Size and number of lymph nodes (LNs) were reported to be associated with the prognosis of stage II colorectal cancer (CRC). The purpose of this study was to determine the prognostic role of the size of LNs (SLNs) measured by computer tomography (CT) and the number of retrieved LNs (NLNs) in the relapse-free survival (RFS) and overall survival (OS) among stage II CRC patients.Consecutive patients diagnosed with stage II CRC at Fudan University Shanghai Cancer Center (FUSCC) from January 2011 to December 2015 were reviewed, and 351 patients were randomly divided into two cohorts for cross-validation. The optimal cut-off values were obtained using X-tile program. Kaplan-Meier curves and Cox regression analyses were conducted for the two cohorts.Data from 351 stage II CRC patients were analyzed. The cut-off values for SLNs and NLNs were 5.8 mm and 22, respectively, determined by the X-tile in the training cohort. In the validation cohort, Kaplan-Meier curves demonstrated SLNs (P = 0.0034) and NLNs (P = 0.0451) were positively correlated with RFS but not with OS. The median follow-up time in the training cohort and the validation cohort were 60.8 months and 61.0 months respectively. Univariate and multivariate analysis revealed that both SLNs (training cohort: Hazard Ratio (HR) = 2.361, 95% Confidence interval (CI): 1.044–5.338, P = 0.039; validation cohort: HR = 2.979, 95%CI: 1.435–5.184, P = 0.003) and NLNs  were independent prognostic factors for RFS whereas not for OS.SLNs and NLNs are independent prognostic factors for patients with stage II CRC. Patients with SLNs > 5.8 mm and NLNs ≤ 22 are apt to have higher risk of recurrence.The online version contains supplementary material available at 10.1186/s12885-023-10935-x. Globally, colorectal cancer (CRC) is the third most common cancer and ranks second in mortality among all cancers, imposing a heavy burden on patients and society . The chaAs an early disease, stage II CRC comprises approximately one-quarter of CRC. It’s been reported that the size of retrieved LNs may be a prognostic factor in stage II CRC , 6–8.HowAlthough American Joint Committee on Cancer (AJCC) guideline recommends the assessment of at least 12 LNs for stage II CRC , the numIn this study, we retrospectively investigated patients with stage II CRC at Fudan University Shanghai Cancer Center (FUSCC) to determine the optimal cut-off values and evaluate the prognostic significance of SLNs and NLNs.This study retrospectively reviewed 6896 consecutive patients at FUSCC from January 2011 to December 2015. The inclusion criteria were as follows: 18–80 years old; pathologically confirmed primary colorectal adenocarcinoma, mucinous adenocarcinoma, or signet-ring cell carcinoma; stage II according to the 8th edition of the AJCC/UICC TNM staging system; and receiving radical resection of the primary tumor. Exclude the following patients: emergency surgery for acute bowel obstruction, bleeding, or perforation; evidence of distant metastases; neoadjuvant therapy or radiotherapy; history of other malignancies; radiological or follow-up data unavailable. Enrolled 351 patients were randomized into two cohorts at a ratio of 1:1 by a random number table: training cohort and validation cohort, for cross-verification. The training cohort was used to determine the best cut-off point and the validation cohort was used to verify the cut-off value.This study was approved by the Institutional Review Board of FUSCC and did no harm to patients. All patients provided informed consent. All retrospective data were retrieved from the FUSCC database. Patients were followed up regularly according to Chinese Society of Clinical Oncology (CSCO) guideline. The follow-up data, including recurrence and death, were registered in the Clinical Statistics Center of FUSCC, through the hospital medical records follow-up platform or direct contact with patients via phone or email. Patients alive at the date of analysis were censored at the date of last follow-up.All patients underwent enhanced abdominal or pelvic CT scan according to standard clinical protocol, which were performed on Sensation 64  or Brilliance  systems. The imaging condition were as follows: 120 kV, 200mA, 5 mm slice thickness, 1.4 or 0.9 pitch, 5.0 mm increment, 512 × 512 matrix and 4.11 cm field of view. All CT enhanced images were collected after 70-75s iodine-based intravenous contrast agent injection. All CT images were retrieved from the picture archiving and communication system (PACS) and exported in digital imaging and communication in medicine (DICOM) format for image segmentation and analysis.www.itksnap.org). The borders of LNs were drawn by excluding adjacent fat, gas, peripheral vascular and normal tissue. To improve the robustness and accuracy of segmentation, an expert radiologist  finally verified and corrected the margins of LNs in the consensus. Then, they measured SLNs independently and any discrepancies were resolved by consensus. To ensure reproducibility, 50 cases were selected and redrawn to test intra-observer consistency using the intraclass correlation coefficient (ICC), with a value of ICC greater than 0.75 indicating good agreement in feature extraction. To ensure the accuracy of LNs masking, the LNs mask was evaluated by the chief radiologist, T.T.), according to the same guideline used to define the boundary of LNs. The parameters measured by the observers showed good agreement, with a mean ICC of 0.88.3D regions of interests (ROIs) of LNs were semiautomatically segmented on the venous phase images by an experienced radiologist , using ITK-SNAP software  and GraphPad Prism version 7 . X-tile program   was usedThis study consecutively enrolled 351 patients at FUSCC from January 2011 to December 2015. These patients were randomly divided into two cohorts: the training cohort (n = 176) and the validation cohort (n = 175). Details of the enrollment process are presented in the flow diagram  and the median follow-up time was 60.8 months . There were 32(18.2%) patients who suffered from recurrence , and 15(8.5%) patients died. The 3-year RFS and OS rates were 90.3% and 94.8% respectively, and the 5-year RFS and OS rates was 81.6%, and 91.0% respectively. In the validation cohort, the median age of patients was 61 years old  and the median follow-up time was 61.0 months . 42(24.0%) patients suffered from recurrence, and 19(10.9%) patients died. The 3-year RFS and OS rates were 88.0% and 93.6% respectively, the 5-year RFS and OS rates were 77.5%, and 88.9% respectively. Sex, histology, tumor location, T stage, adjuvant therapy, pathological grading, venous/perineural invasion, preoperative carcinoembryonic antigen (CEA) level, and MMR (Mismatch Repair) status in the training cohort and validation cohort are shown in Table 2 values at any possible cut-off point by log-rank test for survival and selected the optimal cut-off value according to the highest χ2 value and the minimum P value. Then, the determined optimal cut-off values were verified in the validation cohort.SLNs and NLNs in the training cohort were used to determine the optimal cut-off value using X-tile program. The program calculated associations and χχ2 = 4.4555, Fig. The optimal cut-off value of SLNs in the training cohort was 5.8 mm (χ2 = 4.5906, Fig. The optimal cut-off value of NLNs in the training cohort was 22 (In the validation cohort, SLNs was a significant prognostic biomarker for RFS P = 0.0034, Fig. , Fig. 4ANLNs was also correlated with both RFS P = 0.0451, Fig. , Fig. 1BMerged survival curves, divided via SLNs and NLNs, indicated that combined division by SLNs and NLNs could serve as a prognostic marker for patients as exhibited in Fig. Cox regression analysis was performed for RFS and OS in both cohorts. In the training cohort, univariate analysis showed that SLNs and NLNs (P = 0.048 and 0.043, respectively, Table To minimize the variation of SLNs, we took the largest short-axis diameter of all LNs as SLNs for each patient, which was measured by CT using a reproducible method and also applied in clinical practice . The expThere is incongruity in the optimal LN evaluation for stage II CRC. According to the current guideline, at least 12 lymph nodes are required to ensure accurate staging . HoweverStage migration caused by missed lymph node metastases was a prevailing theory for the association between LNs yield and survival, which was increasingly challenged by the explanation of immune response. In thisTherefore, to give a potential explanation of our findings, we hypothesized that patients with “large and decreased” LNs might be a surrogate marker for exhausted immune response resulting in inferior RFS compared with those with “small and increased” LNs among stage II colorectal cancers, which indicated that initial immunologic heterogeneity of CRC determined their distinct immune response pattern and pre-metastatic immune microenvironment.Although this study has provided a new finding, we are aware that our research has some limitations. Firstly, this study is retrospective and subject to all limitations of retrospective design. Secondly, our research is a single-center cohort study. While we performed internal cohort verification to avoid overinterpretation, multiple-cohort validation is more appropriate to verify whether the findings of this study are generalizable.In conclusion, this study explored the optimal cut-off values of SLNs and NLNs for stage II CRC patients based on the prediction of RFS. We demonstrated that SLNs and NLNs were independent prognostic factors for RFS in stage II CRC patients. Though not all statistically significant, there were similar tendency for OS. Merged survival curve indicated that stage II CRC patients with “small and increased” LNs had the best RFS while those with “large and decreased” LNs were prone to have the poorest RFS, which was the same case for OS despite no significance.Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2Supplementary Material 3


Standard modifiable risk factors (SMuRFs) increase the risk of cardiovascular events in patients with acute coronary syndrome (ACS) and are also strongly associated with obstructive sleep apnea (OSA) in a bidirectional relationship. However, the association of OSA with recurrent cardiovascular events in ACS patients based on the number of SMuRFs remains unclear. Hence, we aimed to elucidate the prognostic implication of OSA in ACS patients stratified by the number of SMuRFs.This was a post hoc analysis of the OSA-ACS study (NCT03362385), including 1927 patients admitted for ACS and undergoing portable sleep monitoring. OSA was defined as an apnea hypopnea index ≥ 15 events/h. The primary endpoint was major adverse cardiovascular and cerebrovascular event (MACCE) including cardiovascular death, myocardial infarction, stroke, hospitalization for unstable angina or heart failure, and ischemia-driven revascularization. Cox proportional hazards model and Kaplan-Meier analysis were used to investigated the relationship between OSA and subsequent cardiovascular events after patients were stratified by the number of SMuRFs.Among 1927 patients enrolled, 130 (6.7%) had no SMuRF, 1264 (65.6%) exhibited 1–2 SMuRFs and 533 (27.7%) presented 3–4 SMuRFs. With the increase of the number of SMuRFs, the proportion of OSA in ACS patients tended to increase (47.7% vs. 51.5% vs. 56.6%), but there was no significant difference between them (P = 0.08). After the stratification of ACS patients via SMuRF numbers and adjustment for confounding factors, fully adjusted Cox regression indicated that OSA increased the risk of MACCE  and ischemia-driven revascularization  in ACS patients with 3–4 SMuRFs.In hospitalized ACS patients, OSA is associated with an increased risk of MACCE and ischemia-driven revascularization among patients with 3–4 SMuRFs. Therefore, screening for OSA should be emphasized in ACS patients with 3–4 SMuRFs, and intervention trials should be prioritized in these high-risk patients.The online version contains supplementary material available at 10.1007/s11239-023-02830-w. Standard modifiable risk factors (SMuRFs), including diabetes, hypertension, hyperlipidemia, and smoking, are routinely targeted strategies for prevention and treatment of cardiovascular disease and are associated with higher risk of cardiovascular events in patients with acute coronary syndrome (ACS) –4. ObstrThis is an ancillary study of the OSA-ACS project (NCT03362385), which is a single-center, prospective, cohort study aimed at investigating the association between OSA and cardio-cerebrovascular events of ACS patients –15. In bStandard modifiable risk factors include current smoking status, hypertension, hyperlipidemia, and diabetes . HypertePortable cardiorespiratory polygraphy  was applied to record the situation of arterial oxygen saturation, nasal airflow, snoring episodes, and thoracoabdominal movements in order to study nocturnal sleep-in stabilized patients. It was considered as a valid test when the polygraph recording was satisfactory and lasted at least 3 h. Airflow absence lasting ≥ 10 s was identified as apnea that was classified into obstructive apnea with thoracoabdominal movement and central apnea without thoracoabdominal movement. Hypopnea was defined as > 30% reduction in airflow for ≥ 10 s accompanied by a > 4% decrease in arterial oxygen saturation. Apnea-hypopnea index (AHI) refers to the cumulative number of apneas and hypopneas per hour during the total recording time. All parameters of sleep study were judged by two independent sleep technologists, while in cases of contradiction, further judgement was performed by a senior sleep medicine consultant. Recruited patients were stratified into OSA group (AHI ≥ 15 events per hour) and non-OSA group (AHI < 15 events per hour). All patients received guideline-directed medical therapy and if necessary, percutaneous coronary intervention or coronary artery bypass grafting was performed. Unless contraindicated, patients discharged from the hospital were administered dual antiplatelet therapy (aspirin plus clopidogrel or ticagrelor) for at least 1 year. Referral to sleep center was offered to patients with moderate to severe sleep apnea (AHI ≥ 15), especially those with excessive daytime sleepiness, in order to achieve further evaluation and intervention.After discharge, follow-up was performed at the clinic or via telephone at 1 month, 3 months, 6 months, 1 year, and then every 6 months. Major adverse cardiovascular and cerebrovascular events (MACCE) were the primary endpoints that included cardiovascular death, ischemia-driven revascularization, myocardial infarction, hospitalization for unstable angina or heart failure, and stroke. Secondary endpoints included components of MACCE, all repeat revascularization, a composite of cardiovascular death, myocardial infarction, or ischemic stroke, and a composite consisting of cardiac events which was the components of the primary end point after excluding stroke.Quantitative data with normal distribution were described by mean ± standard deviation and compared via analysis of variance while non-normal distributed data were described by median (interquartile range) and the difference was analyzed via Kruskal-Walls test. The description of qualitative data was performed by frequency and percentage and the difference was discriminated by chi-square tests. Cox proportional hazards model was utilized to analyze the relationship between OSA and endpoint events after stratification of patients into subgroups according to the number of SMuRFs. Model covariates were determined based on clinical relevance or univariate relationships between variables and outcomes. Finally, we applied 3 models in the study: (1) unadjusted; (2) partially adjusted for age and sex; (3) fully adjusted for age (per 10-year increase), sex, body mass index, prior stroke, prior myocardial infarction, left ventricular ejection fraction (LVEF) less than 40%, systolic blood pressure (per 10 mm Hg increase), and plasma creatinine (per 10 µmol/L increase). Statistical analysis was performed via SPSS  and a two-side P value less than 0.05 was the standard of significant difference.P = 0.004), meanwhile, while with the accumulation of SMuRFs, there was an increase in body mass index (P < 0.001), waist to hip ratio (P < 0.001) and Hs-CRP (P = 0.03). Additionally, as the number of SMuRFs increased, patients with diabetes, hypertension, hyperlipidemia, and prior stroke accounted for a greater proportion and there were more current smokers and alcohol drinkers.The project included 2016 ACS patients undergoing sleep study, of whom 2058 patients completed a sleep study and acquired valid results. After further screening based on exclusion criteria, we eventually enrolled 1927 patients for the final analysis . The median AHI was 14.5 (6.6–23.1), 15.6 (7.0-28.8) and 18.0 (8.5–36.2) events per hour among patients with no SMuRF, 1–2 SMuRFs, and 3–4 SMuRFs, respectively (P = 0.001). Compared with patients without SMuRFs, patients with more SMuRFs exhibited higher oxygen desaturation index (ODI) (P = 0.004) and longer time of arterial oxygen saturation less than 90% (P < 0.001), accompanied by a reduction of nadir arterial oxygen saturation(P < 0.001). Furthermore, patients with more SMuRFs were more probable to be administered the treatment of ACEIs/ARB . Participants were also stratified by OSA to further describe their characteristics, as shown in Supplementary material, table As depicted in Table P = 0.58; Supplementary material, Figure S2). Notably, during the late follow-up period, Kaplan-Meier analysis presented a growing trend towards cumulative incidence of MACCE with increasing number of SMuRFs. Data about crude number of all events in patients stratified by the number of SMuRFs were shown in Supplementary material, Table S3.The median of follow-up for the study was 34.97 (18.67–43.23) months. Kaplan-Meier analysis elucidated no difference in the cumulative incidence of MACCE for patients with different numbers of SMuRFs  and ischemia-driven revascularization , after adjusted for age (per 10-year increase), sex, body mass index, prior stroke, prior myocardial infarction, left ventricular ejection fraction (LVEF) less than 40%, systolic blood pressure (per 10 mm Hg increase), and plasma creatinine (per 10 µmol/L increase). Data about crude number of all events in OSA or non-OSA patients overall or stratified by the number of SMuRFs were shown in Supplementary material, Table S4.The correlation between OSA and risk of cardiovascular events was analyzed by cox regression stratified by number of SMuRFs. As depicted in Tables 3In the study, we found that with the accumulation of SMuRFs, the symptom of OSA presented a more frequent incidence of apnea and hypopnea and a reduced oxygen saturation with a longer duration, which elucidated that patient with more SMuRFs had a worse situation of OSA. In hospitalized ACS patients, OSA is associated with an increased risk of MACCE and ischemia-driven revascularization among patients with 3–4 SMuRFs.Our study founded that the proportion of OSA in ACS patients exhibited an increased trend with the accumulation of SMuRFs (47.7% versus 51.5% versus 56.6%), but there was no significant difference (P = 0.08). Nonetheless, the higher AHI and ODI, the longer time of arterial oxygen saturation less than 90%, and the reduction of nadir arterial oxygen saturation elucidated the potential worsening hypoxia evoked by OSA in ACS patients with more SMuRFs. Hypoxia during sleep is one of the distinguished characteristics of OSA pathophysiological process . The repIt is acknowledged that the accumulation of SMuRFs means an increased burden that is implicated with a greater risk of cardiovascular events . In contAlthough there exists no study on the association between OSA and SMuRFs, previous literatures have probed the impact of OSA on clinical outcomes in patients with heart disease complicated by diabetes, hypertension, or hyperlipidemia . The sevIn our study, OSA was in correlation with an increased incidence of MACCE and ischemia-driven revascularization events in ACS patients with 3–4 SMuRFs after adjusting for confounding factors and hence we think OSA serves as a synergistic risk factor in ACS patients with 3–4 SMuRFs and it is necessary to screen for OSA in ACS patients, especially in whom have more SMuRFs.Although OSA is an independent risk factor for cardiovascular events in ACS patients, the effect of continuous positive airway pressure (CPAP) on secondary cardiovascular protection is controversial. Previous literature elucidated that CPAP showed no reduction in the incidence of cardiovascular events , while FThere existed some limitations in this study. First, we conducted this study in ACS patients while OSA severity was potentially overestimated in any high-risk disease setting. Second, we assessed the OSA with the portable polygraphy which possibly underestimated the severity caused by overestimation of actual sleeping time. Third, this study is based on a prospective cohort study, which inevitably affects the results due to its own disadvantages. Finally, this study was an observational, single-center study recruiting patients from China, which made it necessary to further validate the conclusion in cohort studies and limited the extension to more ethic population.In hospitalized ACS patients, OSA is associated with an increased risk of MACCE and ischemia-driven revascularization among patients with 3–4 SMuRFs. Therefore, screening for OSA should be emphasized in ACS patients with 3–4 SMuRFs, and intervention trials should be prioritized in these high-risk patients.Below is the link to the electronic supplementary material.Supplementary Material 1


It is not known whether LUM/IVA use in children can impact the progression of structural lung disease. We sought to determine the real-world impact of LUM/IVA on lung structure and function in children aged 6–11 years.Data from clinical trials of lumacaftor-ivacaftor (LUM-IVA) demonstrate improvements in lung clearance index (LCI) but not in FEV1, nutritional indices and exacerbations requiring hospitalisation.This real-world observational cohort study was conducted across four paediatric sites in Ireland over 24-months using spirometry-controlled CT scores and LCI as primary outcome measures. Children commencing LUM-/IVA as part of routine care were included. CT scans were manually scored with the PRAGMA CF scoring system and analysed using the automated bronchus-artery (BA) method. Secondary outcome measures included rate of change of ppFEVlumenA ratio) were abnormal at baseline and worsened over the course of the study. In 28 participants, the mean annual change from baseline LCI2.5 (-0.055 (-0.61 to 0.50), p = 0.85) measurements over two years were not significant. Improvements from baseline in weight , height (0.05 (0.02 to 0.09), p = 0.002) and BMI (0.09 (0.03 to 0.15) p = 0.005) z-scores were seen with LUM/IVA treatment. The mean annual change from baseline ppFEV1 (-2.45 (-4.44 to 2.54), p = 0.66) measurements over two years were not significant.Seventy-one participants were recruited to the study, 31 of whom had spirometry-controlled CT performed at baseline, and after one year and two years of LUM/IVA treatment. At two years there was a reduction from baseline in trapped air scores , but an increase from baseline in the % bronchiectasis score . There was no change in overall % disease score . Airway lumen to pulmonary artery ratios (AIn a real-world setting, the use of LUM/IVA over two years in children with CF aged 6–11 resulted in improvements in air trapping on CT but worsening in bronchiectasis scores. Our results suggest that LUM/IVA use in this age group improves air trapping but does not prevent progression of bronchiectasis over two years of treatment.The online version contains supplementary material available at 10.1186/s12931-023-02497-0. Deficient cystic fibrosis transmembrane conductance regulator (CFTR) activity in people with cystic fibrosis (CF) results in chronic airway infection and neutrophilic inflammation , leading1)), reduce pulmonary exacerbation rate and improve body mass index (BMI) in people with CF ≥ 12 years of age and homozygous for Phe508del mutation [Phe508del is the commonest CF-causing mutation . The devmutation . SubsequTo date, only a limited number of clinical trials have used chest CT as an outcome measure . A unifo1 has historically been used as a primary outcome measure in clinical trials in adults and adolescents with CF, it is a less suitable modality to study younger children with relatively well-preserved ppFEV1. Lung clearance index (LCI), derived from multiple breath washout (MBW) has been identified as a more sensitive measure of early lung disease in children with CF when compared with ppFEV1 [1 was demonstrated but a significant improvement in LCI was seen [Although ppFEVh ppFEV1 . MBW canh ppFEV1 . In the was seen . Improvewas seen .One of the key goals of respiratory management in people with CF is to slow the progression of structural lung damage seen with untreated disease, and by extension, prevent disease occurring in those yet to develop it. Previous work has demonstrated a significant correlation between spirometry controlled CT scores and LCI measurements in school aged children with CF . Our hypThis was a two-year real-world cohort study of LUM/IVA at four paediatric sites in Ireland involving children with CF aged 6–11 years and homozygous for the Phe508del mutation. Participants were recruited prior to commencing clinically prescribed LUM/IVA. Local Research Ethics Committees approved the study and informed consent and assent was obtained.2.5 averaged over two years. Secondary outcome measures were change from baseline in ppFEV1, nutritional indices and exacerbations requiring hospital admissions.Children with clinically unstable CF at the time of recruitment or those involved in clinical trials of CFTR modulators were excluded from the study. Clinical data was collected and managed in collaboration with the CF Registry Ireland (CFRI) from one year before and two years after initiation of LUM/IVA. Primary outcome measures were mean change from baseline per annum in spirometry-controlled CT scores and LCIin) and outer (Bout) bronchus diameters were divided by artery diameter to calculate Bin/A- and Bout/A-ratio and wall thickness (WT) was divided by artery diameter to calculate BWT/A-ratio [Spirometry controlled chest CT scans were performed at baseline and then annually for two years in a subgroup of study participants at one site , Crumlin). All staff involved in conducting spirometry controlled CTs were certified by the ECFS CTN lung imaging core facility , at Erasmus Medical Centre, Rotterdam. Spirometry controlled chest CT scanning protocol used the NDD EasyOne® portable spirometer  as outlined previously . CT scan/A-ratio . Airway /A-ratio . AdherenThe study’s intention was to perform multiple breath washout (MBW) testing at baseline and then six-monthly for 24 months (4 follow up measures). However, the collection of follow up LCI measurements was significantly hindered due to the COVID-19 pandemic. As a result, only a limited number of LCI measurements were taken on subjects Table . Partici2.5 results were expressed as the means of all technically acceptable trial results, with a minimum of two acceptable trials required [MBW operators were certified by the European CF Society (ECFS) Clinical Trials Network (ECFS-CTN) LCI core facility at RBHT, and all tests were centrally over-read. MBW was performed using the Exhalyzer®D  in accordance with the testing procedure detailed by the 2013 ERS/ATS consensus statement . MBW tesrequired .2.5, ppFEV1, BMI z-score, height z-score and weight z-score) were analysed using random coefficients mixed models, with fixed and random intercepts and slopes. As measurements were taken at unevenly spaced time points due to the COVID-19 pandemic, time was included in the model as a continuous variable based on study day. Study day was defined as the number of days pre/post initiation of LUM/IVA therapy. The intercepts give the mean at initiation of therapy (baseline). For participants, an interaction with time period indicator variable was included in the model, to compare the slope over time before and after initiation of LUM/IVA therapy. The number of hospitalisations per annum was compared in the period before and after index date using a Poisson regression repeated measures model including time period (before/after initiation of LUM/IVA therapy) and their interaction. The CT parameters  were also analysed using random coefficients mixed models, with fixed and random intercepts and slopes. The BA parameters were analysed using generalised linear mixed models with time, generation and their interaction as fixed effects and generation and lobe as random effects. An autoregressive (AR (1)) structure was fitted to the variance covariance matrix for all repeated measures models. As all of the comparisons that were made were planned, no corrections were applied for multiple comparisons.Each of the continuous responses  had ultra-low dose CT thorax at baseline, year one and year two Table . There w2.5 , p = 0.85) measurements among participants were not significant. Regarding secondary clinical outcomes on the 71 participants, there was a significant change over time in BMI z-scores (0.09 (0.03 to 0.15), p = 0.005), weight z-scores  and height z-scores (0.05 (0.02 to 0.09), p = 0.002) among participants. The mean annual change from baseline in ppFEV1 (-2.45 (-4.44 to 2.54), p = 0.66) measurements among participants was not significant.The data for all remaining clinical outcomes are presented as scatter plots along with the predicted modelled change in clinical outcomes over the study period in Fig. The mean number of hospitalisations per annum was numerically lower after treatment with LUM/IVA, but this was not statistically significant (1.18 (0.85–1.65) versus 0.87 (0.59 to 1.28), p = 0.06).In this study, while the degree of air trapping on CT imaging improved over the two years, bronchiectasis progressed and baseline abnormalities in the ratio of bronchial lumen to artery diameter worsened over the course of the study. We did not detect a significant change in LCI, likely due to the small number of measurements collected on participants. As expected, our study demonstrated improvements in nutritional and growth parameters in children aged 6–11 years with CF treated with LUM/IVA. Similar to the clinical trial in children of this age , we noteImprovements in end organ function with LUM/IVA are modest compared to the improvements noted with previous (Ivacaftor in gating mutations)  and subsIn relation to the impact of LUM/IVA on structural lung disease as measured by CT scores, there are no similar studies to date in children with CF aged 6–11 years. Real-world studies have demonstrated improvements in CT scores in association with Ivacaftor treatment in children aged six years or older with CF and the G511D mutation –31. Morein/A-ratio alone over two years, indicates an improvement in mucociliary clearance and thinning of the airway wall as described in a recent study [We did observe improvements in trapped air scores over two years, but despite this saw a progression of bronchiectasis. This may suggest that LUM/IVA has some effect in relieving small airway obstruction caused by inflammation in small airways but is unable to prevent the worsening of bronchiectasis caused by established or persistent infection and associated inflammation. In order to corroborate our findings, we examined BA dimensions throughout the segmental generations and found widening of the airways of our participants. This method has been shown to correlate well with bronchiectasis detected by PRAGMA CF scoring . The incnt study . The BA nt study  and the nt study . CFTR mont study . Therefo1, but did show an improvement of 0.55 units in LCI values at twelve months among 49 participants over six years of age [1 in those with impaired lung function at baseline but not in those with preserved lung function [1 over one year [1. As this is a cohort of children with well-preserved lung function, our ability to detect significant changes in ppFEV1 was likely limited.While clinical trials have reported improvements in pulmonary function with LUM/IVA in different age groups , 20; thes of age . Other rfunction , 43. In one year . Similar2.5 in children aged 6–11 years with CF over two years. The low numbers, natural variability in disease severity and relatively small effect size suggest that any effect of LUM/IVA has on ventilation inhomogeneity may be insufficient to be detected in a clinical population this size. Most participants only had MBW measurements taken at one or two time points at varying intervals after commencement of LUM/IVA rather than the prespecified four time points over two years as originally planned .Below is the link to the electronic supplementary material.Supplementary Material 1


Diabetic kidney disease (DKD) has mainly been considered as a glomerular disease. Our previous study showed that the progression of DKD was highly correlated with the dysfunction of renal proximal tubular cells. Fermented Cordyceps sinensis (CS), a substitute for natural CS, is a prominent herb widely used in China, and has exhibited excellent efficacy on DKD. However, the underlying mechanisms remain poorly understood.The database analysis was used to identify the main therapeutic targets and pathways of CS involved in DKD treatment. Next, the protective effects of fermented CS on high glucose  induced HK-2 cell injury was validated through cell proliferation and apoptosis assay, including CCK-8, EdU and TUNEL. Finally, quantitative real‑time PCR (qRT-PCR) and western blotting were used to verify key target genes.Our results revealed that 9 main targets  were recognized as key therapeutic targets with excellent binding affinity screened by database analysis and molecular docking. The biological processes were identified by Gene Ontology (GO) enrichment, which appeared mainly involved in the positive regulation of cell proliferation as well as the negative regulation of apoptosis. The verification experiments in vitro revealed that fermented CS significantly attenuated the HG-induced cytotoxicity and apoptosis, and promoted the proliferation of HK-2 cells. Moreover, fermented CS significantly downregulated the expressions of Bax, Caspase-3, VEGFA, P-AKT and P-ERK, and upregulated the expression of PTEN compared with that of HG group.Our results demonstrate that the fermented CS has nephroprotective effects significantly, which functions via promoting proliferation and inhibiting apoptosis of renal proximal tubular cells, likely by targeting Caspase-3, Bax, VEGFA and PTEN. Furthermore, AKT and ERK signaling pathway may be the critical mechanisms underlying the efficacy of fermented CS in DKD treatment.The online version contains supplementary material available at 10.1186/s12906-023-03901-4. Diabetic kidney disease (DKD), is the main cause of end-stage renal failure worldwide. It is a chronic microvascular complication of diabetes mellitus (DM), occurring in 20%–40% of patients with diabetes mellitus . HoweverCordyceps sinensis (CS) is one of the well-known herbs in traditional Chinese medicines (TCM). It is a unique compound naturally formed from the CS (Berk.) Sacc. parasitic on moth larvae and the moth larval bodies. It has protective effects on cardiovascular, liver and endocrine system –9. HowevIn the present study, integrative database analysis and experimental verification were used to reveal the underlying renoprotective mechanism of fermented CS in DKD therapy. Firstly, the potential targets of CS and DKD were predicted by screening various databases. Then the candidate targets and potential mechanisms of fermented CS in the treatment of DKD were identified using functional network and enrichment analysis. Finally, molecular docking and cell experiments were employed to verify these findings.http://tcmspw.com/tcmsp.php). The filtering thresholds of bioactive ingredients were oral bioavailability (OB) ≥ 30% and drug likeness (DL) ≥ 0.18. At the same time, the targets corresponding to active ingredients from CS were mainly gathered from five databases: Pubchem, pharmmaper, Swiss Target Prediction, STITCH and TCMSP.The bioactive ingredients of CS were obtained from the TCMSP (https://www.genecards.org/), OMIM database (https://www.omim.org/) and DisGeNET (https://www.disgenet.org/), CTD, TTD, DRUGBANK. Then, target genes for DKD were obtained by merging the database search results and deleting duplicate targets. Noteworthy, all the targets names were selected by Homo Saipan species and put into the UniProt Knowledgebase (http://www.uniprot.org/) to normalize the gene information. Next, the potential targets of CS mapped to DKD-related genes to further reveal the therapeutic role and candidate targets of CS in DKD.The genes related to DKD were gathered from six existing databases: GeneCards database (http://string-db.org/). Among them, the data of intersection target protein was carried on for constructing the protein–protein interaction (PPI) network diagram using Cytoscape version 3.6.1. Also, the topological features of every node in the interaction network was analyzed to further extract hub genes in the network, which the targets with degree > twofold median were selected as the hub genes. Ultimately, a core subnetwork was built for the ensuing analysis.The protein interaction information of candidate targets was obtained from the String database (https://david.ncifcrf.gov/) to perform GO functional analysis. Notably, a P value less than 0.05 was set as the screening threshold. Then, the top 10 biological process meeting the criterion were displayed as the bubble chart.In order to further reveal the underlying mechanism of CS in DKD therapy, the overlapping targets were imported into the functional annotation tool of Database for Annotation, Visualization and Integrated Discovery (DAVID) 6.8 (http://www.pdb.org/). The binding affinity of compound-target relationship were verified by Autodock Vina software, a freely available open-source packages. The binding models of the docking affinity in the top 5 pairs was visualized by PyMol2.3.0 software and Discovery Studio3.5 software.Molecular docking was applied to explore the predicted binding modes of the screened candidate targets and compounds of CS. The crystal structures of target proteins were downloaded from RCSB Protein Data Bank , 30 mM high glucose (HG) or 30 mM high glucose added to 50 μg/mL fermented Cordyceps sinensis (HG + CS) for 48 h.HK-2, a proximal tubular cell line derived from normal human kidney  was cultured in RPMI 1640 medium supplemented with 11.1 mM D-glucose, 10% fetal calf serum , 100 U/ml penicillin and 100 μ g/ ml streptomycin  at 37℃, 5% COAntibodies used in this study include: antibodies against Bax , Caspase-3 , VEGFA , total ERK , phosphorylated ERK , total AKT , phosphorylated AKT , total JNK , phosphorylated JNK , and GAPDH . All secondary antibodies  were from Jackson ImmunoResearch Laboratories Inc. . Unless indicated, other reagents were from Sigma .The fermented Cordyceps sinensis (Cs‑C‑Q80) is produced by a medicinal strain isolated and extracted from natural CS with artificial fermentation processing. It has similar chemical composition and pharmacological activities to the wild Cordyceps sinensis. It contains nucleosides, amino acids and mannitol, as listed in the standard of the pharmacopoeia of the people’s Republic of China (2015 Edition) . The fer4 cells/cm2. Cells were treated with MG, HG or HG supplemented with fermented CS at various concentrations . At 24, 48, 72 h after the treatment, 10 µL of Cell Counting Kit-8 solution  was added to each well, and the cells were incubated in a humidified CO2 incubator at 37℃ for additional 3 h. Optical density values (OD) at wavelength 450 nm were measured with a microplate reader . The cell survival rate was calculated according to the manufacturers’ instructions.CCK-8 assay was performed to evaluate cell viability. HK-2 cells were cultivated in 96-well plates at a density of 3.0 × 10The proliferation of HK-2 cells was determined by a 5-ethynyl-2-deoxyuridine (EdU) labeling/detection kit  according to the manufacturer's recommendations. HK-2 cells were seeded into 6 well plates containing sterile glass coverslips and stimulated with MG, HG or HG + fermented CS (50 μg/mL) for 48 h. Nuclei were counterstained with DAPI. Immunofluorescence images were viewed and captured using a Leica microscope (at a magnification of 20 or 40), and the ratio of EdU-positive cells (with red fluorescence) to DAPI-stained cells (with blue fluorescence) were used to assess the proliferation activity of HK-2 cells.TUNEL assay was used to analyse the effects of fermented CS on HG induced apoptosis of HK-2 cells. Cells were cultured overnight in 6-well plates and were then exposed to MG, HG or HG + fermented CS (50 μg/mL), respectively. After 48 h, the apoptotic cells were observed under the fluorescence microscope. The apoptosis rate of HK-2 cells was calculated according to the manufacturer’s instructions. Analysis was performed following at least three independent experiments.‑∆∆Ct method. Each experiment was repeated three times to ensure amplification integrity.The mRNA expression of target genes was measured by quantitative real‑time PCR (qRT-PCR). Total RNA was extracted from cultured HK-2 cells with TRNzol-A + RNA isolation reagent (TIANGEN) according to the manufacturer’s instructions. Reverse transcription was conducted with 1 μ g of total RNA and RevertAid First Strand cDNA Synthesis Kit (Fermentas). To detect the mRNAs of target genes, oligonucleotide primers were used as shown in Table Protein levels were measured by Western blotting. Treated HK-2 cells were harvested and collected as cell pellets, and lysed in ice-cold lysis buffer to extract the protein. Their total protein content was measured by bicinchoninic acid assay (Sigma) according to the manufacturer’s instructions. An equal amount of protein from each cell group was processed for SDS-PAGE and electrotransferred to PVDF membranes (Bio-Rad Inc.). Then the PVDF membranes were incubated in blocking buffer with 5% non-fat milk in TBST buffer  at room temperature for 1 h, and then probed overnight at 4 °C with corresponding primary antibodies. It’s worth noting that all the blots were cut prior to hybridisation with primary antibodies to simultaneously incubate different antibodies of similar molecular weight. The membranes were then rinsed with TBST for 3 times, and then incubated with conjugated secondary antibody  at room temperature for 1 h. Antibody-antigen complexes were visualized with Alpha chemiluminescent gel imaging system FluorChem E , and were analyzed quantitatively by densitometry with Image J software . The relative density of immunoreactive bands was normalized against GAPDH.p < 0.05 was considered statistically significant.Experiments were carried out at least in triplicate and data were expressed as mean ± SEM. All data were subjected to statistical analysis using SPSS  19.0 software (from IBM). GraphPad Prism 8.0.1 software was used to draw statistical charts. Student's t-tests was used to compare differences between groups while multiple statistic comparisons were analyzed by one-way analysis of variance (ANOVA) followed by post hoc analysis. A difference with Firstly, using standard conditions with the thresholds of OB ≥ 30% and DL ≥ 0.18 properties, we found a totally of 7 bioactive components of CS via the TCMSP databases, which were Cholesteryl palmitate, Linoleyl acetate, Arachidonic acid, Beta sitosterol, Cerevisterol, Peroxyergosterol and CLR . The proliferation of cells treated with fermented CS restored gradually. This protective effect was significant in the HG + fermented CS group at the concentration of 50 μg/mL at 48 h (p < 0.01), which was selected as the optimal intervention concentration and intervention time of fermented CS for subsequent experimentations.The CCK8 assay was performed to examine the cytotoxic effects of HG and possible protective effects of fermented CS on HK-2 cells. The HK-2 cells were treated with different concentrations of fermented CS  in a HG environment. Additionally, the MG group and HG group were used as control groups. As shown in Fig. p < 0.05, Fig. The EdU assay was also used to assess HK-2 cells proliferation. The results showed that the EdU-positive cells were dramatically reduced in HG-treated cells compared with MG group, while fermented CS treatment increased EdU-positive cells . On the contrary, fermented CS treatment significantly reduced both the expression and the translation levels of VEGF , resulting in a tremendous clinical and economic burden , 16. It vivo , 20. GivTraditional Chinese medicine (TCM) has been recognized to possess satisfactory efficacy in treating DKD as a complementary and alternative medicine . CS has Considering the multicomponent and multitarget characteristics of CS, we used a network pharmacology approach, to investigate the underlying mechanism of CS in DKD therapy. It was predicted that CS could regulate cell proliferation or apoptosis via 9 targets . Based on the results of network pharmacology analysis, cell experiments were used to validate the predicted mechanisms. In the present study, we examined HG-induced toxicity in proximal renal tubular cells in vitro. TUNEL and EdU experiments revealed that fermented CS produced a reversal of apoptosis and proliferation-promoting effect on HK-2 cells induced by HG, suggesting that fermented CS may be effective in the treatment of DKD.Many evidences suggested that the activation of caspase triggers the apoptotic process in various cells , 31. In PTEN is a negative regulator of PI3K/AKT signal pathway, which could specifically block the phosphorylation of PI3K on AKT, and inhibit the activity of AKT, exerting the anti-fibrosis effects . UpregulIt is reported that under the hyperglycaemic state of diabetes, renal tissues are hypoxic, leading to high expression of VEGF and HIF-1 . Recent MAPK family activation is a key modulator of various kinds of cells in the progression of kidney, including tubular epithelial cells. Wei et al. . demonstThere are serval limitations in our research. Firstly, the lack of animal model makes it difficult to completely elucidate the protective efficacy of fermented CS on DKD progression, especially renal proximal tubule cells injury. Secondly, further investigations are needed to reveal the upstream mechanism of the anti-apoptosis effects of fermented CS in DKD. Thirdly, though network pharmacological analysis showed that CS might alleviate DKD through MMP9 and JNK pathways, there was no significant difference between the HG group and the HG + CS group. Therefore, further validation is required with other types of kidney cells, like podocyte. Despite these limitations, no previous studies have investigated the anti-apoptotic effects of fermented CS on renal proximal tubular cells in DKD. Fortunately, the database analysis has become an assessment tool to provide comprehensive insights into the underlying mechanisms of drugs, and further point out directions for our further research.In the current study, we investigated that fermented CS ameliorated cytotoxicity and apoptosis of renal proximal tubular cells induced by HG, and database analysis uncovered the multi-component, multi-target, and multi-pathway potential mechanism underlying the action of fermented CS in DKD. Further, our results illustrated that fermented CS treatment might inhibit the cell apoptosis and improve proliferation through AKT and ERK signaling pathway. Thus, fermented CS as a promising and candidate agent in treatments of DKD, our study provides a new idea for DKD therapy and proposes a robust theoretical basis for the clinical application. However, further study is required to confirm the beneficial effects of fermented CS in vivo as well as more in-depth study of molecular mechanism.Additional file 1: Table S1. The putative targets of CS.Additional file 2: Table S2. DKD related targets.Additional file 3: Table S3. Information for overlapped targets after PPI analysis.


Staphylococcus aureus ST398 can cause diseases in several different animals. In this study we analyzed ten S. aureus ST398 previously collected in three different reservoirs in Portugal . Strains tested against sixteen antibiotics, by disk diffusion or minimum inhibitory concentration, showed decreased susceptibility to benzylpenicillin  and to erythromycin with an iMLSB phenotype (nine strains), and susceptibility to cefoxitin . All strains from aquaculture belonged to the same spa type, t2383, whereas strains from the dolphin and humans belonged to spa type t571. A more detailed analysis using single nucleotide polymorphisms (SNPs)-based tree and a heat map, showed that all strains from aquaculture origin were highly related with each other and the strains from dolphin and humans were more distinct, although they were very similar in ARG, VF and MGE content. Mutations F3I and A100V in glpT gene and D278E and E291D in murA gene were identified in nine fosfomycin susceptible strains. The blaZ gene was also detected in six of the seven animal strains. The study of the genetic environment of erm(T)-type (found in nine S. aureus strains) allowed the identification of MGE (rep13-type plasmids and IS431R-type), presumably involved in the mobilization of this gene. All strains showed genes encoding efflux pumps from major facilitator superfamily , ATP-binding cassettes  and multidrug and toxic compound extrusion  families, all associated to decreased susceptibility to antibiotics/disinfectants. Moreover, genes related with tolerance to heavy metals (cadD), and several VF  were also identified. Insertion sequences, prophages, and plasmids made up the mobilome, some of them associated with ARG, VF and genes related with tolerance to heavy metals. This study highlights that S. aureus ST398 can be a reservoir of several ARG, heavy metals resistance genes and VF, which are essential in the adaption and survival of the bacterium in the different environments and an active agent in its dissemination. It makes an important contribution to understanding the extent of the spread of antimicrobial resistance, as well as the virulome, mobilome and resistome of this dangerous lineage. Infectis (MSSA) . Previous (MSSA) . In facts (MSSA) , with ans (MSSA) . Howevers (MSSA) , not onls (MSSA)  and pig s (MSSA)  uncover n death) . On the n death) . Closer us ST398 .spa type, making it suitable for this type of studies  gives a more complete and discriminative information than traditional methods, like Multilocus Sequence Typing (MLST) and  studies . Aquacul2.2.1.S. aureus ST398 previously collected in three different reservoirs in Portugal: humans , gilthead seabream from aquaculture  and a dolphin  disk diffusion  for the following antibiotics: cefoxitin , ciprofloxacin , levofloxacin , moxifloxacin , rifampicin , mupirocin , and fusidic acid ; (2) minimum inhibitory concentration (MIC) through E-test®  or in-house broth microdilution for the following antibiotics: daptomycin (0.016–256 μg/ml), linezolid (0.5–2 μg/ml), teicoplanin (0.016–256 μg/ml), and vancomycin (0.016–256 μg/ml); (3) E-test® for benzylpenicillin (0.016–256 μg/ml); and (4) VITEK® 2  for the following antibiotics: erythromycin (1–8 μg/ml), tetracycline (0.5–2 μg/ml), tigecycline (0.25–1 μg/ml), and fosfomycin (8–32 μg/ml). VITEK® 2 was also used to detect inducible clindamycin resistance. All antibiotic susceptibility tests were performed and interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST 2022) guidelines2.3.Genomic DNA was extracted with MagNA Pure 96 Instrument  and quantified by Qubit Fluorometric Quantitation , according to manufacturer’s instructions. Libraries from 1 ng of genomic DNA were prepared, in two different sets, using the dual-indexed Nextera XT Illumina library preparation, before cluster generation and paired-end sequencing (2 × 150 bp) on a NextSeq 550 Illumina platform , according to manufacturer’s instructions.2.4.de novo using CLC Genomics Workbench version 21.0.3 , with default parameters, as previously described , according to quality criteria (limit = 0.05), and assembled escribed . Online escribed ], predicescribed ] and spayper 1.0 ], investyper 1.0 ], viruleyper 1.0 ], plasmiyper 1.0 ], and otyper 1.0 , SCCmecFyper 1.0 ], and toyper 1.0 ]. The Coyper 1.0 ] was alsyper 1.0 . All anaed genes .2.5.The genomes of the ten strains included in this study were deposited in GenBank under BioProject number PRJNA795413 and accession numbers JAKFBA000000000 (INSaAq36), JAKFAZ000000000 (INSaAq61), JAKFAY000000000 (INSaAq69), JAKFAX000000000 (INSaAq83), JAKFAW000000000 (INSaAq134), JAKFAV000000000 (INSaAq156), JAKFAU000000000 (LV31741/11), JAKFAT000000000 (INSa869), JAKFAS000000000 (INSa910) and JAKFAR000000000 (INSa934). More information about number of reads/bases/contigs, consensus length, average coverage and contigs N50 is available in 3.S. aureus ST398 strains studied, from different reservoirs, are very similar to each other, not only regarding ARG and VF, but also MGE, demonstrating the importance of the comparative study of different compartments. In S. aureus ST398 from aquaculture belong to the same spa type, t2383, already isolated in food-producing animals  and in humans, associated with an outbreak in a residential care facility , and from the strains recovered from the dolphin and gilthead seabreams  and the erm(T)-type gene. The genetic environment of erm(T)-type gene in all the positive strains (erm(T)-type gene, the genetic environment was composed by a rep gene (involved in replication), a gene encoding a metalloregulator ArsR/SmtB family transcription factor , cadD gene (that encodes for cadmium resistance transporter CadD) and ermCL gene (that regulates the expression of erm genes); downstream the gene, a plasmid truncated replication protein was identified (S. aureus ST398 recovered from a dolphin (LV31741/11) that presented an IS431R upstream the rep gene. All erm(T)-type genes were predicted to be located in plasmids, that may have played a role in the transmission between the different host species and environments. Six of the seven benzylpenicillin resistant strains with animal origin were positive for blaZ gene (except for INSAq83 isolated from muscle of gilthead seabream) and just one was predicted to be located in a plasmid (INSaAq156 isolated from gills of gilthead seabream). When analyzing the contigs that contained blaZ gene, we verified that the only difference between the strains from the gilthead seabream (excepting for INSaAq156) and the dolphin was the presence of phage’s genes in strains from aquaculture origin, namely Staphy_3MRA for INSaAq36, INSaAq61 and INSaAq134, and Staphy_53 for INSaAq69  as “critically/highly important antimicrobials” and commonly used in humans, aquaculture settings and veterinary medicine . In our  strains , was theentified . The onlINSaAq69 . The exalaZ gene  allow uspression . All strS. aureus studied (excepting INSa934 from human origin) presented mutations F3I and A100V in glpT gene and D278E and E291D in murA gene , all were susceptible to fosfomycin  as described by others , ATP-binding cassette  and multidrug and toxic compound extrusion  families, not only responsible for the decreased susceptibility to antibiotics/disinfectants, but also tolerance to heavy metals. These genes were all predicted to be in the chromosome.Excepting for one S. aureus ST398 studied and comprised genes related to host immune evasion (scn and chp), exoenzymes (aur) and toxins  production and adherence (eno-type). Genes scn and chp (located upstream scn gene) compose the immune evasion cluster (IEC) from type C , responsible for preventing opsonophagocytosis and killing of S. aureus by neutrophils  gene, conferring methicillin and tetracycline resistance, respectively, due to the use of broad-spectrum cephalosporins and tetracycline antibiotics in food producing animals. Posteriorly, this lineage was reintroduced in humans, followed by the reacquisition of φSa3 prophage, which usually harbors the genes encoding IEC. However, the boundaries between animal and human CC398 lineages are fading, with recent studies detecting livestock-associated MRSA (LA-MRSA) isolated in humans and positive for IEC genes; these observations may also indicate that the high evolutionary rate of MRSA in terms of virulence and genome content may cause the emergence and spread of more human-adapted strains with more virulent characteristics, which may be happening in MSSA as well -type gene found in this study. This acquisition of new genetic material allows bacteria to adapt and survive in different environments and to the selective pressures exerted by the use of antibiotics , as well as the WGS as a high-throughput technology, makes an important contribution to the scientific community and clinical practitioners to understand the extent of the spread of AMR, and the virulome, mobilome and resistome of this dangerous bacterium. The results obtained can help to recognize ways to break transmission routes and prevent the spread of S. aureus ST398 in various reservoirs, apparently related or not. We also show that aquaculture has received very little attention when compared to terrestrial animals, however it may pose a potential danger to human health, demonstrated here in relation to the spread and/or acquisition of clinically relevant bacterial determinants.To our knowledge no other country reported the presence of the The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/VS performed microbiological and molecular experiments, data analysis namely bioinformatics, and wrote the original draft of the manuscript. VM performed preliminary bioinformatics analysis. NB acquired laboratory data. EF acquired laboratory data and performed microbiological experiments. LC acquired laboratory data. MC acquired funding, conceived, and designed the study, supervised investigation, validated data analysis, reviewed and edited the manuscript. VS, VM, NB, EF, LC, and MC reviewed the manuscript and approved the final version. All authors contributed to the article and approved the submitted version.VS has her Ph.D. fellowship granted by FCT (Fundação para a Ciência e a Tecnologia) with the reference SFRH/BD/133100/2017 co-financed by European Social Fund and the Operational Program for Human Capital (POCH), Portugal. This work was financial supported with funding from FCT/MCTES (UIDB/00211/2020) through national funds.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.


Calves were separated from dams immediately after birth and fed commercial colostrum and milk replacer until euthanasia at 30 h post-initial colostrum feeding. The microbiota of all samples was assessed using 16S rRNA gene sequencing and qPCR. Calf serum was subjected to multiplex quantification of 15 bovine cytokines and chemokines. Our results indicated that the hoof, eye, liver, lung, nasal cavity, and vagina of newborn calves were colonized by site-specific microbiota, whose community structure differed from the ruminal-associated communities . The ruminal fluid microbial community was the only one that differed by treatment (p < 0.01). However, differences (p < 0.05) by treatment were detected in microbial richness (vagina); diversity ; composition at the phylum and genus level ; and in total bacterial abundance (eye and vagina). From serum cytokines evaluated, concentration of chemokine IP-10 was greater (p = 0.02) in VTM calves compared to control calves. Overall, our results suggest that upon birth, the whole-body of newborn calves are colonized by relatively rich, diverse, and site-specific bacterial communities. Noticeable differences were observed in ruminal, vaginal, and ocular microbiota of newborn calves in response to prenatal VTM supplementation. These findings can derive future hypotheses regarding the initial microbial colonization of different body sites, and on maternal micronutrient consumption as a factor that may influence early life microbial colonization.Early life microbial colonization and factors affecting colonization patterns are gaining interest due to recent developments suggesting that early life microbiome may play a role in Developmental Origins of Health and Disease. In cattle, limited information exists on the early microbial colonization of anatomical sites involved in bovine health beyond the gastrointestinal tract. Here, we investigated 1) the initial microbial colonization of seven different anatomical locations in newborn calves and 2) whether these early life microbial communities and 3) serum cytokine profiles are influenced by prenatal vitamin and mineral (VTM) supplementation. Samples were collected from the hoof, liver, lung, nasal cavity, eye, rumen (tissue and fluid), and vagina of beef calves that were born from dams that either received or did not receive VTM supplementation throughout gestation ( The global cattle industry is continuously facing complex and multifaceted challenges. These challenges include: 1) increasing antimicrobial resistance, 2) growing concerns over the ecological footprint of meat and milk production, and most importantly, 3) meeting the elevated consumer demand for beef, which is projected to increase as the global population increases. Therefore, it is essential that the cattle industry continues to work toward improving feed utilization, reproductive efficiency, and host resilience against infectious diseases, while also reducing the environmental impact of beef and dairy production. Host genetic selection has been a primary target for improving animal health and productivity over the last several decades resulting in tremendous progress in both dairy and beef cattle production systems. However, only recently has the microbiota that colonize various mucosal surfaces of cattle become a target for manipulation/engineering to improve animal health and production .The microbial communities residing within the respiratory , gastroiin utero. This, coupled with rodent studies which demonstrate that fetal metabolic and nervous system development is impacted by the maternal microbiota during pregnancy  supplementation during pregnancy is an active area of research to identify the link between maternal nutrition and developmental programming of offspring in both humans  and cattIn the present study, we evaluated whether the bacterial communities colonizing seven different anatomical sites in newborn calves including the hoof, liver, lung, nasal cavity, ocular surface, rumen, and vagina are influenced by maternal VTM supplementation throughout the entire gestation. These anatomic sites were selected considering the importance of the microbiome in each of these sites to bovine health and productivity. We also compared the bacterial communities from these different anatomical locations to provide a holistic view of the whole-body biogeography of the bacterial microbiota in the newborn calf. Finally, blood cytokine profiles were compared between VTM and control calves.All experimental procedures were approved by the North Dakota State University  Institutional Animal Care and Use Committee (protocol ID: A21047).n = 7); or the basal diet supplemented with a vitamin and mineral supplement . The VTM supplement was provided by top dressing 113 g of a loose mineral product  on top of a total mixed ration (TMR) at the time of feeding  of female sexed semen from a single sire. On day 35 post-AI, transrectal ultrasonography was used to determine pregnancy establishment; and this was repeated on day 65 post-Al to confirm that fetuses were female. Later in gestation, heifers were transported to the NDSU Beef Cattle Research Complex  where they were group-housed and fed individually using the Insentec BV feeding system . The basal diet (dry matter basis) was composed of 25% corn silage, 66% alfalfa hay, 4% modified corn DDGS, and 5% corn-based premix at 17.5% crude protein and fed for Heifers calved in group pens and calves were immediately removed from the dam without nursing and moved to individual pens. Within 2 h of birth, calves were fed 1.5 L of commercial colostrum replacer that contained 150 g of IgG  and was delivered using an esophageal tube. Calves were transported (3 km) from the BCRC to the ANPC and housed indoors and in clean individual pens. Calves were fed 1.9 L of milk replacer  via esophageal feeder at 12 and 24 h after the initial feeding. At 30 h after initial colostrum feeding, calves were euthanized by captive-bolt stunning followed by exsanguination. All sample collections described below were done immediately after euthanasia. Of note, given the logistic challenges associated with calf euthanasia, necropsy, and sampling immediately after birth, as well as unpredictable timing of calving, euthanasia was performed 30 h after initial colostrum feeding. To remove the confounding effects of dam colostrum and/or milk and milk composition, and microbial transfer from the dam to calf via nursing and direct contact, all calves were separated from the dam immediately after birth and housed individually and fed with colostrum and milk replacer.Samples from the hoof and vagina  were colImmediately after euthanasia, the calf was dissected for sample collection. Ruminal fluid was collected from the undeveloped rumen using a sterile 20 mL Luer-Lok syringe with a sterile 18-gage needle and was immediately transferred into 50-mL Falcon tubes and placed on dry ice. A small portion of the ruminal tissue  was dissected and placed in a sterile microfuge tube and placed on dry ice. The liver and lung were removed, and tissue samples were obtained using a sterile scalpel that was disinfected with an alcohol prep pad between each sample. Tissue samples were placed into individual sterile petri dishes and rinsed with sterile phosphate-buffered saline (PBS) before being aliquoted into sterile microfuge tubes and 1.0 mL stocks of brain heart infusion (BHI) broth containing 20% glycerol. Samples were then placed on dry ice, transferred to the laboratory, and stored at −80°C until DNA extraction.g for 5 min. Up to 400 μL of supernatant was removed and placed in a new sterile tube. Then 400 μL of 100% ethanol was added and vortexed. All subsequent steps were followed according to the DNeasy Blood and Tissue Kit protocol. Of note, this blood and tissue kit coupled with the enzymatic lysis and bead beating method was used to effectively lyse cells and maximize the DNA extraction from both Gram-positive and Gram-negative species for bacterial community analysis  according to manufacturer instructions with minor modifications. Briefly, the cotton swab tips were submerged in individual aliquots of enzymatic lysis buffer mixture until thoroughly soaked. The enzymatic lysis buffer , also contained 100 mg/mL lysozyme and 25,000 U/mL mutanolysin. The cotton bud was removed from the swab handle using sterilized tweezers and placed back into the enzymatic lysis buffer tube. The samples were then thoroughly vortexed to ensure that the entire cotton swab was submerged and then incubated for 1 h at 37°C at an agitation rate of 800 rpm. Following this incubation, 25 μL of proteinase K and 400 μL of Buffer AL (without added ethanol) were added to the samples and vortexed. The samples were then incubated for 30 min at 56°C with agitation at 800 rpm. Approximately 400 mg of 0.1 mm zirconia-silica beads were then added to the samples and placed in a FastPrep-24 Classic bead beater  and mechanically lysed at 6.0 m/s for 40 s. Samples were then centrifuged at 13,000 × analysis .g for 5 min. The supernatant was transferred into a sterile microfuge tube and the remaining steps were performed according to manufacturer instructions.DNA extraction of the ruminal fluid was performed using the Qiagen DNeasy PowerLyzer PowerSoil kit  following the manufacturer’s instructions with modifications previously described . DNA wasTo address the possibility of contamination at the time of sample collection, negative control swabs were collected. A sterile cotton tipped swab (Puritan) was exposed to the ambient air of the tissue collection room and swirled around to capture airborne contaminants. Additionally, a sterile cotton tipped swab was used to sample the scalpel blade after it had been cleaned and disinfected with ethanol to collect any contaminants surviving the sterilization process between sample collections. DNA from these negative controls were extracted and subjected to 16S rRNA gene sequencing. To account for any contamination introduced during the DNA extraction process, sterile molecular biology grade water  was included as a negative extraction control when a fresh kit was opened. Additionally, a sterile cotton-tipped swab and sterile water were included in an extraction to account for any DNA originating from the cotton tip or wooden handle of the swab. These control samples were included in the qPCR and 16S rRNA gene sequencing.Amplification and sequencing of the 16S rRNA gene was performed as described previously . A totalBurkholderia-Caballeronia-Paraburkholderia) (Primer sequences were removed from the 16S rRNA gene sequences using Cutadapt v. 4.1 . Reads wolderia)  and not olderia) . The numolderia)  and vegaolderia) . To acco2–108 gene copies) were generated using the pDrive cloning vector (Qiagen Inc.) containing the PCR product from the 16S rRNA gene. All qPCRs were performed in duplicate with standards (1 μL) and no-template controls (1 μL of nuclease-free H2O), as well as a positive control . A melt curve analysis was performed following qPCR amplification to ensure that only target genes were amplified. The copy number was normalized to 30 ng of input DNA.Quantitative PCR (qPCR) was used to assess the number of 16S rRNA gene copies in each sample using the method we described previously . Briefly™ 200 system  by Eve Technologies Corp. . Fifteen markers were simultaneously measured using the Eve Technologies Bovine Cytokine 15-Plex Discovery Assay  according to the manufacturer’s protocol. The 15-plex assay consisted of IFNγ, IL-1α, IL-1β, IL-4, IL-6, IL-8, IL-10, IL-17A, IL-36RA, IP-10, MCP-1, MIP-1α, MIP-1β, TNFα, and VEGF-A. Assay sensitivities of these markers range from 0.05–66.51 pg./mL for the 15-plex. Of note, selection of the list of cytokines tested was mainly based on the availability of the maximum number of cytokines in a Discovery Assay specific to bovines. The list encompasses both pro and anti-inflammatory cytokines and chemokines. Individual analyte sensitivity values are available in the MilliporeSigma MILLIPLEX MAP protocol.Blood samples were collected from each calf at the time of exsanguination using 10.0 mL serum vacutainer tubes , and the serum was separated by centrifugation at 1,500 x g for 20 min at 4°C. Serum was then aliquoted into individual microfuge tubes and stored at −20°C until processing. A 120 μL aliquot of serum was subjected to multiplexed quantification of 15 bovine cytokines and chemokines. The multiplexing analysis was performed using the Luminexp-values for multiple comparisons. The number of observed ASVs, Shannon diversity index and the estimated bacterial abundance by qPCR among the eight different sample types were compared using the generalized linear mixed model estimation procedure (PROC GLIMMIX) LSMEANS statement. . The Shapiro–Wilk test was used to determine whether a dataset follows a normal distribution. Differentially abundant genera between the two groups of prenatal supplementation groups were identified using MaAsLin 2 v. 1.10.0  with vegan in R. Pairwise comparisons of the Bray-Curtis dissimilarities between sample types and treatment groups were done using the pairwiseAdonis v. 0.4 R package with the Benjamini-Hochberg procedure used to correct . 1.10.0  in R. OnAfter processing and quality filtering, the average number of sequences per sample were as follows: 185,261 ± 10,017 (SEM) (hoof), 89,852 ± 10,847 (liver), 369,539 ± 42,475 (lung), 254,352 ± 32,171 , 264, 921 ± 20,423 (ocular), 269,858 ± 16,906 , 400,929 ± 30,425  and 223,692 ± 15,009  . From th2 = 0.10; p = 0.188) (p = 0.098) to have reduced inverse Simpson’s diversity compared to control calves whose dam did not receive VTM supplementation.Overall, the epithelial surface of neonatal calf hooves were colonized by a relatively diverse and rich microbial community –G. Commu= 0.188) , richnes= 0.188)  or Shann= 0.188)  of the hp > 0.1) between the two groups of calves , liver abscesses, and infectious bovine keratoconjunctivitis (IBK) were among the top 10 most relatively abundant genera in the hoof microbiota (p = 0.224)  .Staphylococcus (41.6%), Corynebacterium (6.1%), Streptococcus (4.6%), Bacillus (4.2%), and Acinetobacter (3.9%) were the most relatively abundant genera in the liver microbiota. Microbial community structure   as well as the relative abundance of phyla  , richnesof phyla  and genep > 0.1). The nasal cavity was harbored by the respiratory pathogen-containing-genera Mannheimia and Moraxella, but their relative abundance did not differ between the VTM and control calves (p > 0.05). A total of 18 different phyla was detected with Firmicutes (68.7%), Proteobacteria (20%), and Actinobacteriota (7.4%) accounting for more than 95% of the total nasal microbiota. Streptococcus (39.5%) and Staphylococcus (11.1%) were the most dominant genera.A diverse and unique microbial community was detected in the nasal cavity and lung tissue of the newborn calves . The nasp ≥ 0.21). However, the relative abundance of the phylum Proteobacteria was lower  . The top four bacterial genera present in the ruminal fluid were Streptococcus, Escherichia-Shigella, Klebsiella, and Lactobacillus, respectively, (Escherichia-Shigella (p = 0.0002) was reduced in VTM calves compared to control calves (There were only 97 (± 5) total observed ASVs present in the ruminal fluid which were assigned to 7 bacterial phyla. Firmicutes (81.1%) and Proteobacteria (18.8%) accounted for nearly all (99.9%) of the 16S rRNA gene sequences. A significant effect of prenatal VTM supplementation on the microbial community structure of the ruminal fluid microbiota was detected (= 0.002) . Diversictively, . The rell calves . The abul calves  was not 2R = 0.11; p = 0.106; p = 0.116; p = 0.238; p ≤ 0.027; p ≤ 0.002) relative abundance of Actinobacteriota and Bacteroidota phyla compared to the control calves. The top four genera present in the ruminal tissue samples included Streptococcus, Klebsiella, Escherichia-Shigella, and Clostridium sensu stricto 1  while Escherichia-Shigella (p = 0.0009) was reduced in in VTM calves  in the VTM calves, and no differentially abundant genera between the two groups were detected. The total bacterial concentration in the ocular samples was lower in the VTM calves  and diverse microbial community dominated by the phyla Firmicutes (79.7%), Proteobacteria (17.1%), and Actinobacteriota (7.0%). Calves born from VTM supplemented dams had greater microbial diversity  did not differ between groups (p = 0.031) was greater   and diverse (26 different phyla) microbial community. While microbial community structure  .All the top 40 bacterial genera and their abundance in all 8 sample types are provided in 2 ≥ 0.12, p ≤ 0.003). The structure of the respiratory  tract microbiota was similar to that of ocular, vaginal, and ruminal fluid microbiota (p > 0.11) . Firmicutes, Actinobacteriota, and Proteobacteria were the dominant phyla across all sample types, but their relative abundance varied by location  . The oculocation . The rumlocation .Corynebacterium, Macrococcus, Facklamia, and Peptostreptococcus compared to all other sample types (p < 0.05). Streptococcus and Clostridium sensu stricto 7 were more relatively abundant in the ocular microbiota and the liver tissue-associated microbiota contained a greater relative abundance of Staphylococcus spp. The genus Klebsiella and Escherichia Shigella were more abundant in ruminal tissue, ruminal fluid, and vaginal microbiota.Fifteen genera that were differentially abundant between different sampling sites are presented in Acinetobacter, Bibersteinia, Escherichia-Shigella, Klebsiella, and Streptococcus genera were more abundantly present in nasal, ocular, ruminal fluid, ruminal tissue, and vaginal samples. Six ASVs within the Aerococcaceae family and Facklamia genus were exclusively present in ocular samples. Overall, most of these abundant ASVs were absent or only sparsely present in liver tissue.According to the heatmap of the 100 most abundant ASVs , there was considerable inter-individual variation in both the prevalence and abundance of the major taxa present in the hoof, liver, lung, nasal, ocular, ruminal fluid, ruminal tissue, and vaginal samples. No single taxon was identified as being the most abundant between all 8 sampling locations. Several ASVs within the p = 0.02) while IL-4 (p = 0.056) and IL-17A (p = 0.087) tended to be reduced in VTM calves compared to control calves.Among the 15 tested serum cytokines , concentrations of three cytokines were divergent in response to prenatal VTM supplementation . The conin utero microbial colonization  between the VTM and control groups  (Moraxella (BRD and IBK), and Fusobacterium  . Interestingly, we identified several bacterial genera that include pathogens such as ca; BRD) , Moraxelmatitis)  in the hmatitis)  and humamatitis) .Lactobacillus, or potentially pathogenic genera including Moraxella and Mannheimia, in almost all anatomical sites highlight that there may be transfer of microbes between different body sites. The same bacterial species present in multiple body sites in calves/cattle may indicate site-specific functions and adaptation. Such body site-specific genome adaptations have been reported in Lactobacillus spp. , diet, and environment are involved in shaping the respiratory , gut Oh and reprlus spp. . ShotgunNutritional supplementation including VTM supplementation during pregnancy  and mateAlthough maternal VTM supplementation during pregnancy had minimal impact on the nasal, lung, liver, and hoof microbiota of the neonatal calf, the ruminal, vaginal, and ocular microbiota appeared to be affected. The respiratory , ocular, vaginal, and ruminal fluid microbiota were most similar while the hoof and liver microbiota were most dissimilar to the other microbiota. Some genera, such as those that include pathogens associated with BRD, liver abscesses, IBK, and digital dermatitis were present in almost all anatomical sites evaluated. Overall, our study indicates that the gut, hoof, ocular, liver, respiratory, and reproductive sites of newborn calves are colonized by a diverse, dynamic, and site-specific microbiota, and that prenatal VTM supplementation may influence the gut, vaginal and ocular microbiota of newborns, and have minimal influence on blood cytokine profiles. These findings are important for directing future research on developing maternal nutrition and microbiome targeted approaches to improve offspring microbiome development, feed efficiency and disease resilience.Raw sequence data are available from the NCBI Sequence Read Archive under BioProject accession PRJNA890307. Other data that supports the findings of this study are presented within the paper and its The animal study was reviewed and approved by All experimental procedures were approved by the North Dakota State University  Institutional Animal Care and Use Committee (protocol ID: A21047).SA and CRD: conceiving the idea, designing the study, and providing supervision.CRD, KKS, JLH, FB, and ABM: cattle management. SA, CRD, KCS, JLH, KAB, JDK, and FB: animal care and sample collections. KNS and SML: sample processing. DBH, KEG, SA, and SML: data processing, bioinformatics, and statistical analysis. SML and SA: manuscript writing. SML, SA, DBH, KEG, CRD, and KCS: manuscript review, editing, and finalizing. All authors contributed to the article and approved the submitted version.Microbiological work presented in this study was financially supported by the North Dakota Agricultural Experiment Station as part of a start-up package for S.A. Animal procurement and management portion of the project was supported by the North Dakota State Board of Agricultural Research and Education (SBARE), the Central Grasslands Research and Extension Center, and the ND Agriculture Experiment Station.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.