Bacillus subtilis prior to this study. Here, we integrated enzyme kinetic and proteomic data to construct the first genome-scale ecModel of B. subtilis (ecBSU1) using the ECMpy workflow. We first used ecBSU1 to simulate overflow metabolism and explore the trade-off between biomass yield and enzyme usage efficiency. Next, we simulated the growth rate on eight previously published substrates and found that the simulation results of ecBSU1 were in good agreement with the literature. Finally, we identified target genes that enhance the yield of commodity chemicals using ecBSU1, most of which were consistent with the experimental data, and some of which may be potential novel targets for metabolic engineering. This work demonstrates that the integration of enzymatic constraints is an effective method to improve the performance of GEMs. The ecModel can predict overflow metabolism more precisely and can be used for the identification of target genes to guide the rational design of microbial cell factories.Genome-scale metabolic models (GEMs) play an important role in the phenotype prediction of microorganisms, and their accuracy can be further improved by integrating other types of biological data such as enzyme concentrations and kinetic coefficients. Enzyme-constrained models (ecModels) have been constructed for several species and were successfully applied to increase the production of commodity chemicals. However, there was still no genome-scale ecModel for the important model organism  Bacillus subtilis is a model organism of choice for the industrial production of various valuable compounds, such as biopolymers and proteins, due to its excellent capacity for protein secretion, good growth characteristics, distinct endogenous metabolism, and robustness in industrial fermentation [B. subtilis have been successfully applied to guide the production of riboflavin [B. subtilis GEM was published in 2008 [entation . Genome-boflavin , isobutaboflavin , 2,3-butboflavin . The fir in 2008 , and sev in 2008 ,7,8, whiEscherichia coli [Saccharomyces cerevisiae [Aspergillus niger [Corynebacterium glutamicum [B. subtilis [B. subtilis (ec_iYO844) only integrated enzyme kinetic parameters for 17 reactions located in the central carbon metabolism using the GECKO method, but this model allowed more accurate prediction of the flux distribution and growth rate of wild-type and single-gene/operon deletion strains compared to the GEM [By contrast, enzyme-constrained models (ecModels) introduce enzyme kinetic information into a GEM, thus reflecting the protein resource limitation faced during cell growth, enabling them to identify the rate-limiting enzymes in the pathway and further guide rational metabolic engineering. As a consequence, ecModels have been successfully applied to guide the production of L-lysine , poly-glhia coli ,12,15, Srevisiae , Aspergius niger , Corynebutamicum  and B. ssubtilis . The fir the GEM .B. subtilis (ecBSU1), using an updated ECMpy workflow [B. subtilis on different carbon sources, simulate the overflow metabolism, and explain the trade-off between biomass yield and enzyme usage efficiency. Finally, we predicted the target genes of B. subtilis for enhancing the production of industrial chemicals , and the predictions were in good agreement with the literature.In this study, we first systematically updated the iBsu1147 model through GPR update, biomass reaction standardization, etc., after which we established a comprehensive collection of parameters that affect the prediction accuracy of the ecModel , and constructed the first genome-scale ecModel of workflow . Subsequkcat and molecular weight (MW) of an enzyme affect the prediction accuracy of the ecModel [kcat, and the GPR relationship affects the accuracy of MW calculation. Accordingly, we systematically corrected the EC number and GPR relationships. We used the GPRuler tool [R (Revised iBsu1147).The model iBsu1147, which has the most reactions and genes, was selected as the initial model for the integration of enzymatic constraints. Since the iBsu1147 model was released in 2013, we performed quality control on the model, covering substrate utilization, redox balance, energy balance, biomass reaction standardization, and mass balance. Our previous results shown that the  ecModel . For exaler tool  and protler tool  for detaler tool  IDs  and j-th protein (g_num represents proteins expressed in the whole proteome).The molecular weight (MW) of each enzyme was downloaded from the UniProt database according to the gene ID, and the quantitative subunit information was obtained by parsing the ‘Interaction information’ in UniProt . For exae BRENDA  and SABIe BRENDA  databasehe PAXdb  databaseR following the ECMpy workflow and named ecBSU1 , so that each reaction only has one corresponding enzyme. Next, we calculated the MW of each enzyme. For reactions catalyzed by enzyme complexes, we used the total sum of proteins in the complex (j is the number of jth subunits in the complex). Finally, a new enzymatic constraint  and AutoPACMEN was a direct 10-fold expansion without an automated process. In contrast, ECMpy identify the most likely wrong parameters and corrected them through an automatic process based on the cost of enzymes. The process includes the following steps: First, we calculated the enzyme cost of each reaction in the pathway with biomass maximization as the objective , as described in GECKO 2.0 [To improve the agreement of model predictions with experimental data, the enzyme-constrained model required further adjustment of the original ive    , scipy, kcat data in the workflow, ultimately affecting the prediction accuracy of the ecModel. Consequently, we replaced the old EC numbers in the model based on BRENDA, updating a total of 38 reactions , the addition of 4 new reactions , and the modification of GPR relationships for 5 reactions .EC numbers affect the extraction and assignment of eactions . MW is aeactions . For exaBSU31360 and BSU31370′, which has 74% sequence similarity. By further searching the BioCyc database for verification, we found that the protein encoded by BSU31370 is a bifunctional enzyme that catalyzes two reactions , whereas the protein encoded by BSU31360 catalyzes only the NADPH-dependent butanol dehydrogenase reaction. They are more likely to be two isoenzymes, and therefore the correct GPR relationship for BTS should be ‘BSU31360 or BSU31370′. Finally, 1736 reactions, 1459 metabolites, and 1155 genes were included in iBsu1147R.We also observed that there were 58 reactions in the model for which the “and” relationships were not identified by the GPRuler tool, and only 9 reactions had more than 25% similarity . By searR model in terms of substrate utilization, redox balance, energy balance, biomass reaction standardization, and mass balance, we found that all these aspects led to abnormalities in the metabolic pathways generated by the simulation. The boundary of 6 reactions was modified in terms of substrate utilization . For example, experiments have shown that B. subtilis can grow using malate [After running quality control of the iBsu1147g malate , so the g malate  to avoidR model and found that the original molecular mass of the biomass was 1.025 g/mmol, and H+ was missing in the right side of the biomass equation (the coefficient was 105) produced by the hydrolysis of ATP for growth-associated maintenance energy. After correction, the molar mass of the biomass became 0.92 g/mmol. Subsequently, we examined the precursor metabolites and found that the molar masses of protein, DNA, and RNA were 0.86 g/mmol, 0.95 g/mmol, and 0.95 g/mmol, respectively. We normalized the coefficients so that the molar masses of all components were 1 g/mmol, and the molar masses of the biomass also became 1 g/mmol. The details of all the modified reactions are listed in In addition, the molar mass of biomass and its components  was 1 g/mmol, and deviations from this value will result in errors in the calculated specific growth rate. Using the BiomassMW algorithm , we examR contains different IDs for metabolites and reactions, including KEGG IDs and ModelSEED IDs. To meet the input requirements of AutoPACMEN, we converted the KEGG IDs of 1007 metabolites and 785 reactions, as well as the ModelSEED IDs of 265 metabolites and 542 reactions into BiGG IDs. In addition, we kept the original IDs for reactions and metabolites that were not included in the BiGG database.Finally, we found that iBsu1147catk values ” with the highest measurement coverage and evaluation score, and calculated f = 0.588 g enzyme/g protein according to Eq. 1. Finally, the initial B. subtilis ecModel (ecBSU1) contained 1155 genes, 1459 metabolites, 3307 reactions, and 2331 enzyme kinetic parameters, with a total enzyme bound of 0.165 g enzymes/gDW.We used AutoPACMEN to match 2331 6.17 s−1 B. In tot6.17 s−1 C. The enkcat values , as was reported for ecYeast7 [catk correction. We found that the maximal growth rate calculated by ecBSU1 with glucose as substrate was 0.092 h−1 , as 17 reactions with catk values were unidirectional, resulting in its ability to simulate growth using only glucose as a substrate  close to the experimental value [−1 h−1 acetate and 3.93 mmol gDW−1 h−1 pyruvate). The difference in overflow products is mainly due to the limitations of the current ecModel, as the optimization process of the model only supports the pathway that generates the lowest enzyme cost, so that the current ecModel will only generate one overflow product.Further in-depth analysis using the ecModel revealed that the addition of the enzyme kinetic constraint information allowed ecModel to simulate the overflow of by-products from cells at high substrate concentrations. For example, the ecBSU1 results showed that it severe overflow metabolism would occur when utilizing malate at 26.51 mmol gDWal value . HoweverB. subtilis based on the enzyme cost of reactions. We selected 10 products for analysis from 51 commercial chemicals produced using the B. subtilis platform summarized in the literature [−1 (10% of the maximum) and performed reaction enzyme cost calculations with each of these 10 products as the objective function, respectively. Subsequently, a literature search was performed to validate the top 15 reactions in terms of enzyme cost in each pathway.GEMs play a guiding role in predicting metabolic engineering targets. In this work, we predicted potential target genes for the synthesis of several important chemicals  in terature . The proribA, ru5p__D_c --> db4p_c + for_c + h_c) and carbamoylphosphate synthetase , respectively  caralyzes a limiting step for the synthesis of the important precursor dihydrofolate (DHF), and co-overexpression of folC, pabB, folE, and yciA resulted in an additional 66.8-fold improvement of the 5-methyltetrahydrofolate titer, which reached 960.27 μg/L [We found that most of the predicted potential targets for the 10 products have been reported in the literature . Among tectively . Experimp to 25% . In addither 31% . For 5-m.27 μg/L . TherefoDuring the construction of traditional GEMs, not much attention is paid to whether the GPR relationship is “and” or “or”, but the correctness of these two relationships directly affects the simulation accuracy of ecModels. In this study, we systematically examined and corrected the GPR relationships in iBSU1147 by combining the GPRuler tool and protein homology. In addition, we systematically updated the EC numbers, carbon source utilization pathways, biomass reactions, mass balance, energy balance, and redox balance in the iBsu1147 model. This quality checking and correction process has far-reaching implications for improving the quality of GEMs and the construction of high-quality ecModels.R, we constructed ecBSU1 based on the ECMpy approach, in which 2331 of 3307 reactions were integrated with enzyme constraints according to AutoPACMEN, and the kcat coverage reached 76.4% after excluding 254 exchange reactions. The coverage of enzyme parameters was much higher than in ec_iYO844, which integrated only 17 reactions  located in the central carbon metabolism with the addition of catk values. However, enzyme kinetic data are sparse, and measured turnover rates are normally available for only a small fraction of metabolic reactions even in well-studied organisms [catk data for most of the reactions in the model, only 163 reactions had catk values derived from B. subtilis.Using iBsu1147rganisms . Therefokcat correction for 28 reactions. The ecBSU1 corrects the problem that the growth rate of GEMs increases indefinitely with the increase of the carbon source utilization rate. Next, ecBSU1 and iBsu1147R were, respectively, used to simulate the growth rate of B. subtilis on different substrates, and the results showed that the accuracy of ecBSU1 was much better than that of iBsu1147R at the high growth rate stage with enzyme constraints. In addition, the overflow metabolism of B. subtilis was explored using ecBSU1, which showed a physiological state of overflow metabolism in the presence of excess substrates, and demonstrated a trade-off between biomass yield and enzyme usage efficiency.Due to the diversity and incomplete coverage of enzyme parameters in the database, the initial ecModel was over-constrained, but it was able to accurately predict cellular phenotypes after B. subtilis for the synthesis of several commercial chemicals, and the reactions with the highest enzyme consumption were identified as metabolic engineering targets, which was in good agreement with the literature. This provides a new strategy and theoretical basis for metabolic engineering.Thus, ecBSU1 can be used to guide the rational design of microbial cell factories from a new perspective. The simulation results of GEMs usually only contain reaction fluxes, which cannot distinguish the physiological characteristics of each reaction. By contrast, ecBSU1 combines the kinetic characteristics of each reaction based on fluxes, thus demonstrating the enzyme consumption of each reaction, which can assist us to locate the kinetic bottlenecks of different metabolic states. We simulated the enzyme consumption of kcat prediction for metabolic enzymes from any organism merely from substrate structures and protein sequences [kcat values for more than 300 yeast species. In addition, the integration of enzyme constraints greatly improves the predictive power of GEMs and brings the model predictions closer to the experimental measurements, but the biological system is too complex and to be fully described by enzymatic constraints alone. Therefore, it is necessary to integrate more biological data into novel composite constraints, which can include data on thermodynamics [Currently, ecBSU1 has a typical limitation also found in other ecModels, as the implementation of the enzyme abundance constraint is highly dependent on precise kinetic parameters and abundance data for each enzyme , both ofequences . Using tdynamics ,46 and rdynamics , or consdynamics .B. subtilis on a genome-wide scale, which significantly improved its metabolic phenotype prediction ability. The resulting model can be used to explain metabolic overflow phenomena and predict metabolic engineering targets for the synthesis of commercial chemicals in B. subtilis. This study has guiding significance for the rational design of microbial cell factories and provides an important integrated metabolic network model of B. subtilis. Finally, it also offers insights for the improvement of GEMs of other strains, so that the role of such models in the development of synthetic biology can be broadened in the future.This work integrated enzymatic constraints into the GEM of


Internet addiction disorder (IAD) is a global issue that has resulted in a slew of physical and emotional consequences. Studies have indicated that health risk behaviors might be the risk factors for IAD. The published literature on the correlation between the two is lacking. Therefore, we conducted a comprehensive analysis to understand better the link between IAD and health risk behaviors among adolescents and young adults.I2 statistics and Cochran's Q statistics. All analyses were conducted by using Stata version 15.0.We searched ten electronic databases for relevant articles. Data were extracted based on IAD and health risk behaviors . We calculated odds ratios (ORs), a pooled correlation coefficient (r) and 95% confidence intervals (CIs). A fixed-effect model was applied to summarize the pooled effects. Heterogeneity was examined using r = 0.35; 95% CI 0.32–0.37) and smoking , and was associated with an increased risk of suicidal behavior , drinking , and smoking  among adolescents.A total of 16 studies and 61,823 participants were included in this study. Meta-analysis showed that IAD was positively correlated with drinking (We found significantly increased risks of suicidal behavior, drinking, and smoking in adolescents and young adults with IAD. These findings are important to expand our understanding of IAD and have great guiding significance for preventing health risk behaviors of adolescents and young adults.https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42021257729, identifier: PROSPERO CRD42021257729. Internet addiction disorder (IAD) is considered as an inability of individuals to control their internet use, resulting in marked distress and functional impairment in daily life such as psychological, social, academic, and professional problems . The oveTo date, the diagnostic criteria, and classification of IAD are still controversial, and its assessment instruments are inconsistent due to sociocultural and socioeconomic factors. Some scholars have argued that using traditional addiction criteria to define the boundaries of the IAD does not highlight the uniqueness of the behavior itself , 7. It hNotably, internet gaming disorder (IGD) is an important subtype of IAD . IGD as Risky behavioral lifestyles, such drinking, smoking, suicidal behavior, gambling and drug abuse are closely related to the healthy development of adolescents and young adults. Previous research reported a positive association between IAD and adolescents Risky behavioral lifestyles, such drinking, smoking, suicidal behavior, gambling and drug abuse are closely related to the healthy development of adolescents and young adult's risk behaviors , 15. GanTaken together, the data from this series of studies have been useful in demonstrating that IAD is associated with health risk behaviors. However, due to the limitations of the sample sizes and regional effects in many studies, their results were different to some extent. Thus, it is necessary to evaluate the relationship between IAD and health risk behaviors. The findings will lay the foundation for studying the characteristics of IAD among adolescents and young adults and its associated correlates. The results may provide evidence to support the development of more effective interventions.https://www.crd.york.ac.uk/prospero/#recordDetails.This systematic review and meta-analysis were conducted following the Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA) , 25 guidWe searched ten international electronic databases including PubMed, Embase, Web of Science (WOS), the Cochrane Library, PsycINFO, ERIC, China National Knowledge Infrastructure (CNKI), Chinese Biomedical Literature Database (CBM), Technology Periodical Database (VIP) and Wan Fang Database. We also performed a manual search of the gray literature listed in the bibliography, including dissertations and conference papers. This search was updated on Aug 2021. The detailed search strategy for PubMed is provided in Studies were included if they fulfilled the following criteria: (1) used a validated scale to assess IAD; (2) reported the relationship between IAD and health risk behaviors; (3) were observational studies published up to Aug 2021; (4) had a Pearson or Spearman correlation coefficient (r) or odds ratios (ORs) available; (5) were published in Chinese or English.Case reports, meeting abstracts, review papers, commentaries, and those with inadequate information were excluded.Participants were adolescents and young adults aged between 12 and 25 with a standard diagnosis of IAD and associated health risk behaviors. There were no restrictions on gender or race.Studies needed to have clearly reported smoking, drinking, suicidal behavior and other health risk behaviors. For the purposes of this review, health risk behaviors were defined as related behaviors reported at least once in the previous 30 days. That is, a study presenting results in terms of a range of frequencies (“often” and “frequently”) would be chosen for inclusion.Since this meta-analysis was based on published articles to investigate the relationship between IAD and health risk behaviors, there were no comparison or intervention groups.Studies must have included IAD as an independent variable and health risk behaviors as dependent variables for identified associations. Studies that did not adjust for these variables in the outcome analysis were included in this study. Health risk behaviors were measured by the related scales or self-report questionnaires. IAD was measured by Young Diagnostic Questionnaire for IAD (YDQ) , ChineseTwo reviewers independently (JW and QH) completed the title, abstract and full text screening. Items were extracted from the eligible studies including: first author, publication year, country, sample size, age, gender, measures of IAD and health risk behaviors, Pearson or Spearman correlation coefficient (r) or ORs between IAD and health risk behaviors. Disagreements were settled by consensus and discussion with a third reviewer (YT).The Agency for Healthcare Research and Quality (AHRQ), which contains 11 items, was used to evaluate the quality of the cross-sectional studies. The answer “yes” scored 1, while “no” or “unclear” scored 0. Scores of 8–11 were classified as high-quality research, and scores of 4–7 were classified as moderate quality .r)−ln(1−r)] where “ln” is the natural logarithm . “Fisher's z transformation” was used to convert Spearman's correlation coefficients into the normal distribution. The formula for the transformation is z = 0.5[ln, Switzerland (n = 1), Iran and several other middle eastern countries (n = 1), Ethiopia (n = 1), Lebanon (n = 2), Spain (n = 2), and China (n = 8). Although several studies did not specify the age range of the participants, they mentioned the educational stage of the participants, and the majority of them were university students. IAT is the most commonly used diagnostic tool for IAD, followed by YDQ. Regarding the measures of health risk behaviors, most of them were questionnaires/scales drawn up based on the cultural background of the country.All included studies scored above four points, ranging from 5 to 7 .I2= 19.8%, P = 0.288). The combined correlation coefficient(r) was   with slight heterogeneity . We conducted sensitivity analyses to identify the source of the heterogeneity. The result indicated that heterogeneity was significantly reduced when we removed the study by Zhang  and the heterogeneity disappeared  (P = 0.844)  . The comby Zhang . We foun= 0.509) . No appa= 0.844) .I2 < 50%), and a fixed-effects model was applied. The sizes of the pooled effect of IAD on suicidal behavior, drinking and smoking were   , (OR= 1.65–2.30)  and (OR 65–2.30) , respectOur meta-analyses showed that IAD was positively associated with smoking and drinking. We found that IAD was significantly associated with an increased risk of suicidal behavior, drinking and smoking.The present review found that adolescents and young adults with IAD have a significantly higher risk of suicidal behavior than others, in line with the literature –52. A toThe internet is a virtual space with identity concealing that can provide an online sharing community, coupled with the poor self-control of adolescents and young adults, which makes them more likely to develop IAD , 39, 56.There were 13 studies with 55,927 participants included that evaluated the relationship between IAD and drinking and smoking. The results suggested that there was a average positive correlation between IAD and drinking, and a poor positive correlation with smoking. Analysis based on ORs showed that IAD was significantly associated with an increased risk of drinking and smoking.Considering the differences in sociocultural and economic background among populations, there is no standard recognized assessment tool for health risk behavior assessment at present, and many studies are based on self-report questionnaires prepared by researchers . Some studies included data on engaging in at least once health risk behaviors in the past 30 days , 45, 48,Additionally, scholars have found that smoking and drinking usually cluster since they may serve the same purpose socially and psychologically , 59. ResIAD has become an important global mental health issue, and is closely associated with an increased risk of drinking and smoking. Suicidal behavior is a risky behavior that interacts with and influences IAD. Adolescence, is at a stage when individuals are easily influenced by various complex and tempting social environments, forming a series of health risk behaviors . TherefoThe limitations of this review were as follows: first, there were significant differences among reports on health risk behaviors due to the huge sociocultural context among different racial/ethnic groups that had implications for the current review. Second, suicidal behaviors includes suicidal ideation, suicide planning and suicide attempts. Since suicidal ideation is a strong risk factor for suicidal behaviors , we extrAdolescents and young adults with IAD are at higher risk for suicidal behavior, drinking and smoking. Mood disorders are important mediators between IAD and suicidal behavior, and may be a target for effective intervention. In addition, it is important to explore the neural mechanisms of IAD/IGD and smoking, drinking and other substance dependence for the prevention and intervention of those problematic behaviors. The findings of this review are important for educational and psychological practitioners, parents and schools, and may provide guidance for the prevention and intervention of IAD and health risk behaviors.The original contributions presented in the study are included in the article/JW and QH conceived the idea. JW drafted the manuscript. YT, WP, and HL were involved in the interpretation of the study findings. TZ and YW reviewed the manuscript and provided comments. All authors contributed to the article and approved the submitted version.This research was funded by the Natural Science Foundation of China (81072852 and 81574047), the Key R&D Project of Sichuan Province (2019YFS0175), the Xinglin Scholar Research Promotion Project of Chengdu University of TCM (XSGG2019007), and the Training Funds of Academic and Technical Leader in Sichuan Province.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The geriatric nutritional risk index (GNRI) is a commonly used method to assess nutritional risk for predicting potential surgical site infections (SSI) in cancer patients. This study aims to create and verify a simple nomogram and a dynamic web-based calculator for predicting the risk of SSI among gynecologic oncology patients.A retrospective evaluation was conducted on patients who were admitted into a tertiary hospital in China with confirmed diagnosis of gynecologic cancer between 01 August 2017 and 30 November 2021. A two-piecewise linear regression model with a smoothing function was used to investigate the non-linear association between GNRI and SSI to determine the ideal cut-off point. Three models were developed on the basis of different variables to predict SSI in gynecologic oncology patients. Through a nomogram the concordance index (C-index), the Akaike information criterion (AIC), and the integrated discrimination index (IDI) were used to determine the final model. Finally, the performance of the nomogram was validated using the 1,000-bootstrap resamples method and analyzed using C-index, GiViTI calibration belts, and decision curve. Also, a user-friendly dynamic web-based calculator was developed.A total of 1,221 patients were included in the analysis. A non-linear association could be observed between GNRI and SSI risk with a GNRI cut-off value of 101.7. After adding GNRI to Model 2 , the AIC value decreased, the C-index value increased and IDI increased significantly. The nomogram C-index in the development cohort and internal validation cohort demonstrates a moderate-high degree of discrimination. The GiViTI calibrated belt showed a good agreement between the observed and predicted probabilities of SSI. The decision curve validates the clinical feasibility of the nomogram with a threshold value between 0 and 49%.The GNRI cut-off value of 101.7 allowed for appropriate stratification of patients into distinct SSI risk groups. This study found that including GNRI in the above nomogram (Model 2) would enhance its potential to predict SSI in gynecologic oncology patients. With an incidence rate of approximately 5–35% following a gynecologic oncology surgery, surgical site infections (SSIs) are considered one of the most prevalent postoperative complications reported among patients globally –4. Also SSI is the outcome of a complex combination of factors relating to the patient, environment, and to the surgery itself . Recent The primary objectives of this study are to (1) investigate potential associations between the GNRI and the risk of SSI in gynecologic oncology patients; (2) investigate the predictive value of including the GNRI in the study models; (3) to develop and validate a nutrition-based nomogram that incorporates the GNRI and clinical risk factors to predict SSI in gynecologic oncology patients.To establish the development cohort herein, a total of 1,561 consecutive gynecologic oncology patients who underwent surgery at Wenzhou People’s Hospital between 01 August 2017 and 30 November 2021, were retrospectively evaluated . The folExpert judgment, extensive literature studies, and the availability of clinical data were used to select all possible candidate predictors for potential operative site infections in gynecologic oncology patients –9. The fPreoperative peripheral blood samples were drawn from patients no more than 7 days prior to surgery. The comorbidity burden was assessed using aCCI . FurtherThe SSIs were characterized according to the definitions set by the Centers for Disease Control . The SSIt-test or Mann-Whitney U test was employed to compare continuous variables expressed as mean ± standard deviation (SD) or median [interquartile range (IQR)]. All statistical analyses were analyzed using the R software (version 3.6.3)1 and a two-tailed P-value of ≤ 0.05 was considered to be statistically significant.This study follows the guidelines outlined in the Transparent Reporting of a Multivariable Prediction Model for Individual Prognosis or Diagnosis (TRIPOD) statement . To compPrior to data analysis, the distribution of data and missing values for all predictors were examined. The percentage of missing data varied between 0 and 16.5% for each candidate predictor variable. Furthermore, to maximize statistical power and decrease selection bias caused by missing data, we performed multiple imputation with the Multivariate Imputations by Chained Equation (MICE) package in R  24)..24).Based on clinical factors such as MFS score, preoperative LOS, operative time, estimated blood loss, and GNRI, we established a model capable of predicting SSIs in gynecological surgery. The incidence of operative site infection in gynecological surgery is estimated to be 5.3% in this model. The multivariable logistic model included five predictor parameters and had a C statistic of 0.77. We performed a power analysis using the formula developed by Riley et al.  and obta2), normal weight (BMI 18.5–24.0 kg/m2), and overweight/obese (BMI ≥ 24 kg/m2). To categorize the patients, the cut-off point for laboratory values was determined as the upper or lower limit of the normal value. The cut-off point for glucose, albumin, ALT, total bilirubin, platelet count, hematocrit, TLC, WBC was 110 mg/dL, 3.0 g/dL, 40 U/L, 1.1 mg/dL, 350 × 109/L, 36%, 0.8 × 109 /L, and 10 × 109 /L, respectively. The two-piecewise linear regression model with a smoothing function was performed using the GAM package in R to investigate the non-linear relationship between GNRI, preoperative LOS, operative time, estimated blood loss, and SSI. The cut-off point was determined through trial and error, which included selecting turning points within a pre-defined interval and then selecting the ideal cut-off point that produced the highest model likelihood. Furthermore, we ran a log-likelihood ratio test to compare the one-line linear regression model and the two-piecewise linear model.All participants were divided into three groups using BMI for Chinese men and women as the criteria : underweOn the basis of different inclusions of variables, we have developed three models to predict the potential operative site infections in a gynecologic oncology surgery:(a) candidate predictor for Model 1 was limited to only the NNIS risk index; (b) candidate predictors for Model 2 included all factors indicated in Based on the regression coefficients of the chosen independent variables, a simple nomogram was established by R package “regplot” to predict the SSIs in gynecologic oncology patients.P-value > 0.05) suggests that there was no evidence of poor fit of the developed model. In addition, the existing predictors were also evaluated for their accuracy and suitability. A variety of statistical techniques, including the Cook’s distance, the studentized residuals, the variance inflation factor (VIF), and the hat value were used to identify the outliers, collinearity, influential observations, as well as data with high leverage.In order to obtain an unbiased assessment of model performance, 1,000-bootstrap resamples were used for internal validation . DiscrimThe current study utilizes the decision curve analysis (DCA) to evaluate the clinical efficacy of the established nomogram . DCA is pt represents the threshold probability at which the expected benefit of intervention-all-patients is equal to the expected benefit of intervention-none.where, Among the 1,561 cohort participants, a total of 340 patients of which were individuals who were under 18 years of age , patients with the length of stay less than 48 h , patients who were postoperatively hospitalized for less than 24 h , pregnant patients , and as to not introduce bias due to hospital readmissions, another 170 patients (10.9%) were excluded from the study. Finally, the final analysis consisted of 1,221 participants . Among tBefore and after adjusting for the factors that impact the association between GNRI and SSI, the results of the logistic regression analysis both revealed that GNRI remained strongly linked with SSI when examined as continuous variables . FurtherMoreover, we used a smoothing function to investigate the non-linear relationship between additional continuous variables , and SSI in a two-piecewise linear regression model in which the results demonstrated a linear relationship between preoperative LOS and the risk of SSI. In contrast, a non-linear relationship could be seen between the duration of surgery and estimated blood loss with the risk of SSI . Based oUsing the LASSO method, five predictive features with non-zero coefficients  were screened from the 36 candidate predictors , and six2. For instance, the estimated SSI risk was assessed to be approximately 12% , indicating that the observed and predicted probabilities of SSI in the prediction model were in good agreement. Because the VIF in all predictors was less than 5, no multicollinearity was seen. Moreover, there were also no observable changes and high leverage cases since both Cook’s distances and hat values were no more than 0.1 and 0.2, respectively , we developed a simple yet efficient nomogram to predict surgical site infection in gynecologic oncology patients to aid in medical decision-making. In both the training and validation cohorts, the nomogram demonstrated excellent calibration and discrimination performance. Furthermore, as compared to Model 1 (only NNIS risk index included) and Model 2 (without GNRI), the nomogram based on GNRI demonstrated higher prediction reliability, accuracy, and net benefit. This nomogram can assist physicians in making more precise clinical judgments. By only incorporating these five factors in our nomogram and accounting for varied proportions of those variables, we were able to simply and effortlessly provide an accurate forecast of a patient’s likelihood of acquiring SSI in a systematic and thorough manner. In addition, the serum albumin level, height, as well as the patient’s body weight that make up the GNRI in this study, were routinely assessed before surgery and did not necessitate a special examination, highlighting its feasibility in clinical settings. The GNRI was initially designed to assess malnutrition, as well as its associated morbidity and mortality in elderly patients . NumerouP = 0.02). However, by multivariate analysis, the above association was not observed in the laparoscopic surgery group . For this particular model, users may easily acquire the SSI prediction probability simply by inputting or selecting a variable in the graphical user interface. Consequently. healthcare providers may conduct a preliminary assessment of the risk of development of SSI in gynecologic oncology individuals and closely monitor those who are more susceptible. A subset of people who would benefit the most from more regular examinations is the high-risk population. In addition, interventions  can be taken more aggressively in high-risk individuals.There are, however, several limitations that must be taken into account. To begin, this is a retrospective single-center research, which inevitably has limitations due to its methodology and sample size. Therefore, further research is needed to externally validate the suggested nomogram despite the fact that we employed 1,000-bootstrap resamples for internal validation. Secondly, knowing that the cause of SSI is multifactorial, our research was primarily concerned with patient-related and surgical variables, excluding other elements like the operating room setting. Thirdly, there were too many missing data (≥50%) or too few positives in this study to incorporate previously established risk factors for SSI, such as current smokers and intraoperative hypothermia. Fourthly, while each case of SSI was thoroughly evaluated and co-confirmed by a physician and a senior infection control practitioner to avoid misclassification bias in the current investigation, misdiagnosis and missed diagnoses could still have occurred. Finally, even though various assessment tools, such as Barthel Index, Morse Fall Scale, weight, and height, were evaluated by competent nurses in our hospital, we could not rule out the possibility of measurement bias as a result of the screening being performed by different nurses.The current research investigates the use of preoperative GNRI as an independent predictor of SSI in gynecologic oncology patients. We found that a cut-off value of 101.7 for the GNRI allowed for appropriate stratification of patients into distinct SSI risk groups. Moreover, we established and validated a nomogram based on the GNRI to estimate the risk of SSI in gynecologic oncology patients, and proved that incorporating the GNRI into the model may further increase its prediction power.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 the Ethics Review Committee of Wenzhou People’s Hospital. Written informed consent for participation was not required for this study in accordance with the National Legislation and the Institutional Requirements.ZC and MZ analyzed the data and drafted the manuscript. ZX, QY, WX, SG, LC, LQ, JJ, and HWu collected and analyzed the data. XL and HWa conceived and designed the study. All authors read and approved the final manuscript.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.


Background and Objectives: Plantar fasciitis (PF) is a prevalent musculoskeletal disease, with inflammation at the origin of the plantar fascia, that affects sedentary people, particularly middle-aged women. Foot pain and functional limitations lead patients to seek treatment. Investigate the therapeutic effect of conservative treatment combining a custom insole with minimalist flexible shoes and the shoes alone in a gait-training protocol, in the short and long term, in women with PF. Materials and Methods: Design: A randomized, controlled, and single-blind trial. Setting: Biomechanics laboratory. Participants: 36 women, 26 with acute PF and 10 controls. Intervention: Gait-training protocol wearing the minimalist shoes alone , with a custom insole in the shoes , and control . Evaluations were performed at baseline (T0) and after three (T3) and six (T6) months. The intervention had a duration of six months . Primary outcomes were rearfoot pain , the Foot Function Index (FFI), Foot Health Status Questionnaire (FHSQ-Br), and 6 min walk test (6MWT). The secondary outcomes were plantar pressure distribution during gait, measured by the pressure platform, and foot posture. Results: The CIG was effective for reducing pain and improving the FPI after T6 compared to CG. The FPI, FHSQ-Br and 6MWT demonstrated improvements after T6 in both the CIG and SG, compared to the CG. After T6, contact area (rearfoot) and maximum force (forefoot) reduced with CIG. Maximum force (midfoot and rearfoot) reduced with CIG and SG, as did peak pressure (forefoot and midfoot) in relation to CG. Conclusions: A customized insole associated with minimalist flexible shoes during a gait-training protocol can be recommended as a more effective treatment than minimalist flexible shoes alone over the short and long term, for reduction in calcaneus pain, increased function and foot health, and improved walking through reduced plantar load in women with PF. Plantar fasciitis (PF) is a prevalent rheumatic musculoskeletal disease, with inflammation at the origin of the plantar fascia , which pFoot pain and functional limitations are dysfunctions of this disease that lead patients to seek and undergo some conservative treatments. Plantar overload, arch index, and rearfoot pronation have been strongly related to PF severity, i.e., fragmentation and degeneration of the collagen fibers of the plantar fascia ,12,13,14Although the currently available pharmacotherapy with corticosteroid injections is sufficient to control pain ,20, it iIn this rationale, some studies have shown the therapeutic effects on gait of an insole  to reduce pain and plantar load on the calcaneus of patients with PF ,36,37,38The advantages of this type of mechanical treatment are its effectiveness in relieving the plantar overload on the feet, easy accessibility for the patient, and a lower chance of clinical complications compared to surgical treatment ,10. EvidMore recently, the use of minimalist flexible shoes was shown to effectively reduce plantar overload, in the short and long term, in inflammatory diseases caused by impact forces ,42,43,44The design of the study was a randomized clinical trial with concealed allocation, blinding of assessors, and intention-to-treat analysis (six-month intervention) in women diagnosed with PF. The patients with PF allocated to the intervention groups received either the minimalist flexible shoes (SG) or custom orthopedic insoles combined with minimalist flexible shoes (CIG) on the first day and were required to wear them for three and six months consecutively, as the gait-training protocol. The patients allocated to the control group (CG) did not receive any intervention, only the standard protocol with guidelines on shoes for walking. The patients were assessed in the baseline condition (T0) and after three (T3) and six months (T6) after the intervention program.n = 12) and CIG (n = 14) and ten healthy women without PF to the CG (n = 10) (The patients were recruited from two settings: (a) health center; (b) orthopedic and rheumatology clinics at the University Hospital. The diagnosis of PF was confirmed by a physical examination performed by an orthopedist and clinical exams: X-ray and ultrasonography  ,44. A to(n = 10) .2, diagnosis of PF, without a history of any surgical process in the knees, ankles, and hips or muscle injury in the previous six months, and without any diagnosed neurological and rheumatological disease [The inclusion criteria were: female volunteers, aged between 30 and 55 years , body ma disease ,43, as w disease . The patients provided written consent, based on ethical approval from the Human Research Board of the local University (number: 1.074.141). Data access and storage comply with the National Health and Medical Research Council guidelines. This trial is registered in Clinical Trials (number NCT 03040557).The randomization schedule was prepared using Clinstat software by an independent researcher who was not aware of the numeric code for the control and intervention groups. A numeric block randomization sequence was placed in opaque envelopes. After the patients’ agreement and assignment to participate in the research, the allocation into the groups was made by another independent researcher, who was also unaware of the codes. Only the physiotherapist responsible for the clinical trial knows who is receiving the intervention.The clinical examinations were carried out by an orthopedist who was blind to the patient’s allocation. One physical therapist was responsible for pain, functional, and biomechanical assessments. Another physical therapist was responsible for follow-up and supervision of the intervention groups, providing instructions by telephone. Both physical therapists were blind to treatment allocation until the analysis had been completed .The primary outcomes were calcaneus pain, measured by the Visual Analogue Scale (VAS), and feet functionality using the Foot Function Index (FFI), Foot Health Status Questionnaire (FSHQ-Br), and 6 min walk test (6MWT). The FFI evaluates the foot functional limitation. It contains 23 questions subdivided into three domains: foot pain, difficulty, and functional limitations. The final score varies from 0 to 100; the higher the score, the worse the functional condition . The FHS® Sensor, LorAn Engineering Inc., Bologna, Italy), incorporating capacitance transducer sensors (4 sensors/cm2), and sampling at a frequency of 100 Hz. The patients walked freely along a 20 m walkway, with the pressure platform placed in the center of the walkway. The patients were asked to walk in a natural way (as performed in their daily lives) [2), maximum force (N), and peak pressure (kPa) over the four plantar areas of the foot, medial and lateral rearfoot (30% of the foot length), midfoot (30% of the foot length), and forefoot and toes (40% of the foot length), were registered [The Secondary outcomes were plantar pressure distribution during walking using a pressure platform (Lorany lives) ,44. The gistered ,37,38,39gistered . The gait-training program intervention was based on the daily use of the minimalist flexible shoe (SG) alone for three and six months, for six hours a day, seven days a week (at least 42 h per week). The minimalist flexible shoe  used was a low-cost women’s double canvas, flexible, flat, walking shoe, without heel drop, with a 5 mm anti-slip rubber sole and a 3 mm internal wedge of ethylene vinyl acetate. The shoe has a mean weight of 0.172 ± 0.019 kg, with a mass of between 91 g and 182 g, depending on the shoes size  Figure .The patients allocated to the intervention received custom orthopedic insoles combined with the minimalist flexible shoe (CIG), also used daily. The customized insoles were flat, total-contact, weighed 31 g, were made of ethylene–vinyl acetate-EVA, were 3 mm thick in the forefoot with the support element under the plantar arch, and 7 mm in the rearfoot with the support element under the heel (wedge for the heel region), determining the two-point mechanical design. The insoles were manufactured for each patient individually, made with the patient standing on a foot foam mold, from which the EVA insole was fashioned. The gait-training protocol intervention was for three and six months, for six hours a day, seven days a week . Every tThe patients allocated to the control group (CG) did not receive interventions, only the standard protocol, with guidelines on shoes for walking. During the intervention period, patients from the CG were asked not to wear minimalist flexible footwear or similar minimalist shoes and customized insoles, but they continued to receive their health care and painkiller medication at the hospital. At the end of the intervention period, all CG patients also received a free pair of minimalist flexible shoes with or without the customized insole.The sample size was performed considering calcaneus pain as the primary outcome, with an effect size of 0.30 (moderate). In total, 36 women were needed to provide an 80% power to detect a moderate effect difference between groups, considering a significance level of 0.05. The power of an experiment is the probability that the effect is detected. It is usually arbitrarily set to 0.8 or 0.9 . Note that power, symbolized as β, is the chance of obtaining a false-negative result .The tests were based on intention-to-treat analysis, using the two-way ANOVA test to detect treatment–time and inter-group interactions (α = 5%). The effect size, using Cohen’s D, was measured between T0 and after six months (T6) of intervention, considering values of 0.20–0.39 as small, 0.40–0.79 as medium, and >0.80 as large effect sizes.Overall, 60 women initially demonstrated interest in participating , among wThe interventions with SG or CIG promoted a reduction in calcaneus pain and functionality in the women with PF. However, the insole (CIG) was more effective in reducing pain when compared to the shoe alone (SG), after three and six months, with a medium effect size. Improved foot function was also positive with CIG and SG after three and six months. Considering the inter-group assessments, the CIG was better at increasing foot function when compared to the SG and CG after six months of treatment. Another important finding was the decrease in FPI after three and six months with both the SG and CIG . Considering the FSHG-Br, a significant increase was observed after six months with both the insole (CIG) and shoe (SG); however, a medium effect size was observed for the CIG and small for the SG. Inter-group, both the insole and shoe showed improvement only after six months when compared to the CG. Improvements in pain were observed after three and six months with the CIG and the SG, with a large effect size after the interventions. Inter-group, both the CIG and SG showed effective improvements after six months when compared to the CG. The foot function and general foot health demonstrated increases after three and six months in both the CIG and SG, with a medium and large effect size. Inter-group, the CIG and SG did not show improvement for foot function and health compared to the CG after three and six months. With respect to the shoe, the CIG and SG showed effectiveness after three and six months, with a large effect size for the CIG and small for the SG. Inter-group, both the CIG and SG showed improvements compared to the CG . After three and six months with the CIG and SG, the women presented improvements in the six-minute walk test, with a large effect size for the CIG and moderate for the SG. Inter-group, the CIG and SG were different when compared to the CG after three and six months .The biomechanical parameters of plantar pressure distribution after interventions in women with PF revealed that : (A) ConThis was the first clinical trial to compare the therapeutic effectiveness of a custom orthopedic insole in conjunction with a minimalist flexible shoe and the shoe alone. A gait-training protocol with the CIG and SG followed up after three and six months, used for six hours a day, seven days a week (42 h/week), was shown to be effective to improve pain and functionality, and reduce plantar pressure in women with PF, with greater emphasis on the use of the insole (CIG).The main results showed that the custom insole (CIG) and minimalistic shoe (SG) were beneficial for decreasing foot pain. The CIG was more effective in reducing pain compared to the SG after three and six months. Calcaneus pain during gait and other activities of daily living prompts patients with PF to seek conservative mechanical treatment in combination with other therapies, such as pharmacological and physical therapy . EconomiWhat distinguished this study was to show the greater therapeutic efficacy of the combination of a customized insole with a minimalist flexible shoe, as a low-cost intervention to decrease heel pain in women with PF compared to the shoe alone  after the intervention periods of three and six months, considered short and long term. These findings are in agreement with scientific reports on the effectiveness of inexpensive flexible shoes for pain reduction in inflammatory and degenerative diseases, such as knee osteoarthritis ,42,43,44Foot function improved with both the use of the insole (CIG) and minimalistic shoe (SG) after three and six months, but the CIG showed an effective improvement when compared to the SG and CG after six months of treatment. In the clinical routine, a practical guide from the American College of Foot and Ankle Surgeons recommends non-steroidal anti-inflammatory drugs, stretching, and ankle mobility exercises as the initial steps for the conservative treatment of PF, as well as orthoses and/or insole prescription, as effective conservative secondary treatment options ,50,51. TAccording to the literature, the etiology of PF is still unknown; however, it is believed to be multifactorial, with changed biomechanics and delayed healing as likely contributing risk factors ,3. A recLandorf et al. , compariA study using three-dimensional analysis with an infinite element model of the ankle–foot segment revealed that elevated heel height  can significantly change foot biomechanics, especially the strain of force on the anterior talofibular ligament (ATL) and plantar fascia during balance and gait. The authors concluded that the tension force on the plantar fascia decreased at moderate heel height . These fIn the current study, the strain force on the ATL increased with the increase in heel height, while the tension force on the plantar fascia decreased at moderate heel height . PatientThe limitation of this study was that we did not record the plantar fascia thickness by ultrasound over the six months of follow-up with use of the insole and minimalistic shoe (interventions with gait-training protocol); however, our main concern, in this first instance, was to evaluate variables related to pain, function, and calcaneus plantar load in women with PF. Future studies monitoring the plantar fascia thickness may help health professionals to better understand the response strategies to therapeutic mechanical treatment of the disease.A customized insole associated with a minimalist flexible shoe during a gait-training protocol can be recommended as a conservative mechanical treatment, which is more effective than the minimalist flexible shoe alone, over the short and long term, for reducing calcaneus pain, increasing function and foot health, and improving walking through reduced plantar load in women with PF.


The COVID-19 pandemic has hugely impacted many different aspects of human health, and vaccination is one of the most effective weapons to manage it. However, many different factors, such as age, gender, comorbidities and lifestyles, play a role in the response to infections and vaccines. We carried out this study to evaluate the potential role played by some individual factors in the production of anti-COVID-19 antibodies in the light of personalized and future vaccinology. We conducted an observational study consisting of a retrospective phase, exploiting previous data about anti-COVID-19 antibody responses, with a prospective phase to investigate individual variables through the use of a questionnaire. The antibody response after the COVID-19 vaccination was inversely related to old age, increased BMI and the number of smoking years, while a positive correlation was found with moderate alcohol consumption and especially with circulating levels of vitamin D, as clearly shown by the multivariate regression analysis. Our study showed that a number of variables are involved in the COVID-19 vaccine antibody response. These findings are very important and can be considered in the light of a future and personalized vaccinology. The COVID-19 pandemic has exerted a huge and complex impact on many different aspects of human health and society, not only in terms of morbidity and mortality, with more than 6 million deaths worldwide, but also with regard to economic and environmental aspects . VaccinamRNA vaccines have been the most important typology of performed and tested vaccines against COVID-19 . SpecifiHowever, many different factors can play a role in the individual response to vaccines. Age, gender, comorbidities and lifestyles, such as smoking habits, alcohol abuse and physical activity, are well-known factors that have an impact on immunity to natural infections and vaccinations . The immFinally, vitamin D circulating levels have been associated with good immune responses to natural infections and vaccinations ,17. VitaThe purpose of this study was to evaluate the potential role played by some demographic, anthropometric, behavioral, clinical and laboratory parameters in the production of antibody responses of a group of healthcare workers (HCWs) vaccinated with the complete primary cycle of anti-COVID-19 Pfizer/BioNtech’s BNT162b2 vaccine, in the light of a more and more personalized and future vaccinology.We conducted an observational study to investigate the role of some variables on the antibody response to the COVID-19 vaccine. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the local ethics committee .The study involves a combination of a retrospective phase, exploiting previous data, with a prospective phase that aims to investigate demographic, clinical, behavioral and nutritional variables on already existing samples.Specifically, we selected 200 people from a big sample of healthcare workers subjected to the primary cycle of the anti-COVID-19 Pfizer/BioNtech’s BNT162b2 vaccine, carried out between January and May 2021 in the University Hospital “G. Martino” of Messina, Italy. These selected people then underwent an evaluation of their antibody responses as part of routine health surveillance three weeks after the third dose of the vaccine. They possessed variable antibody responses and voluntarily adhered to the study. All the participants were asked to sign an informed consent for the study participation and the use of their serum/plasma samples stored at −80 °C in the virology laboratory of our hospital, followed by the self-administration of a questionnaire for the collection of information useful for evaluating any factors involved in the antibody response, such as their age, gender, body mass index (BMI), vitamin D intake, any comorbidities as well as persistent lifestyle aspects such as their smoking habits, alcohol consumption and physical activity. The information relating to lifestyles was collected using the indicators provided by the Italian PASSI surveillance system, recognized as a system of national importance .The antibody response was evaluated, after centrifugation at 4000 rpm for 10 min, by CLIA test (ChemiLuminescence ImmunoAssay)  consisting of a quantitative assay for the determination of IgG antibodies against SARS-CoV-2 S1/S2 antigens. Specifically, values <12 AU/mL were considered negative.After excluding people who have contracted the COVID-19 infection and those who made use of vitamins and micronutrients, we proceeded to evaluate, on the same samples on which the antibody response had been determined, the quantitative determination of 25(OH)D3 plasma levels by high-performance liquid chromatography (HPLC) with a Bio-Rad 25(OH)D3/D2 kit  according to the manufacturer’s instructions. Separation of 25-OH-vitamin D3 and internal standard took place on a reversed-phase cartridge, followed by subsequent UV detection (λ = 265 nm) and quantitative evaluation [The antibody titer was then correlated with biological parameters such as sex and age, comorbidities, behavioral factors and levels of vitamin D.t-tests. Finally, a multivariate analysis was carried out to evaluate the predictive capacity of the detected variables  on the “antibody response” outcome. Analysis was performed by using the “a priori” model  of multiple regression. Significance was assessed at the p < 0.05 level.Statistical analyses were performed using Prism 4.0 software . All the data obtained on the samples under study were subjected to a preliminary descriptive analysis aimed at summarizing the collected information. Pearson’s correlation test was used to determine any correlations between the studied variables. Stratified data were statistically analyzed using one-way ANOVA and The total number of the sample, the mean age, the gender percentages and all the information collected on the study participants related to antibody responses, body mass index (BMI), vitamin D levels, comorbidities and persistent smoking habits, alcohol consumption and physical activity are summarized in Of the initial sample number, 152 people were enrolled in the study. The mean antibody response of the primary vaccination cycle was 214.51 AU/mL (CI 95%: 187.4–245.2). Men had a slightly higher mean antibody response than women. However, no statistically significant difference was found in the antibody response between sexes, given a little difference of only 20 AU/mL in the antibody response.p = 0.0439) was found between the antibody response and the age of the sample (p = 0.022).Conversely, a significant reverse correlation , no significant correlation was found with the antibody response. Dividing the sample into three groups on the basis of the following BMI values: 18.5–24.99 = normal weight, 25–29.99 = overweight, ≥30 = obese , an intep = 0.0425).According to comorbidities, 33.33% of the participants declared their presence. Specifically, autoimmune/inflammatory diseases and hypertension were the most frequently declared ones. In this regard, a significant difference was found in antibody response in people with and without comorbidities  (t = 2.0p = 0.0484). By dividing the sample into three groups, non-drinkers (54.35%), occasional drinkers (23.91%) and moderate drinkers (21.74%), there were significant differences between groups   .According to physical activity, 73.33% of the subjects carried out moderate (42.22%) or light (31.11%) physical activity during work; moreover, 31.11% and 46.67% practiced intense and moderate physical activity, respectively, in their free time. However, no correlation was found between the antibody response and physical activity.p = 0.0136) was found, likely due to the confounding factor of age  was found between antibody response and vitamin D levels. The mean value of circulating vitamin D levels was 23.10 ng/mL (CI 95%: 21.24–24.97). Stratifying the sample according to the thresholds indicated as severe deficiency (<10), moderate deficiency (10–20) and optimal levels (21–75) [t-test  (A significant positive correlation ( (21–75) , an incr 0.0375) A,B.p = 0.026), while no effects were shown by the demographic, anthropometric, clinical and behavioral variables , as they are one of the categories more at risk of contracting COVID-19 ,31, consOne of the most important parameters that determine antibody response is certainly age, which represents an important response endogenous factor to natural infections and vaccinations. Specifically, as shown by our results and by previous scientific evidence, a natural decrease in the antibody response can be found with increasing age due to the physiological process of immunosenescence . This prAnother important factor affecting the development of post-vaccine antibodies is gender. Although no correlation emerged in this study, our previous study  showed aEven if no significant statistical difference was present between BMI and antibody response, our results found in this parameter a factor influencing antibody response after vaccination in terms of a decrease in antibody production with the increase of BMI. In line with this result, BMI has been identified as one of the factors influencing post-vaccine antibody production . MoreoveIn addition, the presence of comorbidities influenced, in a negative way, the antibody response after vaccination. This finding is very important considering that some chronic pathologies, such as diabetes, cardiovascular diseases and autoimmune diseases, are particularly spread worldwide, especially in people of a certain age. This should be taken into particular consideration in terms of vaccination policies because we cannot consider people affected by comorbidities in the same way as healthy people. In this regard and in this kind of people, a personalized vaccinology approach should be stressed and improved.A key role in the antibody response to vaccination has been identified in lifestyles, which influence the immune response to both natural infections and vaccinations. For example, concerning cigarette smoking, we found an inverse correlation between immune responses and years of smoking. Indeed, smoking has been shown to impair the overall immune response and the ability to form memory cells, necessary for long-lasting immunity, by causing a decrease in vaccine-induced IgG antibody levels ,48. In fAnother very interesting finding of our study is the positive correlation found between the antibody response to the vaccination and the moderate consumption of alcohol. In fact, the consumption of alcoholic beverages can generally affect the immune response. Actually, there is strong evidence that chronic alcohol abuse is associated with increased immunosuppression and susceptibility to both bacterial and viral infections . ConversHaemophilus influenzae type b oligosaccharide conjugated to diphtheria toxoid vaccine and hepatitis B surface antigen (HBsAg). For all these reasons, it is reasonable to think that vitamin D supplements can play a crucial role in preventing respiratory infections, especially in people highly deficient in this metabolite, and in increasing immune responses after vaccinations [Finally, a key role in the antibody response to vaccination has also been identified in vitamin D levels, a steroid hormone whose activity is mediated by the nuclear vitamin D receptors (VDRs) that, after activation and dimerization following the link with the vitamin, translocates to the nucleus to link the vitamin D receptor element (VDRE), a genetic element that regulates the expression of several host genes, some of them involved in immune responses like beta-defensin, which can directly split the outer viral membrane, and cathelicidins, which is involved in the activation of macrophages, dendritic cells and neutrophils ,58. Furtinations . MoreoveOur study showed that a number of variables are involved in the COVID-19 vaccine antibody response, some of them in a positive way and others in a negative way. Specifically, the antibody response after the primary cycle of mRNA COVID-19 vaccination was inversely related to old age, increased BMI and the number of smoking years, while a positive correlation was found with moderate alcohol consumption and especially with circulating levels of vitamin D. These findings are very important and can be considered in the light of a future and personalized vaccinology that can allow us to predict the efficacy of vaccination in a very accurate way in different groups of people depending on their demographic, anthropometric, clinical and laboratory parameters. These results could allow reaching higher vaccination coverage rates through increasing public confidence in vaccines following the improvement of their immunogenicity and the reduction of adverse event rates, with a reduction or elimination in the morbidity and mortality related to vaccine-preventable diseases.


To systematically evaluate the guidelines for endometriosis published in the past decade, and to provide reference for the selection of guidelines for endometriosis through quality evaluation and comparison.PubMed database, Embase database, evidence-based medicine clinical practice guidelines (CPG) database and the National Institute for Health and Clinical Excellence in the United Kingdom were searched by computer from December 2012 to December 2020 to retrieve published endometriosis CPG published by professional institutions or organizations. The search languages are English and Portuguese. Two researchers evaluated the quality of included CPG according to appraisal of guidelines for research and evaluation (AGREE II). The evaluation includes 6 areas: scope and purpose, participants, rigor of formulation, clarity of expression, applicability and independence. The recommendation level of CPG is determined by the distribution of standardized scores in the above 6 areas.A total of 8 articles on endometriosis CPG were included, including 5 guidelines and 3 consensuses, covering 5 countries in 2 continents; the publication year was 2013 to 2020. The average standardized scores of the scope and purpose, participants, rigor, clarity, applicability and independence of CPG were 77.1%, 52.8%, 50.5%, 86.8%, 31.3%, and 36.5%, respectively. Among the 8 CPGs, 1 was grade A (recommended), 5 were grade B (recommended after improvement), and 2 were grade C (not recommended). Seven CPG recommendations were based on expert consensus, and one was developed through detailed literature retrieval, analysis and evidence rating evaluation. There was little difference between the guidelines in terms of treatment-related recommendations.The quality of endometriosis CPG released in 2013 to 2020 is quite different, and some CPGs are not ideal in terms of rigor, applicability and independence. The guidelines issued by NICE in 2017 are A-grade recommendations. The standardized scores in various fields are high, and the formation process of CPG is the most standardized, which is worth learning and reference. Women with endometriosis usually have a series of pelvic abdominal pain symptoms, including dysmenorrhea, difficulty in intercourse, large amount of bleeding during menstruation, non-menstrual pelvic pain, ovulation pain, difficulty in urination and chronic fatigue. Endometriosis is also associated with infertility. There is a strong correlation between the severity of the disease and the impact on women’s fertility, due to impaired ovarian function, ovarian endometriosis cysts and subclinical pelvic inflammation, which may reduce the quality of oocytes and reduce the endometrial receptivity to implantation. As a common chronic gynecological disease requiring long-term management, the current guidelines and consensus related to endometriosis have been up to a hundred departments, which brings great difficulties to clinicians “decision-making and patients” informed choice. Therefore, it is very important for both doctors and patients to select high-quality guidelines or consensus as clinical diagnosis and treatment guidance.Endometriosis (endometriosis) refers to the occurrence, growth, infiltration, and repeated bleeding of endometrial tissue outside the uterus, which in turn leads to pain, infertility, nodules or masses. It is a common and frequently-occurring disease in women of childbearing age. In this study, the internationally recognized guideline evaluation tool-appraisal of guidelines for research and evaluation (AGREE II) was used to evaluate the quality of the included CPGs through a comprehensive retrieval of foreign published CPGs specifically for endometriosis, so as to provide further reference for clinicians.Clinical practice guidelines (CPG) are designated and issued by professional academic organizations or departments based on the best evidence and combined with clinical practice to help clinicians and patients make clinical decision-making guidance, including guidelines, consensus, recommendation, etc, which can not only improve the diagnosis and treatment effect, but also make full use of medical resources.http://www.g-i-n.net/), National Institute for Health for Health and Care Excellence website (https://www.nice.org.uk/guidance), National Guideline Clearinghouse (https://guidelines.gov/), Scottish Intercollegiate Guidelines Network (http://www.sign.ac.uk/), Clinical Practice Guidelines Portal website , New Zealand Guidelines Group website , BCGuidelines website , AQuMed Database website (http://www.aezq.de/aezq/publications). In addition, we searched Google Search Engine and checked the references of all the related guidelines to include more potential guidelines. All guidelines were based on previous published studies, thus no ethical approval and patient consent are required.PubMed database, Embase database, evidence-based medicine CPG database, National Institutes of Health and Clinical Medical Optimization were searched online from December 2012 to December 2020 with search terms of “endometriosis,” “guidelines,” “guidance,” “consensus.” We also searched the websites of guideline development organizations: Guidelines International Network Web site , topics, publication years, publication organization, guidelines development methods, evidence level evaluation methods and other basic information.Each included CPG was evaluated by 2 trained personnel for AGREE II. According to the “Instructions for Clinical Guidelines Research and Evaluation System II (Chinese version),” 23 entries in the following 6 fields were evaluated, including scope and purpose (3 entries), stakeholder involvement (3 entries), rigor of development (8 entries), clarity of presentation (3 entries), applicability (4 entries) and editorial independence (2 entries). Finally, a comprehensive evaluation was carried out according to the scores. Two researchers rated each item from 1 (very disagree) to 7 (very agree). Standardized scores in various areas = /(highest possible rating - lowest possible rating) × 100%. The higher the standardized score of a field, the more complete the reporting, reflecting the better the method of developing guidelines in the field. Combined with the scores of each field of the guide, the recommended level of the guide is divided into 3 grades, and grade A is recommended, that is, the standardized scores of the 6 fields are ≥60.00%; grade B is recommended after modification and improvement, that is, the standardized score of the field is <60.0%, and the standardized score of ≥3 fields is ≥30.0%; grade C is not recommended, that is, the number of fields with a standardized score of <30.0% in 6 fields was ≥3. it indicates that the consistency is high and formal experiments can be carried out.Before the formal evaluation, the 2 researchers used AGREE II to perform pre-experimental scoring on the randomly selected 2 guidelines to test the reliability of the results. The scoring results were statistically analyzed by SPSS 20.0 software, and the consistency of the evaluation results was investigated by intra-group correlation coefficient (ICC). When the ICC value is 0.81 to 1.00,A total of 906 relevant literature were obtained through database retrieval. According to the inclusion and exclusion criteria, 8 CPGs were finally included after excluding duplicate articles Fig. .Among the 8 CPGs included in this study, 5 guidelines and 3 consensus. Covering 5 countries of 2 continents; published in 2013 to 2018, with 6 first editions and 2 updated editions; 7 of them were conducted evidence quality rating and recommendation intensity evaluation based on the Grading of Recommendations  method. In addition to the 2016 Portuguese guidelines, the 2017 WES endometriosis classification guidelines, and the 2018 Japanese guidelines, the remaining 5 CPGs are all about the diagnosis, treatment (drugs and surgery), and pain management of endometriosis in the reproductive system. Four articles also cover the treatment of patients with endometriosis and infertility. The 2014ESHRE guideline referred to the primary prevention of endometriosis, while the 2014 German guideline defined the secondary prevention of endometriosis and made recommendations  and 0.91 (0.79–0.96), indicating that the consistency of the evaluation of the 2 researchers was good.The average standardized scores for the scope and purpose, stakeholder involvement, rigor of development, clarity of presentation, applicability and editorial independence of the 8 CPGs included were 1 in grade A (recommendation), 5 in grade B (recommendation after improvement) and 2 in grade C (no recommendation), respectively  and 4 (clarity of presentation) were ≥60%, and the average scores in fields 5 (applicability) and 6  were <30%, indicating that most of the CPGs for endometriosis did not pay enough attention to these 2 fields.Main shortcomings: The average score of field 3 (rigor of development) was 50.5%, because the lack of high-quality and multi-center clinical research data led to the formulation of most CPGs based on clinical experience and consensus of experts; no detailed inclusion and exclusion criteria; there is no clear description of the method for forming recommendations; some CPGs do not even specify the strength and limitations of the evidence. The average score of filed 5 (applicability) was 31.3% because most CPGs did not mention the promotion and impediments to application; economic cost and health budget were not mentioned. The general score of field 6  is not ideal. Although each CPG has a statement of interest conflict, the wording of most guidelines is not clear about the point that the views of sponsors do not affect the content of the guidelines. In particular, Japan’s CPG has barely elaborated on this field. Overall, the progress of endometriosis CPG in the past decade was limited, especially in the recommendation of diagnosis and treatment strategies.The limitations of this study are as follows. The limitations of research tools: there are no primary and secondary divisions in the 6 fields of AGREE II, which leads to the fact that some CPG levels may not be consistent with the actual. In fact, the quality of GDP cannot be evaluated only by the level, which is also clearly pointed out in the use instructions of AGREE II. Language limitation: This study was limited by language, only English and Portuguese CPG were included, and no further study was conducted on CPG in other languages. AGREE II is only an evaluation system, which is subject to the subjective influence of design developers. It is not necessarily suitable for all regions and countries, and it needs to be refined and updated. The 2017 NICE guide has high scores in 6 fields, and is the only CPG published on the basis of literature retrieval and evidence grading assessment. It provides a detailed description of economic and social costs, promotion and impediments, and provides a detailed comparative analysis of drug treatment costs, which is worthy of promotion and reference.In this study, the diagnosis and treatment strategies and methods recommended by the 8 endometriosis CPGs were not significantly different, but in fact, many problems were controversial in the process of CPG production or in the diagnosis and treatment of patients with endometriosis.Conceptualization: Yan Lei.Data curation: Yan Lei, Xin Du.Formal analysis: Yan Lei, Dejun Chen.Funding acquisition: Hongmei Lian.Investigation: Xin Du, Dejun Chen, Yue Gao.Methodology: Yan Lei.Project administration: Yue Gao, Hongmei Lian.Software: Dejun Chen, Hongmei Lian.Supervision: Yue Gao.Validation: Yue Gao.Visualization: Yue Gao.Writing – original draft: Yan Lei.Writing – review & editing: Xin Du, Hongmei Lian.


Oral submucous fibrosis (OSF) is a potentially malignant condition of the oral cavity characterized by progressive fibrosis of the submucosal tissues. OSF is typically associated with the use of betel quid (BQ), a chewing package made of natural products , with or without smokeless tobacco. BQ ingredients contain pro-carcinogenic bioactive compounds, but also potentially protective biomolecules, and we have shown recently that the chemical properties of different BQ recipes vary, which may explain the unequal prevalence of OSF and oral cancer in BQ users in different geographical regions. Hence, this scoping review was aimed at evaluating the existing literature regarding different BQ compounds and their association with OSF. The repository of the National Library of Medicine (MEDLINE/PubMed), medRxiv databases, Google scholar, Baidu scholar, CNKI, and EBSCO were used to search for publications that investigated the association between BQ chewing and OSF up to November 2021. The search terminology was constructed using the keywords “betel quid” and “oral submucous fibrosis”, and their associated terms, with the use of Boolean operators. The search was conducted under Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) guidelines, together with clear inclusion and exclusion criteria. The review showed that the risk of developing OSF varied between different BQ recipes, and that chewing BQ mixtures containing betel inflorescence (BI) significantly increased the risk of OSF, as did the addition of tobacco. Conversely, the use of betel leaf in the mixture was likely to be protective, which may be due to the presence of polyphenols. Although further research is needed to determine the effect of individual BQ ingredients in the development of OSF, our pilot results provide the scope and rationale for informing future chemopreventive strategies for OSF and oral cancer in BQ chewers.  Globally, approximately 600 million people chew betel quid (BQ). Three fourths of its consumption is concentrated in the Pacific Islands, South Asian, and Southeast Asian countries . BQ chewPiper betle) stem and inflorescence (BS and BI), and various additives . Although OSF is multifactorial in origin, there is strong evidence that areca nut (betel nut) chewing is the main etiological factor. Areca nut (AN) has been classified as an independent group 1 human carcinogen and is invariably chewed as a betel quid (BQ) in the Asia-Pacific region as well as in Asian migrant communities throughout the world ,9. Whilsdditives . Traditidditives . Intrigudditives , thus prThe aim of the present study is to systematically evaluate the existing literature regarding different BQ ingredients and their association with the onset of OSF. We believe that our findings, if developed further, will have important public health implications for the prevention of OSF and oral cancer.The protocol for this structured scoping review was done in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) guidelines . This stThe articles that were included in this study had the following criteria: (1) they were based on humans; (2) they were written in the English language; (3) the BQ habit was practiced with or without tobacco; (4) the odds ratio (OR) or relative risk (RR), together with their corresponding 95% CI, were used as measures of an association between the BQ habit and OSF. Articles were excluded for the following reasons: (1) studies involved non-human samples; (2) studies were meta-analyses, systematic reviews; (3) studies in which an OR measured the association between BQ habit and oral potentially malignant diseases, and did not mention OSF exclusively. No time restriction was applied to the search. The selection process was divided into three phases. In the first phase, the inclusion and exclusion criteria were used to select appropriate papers. In the second phase, papers were reviewed based only on their titles and abstracts under the guidance of the senior author (NC). Adhering to the Cochrane guidelines, a lenient policy was adopted during the selection process where there was any uncertainty. In the third phase, the full texts of publications meeting the inclusion criteria were analyzed. In cases of disagreement or ambiguity, a meeting was held to discuss the decision of inclusion, with the senior author (NC) to determine the final outcome due to seniority and expertise in the field. At the end of the three phases, the papers fulfilling all of the inclusion criteria were included in the qualitative analyses.The following parameters were extracted from each study: name of the first author, year of publication, presence of tobacco, areca nut, betel leaf, slaked lime or other ingredients in the BQ mixture, type of study, the region where the study was conducted, sample size, and the OR/RR.Details of the selection process are shown in Of the remaining 22 articles, 11 papers were excluded because they reported ORs that were not exclusive to OSF; the remaining 11 papers were deemed to be eligible for detailed assessment . An overp < 0.01, RRno-AN-use = 1.0; p < 0.01) [p < 0.0001)); further, males had a higher risk than females ; RRfemales = 61 ) when comparing AN chewers with non-AN chewers [Hazare et al. showed that the RR of OSF increased significantly with increased frequency of chewing AN; the consumption of AN at any frequency increased the risk of OSF development by approximately 50 times  . These rp = 0.058)) [p = 0.0114); ORfemales = 1.29 ) [p = 0.078)) [Variations of these findings have also been reported. Ariyawardena et al. reported that chewing AN alone had a higher but not significant risk of developing OSF ) . By contPan masala consists of a combination of unripe AN, catechu, and slaked lime, with the potential for the addition of flavors and spices . NotablyMehrotra et al. reported that the OR of OSF with pan masala use was 3.01 (95%CI: 1.23–7.36) , whereasGutkha is similar to pan masala; it has the same ingredients but with the addition of powdered tobacco and the use of ripe AN . The thrp-value < 0.001 [Khan et al. reported that OSF associated with smokeless tobacco was most prevalent and that gutkha was the most commonly used smokeless product (26.3%). Khan et al. reported an odds ratio of 17.704 (95% CI: 4.85–64.58) for developing OSF with a  < 0.001 . These f < 0.001 . In addi < 0.001 . Taken together, the data indicate that the use of a tobacco-containing smokeless product is associated with an increased likelihood of developing OSF. p-value of 0.62 [p-value < 0.05 [Ariyawardana et al. analyzed the BQ mixtures with BL and AN, and identified an OR of 3.08 (95% CI: 0.31–30.36) for developing OSF with a  of 0.62 . Additioe < 0.05 .For BQ mixtures without tobacco, Ariyawardana et al. reported an OR of 1 95%CI: 0.24–4.16), indicating no statistically significant difference between the control group and BQ mixture (BL + AN + slaked lime) group ; by cont–4.16, inFor the unspecified BQ mixtures, 13 datasets of OR/RRs were available and the majority of studies were mainly undertaken in India and Taiwan. Although the mixtures were unspecified, it is important to note that most BQ mixtures from India contain tobacco whilst BQ mixtures from Taiwan mainly consist of AN, slaked lime, and piper BI with or without BL. Among the 13 datasets, 1 set reported a weak association between unspecified BQ and OSF , 4 sets p > 0.05); this could be due to the smaller sample size in male stem quid chewers (n = 13). Stem quid is a variant of quid in Taiwan and is a combination of unripe AN, BS, and slaked lime . Yang etp > 0.05); there was no OR value computed for males in this study, possibly due to the insufficient sample size [Lao-hwa is another variant of BQ that is used in Taiwan. It has a similar composition to BQ except that it uses BI instead of BS . Yang etple size .The aim of this scoping review was to examine whether there was an association between different BQ ingredients and the development of OSF. Tobacco has been confirmed to be associated with a significantly increased risk of OSF development. Pan masala, which does not contain BL, BI, BS, or lime had the second highest RR, and AN alone was reported to have the highest RR for development of OSF. The results also suggest that the use of BI is associated with higher risk of developing OSF, whereas BL might reduce the risk when added to AN. The addition of BL, BI, BS, and/or lime to AN would require more research to determine its mechanism and association with OSF development. The majority of studies in this report showed that BQ mixtures containing tobacco had increased risk for OSF compared to controls ,17,20,21Consumption of AN is the primary risk factor for development of OSF as this condition is rarely observed in non-AN chewers . AN, witn = 74 OSF patients); 4 cases and 4 controls for the BQ mixture with lime, and 3 cases and 1 control for the BQ mixture without lime. The addition of slaked lime to BQ mixtures or AN alone has been shown to cause microabrasions, and to facilitate the diffusion of potent chemicals and irritants into the oral mucosa . Slaked Piper betle leaf [Yang et al. showed that BQ mixtures containing BS (stem quid OR = 3.28 and 4.26) had a higher risk than mixtures containing BI (Lao-Hwa OR = 2.8) . Furthertle leaf , there atle leaf . We are tle leaf . The postle leaf . Whilst BQ increases the risk of developing OSF (OR = 17.65 and 7.01) more than stem quid (OR = 3.28 and 4.26) , little Genetic susceptibility is also an important factor to take into consideration whilst analyzing the effect of different BQ mixtures on the development of OSF. As only a relatively modest percentage of BQ chewers develop OSF, this suggests that there is a genetic component to disease pathogenesis that may predispose the individual to developing OSF . SuggestThere are various limitations in the studies that have been included in this report. In terms of internal validity, the Khan et al. report is a retrospective cohort study and may have been subjected to a selection bias of controls, a recall bias, and a misclassification or information bias . Maher eApart from the various combinations of ingredients that can be chewed in BQ, the preparation of AN itself can also contribute to the pathogenicity of the quid. For example, it has been shown that the polyphenols content in AN decreases as the nut matures , that roWe acknowledge that there may also be several limitations to the external validity of the present report. For example, cigarette smoking and alcohol drinking are independent risk factors for OSF , and thePiper betle) leaf, when added to the chewing mixture, may be protective. Furthermore, pan masala and AN alone are associated with significant risk of OSF development. Despite its known potential to cause OSF and other detrimental health effects, BQ and AN chewing is common in the Asia-Pacific region. AN, like tobacco, is addictive , and BQ Unlike tobacco, for which the WHO Framework Convention on Tobacco Control provides evidence-based policies for reducing tobacco use, no global policy exists for the control of BQ and AN use . SimilarIn summary, our scoping review shed light on the heterogeneity of BQ, and identified mixtures and ingredients that are associated with higher or lower risk of developing OSF. Although this was a pilot study, there is clear scope and indications for further research related to the constituents of BQ and OSF risk. To date, there is little, if any work on the possible protective effects of BQ ingredients, and this study’s results could provide an alternative route for reducing the incidence of OSF and OSCC in BQ users.