 and Rp = 3.4) [I = 0.35). This value is higher than that obtained by Reginaldo et al. (I = 0.26) for Brazilian Euphorbiaceae using ISSRs markers [E. resinifera populations is in agreement with the general trend for allogamous and long-lived woody perennial species (Ht = 0.28) and for angiosperm species (Ht = 0.28) [E. resinifera were largely differentiated  which could be due to restricted gene flow (Nem = 0.77) between populations. This finding is in concordance with that observed for other herbaceous outcrossing perennial plant species (Gst > 0.20) [Euphorbia telephioides (0.10) using allozyme markers [Euphorbia fauriei in Korea  and Euphorbia jolkinii in Taiwan  [E. resinifera populations.DNA markers have become a useful tool for evaluating the genetic diversity of many different plant species. In this work, ISSR markers were used to assess the genetic diversity of ctively) , in Saudctively) , and for= 57.7%) . However markers  and by D markers . In addip = 3.4) . Moreove markers . Besides = 0.28) . The wil > 0.20) . The Gst markers . Similar = 0.77) . The indThe analysis of molecular variance (AMOVA) showed that 75.56% of the diversity accounted for within population leaving 24.44% for among populations. The existence of high genetic variability within population should help the population to cope with local environmental changes. This result implies that sampling from a small number of populations, particularly those with high intrapopulation variability, is sufficient for species in situ and/or ex situ conservation. Bin El Ouidane (BIN) and Bzou (BZO) populations with high intrapopulation variability are more convenient for this purpose.E. resinifera populations. Strangely enough, genetic distances were not correlated to the difference of altitude between the populations . A similar result was reported for Moroccan walnut using SSR marker [r = 0.507; P < 0.0001). This result suggests that the population structure follows a model of isolation by geographic distance. Consequently, our finding suggests that more closely situated populations tend to be more genetically similar to one another.Moreover, hierarchical AMOVA revealed a very low genetic differentiation between the two groups of mountain range (FCT = 0.066), even though that it is slightly significant, which suggests that mountain range type have had little influence on structuration’s populations. These findings give choice to sampling from populations of Middle or High Atlas Mountains for conservation and breeding species. Likewise, when assembling the populations according to their mountain versant type and altitude, a very low genetic differentiation was obtained between respective groups , indicating that mountain versant type and altitude did not have an effect on population structure of the Moroccan R marker . Also, oR marker , 59, proE. resinifera populations in Morocco using ISSR markers. The results of this study confirmed that ISSR markers could be powerful tools for detecting genetic diversity among and within E. resinifera populations. The level of genetic diversity was high, and the genetic variation mainly existed within populations. The results led to structure of populations in two gene pools independently of mountain range type, mountain versant type, and altitude. Based on these results, it could be sufficient to sample from a few populations, particularly those most genetically diversified, for any in situ and/or ex situ conservation strategy.The present study is the first work aiming to evaluate the genetic diversity and structure of


This study compares the complementary information from semi-quantitative EEG (sqEEG) and spectral quantitative EEG  to detect the life-long effects of early childhood malnutrition on the brain.N = 202) from the Barbados Nutrition Study (BNS) were used to examine the effects of protein-energy malnutrition (PEM) on childhood and middle adulthood outcomes. sqEEG analysis was performed on Grand Total EEG (GTE) protocol, and a single latent variable, the semi-quantitative Neurophysiological State (sqNPS) was extracted. A univariate linear mixed-effects (LME) model tested the dependence of sqNPS and nutritional group. sqEEG was compared with scores on the Montreal Cognitive Assessment (MoCA). Stable sparse classifiers (SSC) also measured the predictive power of sqEEG, spectral-qEEG, and a combination of both. Multivariate LME was applied to assess each EEG modality separately and combined under longitudinal settings.Resting-state EEGs (p < 0.001); age (p = 0.01) was also significant, with no interaction between group and age detected. Childhood sqNPS (p = 0.02) and adulthood sqNPS (p = 0.003) predicted MoCA scores in adulthood. The SSC demonstrated that spectral-qEEG combined with sqEEG had the highest predictive power (mean AUC 0.92 ± 0.005). Finally, multivariate LME showed that the combined spectral-qEEG+sqEEG models had the highest log-likelihood (−479.7).The univariate LME showed highly significant differences between previously malnourished and control groups (This research has extended our prior work with spectral-qEEG and the long-term impact of early childhood malnutrition on the brain. Our findings showed that sqNPS was significantly linked to accelerated cognitive aging at 45–51 years of age. While sqNPS and spectral-qEEG produced comparable results, our study indicated that combining sqNPS and spectral-qEEG yielded better performance than either method alone, suggesting that a multimodal approach could be advantageous for future investigations.Based on our findings, a semi-quantitative approach utilizing GTE could be a valuable diagnostic tool for detecting the lasting impacts of childhood malnutrition. Notably, sqEEG has not been previously explored or reported as a biomarker for assessing the longitudinal effects of malnutrition. Furthermore, our observations suggest that sqEEG offers unique features and information not captured by spectral quantitative EEG analysis and could lead to its improvement. These differences can be reliably determined using GTE items from visual inspection of the EEG.– Increased sqEEG abnormalities in childhood and adulthood predicted accelerated cognitive decline up to 40 years later in middle adulthood.– A multimodal approach combining spectral-qEEG and sqEEG yields the most accurate results distinguishing PEM from controls.– sqEEG contributes additional information about PEM effects on EEG, which spectral-qEEG does not capture.1.Recent research has demonstrated that exposure to adverse childhood experiences during critical developmental phases can negatively impact both physical and mental health . ChildhoOur research group has addressed this issue by examining the use of the Electroencephalogram (EEG) as a viable technology to investigate the neurodevelopmental consequences of protein-energy malnutrition (PEM) in the first year of life . This woBefore summarizing our prior EEG results for these BNS cohorts, it is necessary to note that two different modalities can extract potential EEG biomarkers:Spectral quantitative EEG  quantifies the EEG through the power spectrum of activity at the channels or the sources. Our group has contributed to developing this technique for several decades  and demoSemi-quantitative EEG (sqEEG) involves scores by expert subjective evaluations of EEG with standardized ratings that are later fully quantified employing statistical techniques. Such scoring of expert evaluation is a common technique in psychometrics  and was pioneered in imaging . The useGlobal Brain Consortium Project: Global EEG Norms).A current and trenchant discussion in clinical neurophysiology com compares the relative merits of spectral-qEEG and sqEEG. As the existence of automatic EEG measures, such as spectral-qEEG, might supplant sqEEG. However, this is not the case. While spectral-qEEG is considered an essential complement to visual inspection of the EEG, visual inspection of the EEG is still considered the gold standard in clinical neurophysiology and is constantly being revised . EffortsOne of the hypotheses underlying this paper is that sqEEG reflects, in addition, to rough background frequency information, additional features in the “grapho-elements” that arise from nonstationary and nonlinear harmonic signal relations, to which spectral analysis is blind. Rather than contraposing spectral-qEEG and sqEEG, we believe it essential to document differences between the two EEG modalities, thus enabling the discovery of additional features needed to improve spectral-qEEG. Beyond adding multivariate cross-spectral EEG information to qEEG, as in , an arseWe previously reported that spectral-qEEG has provided biomarkers that stably and accurately classified between PEM and control groups at 5–11 years of age with an area under the ROC curve (AUC) of 0.83 and 0.82 for the scalp  and sourThe current study aims to address the failure to systematically compare spectral-qEEG and sqEEG measures of early malnutrition throughout the lifespan. To address this research gap, we contrast changes in both EEG modalities . Importantly, we validate these differences by comparing them with cognitive outcomes in adulthood as measured by the Montreal Cognitive Assessment (MoCA), a well-established scale for measuring mild cognitive impairment . We also2.In this paper, we employ different symbols and acronyms, and their explanations are available in 2.1.N = 129, 52 females, 77 males) were healthy classmates of the PEM children matched by age, sex, and handedness who met the same inclusion criteria but had no histories of malnutrition. After discharge from the hospital, all PEM children were enrolled in a government-supported intervention program from ages 0–12 years, which provided food subsidies, nutrition education for the primary caretaker, a 2–3 day/week nursery program, routine medical care, and regular home visits  on the Human Development Index in 2021-2022 . Malnoure visits . These p2.2.n = 258) when the subjects were between 5 and 11 years of age. However, only 108 original EEG recordings  were available during follow-up , the Cuban Neurosciences Center (2017/02/17/CNEURO), and the Barbados Ministry of Health and Wellness. All adult participants provided written informed consent and were compensated for their time and travel expenses.2.3.2.3.1.The original 1978 EEG recordings were obtained using the DEDDAS EEG equipment (sampling rate 100 Hz). The protocol has been described elsewhere in detail . For theData was collected and visually inspected by two neurophysiologists, who were blind to the subjects’ malnutrition history. Nineteen surface electrodes were placed  according to the international 10–20 system, referenced to linked earlobes. The electrode impedance was kept below 5 kΩ. Subjects were seated on a comfortable reclining armchair in a quiet, air-conditioned room and were instructed to stay awake. Resting-state EEGs were recorded for 8–10 min. Note that in 1978 only eye-closed EEGs were collected; in 2018, the EEG was recorded under different conditions . Subjects were monitored regularly by the neurophysiologist for their wakefulness. Throughout the recording, subjects were also asked to report if they were awake.The raw EEG data is denoted as For both sqEEG and spectral qEEG, visual inspection was used to remove bad channels, interference by power lines interference, EKG, EMG. teeth grinding, eye or body movement, and high electrode impedance.Spectral qEEG artifact rejection was supplemented by independent component analysis (ICA)and the AAR plug-in from the EEGLAB 13.6.5b toolbox . AdditioEEG Pre-processing resulted in clean data  denoted 2.3.2.Two clinical neurophysiologists from the Cuban Neuroscience Center evaluated the  studies . They we studies  denoted  0 and 5 .Frequency of rhythmic background activity 0= > 8 Hz, 1 = 7-8 Hz, 2 = 6-7 HzDiffuse slow activity 0 = None, 1 = Slow theta, 2 = Intermittent theta + sporadic delta, 3 = Intermittent theta + intermittent deltaReactivity of rhythmic background activity0 = Normal, 1 = Decreased with eye-opening, 2 = Absent with eye-openingFocal abnormality 0 = No focal abnormality, 1 = Slight unilateral abnormality, 2 = Slight bilateral abnormality, 3 = Severe unilateral + Slight contralateral, 4 = Severe bilateral, 5 = MultifocalParoxysmal activity 0 = None, 1 = Paroxysmal slow activity, 2 = Spikes, 3 = Spike and wave (one Spike followed by a delta frequency wave)Sharp wave activity .0 = None, 1 = Sporadic sharp waves, 2 = Frequent sharp waves.For the 1978 analyses, only five GTE items were used since the “Reactivity of rhythmic background activity” must be evaluated by comparing the resting state to other conditions which were not included in this earlier study.2.3.3.The Montreal cognitive assessment (MoCA) was administered to participants in middle adulthood (at 45–51 years) by a researcher blind to the participant’s nutrition group. The MoCA comprises seven subscales, assessing visuospatial and executive functions, naming, attention, language, abstraction, memory, and orientation. It is a validated screening measure for mild cognitive impairment (MCI) . The hig2.3.4.Childhood standard of living was measured at three time points in childhood and adolescence using the 50-item Barbados Ecology Questionnaire , which a2.4.2.4.1.N = 202 (108 in 1978 and 94 in 2018). However, the number of repeated measures doubled when judging the effect of differences between evaluators. We assessed alternative IRT factor models based on item loadings and trace plots and optimized them using model fit indices such as Akaike’s Information Criterion (AIC) and the Bayesian Information Criterion (BIC).Using Item Response Theory (IRT), we obtained a latent variable underlying all GTE items for sqEEG. This approach is used as the indicator variables were categorical and not continuous. A polytomous IRT was implemented as the responses ranged between 0 and 5 . The anaspection . When weSi,age,⊙ . Conside2.4.2.We processed the EEG recordings Furthermore, normative EEG norms  were appanalysis . The det2.4.3.lme function from the nlme R package (We incorporated the latent variable E) model  to exami package . We mode2.4.3.1.As mentioned before, when describing data collection, EEG recordings in 2018 were limited by funding constraints, producing a missing data issue. In order to assess the validity of the ignorability assumption, we conducted a sensitivity analysis. The ignorability assumption is based on the notion that all confounding factors should be adjusted-for to infer from the regression coefficient as average effects. This assumption makes the treatment assignment ignorable, like a completely randomized experiment. However, the missing data mechanisms, such as Missing Not at Random (MNAR), can violate this assumption. To evaluate the effects of missing data mechanisms on the LME model estimates, we used the R package ISNI  to apply2.4.4.To validate the r scores . The MOC2.4.5.In order to evaluate the effectiveness of er (SSC) , as show2.4.6.Screened variables from the stable sparse classifier (SSC) were employed as stable biomarkers to test for the nutritional group differences. The selected variables for These new variables were utilized to implement multivariate LME models . The ideFurthermore, we created two other models from the main MLME model. These models were implemented such that coefficients related to one of the modalities were forced to be zero. We have implemented these models via MLME to make these models comparable in terms of model fit indices and to evaluate the optimal biomarkers under longitudinal settings.R ANOVA function was applied for model comparison. The ANOVA function is a statistical tool used to compare multiple models and determine if the difference in fit between them is significant. The ANOVA functions work with log-likelihood values and Log Likelihood ratio (L ratio). The log-likelihood is the probability of observing the data given the model parameters and measures how well the model fits the data. The higher the log-likelihood, the better the model fits the data.On the other hand, the L ratio measures the improvement in model fit when comparing two nested models. It is calculated as the difference in log-likelihoods between the two models. Moreover, the Log-Likelihood of the different models is compared using a chi-squared test to determine if the difference is statistically significant. If the value of p of the chi-squared test is less than 0.05, in that case, we reject the null hypothesis that the models are equally good fits and conclude that one model is a better fit than the other based on the log-likelihood values.3.3.1.3.2.2 with Yates correction = 5.79, p = 0.016).Visual inspection of the EEG recordings using the GTE items demonstrated a higher rate of abnormalities in the PEM group compared to the controls in both the 1978 and 2018 datasets, with more abnormal findings reported at the older ages in 2018 than in children (1978). The total abnormalities found at ages 5–11 years have been previously reported . At ages3.3.In this study, we initially employed an Item Response Theory (IRT) model with five items to investigate the relationship between the GTE  items and the latent factor To optimize the model, we excluded the “Background Frequency” item and collapsed the response categories for “Sharp Waves” and “Paroxysmal Activity.” This change resulted in a more favorable model with improved fit indices (AIC 2494.16 and BIC 2554.29). The results of the optimized model are presented in 3.4.The statistical model used to assess group differences is as follows:p = 1). Consequently, we proceeded with the simpler model that only included participant effects. Additionally, the interaction between Group and Age was not significant (p = 0.81), suggesting that the nutritional group differences remain constant over the lifespan .We compared two models (1) that included random effects for subjects only and (2) random effects for both subjects and evaluators. However, the ANOVA results indicated no significant difference between the model that only considered participant-based random effects and the other model that included participants plus evaluator effects (p < 0.001), indicating a strong association between malnutrition history and the outcome variable, p < 0.001), while Sex and Standard of Living (Ecology factor) did not demonstrate any significant association with As presented in 3.5.We initially conducted a comparison between the Total MoCA scores of two groups  where the former had a mean score of 24.74 (±4.23) and the latter had a mean score of 28.04 (± 3.16). The results showed a significant difference between the two groups, with higher scores in the control group indicating less cognitive impairment. To validate the Neurophysiological status latent score (To assess if p = 0.02), indicating that early EEG abnormalities associated with malnutrition predict adult cognitive impairment. Interestingly, this association was also observed in middle adulthood with p = 0.003), indicating that the relationship between childhood As shown in 3.6.We applied the stable sparse classifier (SSC) to predict biomarkers for two different time points: childhood and adulthood. As a prediction method, the classifier was not designed to handle longitudinal data. SSC was employed under three different settings to compare the prediction accuracy of both methods independently and when combined. The implemented models area joint model for In contrast, the performance of the sparse stable classifier in middle adulthood (for ages 45–51 years) was slightly lower, with mean AUC values ranging from 0.72 ± 0.01 . This suggests that adding 4.Our findings indicate noticeable distinctions between protein-energy malnutrition (PEM) and the control (CON) groups. Specifically, we observed a higher frequency of abnormal Grand Total EEG (GTE) item scores in the PEM group compared to control during both childhood and middle adulthood. The Our sqEEG-based approach utilized Item Response Theory (IRT) to generate latent Among the GTE items, two significant contributors to the IRT-based latent neurophysiological status were “Focal abnormalities (0.98)” and “paroxysmal activity (0.73).” Focal abnormalities are characterized by an increase in bilateral and multifocal abnormalities in the EEG recordings, which contradicts the expected normal maturation process of the brain . On the p < 0.001). The sensitivity index for missingness yielded robust results, which aligns with our previous findings (p = 0.01). However, the interaction between Group and Age was insignificant (p = 0.81), suggesting that the observed differences in nutritional groups remained consistent across the lifespan, with no reversal of the effects of malnutrition as subjects matured. The non-significant interaction term between age and nutritional group indicates agreement between the two studies.The standardized IRT scores based on two evaluators at each time point were analyzed using a Linear Mixed Effect (LME) model, which revealed no significant differences when incorporating evaluators as random effects. The additional random effects did not improve the model’s performance, thus indicating the absence of evaluator bias. The LME results revealed highly significant differences between the groups . However, the composite sample employed for this analysis (N = 149) represents 58% of the total cohort, which is substantial in comparison with other international longitudinal studies  and spectral quantitative EEG (qEEG). This study found that increased sqEEG abnormalities during childhood predicted an accelerated cognitive decline that persisted for over five decades into middle adulthood. Both the MoCA and qEEG results corroborated these findings. Overall, the study highlights the distinct features of both sqEEG and spectral-qEEG and underscores the value of utilizing a combined approach to develop an effective predictive biomarker for malnutrition-related cognitive impairment in children who are at high risk. It also highlights the need for an augmented qEEG measure set, including functional connectivity and nonlinear/nonstationary features.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Informed consent was provided by parents of all participants when they were initially enrolled in the study under Protocol E1962, approved by the Boston University Medical Center Institutional Review Board and the Ethics Committee of the Barbados Ministry of Health. Current oversight (in adulthood) is provided by the Massachusetts General Brigham IRB (Protocol No. 2015P000329), the Cuban Neurosciences Center (2017/02/17/CNEURO), and the Barbados Ministry of Health. All adult participants provided written informed consent and were compensated for their time and travel expenses. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.PV-S, MB-V, and JG conceived, organized, and executed this project in collaboration. JG contributed as co-founder and director of the 50+ year Barbados Nutrition Study. AC-R, CS-M, AR, JG, CB, and SA were responsible for data acquisition. AC-R, TV-A, YG, QT, VB, and AR undertook the data curation and scoring of the GTE. PV-S, IM, LG-G, MB-V, FR, and JG developed the methods. FR undertook the statistical analyses, with the participation of AC-R and JB-B. FR, UR, AR, JG, MB-V, and PV-S prepared the manuscript. MB-V, PV-S, and JG acquired the funding. All authors contributed to the article and approved the submitted version.


Previous studies have indicated a decrease in T regulatory cells (Tregs) among patients with steroid-resistant nephrotic syndrome. CCL22 and Leptin influenced the immune function of Tregs through their respective pathways. This study aimed to compare patients with steroid-sensitive nephrotic syndrome (SSNS) and steroid-resistant nephrotic syndrome (SRNS) in terms of CCL22 and Leptin levels.This prospective study included 117 children diagnosed with idiopathic nephrotic syndrome (INS). Peripheral blood samples were collected before initiating steroid therapy, and serum levels of CCL22 and Leptin were measured. Patients were categorized into three groups based on their response to steroid treatment. Renal biopsies were recommended for all children diagnosed with INS, with higher acceptance rates in glucocorticoid resistance patients.Based on the response to steroid treatment, 117 children were divided as groups of SSNS (82 cases), frequent relapse nephrotic syndrome (FRNS) (10 cases), and SRNS (25 cases). A total of 41 patients underwent kidney biopsy, 11 cases (13.4%) in SSNS, 7 cases (70.0%) in FRNS and 24 cases (96.0%) in SRNS. 30 cases were minimal change disease (MCD), 9 cases were mesangial proliferative glomerulonephritis (MsPGN) and 3 cases were focal segmental glomerulosclerosis (FSGS). The levels of Leptin were significantly higher in SR patients (1208.1 ± 1044.1 pg/ml) compared to SS patients (515.4 ± 676.9 pg/ml) and controls (507.9 ± 479.8 pg/ml), regardless of the pathological type. CCL22 levels were significantly elevated in SRNS (92.2 ± 157.0 pg/ml), but the difference seemed to be attributed to the specific type of pathology, such as Minimal change disease (MCD) (127.4 ± 206.7 pg/ml) and focal segmental glomerulosclerosis (FSGS) (114.8 ± 22.0 pg/ml). For SRNS prediction, the AUC of Leptin, CCL22, and the joint prediction index were 0.764, 0.640, and 0.806, respectively.Serum levels of CCL22 and Leptin, detected prior to steroid therapy, were associated with steroid resistance in childhood INS. Steroid-resistant nephrotic syndrome (SRNS) is characterized by the lack of response to steroid therapy, resulting in persistent proteinuria after four weeks of standard treatment . Among cStudies have demonstrated an association between low steroid responsiveness and the regulation of T lymphocyte function. Patients with SRNS exhibit a decrease in T regulatory cells (Tregs), whereas an increase in Tregs has been observed in response to effective immunosuppressive or monoclonal antibody therapies . Tregs pLeptin and CCL22 have been identified as potential immunomodulators due to their interactions with Tregs. Leptin has been shown to sustain the activity of pro-inflammatory cytokines and immune cells, while also enhancing the immune response by stimulating M2 macrophages, promoting Th1 and Th17 cells, and inhibiting Tregs , 6. InhiIn order to examine the involvement of Leptin and CCL22 in the development of SRNS, this study prospectively obtained serum samples from children with nephropathy prior to commencing steroid therapy. The patients were classified based on their response to steroid treatment, and the relationship between serum Leptin and CCL22 levels and steroid sensitivity in children with nephrotic syndrome was analyzed.This study was conducted from January 2019 to September 2021 and involved children with idiopathic nephrotic syndrome who had not received prior steroid therapy. The study protocol involving human participants was reviewed and approved by the Ethics Committee.The diagnosis of idiopathic nephrotic syndrome (INS) was based on the presence of edema, 24-h urinary protein excretion of ≥50 mg/kg, morning urinary protein/creatinine of >2 mg, hypoalbuminemia of <25 g/L and the disease of unknown causing. All children with INS received the standard steroid therapy and were classified into three categories, steroid-sensitive nephrotic syndrome (SSNS), frequent relapse nephrotic syndrome (FRNS) and steroid-resistant nephrotic syndrome (SRNS), on the basis of their clinical responses toward steroids. The SSNS group included patients with negative urinary protein for ≤4 weeks in those treated with sufficient prednisone [2 mg/(kg·d) or 60 mg/(m·d)]. The frequent relapse group included patients in whom INS recurred two times or more within half a year, or four times or more within 1 year in the course of the disease. The SRNS group included patients who failed to achieve remission after 4 weeks of daily sufficient prednisone. FRNS and SRNS were collectively referred to as refractory nephrotic syndrome (RNS). Further, the relapse group included patients in whom the quantity of urinary protein was ≥50 mg/kg, or the urinary protein/creatinine (mg/mg) of morning urine was ≥2.0, or the morning urinary protein changed from negative to positive for three consecutive days. The non-relapse group included patients in whom INS no recurred within 1 year after the first complete remission. The infrequent relapse group included patients in whom INS recurred once within 6 months or one to three times within 1 year after the first complete remission. Patients who were not finished four weeks of glucocorticoid therapy, or received other immunosuppressants, monoclonal antibodies, or cytotoxic drugs within four weeks, would be excluded from the study.The clinical features of enrolled patients were recorded from the medical record, such as age, genders, creatinine, estimated glomerular filtration rate (eGFR), 24-h urine protein (Upro), occurred of complications and hypertension. eGFR was calculated according to Schwarz-formula .The blood sample were collected into vacuum sampling vessel containing coagulant  before steroid therapy from the enrolled patients. Serum were separated within 2 h after blood collection and then divided and stored in the refrigerator at 2–8°C immediately. All the serum samples were transferred to an ultra-low temperature refrigerator (−80°C) no more than 8 h. The samples were guaranteed not to thaw until the ELISA test.Renal biopsies were recommended in patients with INS diagnosis. Pathology test should obtain the written informed consent of the guardian first. Renal biopsies were executed by skilled nephrologists, and all biopsies were assessed by pathologists through light microscopy and immunofluorescence. Each kidney sample was observed for the total number of glomeruli, glomerular sclerosis, mesangial proliferation, basement membrane thickening, tubular degeneration and atrophy, interstitial fibrosis, interstitial inflammation, etc.Human Leptin ELISA KIT , Human CCL22 ELISA KIT  were used for patient serum test. Briefly, to assay each protein, serum samples at an optimal dilution were added to a microplate precoated with capture antibody, incubated, washed and followed by addition of capture antibody, horseradish peroxidase and substrate. The absolute levels of serum protein biomarkers were determined using standard curves run on each ELISA plate, and normalized by urine creatinine concentration.t-test was used to assess age differences. Outlier detected by histogram, and replaced with the average in statistical process. Pearson bivariate correlation analysis was used for correlation statistics. A P-value greater than 0.05 indicated no significant difference. After quantifying the ELISA results based on the standard curve, the t-test or Wilcoxon rank sum test was used for between-group comparisons, and analysis of variance or Kruskal-Wallis test was used for comparisons involving more than two groups. Statistical significance was set at P < 0.05. Receiver operating characteristic (ROC) curves were utilized to determine the effective area, sensitivity, and specificity of candidate indexes.The quantitative data with normal distribution was expressed as (Average ± SD), and the classified data indicated with the quantity of each component separately. Statistical analysis was performed using SPSS 22.0. The chi-square test was used to analyze gender differences among the groups, while the P > 0.05). There were 148 complications could be recorded in 117 INS patients, with 12 types. The first three kinds were inflammation (48 cases), hydrops (30 cases) and liver injury (23 cases). There was no difference in the incidence of complications among three subgroups (P > 0.05). The incidence of hypertension was different (P < 0.05), with the highest in SRNS.A total of 117 cases  with childhood INS were included in the study, comprising the subgroups of SSNS (82 cases), FRNS (10 cases), and SRNS (25 cases). Additionally, 40 cases  undergoing health check-ups and confirmed to be free of obvious diseases were selected as the healthy control group. Serum samples were collected before steroid therapy and stored according to the aforementioned protocol. The clinical features of children enrolled in the study were listed in A total of 41 (35.0%) patients underwent kidney biopsy, 11 cases (13.4%) in SSNS, 7 cases (70.0%) in FRNS and 24 cases in SRNS with an acceptance rate up to 96.0%. Minimal change disease (MCD) was the most common pathological type with 30 cases, mesangial proliferative glomerulonephritis (MsPGN) with 9 cases and focal segmental glomerulosclerosis (FSGS) with 3 cases. Pathological types in the three groups with serum concentration of CCL22 and Leptin were shown in P = 0.368), eGFR (P = 0.329), Upro (P = 0.241). At the same between CCL22 and Creatinine (P = 0.060), eGFR (P = 0.570), Upro (P = 0.963). These indicated that changes of Leptin and CCL22 in INS patients had a different significance compared to clinical features.There was no significant correlation between Leptin and Creatinine . The diagnostic performance of serum CCL22 and Leptin in predicting RNS or SRNS was evaluated using the ROC curve shown in Compared to SSNS group, the concentration of serum CCL22 and Leptin significantly increased in SRNS (The present study focused on the serum concentrations of CCL22 and Leptin in patients with idiopathic nephrotic syndrome. By prospectively collecting samples and conducting pre-steroid tests, we observed differences in CCL22 and Leptin concentrations among groups of patients with varying steroid responses. This study demonstrated the potential of CCL22 and Leptin to predict steroid resistance in the early stages of nephrotic syndrome.Tregs play a crucial role in immune tolerance and are recognized as regulators of inflammation in INS. Previous research has shown that patients with steroid-resistant nephrotic syndrome exhibit lower levels of Tregs compared to those with steroid sensitivity . A sloweMeasuring Leptin level is a hot topic now, and more topics are focusing on its measurement as early childhood obesity, early childhood developmental assessment scores , 14. Ourin vitro and in vivo (CCL22 is an important factor in facilitating the migration of Tregs both  in vivo . In our As a small-sample, single-center observational study, our research has certain limitations. Expanding the sample size and conducting multi-center studies would provide more compelling conclusions. Furthermore, further investigations into the mechanisms of other related molecules would enhance the credibility of our findings and pave the way for subsequent explorations of the underlying mechanisms.In conclusion, this study has demonstrated an association between serum concentrations of CCL22 and Leptin, measured prior to steroid therapy, and steroid resistance in childhood idiopathic nephrotic syndrome. Additionally, there were notable variations in CCL22 concentration across different glomerular pathological types, highlighting the need for further investigation into the molecular mechanisms involved.


Among nationally representative cross-sections of women in need of contraception from Burkina Faso, Côte d'Ivoire and Kenya, we aimed to: (1) examine the intersection of past-year physical/sexual intimate partner violence (IPV), emotional IPV and reproductive coercion (RC) and (2) assess the impact of physical/sexual IPV, emotional IPV and RC on women’s contraceptive use outcomes, including current contraceptive use, method type and covert use.The present analysis uses cross-sectional female data collected in Burkina Faso (December 2020–March 2021), Côte d'Ivoire (October–November 2021) and Kenya (November–December 2020).Burkina Faso, Côte d'Ivoire and KenyaAnalytical samples were restricted to partnered women with contraceptive need who completed a violence module .The exposures of interest—past-year emotional IPV, physical/sexual IPV and RC—were assessed using abridged versions of the Revised Conflict and Tactics Scale-2 and Reproductive Coercion Scale, respectively. Outcomes of interest included current contraceptive use, contraceptive method type , and covert contraceptive use, and used standard assessments.Across sites, 6.4% (Côte d'Ivoire) to 7.8% (Kenya) of women in need of contraception experienced RC; approximately one-third to one-half of women experiencing RC reported no other violence forms (31.7% in Burkina Faso to 45.8% in Côte d'Ivoire), whereas physical/sexual IPV largely occurred with emotional IPV. In multivariable models, RC was consistently associated with covert use (Burkina Faso: aOR 2.84 (95% CI 1.21 to 6.64); Côte d'Ivoire: aOR 4.45 (95% CI 1.76 to 11.25); Kenya: aOR 5.77 (95% CI 3.51 to 9.46)). Some IPV in some settings was also associated with covert use ; physical/sexual, Kenya: aOR 2.35 (95% CI 1.33 to 4.17)).Across settings, covert use is a critical strategy for women experiencing RC. Country policies must recognise RC as a unique form of violence with profound implications for women’s reproductive health. Concurrent measurement of reproductive coercion (RC) and intimate partner violence within three national samples  afforded examination of concurrent violence experiences.Given inclusion of comprehensive measures, there was a unique opportunity to examine the interplay of violence experiences and their impact on three contraceptive use outcomes .Findings are cross-sectional, limiting conclusions surrounding temporality of associations.While RC measurement uses the pregnancy coercion subscale from the RC Scale, the most comprehensive measure for RC to date, RC items do not account for a wide range of RC behaviours, and therefore, may underestimate the true burden of RC in these contexts.Intimate partner violence (IPV) is pervasive globally—recent evidence highlights that 27% of women are affected by physical or sexual IPV.Current evidence examining the reproductive health impact of these leading forms of violence has been concentrated within high-income contexts, namely, the USA10Understanding the impact of abusive partner dynamics on women’s reproductive health in sub-Saharan Africa is critical given that sub-Saharan African women experience disproportionate adverse health burdens; specifically, they have the highest global rates of unintended pregnancy,11 13 17Inclusion of comprehensive RC measures within recent population-based surveys allows for the examination of the interplay of violence experiences and their impact on contraceptive use outcomes. To date, analyses examining linkages between unique forms of violence and contraceptive use have been limited given lack of RC and IPV measures concurrently included in national surveys. In three national samples across East and West Africa, this study aimed to: (1) examine the intersection of physical/sexual IPV, emotional IPV and RC and (2) assess the impact of IPV and RC on women’s contraceptive use outcomes .In 2019, Burkina Faso, Côte d'Ivoire and Kenya ranked similarly on the United Nations Development Programme Gender Inequality Index , which administers annual population-based panel and cross-sectional questionnaires at the household, female and facility levels using a multistage cluster design allowing for nationally and regionally representative estimates. Additional details are available at pmadata.org.This analysis uses cross-sectional female data collected in Burkina Faso (December 2020–March 2021), Côte d'Ivoire (October–November 2021) and Kenya (November–December 2020). Sites were selected for the present analysis due to implementation of the PMA GBV module, which allowed for examination of both IPV and RC experiences.All female household members ages 15–49 were eligible for the PMA female survey. In accordance with ethical best practices,Data were collected by trained resident enumerators (REs) using mobile phones equipped with Open Data Kit software; privacy checks were built into the survey. Prior to implementation, all REs completed a GBV-specific training, which emphasised maintaining confidentiality and privacy, asking non-judgemental questions, monitoring for emotional upset  and referring women to support services. In line with best practices,The GBV module was included within select PMA surveys at the request of the Ministries of Health. Results are disseminated to in-country stakeholders at national and local events.One woman per household was randomly selected to complete the GBV module ; participants did not complete the GBV module if there were privacy concerns . IPV and RC items were only administered to married or partnered women for a final sample of 3048 women in Burkina Faso, 1852 women in Côte d'Ivoire and 4355 women in Kenya.The analytical sample was further restricted to women in need of contraception, defined as being sexually active in the last 12 months, wanting to wait more than 12 months to have another child or not wanting any more children, not currently pregnant and fecund, for a final sample of n=1863 Burkina Faso, n=1105 Côte d'Ivoire, n=3390 Kenya. Analytical samples float to accommodate small amounts of missing data (<1%).Three independent variables classified past-year experiences of violence perpetrated by a husband/partner  via behavioural assessment. Past-year IPV was measured via five-item abbreviated version of the Revised Conflict and Tactics Scale-2.Three dichotomous dependent contraceptive use variables  and covert contraceptive use) were examined using standard assessments.Several sociodemographic and relationship characteristics were explored as potential confounders between violence and contraceptive use outcomes using standard assessments,All analyses were stratified by site. Sociodemographic, relationship, and sexual and reproductive health characteristics were examined overall and among those with contraceptive need. The prevalence of past-year RC was calculated overall and by item; prevalence of past-year IPV was calculated overall and by IPV subtype . Overlapping experiences of emotional IPV, physical/sexual IPV and RC were examined via Venn diagrams. Bivariate and multivariable logistic regression models were used to separately examine each contraceptive outcome by experience of violence; specifically, three models were used: (1) bivariate, (2) adjustment for sociodemographic and characteristics only and (3) adjustment for sociodemographic and relationship characteristics and other types of violence. Sociodemographic and relationship characteristics were examined for multicollinearity, and ultimately, the third model was retained given <10% difference in effect estimates between models. All analyses were conducted in Stata V.16 and weighted to account for survey design.In Burkina Faso and Kenya, most women lived within rural areas, whereas in Côte d’Ivoire, residence was split between urban and rural localities . ApproxiAcross sites, emotional IPV was the most prevalent type of violence experienced among women in need of contraception , followed by physical/sexual IPV  and RC  in Burkina Faso only and increased covert contraceptive use in both Burkina Faso  and Kenya . Physical/sexual IPV was associated with covert contraceptive use in Kenya only .In bivariate analysis, RC was associated with covert contraceptive use in all three sites p<0.001; . EmotionIn multivariable models, RC remained consistently associated with increased covert use of contraception across sites (Burkina Faso: adjusted odds ratio (aOR) 2.84; 95% CI 1.21 to 6.64; Côte d’Ivoire: aOR 4.45; 95% CI 1.76 to 11.25; Kenya: aOR 5.77; 95% CI 3.51 to 9.46; Leveraging large-scale, population-based samples, this analysis illuminates the prevalence of RC and overlap with physical/sexual and emotional IPV, and its impact on current and covert contraceptive use in Burkina Faso, Côte d’Ivoire and Kenya. Data illustrate that for women experiencing RC who are in need of contraception, RC often occurred in isolation (31.7% in Burkina Faso to 45.8% in Côte d’Ivoire) or coupled with emotional IPV (16.7% in Côte d’Ivoire to 36.0% in Burkina Faso). Accordingly, only situating RC interventions within IPV programmes and response services may overlook the needs of women who are experiencing this unique, yet detrimental, form of violence. Further, screening for physical or sexual IPV alone will fail to capture most RC experiences.Notably, experience of past-year RC was consistently associated with increased covert use of contraception across sites , however, similar results were not observed for female-controlled method use. Covert use may prove a promising, yet risky safety strategy for women seeking to avert pregnancy and also experiencing RC.Consistent with Demographic and Health Survey data, associations between physical/sexual IPV and contraceptive use were statistically insignificant,Results must be considered in light of limitations. Foremost, findings are cross-sectional, limiting conclusions surrounding temporality of associations—longitudinal work is needed to disentangle whether women are using contraception because of experienced violence or whether the violence is incurred given contraceptive use. Further, while RC measures have improved since this form of violence of first conceptualised, abortion coercion and forced contraceptive use are still not components of the RC scale, limiting our ability to capture a more inclusive range of RC experiences. Our RC measures also did not account for condom use manipulation and thus may undercount the true burden of RC in these contexts. Additionally, pregnant women were not included in the analytical sample, which could further underestimate RC prevalence as these women may be pregnant due to RC. However, including pregnant women in the sample would also lead to a systematic attenuation of the results between RC and contraceptive use given that these women are not in need of contraception. Women in dating partnerships were also excluded from the present analysis due to embedded survey skip logic, though evidence among urban adolescents and young women indicates high prevalence of RC22 23Importantly, policy-makers and family planning and violence service providers must recognise RC and its health impact to holistically support women’s needs. Work to dismantle deep-rooted social norms that dictate spousal permission prior to contraceptive provision must be included in healthcare provider trainings on how to support women experiencing RC. Interventions, such as Addressing Reproductive Coercion in Health Settings in Kenya,


This longitudinal study aimed to examine the association of intrinsic motivation, parental physical activity, and parental support with physical activity maintenance and increase among children and adolescents.A sample of 2,424 children and adolescents in Shanghai, China participated in the two-wave survey before and during the COVID-19 pandemic. The questionnaire measured children and adolescents’ physical activity and intrinsic motivation, as well as their parental physical activity and support (concern for their child and co-activity with their child). Multivariable logistic regressions were performed by groups to examine the associations between these factors and physical activity change.Most children and adolescents exhibited a decline in physical activity participation during the pandemic, as indicated by a mere 15.0 and 8.0% of individuals maintaining and increasing their pre-pandemic levels, respectively. Among the initially active participants, perceived self-choice [OR = 1.341 (95%CI: 1.173–1.533)] and parental concern [OR = 1.922 (95%CI: 1.204–3.068)] predicted maintained physical activity. Increased physical activity was predicted by perceived enjoyment [OR = 1.193 (95%CI: 1.046–1.362)] and parental co-activity (OR = 1.995 [95%CI: 1.095–3.633]).This study provides longitudinal evidence that intrinsic motivation and parental support can have a positive impact when physical activity levels change significantly. Effective interventions targeting multilevel factors are needed to maintain or increase children and adolescents’ physical activity. HoweverUnderstanding the psychological mechanism underlying physical activity behavior is pivotal to implementing effective physical activity intervention in children and adolescents. Self-determination theory (SDT) is a behavioral change theory that has been successfully used for both explaining the motivational dynamics of physical activity behaviors and developing theory-grounded physical activity intervention programs in children and adolescents , 8. SDT via fostering or thwarting motivation  between March 13th and 23rd, 2020, right before the public health emergency response downgraded to Level 2 and the strict lockdown was relaxed. A total of 2,427 students and parents completed the first to the second waves. Participants with invalid responses to physical activity time (> 24 h per day) were excluded. The final sample consisted of 2,424 children and adolescents. The flow chart of participants in this study is presented in The first survey was conducted among 7,544 children and adolescents in the ten schools as well as their parents (approximately 83% participation rate) between January 3In each survey wave, we first distributed information about the study among school head teachers and obtained their permission. The questionnaire was then distributed online by the head teacher to each parent and their children. Written informed consent from each parent was obtained before data collection. The parents and their children were asked to complete two separate parts of the questionnaire. The parents were instructed to assist primary school students in the process.2.2.The questionnaire consisted of a child/adolescent section and a parent section. Physical activity and intrinsic motivation were derived from the child/adolescent section, while parental physical activity and parental support were derived from the parent section.2.2.1.Physical activities were assessed by the Global Physical Activity Questionnaire (GPAQ), which was developed by the World Health Organization (WHO)  and modi2.2.2.Children and adolescents’ intrinsic motivation was assessed by five statements derived from five subscales of the Intrinsic Motivation Inventory (IMI) . The sta2.2.3.The GPAQ, which was originally designed for the adult population , was als2.2.4.Parental support included two aspects of intangible support  and tangible support (co-activity with their child), respectively. Parental concern was assessed by a single question “How much do you concern about your child’s physical activity in your current life?.” Possible responses included “occasionally care,” “frequently care,” and “always care.” Parental co-activity was assessed by another question “How often do you engage in physical activity with your child?.” Possible responses included “Never,” “1 ~ 4 times per month,” and “>1 time per week.”2.2.5.Sex , age, and grade  were reported by children and adolescents. Mother’s and father’s educational level  and family annual income  were reported by parents.2.3.Descriptive analyses were conducted for the baseline physical activity level on sample characteristics. Categorical variables were described by numbers and frequencies. Age was described by mean and standard deviation (SD), while IMI scores and parents’ physical activity were described using median and Q1-Q3 quartiles. Comparisons of all the baseline variables between the active group and inactive group were conducted using the Chi-square tests for categorical variables and the Wilcoxon rank-sum tests for continuous variables.p less than 0.05 was considered significant.Participants were further categorized into active and inactive groups according to their assessments of physical activity level at follow-up. A series of logistic regressions were performed separately in the initially active and inactive participants, and odds ratios  were calculated to compare the active and inactive participants at follow-up. Participants with missing data on parental physical activity, parental concern of their children’s active level, and parental frequency of coactivity with their child (n = 122) were excluded from the analyses. There were no significant differences in the sex or age distribution between the included and excluded participants . The biv2.4.The study was approved by the Ethics Committee of Shanghai Jiaotong University School of Medicine (SJUPN– 201813). Written informed consent was obtained from all the parents.3.3.1.Of the included 2,424 children and adolescents, 1,241 were boys (51.2%) and 1,183 (48.8%) were girls, ranging in age from 6 to 15 [mean age (SD): 11.1 (2.67) years]. At baseline, 1,120 (46.2%) children and adolescents were physically active. Descriptive statistics of baseline sample characteristics according to the physical activity level are presented in p < 0.001), age (p < 0.001), grade (p < 0.001), father’s educational level (p = 0.017), and annual family income (p = 0.014) were associated with physical activity level. Compared to those in the inactive group, the active children and adolescents scored higher on all the items of intrinsic motivation (p < 0.001). In addition, all the parental factors (p < 0.001) were positively associated with children and adolescents’ physical activity levels.The results of chi-square tests showed that sex (p < 0.001), perceived competence (p < 0.001), perceived choice (p < 0.001), and value/ usefulness , while negatively associated with pressure/ tension . The associations between parental physical activity and items of intrinsic motivation were insignificant.Correlations between intrinsic motivation, parental physical activity, and parental support are presented in 3.2.n = 1,120), 168 (15.0%) reported sufficient physical activity participation at follow-up (active maintainers). Of the initially inactive participants , only 104 (8.0%) were active at follow-up (increasers). Patterns of physical activity change during follow-up are presented in At follow-up, 272 (11.2%) children and adolescents were physically active. Of the initially active participants  of initially inactive participants reported sufficient physical activity at follow-up. The pronounced trend toward declining may be attributed to the study time. During the early phase of the COVID-19 pandemic, children and adolescents were unable to adequately maintain their normal physical activity patterns due to home confinement , 30. ConAccording to the hypothetic model, personal motivation and parental factors significantly influence physical activity in children and adolescents , 31. In As indicated by SDT, physical activity behavior is innately driven by intrinsic motivation. Some researchers suggested that while extrinsic motivation could support initial physical activity behavior, intrinsic motivation is necessary for long-term behavioral change . ConsistThe present study also illustrated the positive effects of parental factors on physical activity over time. However, the significance of parental physical activity was completely nullified in the multivariable model. Inconsistent findings have been evidenced on the association between parent and child physical activity. A systematic review indicates that methodological differences between studies could partially explain the heterogeneity in the results, with stronger associations reported using objective measures of physical activity . Moreovevia providing practical assistance, which in turn can lead to increased physical activity levels  were not incorporated. Future research may consider investigating the influence of these supportive parental behaviors on physical activity changes in children and adolescents. Finally, the sample in the present study was limited to Shanghai. Therefore, the present finding should be generalized with caution.5.This longitudinal study focused on the short-term physical activity change during the COVID-19 pandemic among children and adolescents. Our findings demonstrated the significant effects of intrinsic motivation and parental support on physical activity over time. Specifically, perceived self-choice and parental concern about their child’s physical activity predicted maintained physical activity among the initially active children and adolescents, while perceived enjoyment and parental co-activity with their child predicted increased physical activity among the initially inactive children and adolescents. Interventions should capitalize on intrinsic motivation and parental support to promote physical activity and confront the overall decreasing trend during adolescence.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethics Committee of Shanghai Jiao Tong University School of Medicine. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.MX: conceptualization, methodology, and funding acquisition. MX, YL, XG, and EZ: data curation. YL and FH: formal analysis. YL and XG: Writing – original draft. MX, YC, and HL: writing – review and editing. All authors contributed to the article and approved the submitted version.This study was funded by Shanghai Municipal Education Commission , the National Natural Science Foundation of China (grant number 71804110), Shanghai Science and Technology Development Funds (grant number 21QA1405300), Science Foundation for new teachers of Shanghai Jiao Tong University School of Medicine (grant number 20×100040012), and Shanghai Municipal Health Commission (GW-10.1-XK07).We thank all the teachers and students’ parents involved in this study for their contributions and efforts in conducting the survey.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.https://www.frontiersin.org/articles/10.3389/fpubh.2023.1175439/full#supplementary-materialThe Supplementary material for this article can be found online at: Click here for additional data file.