Continuous treatment with azithromycin may lead to fewer acute exacerbations of chronic obstructive pulmonary disease (AECOPD), but little is known of its impact on systemic and functional outcomes in real-life settings.This was a multicenter prospective observational study of patients with severe COPD who started treatment with azithromycin. Tests were compared at baseline and after 3 and 12 months of treatment. These included lung function tests, a 6-min walking test (6MWT), and enzyme-linked immunosorbent assays of serum and sputum markers, such as interleukins , tumor necrosis factor receptor 2 (TNFR2), and inflammatory markers. Incidence rate ratios (IRR) and their 95% confidence intervals (95% CI) are reported.Of the 478 eligible patients, the 42 who started azithromycin experienced reductions in AECOPDs  and hospitalizations . Treatment was also associated with significant improvement in the partial arterial pressure of oxygen  at 12 months. While TNFR2 was reduced significantly in both serum and sputum samples, IL-13 and IL-6 were only significantly reduced in serum samples. Moreover, an elevated serum and sputum IL-8 level significantly predicted good clinical response to treatment.Continuous azithromycin treatment in a cohort of patients with severe COPD and frequent exacerbations can significantly reduce the number and severity of exacerbations and improve gas exchange. Treatment changes the pattern of microorganism isolates and decreases the inflammatory response. Of note, IL-8 may have utility as a predictor of clinical response to azithromycin treatment. Acute exacerbations of COPD (AECOPD) affect not only the natural history of the disease but also quality of life, the number of hospitalizations, and mortality rates . ExacerbRandomized controlled trials have demonstrated the efficacy of prophylactic azithromycin in reducing AECOPDs, and as such, current clinical guidelines recommend its use in patients with frequent exacerbations despite optimal inhaled therapy –7. HowevAntibiotic prophylaxis in AECOPDs may increase the risk of selection for resistant bacteria, facilitating the spread of antimicrobial resistance. Even though azithromycin is usually well-tolerated drug, undesirable side effects can still result from polypharmacy, advanced age, and comorbidities in real-world settings. Moreover, the therapeutic response can vary depending on the characteristics and baseline statuses of patients. Investigating what factors predict a favorable response, both in the number of exacerbations and their functional effects, is of particular clinical interest.This study aimed to analyze the systemic and pulmonary functional changes associated with severe COPD in patients with frequent exacerbations after continuous azithromycin treatment, their effect on microbiological isolates in residual exacerbations, and the potential involvement of inflammatory and immunomodulatory mechanisms.2.2.1.This was a multicenter, observational, and prospective study of patients with severe COPD and frequent exacerbations who regularly attended comprehensive care programs in the respiratory day hospitals of three university hospitals in Barcelona. The participating hospitals were Hospital Universitari de Bellvitge (HUB), Hospital Parc Taulí (HPT), and Hospital Germans Trias i Pujol (HGTiP). All patients who started treatment with azithromycin according to clinical guideline recommendations and practice  were inc2.2.COPD was defined according to the GOLD recommendations: a history of smoking (>10 pack-years) and an FEV1  to FVC  ratio < 70% . By clinAll patients signed an informed consent form in accordance with the principles outlined in the Declaration of Helsinki, and the local ethics committee approved the study .2.3.All included patients started treatment with azithromycin 250 mg three times per week for 1 year. Sociodemographic data, anthropometrics, COPD history, and symptom evaluations were collected in all patients. Symptom tests included the COPD Assessment Test (CAT), London Chest Activity of Daily Living Scale (LCADL) , and the2.4.g, and the supernatant was taken for analysis.Serum samples were obtained from venous blood. After allowing the sample to stand at room temperature for 2 h, it was centrifuged for 15 min at 1000 × g for 15 min. The supernatant was stored at −80°C for determinations.Sputum was separated from contaminating saliva by macroscopic examination, before being weighed and homogenized with 4 volumes of dithiothritol . After 15 min at room temperature, the same volume of phosphate-buffered saline was added and the whole mixture was filtered and centrifuged at 600 × Analysis was performed by enzyme-linked immunosorbent assay (ELISA). Cytokine concentrations  were measured in serum samples and supernatant sputum samples using a multiplex immune-bead assay . TNR2 was analyzed with a Human soluble TNF Receptor 2 ELISA Kit . All procedures were performed according to the manufacturer’s instructions, centralized in one of the centers.2.5.2 atmosphere (blood and chocolate agar) or ambient air atmosphere (MacConkey agar). Bacterial colonies were sub-cultured for identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry .Sputum samples were obtained spontaneously at visits and during AECOPD episodes before starting any antimicrobial treatment. Only good quality sputum samples were considered  following the microbiological routine . Briefly2.6.t (parametric), or Mann–Whitney (nonparametric) tests, applying the Bonferroni method if the Kruskal–Wallis test found significant differences. A negative binomial regression model was used to study the number of exacerbations after treatment, reporting the incidence rate ratio (IRR) and 95% confidence interval (95%CI). Different predictive statistical models were fitted for each analytical or clinical variable of interest, adjusted by age and Charlson index. A value of p of 0.05 was considered statistically significant. IBM SPSS version 22  was used for all analyzes.Data are expressed as means ± standard deviation (SD) or medians [25th–75th percentile] for continuous data and as frequencies (percentage) for categorical data. Multiple comparisons were evaluated by chi-squared , student 3.3.1.2-agonists (LABAs) and long-acting muscarinic antagonists (LAMAs), 39 (93%) inhaled corticosteroids (ICS) and nine (21%) long-term oxygen therapy. Participants corresponded to group E of the current GOLD classification (1 44.5% ± 13.8%), an average mMRC dyspnea symptom score of 2, and a median of 5 (4–6) exacerbations during the previous year , diarrhea (n = 1), and personal preference (n = 1).Of the 478 eligible patients with severe COPD in three respiratory day hospitals, we included 42 (8.8%) who started azithromycin (ous year . Patient3.2.2) was observed after treatment with azithromycin at 3 and 12 months compared with the baseline visit . This was also associated with a significant improvement in oxygen saturation both at rest and during exercise. However, symptoms (except for a slight improvement in CAT and LCADL scores), the 6MWT distance, and lung function did not differ significantly after 12 months  in the year before starting azithromycin compared with only 65 during treatment  and Moraxella catarrhalis (p = 0.0031) . Microorganisms otherwise increased in the upper respiratory tract, and sputum isolates of Pseudomonas aeruginosa and Streptococcus pneumoniae did not change.A total of 95 microorganisms were obtained during the 188 AECOPD in the year before starting treatment with azithromycin, which decreased to only 41 microorganisms during 65 AECOPD throughout the follow-up period. There was a significant reduction in frequent sputum isolates, such as  0.0031) , and a sM. catarrhalis decreased from 26.6 to 2.8% (p = 0.0026) and S. maltophilia increased from 0 to 11.1% (p = 0.0026), with a reset observed in the usual upper respiratory tract bacterial microbiota from 11.4 to 47.2% (p < 0.0001). In severe AECOPD, P. aeruginosa isolates increased from 1 to 3 (p = 0.0075) and S. pneumoniae isolates increased from 0 to 2 (p = 0.0078) after azithromycin therapy.Overall, the pattern of microbiological isolates changed with the intensity of AECOPD from before to after azithromycin therapy . In mode4.This multicenter, prospective, observational study analyzed the systemic and functional real-life effects of continuous treatment with azithromycin in patients with severe COPD and frequent exacerbations managed in respiratory day hospitals. In addition to confirming that azithromycin effectively reduced the rate of moderate to severe COPD exacerbations, we provide the first evidence that treatment also improves gas exchange, both at rest and during exercise, after 1 year of treatment. Of the interleukin profiles analyzed in this study, IL-8 and TNFR2 presented as the major inflammatory mediators in the immunomodulatory response to azithromycin in patients with COPD. Baseline elevations of IL-8 in both blood and sputum samples predicted the therapeutic response and may indicate its potential utility as a biomarker. Changes were also observed in the microbial composition of subsequent AECOPD episodes after treatment, with the identification of potentially pathogenic microorganisms with unusual patterns of intrinsic antibiotic resistance that required treatment. This increases our knowledge about how azithromycin treatment affects the inflammatory response and bacteria selection in the airways, together with the implications for future COPD exacerbations.As expected, continuous azithromycin treatment in patients with severe COPD and frequent exacerbations led to fewer exacerbations up to 66%) after 1 year of treatment, even using a dosage of 250 mg 3 times a week. From the limited published evidence available  and more potentially pathogenic microorganisms with unusual patterns of intrinsic antibiotic resistance that required treatment . This has clear implications when selecting empirical antibiotics for residual AECOPDs in patients receiving continuous azithromycin. Viral infections usually act as the trigger of the exacerbation, but no routine study of viral infections was performed in our study. Further research is therefore required.Despite the significant reduction in exacerbations under continuous azithromycin treatment, some patients still had further AECOPD episodes. Although these patients had fewer hospitalizations, they required more days in hospital to treat severe AECOPD. They also showed changes in the patterns of microbiological isolates in sputum samples after continuous azithromycin treatment, with fewer frequent , who clinical trials might miss, and to study the role of azithromycin on inflammation and bacterial isolates in these cases.In conclusion, continuous azithromycin treatment for patients with severe COPD, comorbidities, and frequent exacerbations not only significantly reduces exacerbation and severity rates after 1 year of treatment but also improves gas exchange. Changes are also seen in microorganism patterns and inflammatory responses after treatment. Of note, high serum and sputum IL-8 levels at baseline also appear to predict a good clinical response to azithromycin and may indicate its potential utility as a biomarker. If proven, IL-8 as could be used to improve patient selection and reduce microbial resistance in clinical practice.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by local ethics committee . The patients/participants provided their written informed consent to participate in this study.EC participated in patient’s inclusion, statistical analysis, figure preparation, data interpretation, and manuscript drafting. DH participated in the study design, patient’s inclusion, statistical analysis, data interpretation, and obtaining funding. CM and AM participated in the study design, patient’s inclusion, and data interpretation. AC-S participated in sputum analysis, figure and table preparation, and data interpretation. XP participated patient’s inclusion and data interpretation. MG-N participated in sputum analysis and ELISA experiments. SM participated in sputum analysis, data interpretation, and critical revision of the manuscript. SS contributed to the study concept and design, patient inclusion, funding, study supervision, data interpretation, manuscript drafting, and critical revision of the manuscript for important intellectual content. All authors contributed to the article and approved the submitted version.The research was funded by the Instituto de Salud Carlos III (grant PI20/00777 and PI16/00977), co-funded by the European Regional Development Fund (ERDF) “A Way to Build Europe” and grants from Menarini to SS and from SEPAR 088/2016, SEPAR 1116/2020 and FUCAP-2017.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.


Lower-extremity endovascular revascularization (LER) is often required for diabetic patients with chronic limb threatening ischemia (CLTI). During the post-revascularization period patients may unpredictably experience major adverse cardiac events (MACE) and major adverse limb events (MALE). Several families of cytokines are involved in the inflammatory process that underlies the progression of atherosclerosis. According to current evidence, we have identified a panel of possible biomarkers related with the risk of developing MACE and MALE after LER. The aim was to study the relationship between a panel of biomarkers - Interleukin-1 (IL-1) and 6 (IL-6), C-Reactive Protein (CRP), Tumor Necrosis Factor-α (TNF-α), High-Mobility Group Box-1 (HMGB-1), Osteoprotegerin (OPG), Sortilin and Omentin-1- at baseline, with cardiovascular outcomes (MACE and MALE) after LER in diabetic patients with CLTI.In this prospective non-randomized study, 264 diabetic patients with CLTI undergoing endovascular revascularization were enrolled. Serum levels of each biomarker were collected before revascularization and outcomes’ incidence was evaluated after 1, 3, 6 and 12 months.During the follow-up period, 42 cases of MACE and 81 cases of MALE occurred. There was a linear association for each biomarker at baseline and incident MACE and MALE, except Omentin-1 levels that were inversely related to the presence of MACE or MALE. After adjusting for traditional cardiovascular risk factors, the association between each biomarker baseline level and outcomes remained significant in multivariable analysis. Receiver operating characteristics (ROC) models were constructed using traditional clinical and laboratory risk factors and the inclusion of biomarkers significantly improved the prediction of incident events.Elevated IL-1, IL-6, CRP, TNF-α, HMGB-1, OPG and Sortilin levels and low Omentin-1 levels at baseline correlate with worse vascular outcomes in diabetic patients with CLTI undergoing LER. Assessment of the inflammatory state with this panel of biomarkers may support physicians to identify a subset of patients more susceptible to the procedure failure and to develop cardiovascular adverse events after LER.The online version contains supplementary material available at 10.1186/s12933-023-01872-x. Peripheral artery disease (PAD) is a frequent complication of type 2 diabetes mellitus (T2DM) . One of Scientific evidence has shown that an inflammatory process underlies the progression of atherosclerosis –12. SeveThe aim of this study is to assess the relationship between the baseline levels of these biomarkers and the outcomes after LER, in particular MACE and MALE, in diabetic patients with CLTI undergoing revascularization.Fondazione Policlinico Universitario A. Gemelli Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) and adhered to the principles of the Declaration of Helsinki. All the individuals agreed to participate in the study and gave informed consent. This clinical protocol was designed as a prospective non-randomized study to verify the relationship between the serum levels of a panel of biomarkers  and the incidence of MACE and MALE after LER performed in diabetic patients with CLTI.This study was approved by the Ethics Committee of the Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy, from December 1, 2019 to January 31, 2022. At the recruitment visit, clinical, laboratory, ultrasound and radiological data of the patients were collected. The diagnosis of CLTI of at least one limb, category 4, 5 or 6 of PAD in accordance with the Rutherford classification [We evaluated consecutive patients with diabetes and CLTI admitted to the Department of Cardiovascular Internal Medicine at fication , and thefication . ExclusiAll patients were taking lipid-lowering therapy to achieve a low-density lipoprotein cholesterol (LDL-C) target of less than 55 mg/dL.At the time of enrollment all patients were on a single antiplatelet drug, and after LER they took dual antiplatelet therapy (aspirin and clopidogrel) for 1 month.Balloon angioplasty and, if indicated, arterial stenting were performed according to standard techniques . The proFor the follow-up, patients were evaluated 1, 3, 6 and 12 months after the LER to assess incidence of MACE and MALE. MACE was defined as composite of myocardial infarction, stroke and cardiovascular death . MALE waBlood sampling of patients was performed at baseline, before LER, after an overnight fast. Fasting glucose (FBG), serum creatinine, total cholesterol, LDL-C, triglycerides, and glycated hemoglobin (HbA1c), were determined. Renal function was calculated using eGFR, according to the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula . Serum wBased on the previous data, we assumed an effect size of 0.20. To have a power (1 – β) of 90% and considering an α error of 5% (0.05), a total of at least 255 patients were required. Data were summarized as means  for continuous variables and counts (percentages) for categorical variables. Demographic and clinical data of the population were compared using chi-square and t-test. We assessed the association between each biomarker and the risk of developing MALE and MACE by means of logistic regression models in which the outcome of interest was entered as the dependent variable and each biomarker was entered as a continuous independent variable. We assessed whether knowledge of the biomarker values would lead to improved prognostic prediction regarding the outcomes of interest by constructing receiver operating characteristic (ROC) curves for a model including only traditional risk factors  and for a model including all the aforementioned risk factors plus all biomarkers as continuous variables. We then compared the areas under the ROC curves using the roccomp function in Stata software; this function tests the equality of two ROC areas obtained by applying two or more test modalities to the same sample or to independent samples. All statistical analyses were performed separately for each of the two outcomes of interest.Statistical significance was established at p < 0.05. All analyses were performed with Stata software version 17.0 for MacOS  and SPSS version 25.0 for MacOS .A population of 264 diabetic patients with CLTI was enrolled and followed up for the entire duration of the study. The mean age of patients was 73.4 ± 8.8 years. Of these, 171 (64.8%) were males. The median duration of T2DM was 16.2 ± 13.7 years. The mean BMI was 26.4 ± 5.0 kg/m2. Comprehensively, we included 63 (23.8%) smokers and 120 (45.5%) former smokers. Among all patients, 204 (77.3%) had arterial hypertension, 243 (92.0%) had hypercholesterolemia, 117 (44.3%) had history of CAD and 99 (37.5%) patients had history of CVD. LDL-C (71.4 ± 28.5 mg/dL) and HbA1c (7.1 ± 1.2%) were only slightly above target according to guidelines in force at the time of recruitment . Taking 2vs. 25.8 ± 4.4 Kg/m2, p < 0.01), already history of CAD  and of hypercholesterolemia , higher creatinine serum levels , compared to patients without MACE. There were no differences between patients with MACE and without MACE in LDL-C and HbA1c levels. Complete clinical data from patients with MACE and without MACE are shown in Supplementary Table 1. Considering baseline biomarkers levels, patients with MACE had higher baseline serum levels, except Omentin-1 levels that were lower in patients with MACE compared with patients without MACE  patients experienced MACE after LER. Patients with MACE had diabetes for longer duration , higher BMI  Table .Supplementary Table 2. Considering baseline biomarkers levels, patients with MALE had higher baseline serum levels, except Omentin-1 levels that were lower in patients with MALE compared with patients without MALE  patients experienced MALE after LER. Patients with MALE were younger (p < 0.01), had more history of hypercholesterolemia (p < 0.01), of CAD (p < 0.01) and of CVD (p = 0.02), and had lower HDL-C levels than patients who did not have MALE (p < 0.01). Furthermore, Rutherford category 4 was more represented in patients undergoing MALE (p = 0.01). There were no differences between patients with MALE and without MALE in terms of diabetes duration (p = 0.67), BMI (p = 0.14), history of high blood pressure (p = 0.41), glycated hemoglobin levels (p = 0.13), LDL-C levels (p = 0.20), Rutherford category 5 (p = 0.97) and category 6 (p = 0.44), and creatinine (p = 0.59). Complete clinical data from patients with MALE and without MALE are shown in The multivariate logistic regression analysis showed that, after adjustments for the cardiovascular risk factors, age, Rutherford stages II-4 and III-6, HbA1c, total cholesterol, CRP, OPG, HMGB-1, and Omentin-1 were independent determinant for MALE after LER in diabetic patients with CLTI (p < 0.01) Table .The baseline ROC model included age, sex, BMI, high blood pressure, diabetes duration, smoking status, Rutherford staging, previous cardiovascular and cerebrovascular events, treatment, total cholesterol, LDL-C, triglycerides, FBG, and HbA1c. The comparison of ROC curves between the baseline model with only clinical and laboratory risk factors and the model including our panel of biomarkers is reported in Fig. Diabetic patients with CLTI undergoing LER have a very high risk of MACE and MALE in the 12-month period following treatment . Best meIt is well established that inflammation plays a key role in the complications of diabetes mellitus . SeveralIn recent years, the CANTOS study raised the attention on the role of IL-1 in cardiovascular diseases. Researchers have shown that the intake of canakinumab, a monoclonal antibody directed against IL-1, that is used in clinical practice for treatment of some rheumatological diseases, significantly reduced the risk of recurrence of cardiovascular events in patients with history of myocardial infarction and persistently high CRP levels, confirming the close link between inflammation and atherosclerosis . To our The multivariate analysis for MACE showed that, after adjusting for the factors of conventional cardiovascular risk and all biomarkers in analysis, IL-6, OPG e CRP levels are independently associated with the outcome. The ROC curve confirmed their high predictive power. These results confirm the association between IL-6, OPG, CRP and cardiovascular outcomes that already emerged in one of our previous study . MultivaIn the past years, the little of awareness in PAD has led to scarce tools for the early diagnosis, progression and prognosis evaluation, resulting in inadequate therapeutic interventions. Non-invasive circulating serum biomarkers may be predictive effective tools in this setting. Several studies have evaluated the possible role of different molecules in the diagnosis and prognosis of PAD . These sThere is a scarcity of scientific research on predictive models for MACE and MALE after LER in diabetic patients with CLTI. In a retrospective study, Stone et al. found that, after LER, elevated pre-procedural CRP levels were associated with MALE, and elevated levels of CRP and brain natriuretic peptide (BNP) were associated with late cardiovascular events . Berger To our knowledge, the present study is the first to demonstrate that a panel of circulating biomarkers easily measurable could predict the incidence of MACE and MALE over 90% in diabetic patients with CLTI after LER.A limitation of this single-center study is the small number of patients included. However, this is a pilot study conducted on a carefully selected population with restrictive characteristics in order to select a population as homogeneous as possible. Based on the limited sample size analyzed, it was not possible to perform a comprehensive analysis of each individual biomarker in relation to individual conventional risk factors. A further limitation of the study is the lack of a formal protocol to rule out possible bone infections (for example using standard radiographic examinations or computed tomography or magnetic resonance imaging of bone), although patients with obvious signs of infections were excluded from recruitment. The aim of this research was to find easily accessible and reproducible biomarkers for the typical diabetic patient with CLTI. The study also did not clarify the effect that the revascularization procedure could have on the levels of analyzed biomarkers. Therefore, it might be helpful to monitor cytokine levels after the procedure and in the follow-up period in order to evaluate the effect of the variations of these on MACE and MALE occurrence. In addition, more conclusive evidence about the real causal relationship between the studied biomarkers and the outcomes after LER in diabetic patients will only be found after clinical multi-centric studies, on larger cohorts and longer follow-up periods. Finally, our data doesn’t offer results that can immediately change the clinical management of patients. However, this type of research is essential to fill the gap that the classic biomarkers  cannot resolve, regarding the chance of predicting the development of MACE and MALE. It is well-established that diabetic patients with PAD and similar risk factors can experience unpredictable vascular outcomes, indicating the presence of unknown or uncontrollable, at least at the moment, risk factors. CurrentOur study showed that elevated levels of IL-1, IL-6, TNF-α, PCR, HMGB-1, OPG and Sortilin and reduced Omentin-1 levels correlate with worse vascular outcomes in CLTI diabetic patients after LER. Hence, they hold significant potential as biomarkers, given their ease of measurement and reproducibility. These parameters can reliably predict the likelihood of complications that a treated patient may encounter. A standardized score that combines conventional cardiovascular risk factors and these biomarkers could enable clinicians to assess patients’ risk profile, identify patients most susceptible to unfavorable outcomes and choose the most appropriate and personalized therapeutic and follow-up approach. While these findings require validation on a larger patient cohort, they indicate progress in the management of this complex and disabling condition.Below is the link to the electronic supplementary material.Supplementary Material 1


Quercus petraea in south-eastern Europe is limited despite the species’ significant role in the re-colonisation of Europe during the Holocene, and the diverse climate and physical geography of the region. Therefore, it is imperative to conduct research on adaptation in sessile oak to better understand its ecological significance in the region. While large sets of SNPs have been developed for the species, there is a continued need for smaller sets of SNPs that are highly informative about the possible adaptation to this varied landscape. By using double digest restriction site associated DNA sequencing data from our previous study, we mapped RAD-seq loci to the Quercus robur reference genome and identified a set of SNPs putatively related to drought stress-response. A total of 179 individuals from eighteen natural populations at sites covering heterogeneous climatic conditions in the southeastern natural distribution range of Q. petraea were genotyped. The detected highly polymorphic variant sites revealed three genetic clusters with a generally low level of genetic differentiation and balanced diversity among them but showed a north–southeast gradient. Selection tests showed nine outlier SNPs positioned in different functional regions. Genotype-environment association analysis of these markers yielded a total of 53 significant associations, explaining 2.4–16.6% of the total genetic variation. Our work exemplifies that adaptation to drought may be under natural selection in the examined Q. petraea populations.Knowledge on the genetic composition of  Forest ecosystems are undergoing unprecedented changes in environmental conditions due to global change impacts (González de Andrés https://efi.int/knowledge/models/efiscen). Among seven groups of tree species, Quercus petraea (Matt.) Liebl. was ranked fourth in terms of economic importance, being of considerable productivity  to explore the number and distribution of loci affecting the variations in the adaptively important traits by FST outlier detection and associations of SNP allele frequencies with environmental variation; and (iv) by annotating these loci, to describe the biological process, molecular function and cellular component, assuming the same function in both the resulted, already annotated model species, and Q. petraea.In this study, we identified and investigated the genetic variation at several stress-response loci in natural sessile oak populations from the Central-Eastern European region. The objectives of the study were (i) to reveal the genetic structure, diversity and differentiation of the sampled populations; (ii) to identify stress-response loci in our RAD sequencing dataset after mapping paired-end reads to the Q. petraea, extending to Bulgaria, Hungary, Romania, Serbia, Bosnia and Herzegovina, Kosovo and Albania  of nia Fig. . Altogetnia Fig. . Samplinnia Fig. . Detailenia Fig. .A variation of RAD sequencing, that is suitable for high-confidence genome-wide SNP loci discovery, double-digest RAD-seq (ddRAD-seq), was used to genotype all individuals  Unix-based HPC server, allocating 40 cores (80 threads) and 38 GB of RAM. Key steps of the bioinformatics pipeline are shown in Fig. Short read sequences (~77 M) were demultiplexed and adaptor-trimmed by using MiSeq Control Software . The resulting sequences  were 3′ and 5′ end-trimmed using the FastQ Toolkit. Reads with a mean quality score <30 and shorter than 200 bp were filtered. Computational processing of short-read data was carried out with Stacks 2.0  were filtered out  ranging from K = 2 to 9. The Python script ‘ChooseK’, included with the fastStructure package, was used to choose the number of groups that maximize the log-marginal likelihood lower bound , observed heterozygosity (Ho), and the inbreeding coefficient (FIS) were calculated for each population and for each genetic cluster in R using the ‘adegenet’ package  (Nei FST distances using the ‘ggdendro’ package in R (de Vries and Ripley Expected heterozygosity (He Goudet  using 10e Goudet . Privatee Goudet . The divFST) Nei  by creatFST) Nei . In addiFST frequency or Bayesian based) can outperform single tests in the detection of loci under directional selection, we applied distinct approaches to see if they converged and detected the same outliers , the ‘LeftTrimFraction’ was set to 0.08, the ‘RightTrimFraction’ to 0.30, and the Hmin parameter was left at the default setting (0.1). The false discovery rate threshold for calculating q-values first was 0.05, as set by default , as an example presented in Table The loci containing the significant SNPs was extracted from the initial dataset and annotated using the NCBI’s BLASTN and BLASTX services (To test for associations of loci (SNPs) with environmental variables , five different regression approaches were applied.r2 value below 0.75 and with a VIF < 10, thus were subsequently used for the association analyses. Selected environmental variables are detailed in Table First, an environmental dataset of 84 monthly, seasonal, and annual variables was created by extracting climate data from WorldClim 1.4  implemented in the ‘gdm’ package  while maintaining a relatively low false detection rate , similarly to Kwon et al. . The GLMQ. robur reference genome with an average success rate of 92.43%  for the SNP data. At this LLBO, we identified three main genetic clusters  and few introduced individuals as a separate group  and among the genetic clusters (FST ranging from 0.022–0.083). Cluster 3 showed the highest differentiation compared to Clusters 1 and 2 (FST = 0.065 and 0.083), while differentiation between Clusters 1 and 2 was much lower (FST = 0.022)  Fig. . The UPG22) Fig. .e = 0.181–0.223; Ho = 0.166–0.210), as detailed in Table e = 0.181; Ho = 0.166; Ar = 1.570). Significant inbreeding depression was not detected (p < 0.05). Private alleles were absent, and only HU3 had a unique allele. Diversity among genetic clusters was similar; only Cluster 3, which contained only the BU4 population, showed slightly lower diversity values  located at different loci, and at six different chromosomes  were considered robust outliers  served as a covariate, and last MLM where the average relationship was estimated by kinship (Q + k). Table By using the nine robust SNPs, a total of 102 significant marker-environment associations were detected, 21 with GDM, 48 with the LFMM, 9 with SFA and 14–10 with the GLM and MLM. However, similar to outlier analysis, we considered only those marker-environment associations that were detected with at least two different methods. Among the 53 jointly detected associations, the significance levels were different and ranged from 0.05 to 0.001.The GDM method found relationships only for four SNPs . In these relationships, PETCQ, prec6_16, bio3_16 were the most frequent, and presented the highest relative importance , prec6_16 (0.167 R2) and aridity (0.189 R2), and explaining 16.6–21.9% of variance . The highest |z | -score values  were found for three SNPs, namely for 227780_278, 277051_56 and 361438_297, in association with PETCQ (6.036 and 6.289), prec6_16 (6.022) and aridity (6.414). The single-factor analysis (SFA) was similar to LFMM, where the LFMM |z | -scores values were high, the SFA adjusted p values ranging from 0.05 to 0.001), however, the same associations detected by MLM, were not significant with FDR and Bonferroni-adjusted significance thresholds. The highest R2 values were detected for 50083_64 and 238771_249 in association with mCBTemp10 (both 0.150 R2). MLM, similarly to LFMM and SFA, revealed the highest significant association between 277051_56 and PETCQ (0.045 R2).The GLM and MLM detected much less numbers of associations (14 and 10), also the significance thresholds were largely different. The GLM detected associations with significant FDR and Bonferroni-adjusted thresholds (R2). SNP 50083_64, 180975_104 and 227780_278 was explained chiefly by PETCQ , 96506_153, 96534_88, 361438_297 and 437832_228 by aridity , 238771_249 by mCBTemp10 (avg. R2adj = 8.2%), 277051_56 by bio8_18 (avg. R2adj = 11.2%). By summarizing the environmental predictors, in decreasing order, aridity (81.7%), PETCQ (79.9%), bio8_16 (51.9%) and prec6_16 (49.7%) explained the highest amount of the genetic variation, thus considered to be the most important  , and this population is located at the eastern edge of the Black Sea refugium and in the eastern part of the current European distribution range of the species. Additionally, individuals of this region are considered to be a distinct subspecies in the Euro+Med Plantbase (http://ww2.bgbm.org/EuroPlusMed/query.asp) and on The Plant List (http://www.theplantlist.org), Q. petraea subsp. iberica (Steven ex M. Bieb.) Krassiln., and Schwarz   selection. Those SNPs that were presented in NCBI databases resided in four different functional regions. Two SNPs found in a gene encoding the uncharacterized protein 115976593 (NCBI ID), one in bHLH162 transcription factor, one in hydroxymethylglutaryl-CoA synthase coding region, and one in a hypothetical protein coding region (possible double-strand damage repair). No biological function can be associated to uncharacterized proteins, however, bHLH transcription factor is related to NPF genes  showed signatures of selection according to differentiation outlier analyses. The observed proportion was lower than obtained in other studies on temperate broadleaf forest trees (Derory et al. p ≤ 0.05 to 0.001. The variations seen in the analyses are due to differences in the methods used to account for demographic history or neutral genetic structure, which results in different statistical strengths for different sampling schemes or complicated demographic situations (De Mita et al. Caution must be exercised when interpreting our results, taking into account the potential for false positive selection signatures (Excoffier et al. FST outlier and environmental association analyses, which are only correlative measures (Ahrens et al. The above list of significant marker-environment associations clearly shows, that most variables are linked to aridity. To validate functional mechanisms underlying selection signatures, it is generally recommended to use annotations, which can provide additional support beyond relying solely on While sessile oak populations are well studied in Europe, in the Central-Eastern European region, including the Balkan Peninsula it is seldom tested with genome-wide genetic markers. The identification of genetic variants within populations of this region may hold the key to facilitating the sessile oak’s adaptation to the deleterious effects of modern climate change, thus underscoring the crucial significance of further investigation in this field. Through our examination, we found molecular evidence indicating that important climate-related factors, such as drought, may have influenced the adaptive divergence of sessile oak populations in the Central-Eastern European region. Moreover, our results suggest that these populations may contain beneficial genetic variants that could aid the species in responding to the rapidly changing climate. In light of these findings, it is vital that European forest tree species conservation and management programmes incorporate the Central-Eastern European sessile oak populations, including facilitated gene flow, into their strategies to combat the significant threat that climate change poses to forest tree populations.Supplementary Material


This study aims to conduct a meta-analysis to assess the effect of virtual reality-based therapy (VRBT) on balance dimensions and fear of falling in patients with multiple sclerosis (PwMS). Secondarily, to determine the most recommendable dose of VRBT to improve balance.PubMed Medline, Web of Science, Scopus, CINAHL and PEDro were screened, without publication date restrictions, until September 30th, 2021. Randomized controlled trials (RCTs) comparing the effectiveness of VRBT against other interventions in PwMS were included. Functional and dynamic balance, confidence of balance, postural control in posturography, fear of falling and gait speed were the variables assessed. A meta-analysis was performed by pooling the Cohen's standardized mean difference (SMD) with 95% confidence interval (95% CI) using Comprehensive Meta-Analysis 3.0.p < 0.001); dynamic balance ; postural control with posturography ; confidence of balance ; and in reducing fear of falling ; but not on gait speed . Besides, the most adequate dose of VRBT to achieve the greatest improvement in functional balance was at least 40 sessions, five sessions per week and 40–45 min per sessions; and for dynamic balance, it would be between 8 and 19 weeks, twice a week and 20–30 min per session.Nineteen RCTs, reporting 858 PwMS, were included. Our findings reported that VRBT is effective in improving functional balance (SMD = 0.8; 95%CI 0.47 to 1.14; VRBT may have a short-term beneficial role in improving balance and reducing fear of falling in PwMS.The online version contains supplementary material available at 10.1186/s12984-023-01174-z. Multiple sclerosis (MS) is an immune-mediated, inflammatory and neurodegenerative chronic disease that causes demyelination and axonal degeneration in the Central Nervous System (CNS) . MS has In the field of neurorehabilitation, along with pharmacological and conventional therapy (CT), virtual reality-based therapy (VRBT) is being employed in the last decade to reduce the impact of disabling sequelae and to improve the quality of life of these patients  and otheIn recent years, several reviews have analyzed the effect of VRBT on balance and/or risk of falls –34. Howe. It was registered in the PROSPERO database (CRD42021256768).A systematic review with meta-analysis was performed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 statement . It was A search was performed, independently, by two authors (ICP and FANE), in PubMed Medline, Web of Science (WOS), Scopus, CINAHL Complete and PEDro (Physiotherapy Evidence Database) and through the references of the retrieved records , independently, screened the titles and abstracts of all the retrieved studies for further examination. Disagreements were resolved by a third author (FANE).The following inclusion criteria were used: (1) RCTs or pilot RCTs with at least two groups; (2) assessing the effect of VRBT on the outcomes of interest (see outcomes section) in comparison to others controls; (3) in PwMS; (4) and studies that provided quantitative data about the outcomes to perform the meta-analysis. The exclusion criteria were: (1) RCTs including patients with different neurological diseases apart from PwMS in the same group; (2) studies reporting statistical data which cannot be meta-analyzed with our software.For each study the following data were extracted: (1) overall study characteristics ; (2) number of groups; (3) sample characteristics for each group ; (4) characteristics of the VRBT intervention ; (5) type of therapy used as control; (6) quantitative results for each variable at the end of the intervention  ; and evaThe variables assessed in this systematic review were three: postural balance, fear of falling and gait speed. Considering that postural balance is a complex function integrated by some dimensions, we independently assessed the functional balance, dynamic balance, confidence of balance and postural control measured with posturography.Risk of bias and methodological quality of the included studies was assessed using the PEDro Scale. This scale is composed by 11 binary items (“yes” if the criterion is met or “no” when the criterion is not met) . The totThe GRADE  approach was employed to assess the level of quality evidence of findings in each meta-analysis, through the assessment of risk of bias, inconsistency, inaccuracy, indirectness and risk of publication bias . With thComprehensive Meta-Analysis version 3.0  [P < 0.1 there exists a risk of publication bias) [P for Q-test and the degree of inconsistency (I2) from Higgins [P < 0.1 and it can be categorized as low (I2 < 25%), moderate (I2 25–50%) or large (I2 > 50%) [Statistical analysis was performed by two authors by using NJ, USA) . A meta-NJ, USA)  with 95%NJ, USA) . SMD proNJ, USA) . In addiNJ, USA) . The risNJ, USA)  using thon bias) . In addion bias) . Accordion bias) . Finally Higgins . Heterog2 > 50%) , 51.In order to assess the contribution of each study to the global effect in each meta-analysis, a sensitivity analysis was performed using the leave-one-out method . In addin = 303), 260 studies were screened by title/abstract. One hundred and ninety-seven studies were excluded by title/abstract whereas 44 did not meet the inclusion criteria. Finally, 19 RCTs [Five hundred and sixty-three references were identified . After removing duplicate records   and dynaAll studies were included in the quantitative synthesis. Table p < 0.001) in favor of VRBT (Table p < 0.001) on BBS is observed in favor of VRBT compared to other controls. A possible risk of publication bias has been identified (Egger p = 0.9 and Trim-and-fill variation of 16%) . Sensitivity analysis did not show variations.Thirteen RCTs , 69, 71 BT Table ; Fig. 2Ap = 0.005) in favor of VRBT when compared with UC [p = 0.001) in favor of VRBT when compared with CT [p = 0.011) in favor of VRBT + RAGT vs RAGT [p < 0.001) was observed in PwMS with moderate disability  [p < 0.001) [p = 0.045) [For specific subgroups comparisons, the analyses of the BBS show: an increase of 3.92 points  , 71; bei< 0.001) , 59; and= 0.045) , 69 (Addp = 0.002) on dynamic balance in favor of VRBT (Table p = 0.24 and Trim-and-fill variation of 40%) . Sensitivity analysis showed a variation of 19% in the effect size with respect to the original SMD when the study of Kalron [p = 0.008).Data from thirteen RCTs , 70, 75 BT Table ; Fig. 2Bf Kalron  was remop = 0.001) in favor of VRBT [p = 0.049) [p = 0.012) [p = 0.007) [p = 0.01) [Compared to CT, the analysis showed a medium effect of VRBT   (Additio= 0.012) , 68, 70;= 0.007) , 65, 70; = 0.01) , 68, 70 p = 0.017) was observed on Sway Area for the EC condition   [Three studies , 62, 69 on Table ; Fig. 3A= 0.004) , 62.Fig.p = 0.048) on CoP excursion with OE has been observed   in favor of VRBT (Table p = 0.001) on the ABC scale in favor VRBT, with no risk of publication bias . Sensitivity analysis did not show variations.Three RCTs , 68, 70 BT Table ; Fig. 4Ap = 0.001) on the ABC scale in favor of VRBT compared to UC [p < 0.001) compared to CT [Subgroup analysis revealed an increase of 10.03 points  (Table p = 0.002) in FES-1 in favor of VRBT. No risk of publication bias was found . Sensitivity analysis showed a variation of 51% in the effect size after removing the study of Kalron [p = 0.03).Three RCTs , 65, 68 5) Table ; Fig. 4Bf Kalron , althougp = 0.4) (Table p = 0.12 and Trim-and-fill change of 46%)  was observed. Sensitivity analysis did not report substantial variations. Subgroup analyses, according specific comparisons, did not show differences between VRBT vs UC , VRBT vs CT , and VRBT + CT vs CT .Seven RCTs , 68, 69 The aim of this systematic review with meta-analysis was to collect all previous RCTs assessing the effectiveness of VRBT to improve balance in its different dimensions and to reduce fear of falling in PwMS. A second aim was to define the optimal dose of the VRBT protocol to improve functional and dynamic balance in PwMS. The present findings suggest that VRBT: (1) improves functional and dynamic balance; (2) increases confidence of balance and postural control assessed with posturography; (3) reduces fear of falling; (4) but, does not improve gait speed in PwMS. Compared to previous reviews –34, the The present results have shown that VRBT is effective improving functional balance (with regard to ADLS) in PwMS. A large effect is also observed for VRBT, with an increase of 3.36 points on the BBS score when compared to other interventions or UC. Regarding functional balance results (assessed with BBS) from previous reviews, we must mention that not the all reviews found significant improvements in VRBT compared to other therapies. While Casuso-Holgado and Santos-Nascimento did not show statistically significant differences between VRBT and CT , 32, ourWe must note that in contrast to the reviews by Casuso-Holgado and Santos-Nascimento , 32, ourRegarding the effect on postural control assessed with posturography, the meta-analysis shows a medium effect on Sway Area in EC condition, and a small effect on CoP excursion in EO in favor of VRBT compared to CT. This points out that VRBT is helpful to reorganize the sensory inputs related with balance . Thus, VRBT leads to an increase of postural control when different sensory inputs are lost. In addition, our findings showed that VRBT does not improve gait speed, in agree to Casuso-Holgado .As shown in the review of Akkan,  the imprMultisensory information is crucial to produce an effective antigravity muscular response in order to avoid destabilization and to maintain balance . TherefoFindings reported in this study are clinically relevant and provide the most appropriate VRBT dose for treating functional and dynamic balance in PwMS. One strength of our findings is that we provide the most optimal dose to obtain the largest improvement on functional and dynamic balance. One strength of our findings, with large interest for clinical practice, is that we report the most appropriate dose of VRBT to obtain the largest improvement for functional , and for dynamic balance . Our findings provide support to the use of VRBT to recover balance in neurological diseases such as MS. The majority of the VR devices employed in the included studies are non-immersive, so the present results may be more valid for interventions based on non-immersive VR devices. As VRBT may be used both, in clinical settings and home, it may be also considered an excellent tool for tele-rehabilitation.Assuming that the results reported in this meta-analysis are clinically relevant, some limitations must be considered however. Thus, the low number of participants per meta-analysis may reduce the accuracy of our findings, although studies involving neurological patients usually have small sample sizes. Furthermore, the small number of studies that assess some outcomes, such as balance confidence, fear of falling or postural control may also reduce the generalization of the findings. In addition, the medium risk of bias in the included studies, resulting from the impossibility of blinding participants and therapists, and assessors in sometimes, increases the selection risk, performance and detection biases. Other limitation is related to the risk of publication bias observed in some meta-analysis, and the impossibility of assessing this variable in some studies, which also reduces the generalization of the findings. Sensitivity analysis surpasses 20%, being another limitation that reduces the precision of our findings. Another limitation is related to the low quality evidence found in some meta-analysis, which can affect to the robustness of our findings. Finally, all the included studies conducted the assessment in the short-time, so it has not been possible to assess the effect of VRBT in the medium- and long-term.This review provides evidence supporting the effectiveness of VRBT to improve postural balance in PwMS. VRBT is better than UC, CT or RAGT to increase the functional balance, being able to exceed the MCID for BBS reported by scientific literature after VRBT. Therefore, VRBT can be considered an excellent strategy for functional balance rehabilitation in PwMS showing moderate disability. To increase functional balance VRBT would be applied during 40 sessions or more, five sessions per week and between 40 and 45 min. Regarding dynamic balance, VRBT shows a small effect, especially in PwMS with only minimal signs of disability. Our findings recommend that the more appropriated dose of VRBT protocols to improve dynamic balance would be between 8 and 19 sessions, with a duration of 20–30 min per session and twice per week. VRBT also improves different parameters related to postural control in EO and EC conditions. In addition, VRBT reduces the fear of falling compared with CT, increases balance confidence associated to ADLS. However, further RCTs studies using a larger sample size and a control of risk of bias are required in order to increase the generalizability of the present findings.Additionalfile1. Supplementary Tables:TableS1. Description of each Test Reported by Included Studies. TableS2. Subgroup Analysis According Disability Status (EDSS) of PwMS in Studies Included. TableS3. Subgroup Analysis According Total Number of Sessions. TableS4. Subgroup Analysis Number of Sessions per Week. TableS5. Subgroup Analysis According Duration of each Session in Minutes.Additionalfile2. Supplementary Figures: FigureS1. Funnel Plot of the Effect of VRBT on Functional Balance. FigureS2. Funnel Plot of the Effect of VRBT on Dynamic Balance. FigureS3. Funnel Plot of the Effect of VRBT on Sway Area with Eyes Closed. FigureS4. Funnel Plot of the Effect of VRBT on Centre of Pressure Excursion with Eyes Open. FigureS5. Funnel Plot of the Effect of VRBT on Confidence of Balance. FigureS6. Funnel Plot of the Effect of VRBT on Fear of Falling. FigureS7. Funnel Plot of the Effect of VRBT on Gait Speed.


Sepsis is a life-threatening disease with a poor prognosis, and metabolic disorders play a crucial role in its development. This study aims to identify key metabolites that may be associated with the accurate diagnosis and prognosis of sepsis.Septic patients and healthy individuals were enrolled to investigate metabolic changes using non-targeted liquid chromatography-high-resolution mass spectrometry metabolomics. Machine learning algorithms were subsequently employed to identify key differentially expressed metabolites (DEMs). Prognostic-related DEMs were then identified using univariate and multivariate Cox regression analyses. The septic rat model was established to verify the effect of phenylalanine metabolism-related gene MAOA on survival and mean arterial pressure after sepsis.A total of 532 DEMs were identified between healthy control and septic patients using metabolomics. The main pathways affected by these DEMs were amino acid biosynthesis, phenylalanine metabolism, tyrosine metabolism, glycine, serine and threonine metabolism, and arginine and proline metabolism. To identify sepsis diagnosis-related biomarkers, support vector machine (SVM) and random forest (RF) algorithms were employed, leading to the identification of four biomarkers. Additionally, analysis of transcriptome data from sepsis patients in the GEO database revealed a significant up-regulation of the phenylalanine metabolism-related gene MAOA in sepsis. Further investigation showed that inhibition of MAOA using the inhibitor RS-8359 reduced phenylalanine levels and improved mean arterial pressure and survival rate in septic rats. Finally, using univariate and multivariate cox regression analysis, six DEMs were identified as prognostic markers for sepsis.This study employed metabolomics and machine learning algorithms to identify differential metabolites that are associated with the diagnosis and prognosis of sepsis patients. Unraveling the relationship between metabolic characteristics and sepsis provides new insights into the underlying biological mechanisms, which could potentially assist in the diagnosis and treatment of sepsis.This human study was approved by the Ethics Committee of the Research Institute of Surgery (2021–179) and was registered by the Chinese Clinical Trial Registry .The online version contains supplementary material available at 10.1186/s12871-023-02317-4. Sepsis is a significant public health concern characterized by a dysregulated host response to infection, resulting in life-threatening organ dysfunction . The gloMetabolomics is a novel approach developed following genomics, transcriptomics, proteomics and lipidomics , 7. It iMachine learning is a key area within the field of artificial intelligence , 14, repThe present study employed liquid chromatography quadrupole time of flight mass spectrometry (LC-QTOF/MS) to analyze the serum metabolites of sepsis patients. Machine learning techniques were then utilized to identify potential biomarkers for the diagnosis and prognosis of sepsis.https://arriveguidelines.org) for the reporting of animal experiments.This human study was approved by the Ethics Committee of the Research Institute of Surgery (2021–179) and was registered by the Chinese Clinical Trial Registry . All the participants provided written informed consent before inclusion in this study. All experimental procedures were approved by the Animal Ethics Committee of Army Medical University, China, and were performed in accordance with the National Institutes of Health “Guidelines for the Care and Use of Laboratory Animals”. The study was carried out in compliance with the ARRIVE guidelines. All methods are reported in accordance with ARRIVE guidelines  of Daping Hospital, Army Medical University, based on the Sepsis-3 criteria for sepsis and septic shock. Additionally, 15 age-matched healthy volunteers were enrolled from the State Key Laboratory of Trauma, Burns, and Combined Injury. The inclusion and exclusion criteria for septic patients are presented in Supplementary Table R = 8.6 cm) for 15 min at 4 °C. The resulting supernatant was transferred to a fresh glass vial for analysis. A quality control (QC) sample was prepared by combining equal aliquots of the supernatants from all the individual samples.A 10 mL peripheral venous blood sample was collected from all participants in both groups. Subsequently, the samples were centrifuged at 3000 rpm for 10 min, and the resulting supernatant was aliquoted into 1.5 mL centrifuge tubes and stored at -80 °C. For analysis, 100 μL of each sample was transferred to an EP tube. To this, 400 μL of extract solution  was added, followed by 30 s of vortexing, 10 min of sonication in an ice-water bath, and 1 h of incubation at -40 °C to precipitate proteins. The sample was then centrifuged at 12,000 rpm , LC–MS/MS analyses were conducted using a UHPLC system  coupled to an Orbitrap Exploris 120 mass spectrometer  with a UPLC HSS T3 column . The mobile phase consisted of 5 mmol/L ammonium acetate and 5 mmol/L acetic acid in water (A) and acetonitrile (B). The auto-sampler temperature was maintained at 4 °C, and the injection volume was 2 μL. The Orbitrap Exploris 120 mass spectrometer was selected for its capability to acquire MS/MS spectra in information-dependent acquisition (IDA) mode, controlled by Xcalibur software (Thermo). In this mode, the acquisition software continuously evaluates the full scan MS spectrum. The ESI source conditions were set as follows: sheath gas flow rate at 50 Arb, aux gas flow rate at 15 Arb, capillary temperature at 320 °C, full MS resolution at 60,000, MS/MS resolution at 15,000, collision energy at 10/30/60 in NCE mode, and spray voltage at 3.8 kV (positive) or -3.4 kV (negative), respectively.The raw data were converted to the mzXML format using ProteoWizard and processed with an in-house program developed in R. This program utilized XCMS for peak detection, extraction, alignment, and integration. Metabolite annotation was performed using an in-house MS2 database called BiotreeDB, with a cutoff set at 0.3 for annotation.In this study, various machine learning algorithms were employed to identify differentially expressed metabolites (DEMs). The random forest (RF) algorithm was utilized to determine the optimum DEMs between the sepsis and healthy control groups. Using the "e1071" R package, the support vector machine (SVM) generated eigenvectors were eliminated to extract the optimal variables for identifying diagnostic DEMs in sepsis. Additionally, a SVM classifier was constructed with tenfold cross-validation. Finally, a Venn diagram was employed to identify the shared DEMs.https://www.gsea-msigdb.org/gsea/) for further investigation. The mRNA expression matrix was downloaded from the Gene Expression Omnibus (GEO) dataset (https://www.ncbi.nlm.nih.gov/geo/). In this study, the GSE57065 dataset was utilized for screening differentially expressed genes (DEGs). The R package "SVA" was employed to normalize the gene expression matrices and eliminate batch effects. Perl scripts were used to convert the dataset probes into corresponding gene symbols.A total of 18 genes related to phenylalanine metabolism were obtained from the Molecular Signatures Database (MSigDB) (n = 30), Sepsis (n = 30), and RS8359-treated Sepsis (n = 30). The RS8359 compound  was administered at a dosage of 5 mg/kg, dissolved in ddH2O, through the tail vein 30 min prior to inducing sepsis in the RS8359-treated group. The Sepsis group received an equivalent volume of ddH2O. The rats were anesthetized with sodium pentobarbital  for the surgical procedure. Cecal ligation and puncture (CLP) were conducted to create the sepsis model following established protocols [The ethics and protocols for the animal experiments were approved by the La-boratory Animal Welfare and Ethics Committee of the Army Medical University . Adult male and female Sprague–Dawley rats weighing between 200-220 g were bred in a facility with filtered positive-pressure ventilation, under a 12:12-h dark/light cycle, with ad libitum access to food and water. The rats were randomly assigned to three groups: Control (http://www.r-project.org). The DEGs were identified using the "Limma" package with a significance threshold of P < 0.05. Receiver operating characteristic (ROC) analysis was performed, and the area under the curve (AUC) was calculated to assess the predictive performance of the classifier.All statistical analyses were conducted using R software . There were no significant differences in gender and BMI among the two groups (p > 0.05). However, we observed significant differences in temperature (p = 0.01), heart rate (p = 0.001), and white blood cell count (p = 0.003) when comparing septic patients to the healthy controls  between the sepsis and control groups, with 327 identified in the positive ion mode and 205 in the negative ion mode , which can increase the metabolite coverage and improve the detection effect. Subsequent data analysis involved separate analyses of the two groups. Principal component analysis (PCA) revealed significant differences in metabolites between sepsis patients (SP) and healthy controls (HC) Fig. A, B. Furode Fig. G. The piode Fig.  E, F. Idode Fig. H.Fig. 1IFigure +), total cholesterol (TC), and B-type natriuretic peptide (BNP).We constructed a Random Forest (RF) model using minimum error regression trees for screening differentially expressed metabolites (DEMs) Fig. A. ThrougWe further screened metabolites by ROC analysis and calculated the area under the curve (AUC). The results showed that the AUC of the 4 key DEMs analyzed by machine learning were both 1.0, which showed an excellent prediction ability for sepsis  between the sepsis and healthy control groups using a threshold of |log2FC|> 1 and p = 0.007), L-Threonic , Cer/AS(d17:3/16:2) , Diethylphosphate , Hydroxycotinine  and Syringol  were identified by multivariate Cox regression analysis , and the ICU mortality was 13.3% (4/30). Univariate Cox regression and multivariate Cox regression analysis were used to identify the prognostic related DEMs. After observing the survival outcome of septic patients within 28 days, DEMs associated with survival were identified by univariate Cox regression analysis in NEG and POS . These DEMs were primarily involved in various metabolic pathways, including amino acid biosynthesis, phenylalanine metabolism, tyrosine metabolism, glycine, serine, and threonine metabolism, as well as arginine and proline metabolism. Machine learning algorithms SVM and RF were utilized to identify four metabolic biomarkers  that are associated with the diagnosis of sepsis. Additionally, an analysis of transcriptome data from sepsis patients in the GEO database revealed a significant up-regulation of the phenylalanine-metabolism-related gene MAOA in the sepsis group. Inhibition of MAOA with the inhibitor RS-8359 resulted in improved mean arterial pressure and survival rate in septic rats. Finally, univariate and multivariate Cox regression analysis identified six DEMs (FA (22:5), L-Thyronic, Cer/AS (d17:3/16:2), Diethylphophase, Hydroxyco-tinine, and Syringol) that are associated with the prognosis of sepsis.Machine learning algorithms have played a crucial role in the mining of metabolomic data . A lot oPrevious studies have shown that bacterial infection can lead to an imbalance of amino acid metabolism . In sepsThis study utilized metabolomics, machine learning algorithms, and prognosis analysis to screen potential metabolic biomarkers associated with the diagnosis and prognosis of sepsis. Moreover, it specifically examined the alterations in phenylalanine metabolism following sepsis and subsequently analyzed the regulatory mechanism of MAOA. However, since this study is considered exploratory and based on a small sample size of very new biomarkers, we opted to use completely healthy individuals as the control group. This decision could potentially lead to an overestimation of the sensitivity and specificity of the biomarkers. In future large sample studies, we will select case-controls as the control group to authenticate the diagnostic efficacy of these biomarkers.The current study utilized metabolomics and machine learning algorithms to identify distinct metabolites associated with the diagnosis and prognosis of sepsis patients. Investigating the association between metabolic characteristics and sepsis offers novel insights into the underlying biological mechanisms, which may potentially enhance the diagnosis and treatment of sepsis.Additional file 1: Supplementary Table 1. The inclusion and exclusion criteria of septic patients.Additional file 2.


To evaluate the prevalence and treatment of postmenopausal women with osteoporosis in recent years, analyze differences between the prevalence diagnosed by physicians and the prevalence detected by bone mineral density (BMD), and observe the trends of prevalence and treatment rate of osteoporosis in postmenopausal women over time are of great value for the management of osteoporosis.This cross-sectional study collected the data of 4012 postmenopausal women from the National Health and Nutrition Examination Survey (NHANES) from 2005 to 2010, 2013 to 2014 and 2017 to 2018. The prevalence of osteoporosis and osteopenia as well as the treatment rate of osteoporosis were analyzed using Mann-Kendall trend test. Subgroup analysis was conducted in different age, race, body mass index (BMI), diabetes, hypertension, or glucocorticoid use groups.P = 0.027) during 2005–2018. The prevalence of osteoporosis in postmenopausal women determined by bone mineral density (BMD) examination reached 9.2% during the five cycles. From 2005 to 2018, the prevalence of physician diagnosed osteoporosis fluctuated in a small range. For osteopenia measured by BMD, the prevalence was 59.6% and a gradual increasing trend was found between 2005 and 2018 . Among patients with physician diagnosed osteoporosis, the treatment rate reached 70.49%. The treatment rate of physician diagnosed osteoporosis was decreased from 2005 to 2008, and further decreased from 2009 to 2018 . The actual treatment rate of osteoporosis patients was 55.53%. During 2005–2018, the actual treatment rate of osteoporosis showed a continuous decline .The overall prevalence of physician diagnosed of osteoporosis was 17.4% and was fluctuated in a small range and remained relatively stable within a certain range (Mann-Kendall trend test: Z = 2.20, Osteoporosis management might be insufficient and more efforts are needed to improve the diagnosis and treatment rates of osteoporosis in postmenopausal women. Osteoporosis is a kind of skeletal disorder characterized by decreased bone quality and bone mineral density . OsteopoRecently, although the United States Preventive Services Task Force (USPSTF) recommends routine osteoporosis screening and bone mineral density (BMD) testing for women aged≥65 years . WhetherIn our study, we planned to explore the trends of prevalence and treatment rate of osteoporosis and osteopenia in postmenopausal women over time based on the data from the National Health and Nutrition Examination Survey (NHANES). The prevalence and treatment of osteoporosis and osteopenia in postmenopausal women during recent years, the differences between the prevalence of osteoporosis and osteopenia diagnosed by physicians and the prevalence detected by BMD, and the trend of prevalence and treatment rate of osteoporosis in postmenopausal women with different ages, races and other characteristics are valuable for the further targeting management.This cross-sectional study collected the data of 5309 postmenopausal women in the NHANES from 2005 to 2010, 2013 to 2014 and 2017 to 2018. NHANES is a program of continuous 2-year-cycle cross-sectional surveys conducted by the Centers for Disease Control and Prevention (CDC). NHANES involved in about 5,000 persons each year from various counties across the U.S., which are divided into a total of 30 primary sampling units (PSUs), of which 15 are visited annually. The NHANES combines interviews, physical examinations, and laboratory evaluations to obtain a large amount of quantitative and qualitative data . All par2 or ≥25 kg/m2), waist circumference (cm), cotinine (ng/mL), cadmium (ug/L), lead (ug/dL), iron (umol/L), mercury (umol/L), 25-hydroxyvitamin D2 , total energy , Day 1 calcium intake (mg), calcium intake in dietary supplement (yes or unknown), total calcium intake (mg), Day 1 vitamin D intake , and vitamin D intake in dietary supplement (yes or unknown) were variables analyzed in this study.Age (years), race , education or Equivalent, some college or AA degree, or college graduate or above), marriage , poverty-to-income ratio (PIR), drinking (yes or no), smoking (yes or no), physical activity (MET· min/week), hypertension (yes or no), diabetes (yes or no), previous fracture (yes or no), parental fracture (yes or no), glucocorticoid use (yes or no), anti-osteoporosis therapy (yes or no), body mass index  ≥6.5%, physician diagnosis or those receiving hypoglycemic therapy. Glucocorticoid use was identified according to OSQ (Ever taken prednisone or cortisone daily?) or whether they had used hormonal medications. Smoke was defined based on SMQ_D, and those have smoked at least 100 cigarettes in the lifetime were considered to be a smoker, otherwise a non-smoker. Physical activity was converted into energy expenditure, which was calculated based on PAQ from the database. Energy expenditure [Metabolic Equivalent (MET)·min] = recommended MET × Exercise time of corresponding activity (min).People who answered “yes” to the question “Have you ever been diagnosed with osteoporosis by a physician?” were classified as physician-diagnosed cases of osteoporosis. The number of physician diagnosed cases of osteoporosis during the 5 cycles was divided by the total number of postmenopausal women in the 5 cycles analyzed in our study to calculate the weighted overall prevalence of physician diagnosed osteoporosis. The number of physician diagnosed osteoporosis patients in 2005–2006, 2007–2008, 2009–2010, 2013–2014 or 2017–2018 was divided by the total number of postmenopausal women in each year to calculate the annual weighted prevalence of physician diagnosed osteoporosis.The number of osteoporosis patients diagnosed by BMD measurement during the 5 cycles was divided by the total number of postmenopausal women in the 5 cycles to calculate the measured weighted prevalence of osteoporosis diagnosed by BMD measurement. The numbers of osteoporosis patients diagnosed by BMD measurement in 2005–2006, 2007–2008, 2009–2010, 2013–2014 or 2017–2018 was divided by the total number of postmenopausal women in each year to calculate the weighted prevalence of osteoporosis diagnosed by BMD measurement.Women who answered “yes” to the question “Have you ever been diagnosed with osteopenia by a physician?” were classified as physician-diagnosed cases of osteopenia. The number of physician diagnosed cases of osteopenia during the 5 cycles was divided by the total number of postmenopausal women in the 5 cycles analyzed to calculate the weighted overall prevalence of physician diagnosed osteopenia. The number of physician diagnosed osteopenia patients in 2005–2006, 2007–2008, 2009–2010, 2013–2014 or 2017–2018 was divided by the total number of postmenopausal women in each year to calculate the annual weighted prevalence of physician diagnosed osteopenia.The number of osteopenia patients diagnosed by BMD measurement during the 5 cycles was divided by the total number of postmenopausal women in the 5 cycles to calculate the measured weighted prevalence of osteopenia diagnosed by BMD measurement. The numbers of osteopenia patients diagnosed by BMD measurement in 2005–2006, 2007–2008, 2009–2010, 2013–2014 or 2017–2018 was divided by the total number of postmenopausal women in each year to calculate the weighted prevalence of osteopenia diagnosed by BMD measurement in each cycle.The number of osteoporosis patients received treatments during the 5 cycles was divided by the total number of physician diagnosed osteoporosis postmenopausal women in the 5 cycles to calculate the weighted overall treatment rate of physician diagnosed osteoporosis. The treatment of physician diagnosed osteoporosis was defined based on the answer of “yes” to the question “Have you ever been treated for osteoporosis by a physician?”, and women who received metabolic agents, bone resorption inhibitors, bisphosphonates, and miscellaneous bone resorption inhibitors. The number of osteoporosis patients received treatments in 2005–2006, 2007–2008, 2009–2010, 2013–2014 or 2017–2018 was divided by the total number of physician diagnosed osteoporosis postmenopausal women in each year to calculate the annual weighted treatment rate of physician diagnosed osteoporosis in each year.The number of osteoporosis patients received treatments during the 5 cycles was divided by the total number of postmenopausal women with physician diagnosed osteoporosis and osteoporosis diagnosed by BMD measurement in the 5 cycles to calculate the actual treatment rate of osteoporosis. Women who answered “yes” to the question “Have you ever been treated for osteoporosis by a physician?”, and those who received metabolic agents, bone resorption inhibitors, bisphosphonates, and miscellaneous bone resorption inhibitors were defined to received treatments for osteoporosis. The numbers of osteoporosis patients received treatments in 2005–2006, 2007–2008, 2009–2010, 2013–2014 or 2017–2018 was divided by the total number of postmenopausal women with physician diagnosed osteoporosis and osteoporosis diagnosed by BMD measurement in each year to calculate the weighted actual treatment rate of osteoporosis.https://wwwn.cdc.gov/Nchs/Nhanes/2005-2006/DXXFEM_D.htm). All participants underwent BMD testing by DXA examinations. BMD was measured via DXA and BMD at the femur neck and total femur was used to calculate the T-score [respondent’s BMD-reference group mean BMD)/reference group standard deviation (SD)]. The reference group for the femoral neck consisted of non-Hispanic White women aged 20–29 years from NHANES III , and the Mann-Whitney U rank sum test was used for comparison between groups. Enumeration data were described by n (%). Chi-square test was used for comparison between groups. All the data were subjected to a weighted manner. The masked variance unit pseudo-stratum was sdmvstra, and the masked variance unit pseudo-primary sampling units was sdmvpsu. The confidence interval (CI) was applied for evaluating the reliability of an estimate. A set of weights WTDRD1 was used when an analysis uses the Day 1 dietary recall data . The set of weights WTDRD1 is applicable to the respondents with Day 1 data. Day 1 weights were constructed by taking the MEC sample weights (WTMEC2YR) and further adjusting for the additional non-response and the differential allocation by day of the week for the dietary intake data collection. The prevalence and treatment rate trends of osteoporosis in postmenopausal women were analyzed using Mann-Kendall trend test. The Z value >0 and P<0.05 indicated an increased trend while the Z value >0 and P<0.05 represented a decreased trend. Subgroup analysis was conducted in different age, race, BMI, diabetes, hypertension, or glucocorticoid use groups. All statistical tests were performed by a two-sided test with α = 0.05. SAS 9.4  was used to calculate weighted prevalence and treatment rates. Prevalence and treatment rates of osteoporosis were plotted using R 4.0.3 .The normality of quantitative data was evaluated by Kolmogorov-Smirnov, and normally distributed measurement data were described as Mean ± standard deviation (SD). Independent sample t test was used for comparison between the two groups. The non-normally distributed measurement data were described as median and quartiles [M , without data on physician diagnosed osteoporosis or not (n = 26), without data on marriage (n = 2), smoking (n = 1), previous fracture (n = 1), parental fracture (n = 89), height (n = 15), weight (n = 1), waist circumference (n = 33) or total energy (n = 106) were excluded. Finally, 4012 subjects were included. Among them, 715 people had physician diagnosed osteoporosis and 3297 people did not self-report to have osteoporosis . There wThe percentage of smokers (45.71% vs 42.62%), previous fracture (4.26% vs 1.01%), glucocorticoid use (14.44% vs 8.49%), and complicated with hypertension (64.52% vs 56.19%) in the physician diagnosed osteoporosis group was higher than non-physician diagnosed osteoporosis group. The mean physical activity (1318.44 MET·min/week vs 1875.40 MET·min/week) in the physician diagnosed osteoporosis group was lower than non-physician diagnosed osteoporosis group .2 vs 27.10 kg/m2 vs 31.20 kg/m2) and waist circumference (88.75 cm vs 93.81 cm vs 102.51 cm) were statistical difference among participants with osteoporosis or osteopenia diagnosed by BMD and normal people. The percentages of previous fracture (5.91% vs 1.26% vs 0.96%) and glucocorticoid use (11.29% vs 9.09% vs 9.70%) were statistical difference among participants with osteoporosis or osteopenia diagnosed by BMD and normal people (The mean age (70.67 years vs 63.75 years vs 58.85 years), BMI (24.60 kg/ml people .P = 0.027). The prevalence of osteoporosis in postmenopausal women determined by BMD examination reached 9.2% during the five cycles. From 2005 to 2018, the prevalence of physician diagnosed osteoporosis fluctuated in a small range. For osteopenia measured by BMD, the prevalence was 59.6% and a gradual increasing trend was found between 2005 and 2018   .P = 0.027). In 2018, the treatment rate of these patients was 59.28%. In order to avoid overestimating the treatment rate of osteoporosis, the actual treatment rate of osteoporosis was calculated. The actual treatment rate of osteoporosis patients was 55.53%. During 2005–2018, the actual treatment rate of osteoporosis showed a continuous decline , and dropped to 47.52% in 2018 . In the subgroup of people aged ≥65 years, the overall prevalence of physician diagnosed osteoporosis and osteoporosis or osteopenia measured by BMD were 26.4%, 17% and 57.3%, respectively , while the prevalence of physician diagnosed and BMD-determined osteoporosis remained relatively stable , and the prevalence of physician diagnosed osteoporosis and osteoporosis diagnosed by BMD exhibited a relatively stable fluctuation. In BMI<25kg/m2 group, the prevalence of physician diagnosed osteoporosis was 22.59%. The overall prevalence of osteoporosis diagnosed by BMD was 17.08%, and a relatively stable fluctuation tread was observed during 2005–2018. The overall prevalence of osteopenia diagnosed by BMD was 63.74% and the prevalence trend was increased from 2005–2018  (In BMI≥25kg/m= 0.027) .In subjects with diabetes, the overall prevalence of physician diagnosed osteoporosis and osteoporosis or osteopenia measured by BMD were 16.12%, 7.4% and 41.3%, respectively. The prevalence of physician diagnosed osteoporosis and osteoporosis or osteopenia showed relative stable fluctuations from 2005–2018. In those without diabetes, the overall prevalence of physician diagnosed osteoporosis and osteoporosis or osteopenia measured by BMD 17.68%, 9.59% and 54.04%, respectively. Relatively stationary trends were found in the prevalence of physician diagnosed osteoporosis and osteoporosis or osteopenia measured by BMD during 2005–2018 .In people with hypertension, the overall prevalence of physician diagnosed, BMD-measured osteoporosis and osteopenia were 19.49%, 10.58% and 49.68%, respectively, and presented relative stable fluctuations during 2005–2018. The overall prevalence of physician diagnosed, BMD-measured osteoporosis and osteopenia were 14.59%, 7.37% and 54.85% in those without hypertension. Relative stable fluctuations were found in the prevalence of physician diagnosed, BMD-measured osteoporosis and osteopenia during 2005–2018 in those without hypertension .P = 0.027), while the prevalence of physician diagnosed and BMD-detected osteoporosis showed relatively stable fluctuations Click here for additional data file.


Haemophilus influenza B (Hib) production. Therefore, new hepatitis B sources are needed. This study aimed to evaluate the immunogenicity of the DTP–HB–Hib vaccine (Bio Farma) that used a different source of hepatitis B. A prospective randomized, double-blind, bridging study was conducted. Subjects were divided into two groups with different batch numbers. Healthy infants 6–11 weeks of age at enrollment were immunized with three doses of the DTP–HB–Hib vaccine after a birth dose of hepatitis B vaccine. Blood samples were obtained prior to vaccination and 28 days after the third dose. Adverse events were recorded until 28 days after each dose. Of the 220 subjects, 205 (93.2%) completed the study protocol. The proportion of infants with anti-diphtheria and anti-tetanus titers ≥ 0.01 IU/mL was 100%, with anti-HBsAg titers ≥ 10 mIU/mL was 100%, and with Polyribosylribitol Phosphate-Tetanus Conjugate (PRP-TT) titers > 0.15 µg/mL was 96.1%. The pertussis response rate was 84.9%. No serious adverse events related to the study vaccine occurred. The three-dose DTP–HB–Hib vaccine (Bio Farma) is immunogenic, well tolerated, and suitable to replace licensed-equivalent vaccines.Satisfying the needs of the national immunization program requires maintaining diphtheria–tetanus–pertussis (DTP)–hepatitis B (HB)– A target of the sustainable development goals (SDGs) for 2030 is to decrease infant and child mortality . The SDGDuring the Coronavirus disease 2019 (COVID-19) pandemic, infant and child mortality rates were higher in lower- and middle-income countries (LMICs) ,6. More Diphtheria has a worldwide distribution but is now uncommon in Western Europe and the USA; however, it is still endemic in many parts of the world, especially in Southeast Asia . In 2017Tetanus is a major cause of death in Asia and Africa, especially in rural and tropical areas where neonatal tetanus  is common. Data from the WHO show that neonatal mortality from tetanus ranges from <5 to >60 per 1000 live births . HoweverBasic immunization in Indonesia was initiated on 1977 through the expanded program in immunization (EPI) . The basHaemophilus influenza B (Hib) vaccine  combines the diphtheria and tetanus toxoids, inactive pertussis bacteria, HB surface antigen, and Hib, and underwent clinical trials in 2013 [The DTwP–HB– in 2013 . Today,  in 2013 . Meanwhi in 2013 ,23,24,25 in 2013 ,23,24,25The true burden of HB, DTP, and Hib is multifactorial. Coordination with all of the stakeholders for the access and vaccine schedule is needed in future strategies. This study was conducted to evaluate the immunogenicity of the Pentabio vaccine, which uses a different source of HB.www.randomization.com e.g., accessed on 13 October 2020. The subjects were divided into two groups based on the source of vaccine , had a history of an allergic reaction that was likely to be triggered by any vaccine component, if they had DTP, HB, or Hib infection, had a history of congenital or acquired immunodeficiency, had uncontrolled coagulopathy or blood disorders, had chronic illness, or immunosuppressive conditions, if they were undergoing immunosuppressive therapy, had received immunoglobulin therapy or blood products before starting or during the study, had received a vaccination other than oral polio and the BCG vaccine, were participating in another clinical study, or had a mother who was HbsAg or HIV positive (by rapid test).Infants were withdrawn from the study if, after vaccination, they experienced acute illness, had an axillary temperature of 37.5 °C, or had received treatment likely to alter their immune response in the 4 weeks before vaccination . Additionally, infants were withdrawn if they were accidentally administered a non-investigational DTwP, HB, or Hib conjugate vaccine after the study vaccination, had encephalopathy and seizures within 7 days following the last injection, had an axillary temperature > 39.6 °C within 3 days following the last injection, inconsolable crying for more than 3 h following the last injection, had any clinically significant allergic reaction suspected to have occurred within 3 days following the last injection, had hypotonic hyporesponsive episodes within the 3 days following the last injection, and had thrombocytopenic purpura.® (Bio Farma) was a DTP–HB–Hib combination vaccine produced in forms of homogenous suspension containing the following: Diphtheria toxoid, tetanus toxoid, inactive Pertussis bacteria (whooping cough), non-infectious pure Hepatitis B surface antigen (HBsAg) and a non-infectious Hib component, which was a resulting conjugation of Haemophillus influenza type B and tetanus toxoid.The recombinant Hepatitis B vaccine was an inactivated HbsAg produced in yeast cells (Hansenula polymorpha), using recombinant DNA technology. It was a whitish liquid that was produced by a yeast cell, genetically engineered in culture and carrying the relevant gene of the HbsAg. The Pentabio®). After a birth dose of HB vaccine, healthy infants, 0–3 days of age at enrollment, were immunized with three doses of DTP–HB–Hib vaccine with an interval of 4 weeks. Blood samples were obtained before vaccination and 28 days after the third dose. Adverse events were recorded until 28 days after each dose.A randomized, prospective, double-blind, two-arm parallel group intervention study was conducted in Bandung city from October 2020 to October 2021. Subjects were divided into two groups: Group A received DTP–HB–Hib using a different source of hepatitis B, and Group B received DTP–HB–Hib (PentabioBlood samples (4 mL) were collected in vacutainer tubes at visit 0 (V0) (cord blood at birth), V2 before DTP–HB–Hib vaccination, and V5 (28 days after the third dose of DTP–HB–Hib). After clotting at room temperature, blood samples were centrifuged at 3000 rpm for 15 min, and sera were aseptically separated into 2 aliquots (1.5 mL each) within 24 h after sampling. Each blood sample was labeled indicating the blood sampling stage , trial code, inclusion number, and subject’s initials. Sera were rapidly stored in a freezer at −20 °C/−80 °C. Temperature was monitored and documented on the appropriate form during the entire trial.The anti-diphtheria, anti-tetanus, and anti-PRP-TT serological responses were tested by the ELISA method, and anti-pertussis was tested by the micro agglutination procedure. Anti-HBs ELISA was tested using a kit reagent from Abbott. Anti-hepatitis B titers were measured by an chemiluminescent microparticle immunoassay architect using a reagent kit on architect i 1000sr.All tests (ELISA and microagglutination), except for anti-HBs, were conducted in the Immunology Laboratory of the Clinical Trial Department of Bio Farma. These tests were already validated by the Clinical Trial Department and approved by the Quality Assurance Division. This lab has been certified for ISO 14001:2004, ISO 9001:2008 and OHSAS 18001:2007. The test for anti-HBs was conducted in Commercial Laboratory, and had been assessed by Quality Assurance of Bio Farma and certificated for ISO 9001 and KAN  Jakarta, Indonesia. Any additional serological analysis on antigen(s) included in the vaccine tested in this trial may be performed if Bio Farma considers it necessary to further document the immunogenicity results of this trial or other trials.The investigator assessed the intensity , duration, and relation of each adverse event during the trial vaccines. Local and systemic reactions, expected or unexpected, occurring within 30 min, 72 h, and 28 days after each injection were evaluated by interviewing the parents on post-surveillance visits V1, V2, V3, and V4. The axillary temperature was measured for three days after each dose in the evening and/or at the time of the febrile peak, and the highest temperature (°C) was recorded in the diary card. The trial team recorded the information in the CRF. Any severe adverse event occurring within 28 days after each injection was also recorded.The sample size is determined based on a 95% confidence interval and the power of the test at 80%. Using the sample size formula for comparing two population proportions, the required sample size would be 110 in each group; this included the anticipation of a 10% dropout. The main evaluation criteria were defined as the percentage of infants with anti-diphtheria and anti-tetanus titers ≥ 0.01 IU/mL, anti-HBsAg titers ≥ 10 mIU/mL, and anti-PRP-TT titers > 0.15 μg/mL 28 days after the last injection of DTP–HB–Hib in Group A. The primary analysis was a comparison of the post-last dose seroprotection rates between groups A and B.For immunogenicity analysis, the seroprotection and/or seroconversion rates at the last post-surveillance visit (V5) were presented as crude rates with 95% confidence intervals (CI) . Meanwhile, the seroprotection and/or seroconversion rates at V1 and V4 were presented as geometric means of titers (GMTs) with 95% CI.For safety analysis, the number and percentage of subjects with at least one local or severe reaction  and their frequency  were assessed.® (Bio Farma) and Pentabio®, while code B represented the recombinant hepatitis B and DTP–HB–Hib (Bio Farma) vaccines using a different hepatitis B source  and seroprotection rates for ≥0.01 and ≥0.1 IU/mL (p = 0.08 and p = 0.529) between the two groups. The percentage of infants whose antibodies increased by ≥4 times in each group was 63.7% and 62.1%, and the proportion of seronegative infants who became seropositive was 95.7% and 100%.After completing the three-dose primary series, nearly all subjects in each group achieved a standard protective concentration of ≥0.01 IU/mL (97.1% in group A and 100% in group B) and ≥0.1 IU/mL (97.4% and 87.4%)  against p = 0.270) and seroprotection rates for ≥0.01 and ≥0.1 IU/mL (p = 1.00 and p = 1.00) between the two groups. The percentage of infants whose antibodies increased by ≥4 times in each group was 22.5% and 32.0%, and the proportion of seronegative infants who became seropositive was 100% for both.The tetanus standard protective antibody concentrations for ≥0.01 and ≥0.1 IU/mL were 100% for both concentrations and groups. There was no significant difference in the GMC values (p = 0.038) after vaccination than before vaccination. The percentage of infants whose antibodies increased by ≥4 times in each group was 98% and 95.1%, and the proportion of seronegative infants who became seropositive was 94.5% and 92.6%.As shown in p = 0.027). The proportion of seronegative infants who became seropositive was 100%. The difference in the levels of anti-HBs GMTs was statistically significant at the V5 group (p = 0.001)  achieved seroprotective antibody concentrations ≥10 mIU/mL against the HB surface antigen after vaccination at birth and after the three-dose primary series . The per= 0.001) .p = 0.974).After completing the three-dose primary series, ≥96% and ≥79% of subjects in each group had seroprotective anti-PRP concentrations of ≥0.15 μg/mL and ≥1.0 μg/mL, respectively . The perMost of the patients did not develop adverse events within 30 min, 72 h, and 28 days after vaccination. Only redness and swelling within 72 h after the first dose of DPT–Hb–Hib was noted. No anaphylactic or severe reactions occurred within 30 min after any dose of the study vaccine.n = 9; Group B, n = 8). Meanwhile, two subjects had ABO incompatibility and omphalitis. Five subjects  were hospitalized for 4–10 days due to bronchopneumonia. Two subjects with Hirschsprung disease, one subject with dengue hemorrhagic fever, and one subject with malnutrition were hospitalized; these conditions were considered as coincidental and unrelated to the study vaccine, as declared by the National Adverse Event Following Immunization committee.From the first dose to 35 days after the third dose, 26 serious adverse events occurred in 11 (10.6%) and 15 (14.5%) subjects in groups A and B, respectively. In 17 subjects, the serious adverse event was neonatal hyperbilirubinemia  before the primary vaccination series. Research in the United Kingdom reported significantly lower antibodies against tetanus toxoid antigens in infants in whom pre-immunization  antibody concentrations were already high . AnotherCases of newborns with high levels of transplacentally acquired anti-tetanus toxoid are common in Indonesia, where programs for the prevention of neonatal tetanus are implemented by vaccinating pregnant women. Neonatal tetanus can be prevented by vaccinating women of reproductive age with the tetanus toxoid either during or outside of pregnancy. For lifelong protection, the WHO recommends that an individual receives six doses (three primary plus three booster doses) of tetanus toxoid . The thrAdditionally, 61.2%, 80.6%, 17.3%, and 1.0% of subjects had seroprotective antibody concentrations against diphtheria, HB surface antigen, Hib, and pertussis before receiving the primary vaccination series. However, the low levels of anti-pertussis antibodies should be given attention. At the age of 2 months and after receiving HB immunization at birth, the HB vaccine only provided protection to 17.3% of the subjects; this increased to 100% after administering three doses containing HBsAg. Protection lasts for at least 20 years and is probably lifelong. The WHO does not recommend booster vaccinations for persons who have completed the three-dose vaccination schedule. However, the WHO recommends that all infants receive the HB vaccine as soon as possible after birth, preferably within 24 h, followed by two or three doses of HB vaccine at least 4 weeks apart to complete the vaccination series . In areaInfants aged 2 months (6–10 weeks) have a high-risk of pertussis because only 1% have antibodies above the protection value; hence, these infants should receive immunization immediately. A previous study demonstrated that the pooled efficacy of the whole-cell pertussis (wP) vaccine against pertussis in children was 78%, but efficacy varied significantly among the vaccines . A studyPublic health concerns before the implementation of Hib immunization programs included pneumonia and meningitis in young children caused by Hib . There wThe results of immunogenicity testing between the control and intervention groups showed similarly favorable results. Meanwhile, the GMT values of anti-HBs from the DTP–HB–Hib (Bio Farma) vaccine using a different hepatitis B source were significantly higher. A study of Pentabio in Iran showed similar results to this study .The incidence of local and systemic reactions decreased with successive doses of primary vaccination . This stAll vaccines were well tolerated; there were no differences in the rates of local and systemic reactions between the two groups. In the Pentabio group, 100% of the children reached protective levels of antibodies (seropositivity) against tetanus and diphtheria, 99% against HB, 98.1% against Hib, and 89.5% against pertussis. This shows that the Pentabio vaccine is safe and immunogenic and can be administered in healthy children . A doublAfter primary vaccination with Pentabio, 99.3% of the subjects showed protective levels of antibodies against diphtheria, tetanus, HB, and Hib. The most common reactogenicity was local reactions, which were mild in severity. The immune responses and reactogenicity were consistent between the three different batch numbers of the Pentabio vaccine. This study demonstrates that the new pentavalent DTP–HB–Hib vaccine (Pentabio) is immunogenic and tolerable, and can be used to support a childhood vaccination program in Indonesia . During In conclusion, our study shows that the three-dose DTP–HB–Hib (Bio Farma) regimen that uses a different source of hepatitis B is immunogenic, well tolerated, and suitable to replace licensed equivalent vaccines based on its immunological and safety profile. No vaccine-related serious adverse events or deaths were reported during the study.