France is a low prevalence country for colistin resistance. Molecular and epidemiological events contributing to the emergence of resistance to colistin, one of the 'last-resort' antibiotics to treat multidrug-resistant Gram-negative infections, are important to investigate.Klebsiella pneumoniae (CRKP) in hospitals in Marseille, France, and to molecularly characterise clinical isolates.This retrospective (2014 to 2017) observational study aimed to identify risk factors associated with acquisition of colistin-resistant K. pneumoniae respectively. Whole-genome-sequences (WGS) of CRKP were compared with 6,412 K. pneumoniae genomes available at the National Center for Biotechnology Information (NCBI).To identify risk factors for CRKP, a matched-case–control (1:2) study was performed in two groups of patients with CRKP or colistin-susceptible K. pneumoniae genomes in NCBI revealed that in chromosomal genes likely playing a role in colistin resistance, a subset of five specific mutations were significantly associated with ST307 (p < 0.001).Multivariate analysis identified male sex and contact with a patient carrying a CRKP as significant independent factors (p < 0.05) for CRKP acquisition, but not colistin administration. WGS of nine of 14 CRKP clinical isolates belonged to the same sequence type (ST)307. These isolates were from patients who had been hospitalised in the same wards, suggesting an outbreak. Comparison of the corresponding strains’ WGS to A ST307 CRKP clone was identified in this study, with specific chromosomal mutations in genes potentially implicated in colistin resistance. ST307 might have a propensity to be or become resistant to colistin, however confirming this requires further investigations. Klebsiella pneumoniae [mcr) is concerning [K. pneumoniae, a study between 2015 and 2017, concerning 18 European countries estimated an overall colistin resistance rate of 5.4% [Since the mid-2000s, carbapenem resistance has led to the revival of colistin, a polymyxin, as a last resort antibiotic  to treatncerning . For K.  of 5.4% . Other s K. pneumoniae, some investigations point to other risk factors in patients, such as previous carbapenemase-producing K. pneumoniae colonisation, corticosteroid administration and prior hospitalisations. The aforementioned risk factors were mainly assessed in Greece [In the literature, previous use of colistin has been reported as a risk factor for colistin resistance. Nevertheless, colistin-resistant bacteria have also been isolated from people who had not previously been treated with this drug  and, forn Greece  and Italn Greece , where tn Greece . In our K. pneumoniae sequence type (ST)307 was first reported in a clinical case in 2009 in Pakistan but subsequent findings indicated that this ST might have already emerged in the mid-1990s [K. pneumoniae carbapenemase; KPC) and it has even replaced the multidrug-resistant international ST258 in some countries such as Canada and Italy [Klebsiella pneumoniae isolates in France found the emergence of several high-risk clones in the hospital environment including ST307 [K. pneumoniae STs reported to be colistin-resistant, those belonging to clonal complexes (CC)258  and CC15 have most frequently been identified [The id-1990s . ST307 hnd Italy . In 2018ng ST307 . Among sng ST307 ,13,14. Oentified .K. pneumoniae (CRKP) isolated from patients hospitalised in Marseille university hospitals between February 2014 and May 2017, to molecularly characterise the strains, and to determine risk factors associated with CRKP acquisition. Because strains of ST307 dominated among the CRKP identified, these were also the object of further focus and epidemiological investigations.The aim of our work was to find colistin-resistant This retrospective study was conducted on patients who had been hospitalised in Marseille university hospitals, France, from February 2014 to August 2017. These four hospitals  have a total of 3,700 beds.K. pneumoniae isolates that are susceptible to less than three families of antibiotics included in our routine antibiotic testing panel, or upon specific request. We selected all K. pneumoniae strains (one per patient) for which a colistin minimum inhibitory concentration (MIC) was > 2 µg/mL (performed by E-test or microdilution method) according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommendations [Our laboratory carries out the microbiological analysis of specimens collected in the four hospitals. Colistin susceptibility testing is performed on ndations . Antimicndations .K. pneumoniae (CSKP) strains isolated during the same study period. In this case–control analysis, controls were matched to cases (2:1) by age, type of ward  and type of sample. Comorbidities/conditions analysed were as follows: organ transplant recipient, chronic renal disease, haematologic or solid organ cancer (excluded if remission > 5 years), chronic pulmonary disease, diabetes mellitus, splenectomy, cerebrovascular disease, liver cirrhosis and heart disease. Modified Charlson scores ≥ 3 [Cases were defined as patients for whom a CRKP had been isolated at least once in one sample, thus including patients colonised and/or infected with CRKP. Patients' clinical histories were obtained from their electronic medical records. Each case was included in the study only once, i.e. at the time of the first CRKP isolation. In order to assess the risk factors for CRKP carriage, cases were compared to control patients colonised or infected with colistin-susceptible ores ≥ 3  were calThe selected variables were compared by the chi-squared test, Fisher’s exact test and the Student’s t-test as appropriate. Univariate analysis was two-sided and a significant p value was chosen at p < 0.05. Multivariate analysis was performed by conditional logistic regression, and only the risk factors with a p value < 0.05 in univariate analysis were used for the multivariate analysis. Statistical analysis was performed using the SPSS software ).K. pneumoniae strains were sequenced with the MiSeq Technology  with a 2 × 250 paired-end run strategy using the Nextera Mate Pair sample prep kit (Illumina). Genome assembly was done using the A5-miseq software (http://sourceforge.net/projects/ngopt/) and annotated with Prokka (https://github.com/tseemann/prokka). Plasmid replicons, virulence and resistance genes were sought with Abricate. Genome files were deposited in the National Center for Biotechnology Information (NCBI) database under the Bioproject number PRJNA520974 (Supplementary Table S1).Genomic DNAs of http://bigsdb.pasteur.fr/klebsiella/klebsiella.html). The wzi locus type was also identified with information from the BIGsdb database. The pangenome of K. pneumoniae isolates was determined using Roary [https://cge.cbs.dtu.dk/services/CSIPhylogeny). An average of 10 SNP (range: 0–30) was chosen as cut-off to define isolates of a same clone as previously described [Multilocus sequence typing (MLST) analysis was performed in silico to determine the ST of collected strains using the Bacterial Isolate Genome Sequence Database (BIGSdb) database (ng Roary . We usedescribed .K. pneumoniae MGH78578 (GenBank accession number: CP000647). The deleterious effect of detected mutations on the function of proteins was investigated using Protein Variation Effect Analyzer . Moreover, we investigated the frequency of the mutations found in PmrA/B, CrrA/B [AcrR/S [K. pneumoniae available on NCBI (last upgrade April 2019) by Basic Local Alignment Search Tool (BLAST) protein (blastp) using as threshold expect (E)-value 10e−5. Next we performed MLST using PubMLST typing schemes by local BLAST on all K. pneumoniae genomes available in NCBI to compare the frequency of different STs in this database.Sequences of proteins involved in colistin resistance were compared with the colistin-susceptible (MIC = 2 mg/L)  referenc, CrrA/B  and AcrR [AcrR/S -23 in alThis study was qualified as an internal study not involving the human person, as it was conducted on the basis of data collected as part of the individual therapeutic or medical follow-up of patients, by the personnel providing this follow-up and for their exclusive use. It was approved by the sites' institutional review boards with a waiver of informed consent and registered under number RGPD/Ap-Hm 2020–02.K. pneumoniae isolated in our laboratory. During this period, 374 determinations of colistin MIC were performed. The median age of the 22 patients was 69 years (range: 53–76) and 18 were males (sex ratio = 4.5). Four strains were isolated in 2014, eight in 2015, seven in 2016 and three in 2017. Strains were recovered from five respiratory, six urine, and five stool samples as well as one blood culture, one bone biopsy, two liquid punctures and two cutaneous swabs. Colistin MIC varied from 4 to > 64 µg/mL (Supplementary Tables S2 and S3).From February 2014 to August 2017, 22 patients carried a CRKP isolate among the 5,304 patients who had at least one The 22 CRKP cases were matched by age, type of ward and sample type with 44 controls carrying a CSKP isolated during the same study period. Univariate analysis of the two groups identified as significant risk factors male sex, length of hospital stay, recent bacterial infection, previous isolation of an ESBL or a carbapenemase producing bacteria and contact with cases carrying a CRKP . FinallyIn the multivariate analysis, male sex (p < 0.05) and contact with a case carrying a CRKP (p < 0.01) remained the only independent factors for CRKP acquisition . Supplementary Table S4).In our study, only five patients had previously received colistin. Three cases with CRKP received at least intravenous colistin, while two patients with CSKP received only aerosolised colistin (K. pneumoniae infection.Twelve cases among the 22 in the CRKP group were infected vs 21 of 44 patients in the control group (p = 0.862). Mortality rate reached 18% (4/22) in the CRKP group vs 25% (11/44) in the CSKP group (p = 0.770). In the CRKP group, one death was attributable to We compared cases with a CRKP infection to those who were just colonised by CRKP. The length of stay in hospital  as well as the delay before the first positive CRKP sample  were significantly shorter in the infected group than in the colonised group. However, while the delay in the colonised group was based on 10 cases, only five of them had tested negative for CRKP at admission. The remainder had not been tested at that time, but were nevertheless used to estimate delay, under the assumption that testing was not performed, due to absence of risk factors requiring this. No other significant difference was observed between the two groups. No specific ST was associated with infection or colonisation.Supplementary Tables S2 and S3.As this work was a retrospective study and all laboratory strains are not permanently stored, only 14 of the 22 CRKP isolates from the microbiology laboratory were available for WGS analysis. Genomes sizes ranged from 5,518,326 bp to 5,912,443 bp with a percentage of GC bases ranging from 56.3 to 57.3%. Genomes were assembled in 74 to 603 contigs with a 19 to 55 coverage. The main features of the different genomes are reported in K. pneumoniae strains, nine belonged to ST307, followed by two to ST15, two to ST101 and one to ST322. Antibiotic susceptibility testing was performed on the 14 sequenced strains (Supplementary Tables S2 and S3).Of 14 colistin-resistant Resistance rate to each antibiotic is presented in OXA-1, blaSHV-28, aac(3)-IIa, aac(6’)Ib-cr, qnrB1, tetAbla, and catB4 genes (Supplementary Tables S2 and S3). All but two (KP5CR and KP8CR) of these strains carried a OXA-48bla. The two ST15 CRKP Ib-cr, aadA2, aph(3’)-Ia, sulI, dfrAbla and catB4. One ST15 strain (KP6CR) was also carrying DHA-1, aac(3)-IIa, qnrB1, qnrB4, mph(A)bla genes. The two CRKP ST101 (KP11CR and KP16CR) had a OXA-1, blaOXA-9, blaSHV-15, blaCTX-M-15, aac(3)-IIa, dfrA, tetDbla and catB4 genes. The strain KP11CR also carried OXA-48aac(6’)Ib, bla genes. The strain KP16CR was also positive for TEM-1, sulII, tetAbla. Finally, the ST322 strain (KP15CR), which was susceptible to all antibiotic but cyclins, was carrying a tetB and a SHV-11bla gene.The nine ST307 CRKP carried The pangenome of the 14 isolates recovered 7,897 genes, including 4,136 genes belonging to the core genome and 3,761 to the accessory genome . A phyloOXA48bla gene that was present in the other ST307 strains.Interestingly, five of the nine ST307 cases  met at different times between 2015 and 2016 in the same ward, i.e. intensive care unit (ICU)1 and ICU2 , one ST2975 and one genome with an unknown ST . The frequency of the five mutations was strongly associated with the ST307 . Unfortunately, colistin susceptibility data were mostly lacking from the metadata of these genomes, preventing any insight into any potential association with colistin resistance.Five isolates (35.7%) had mutations in the analysis . We alsoSupplementary Table S5). A total of 288 genomes (4.5%) had mutations leading to presence of both L213M and T246A in the PmrB protein.Taken one by one, the frequency of each mutation in the 6,412 genomes was as follows: 3.2% (n = 204) of A41T in PmrA, 69.8%  of C68S in CrrB and 3.3% (n = 209) of S76R in AcrS . In terms of the occurrence of each ST or CC among the 6,412 genomes in GenBank, CC258 was most prevalent , followed by ST11 , ST15 , ST101  and ST307   . Such anSupplementary Table S6), though we did not identify colistin administration as a risk factor for CRKP acquisition. It should be nevertheless noted, that in our study, only five patients received colistin (two in the CSKP group vs three in the CRKP group), resulting in relatively weak statistical power.The results of our case–control analysis confirmed those of other studies . In two of these (ST322 and ST15), the mgrB gene was inactivated by an insertion sequence (IS10) and by a mutation leading to a stop codon respectively, while in another (ST307), a substitution occurred at position 53 of PmrA (Resistance to colistin in cr genes . All str of PmrA . RemarkaSupplementary Table S7).Unfortunately, for the moment, we were not able to clearly decipher the molecular mechanism of colistin resistance for the remaining nine strains, two of which were ST101 and seven ST307. Of the latter, it can nevertheless be noted that five  had identical mutations compared to the reference strain MGH78578, but exhibited various colistin MICs. A different level of gene expression might explain these MIC differences, and this could be explored by transcriptomic analysis in further work. The mutations present in these five ST307 isolates  were also present in all nine ST307 sequences recovered from our CRKP cases. Subsequent finding that all 192 ST307 bacterial genomes in NCBI, as at April 2019, also shared these mutations was surprising. Unfortunately, colistin susceptibility data are mostly lacking from the metadata of these genomes, preventing insight into the relation of these mutations with colistin resistance. Further work is needed to study any possible association  in Amsterdam, the Netherlands (13–16 April 2019), ST307 was reported as an emerging clone in several countries including Italy [K. pneumoniae from Kentucky, US [During the 29ng Italy ,28, Russng Italy , South Ang Italy , Spain [ng Italy  and the ng Italy . From thucky, US , three wWe previously reported a case of an osteitis caused by a ST307 CSKP  treated K. pneumoniae ST307 strain suggests, however, that different clones of ST307 have diffused in our hospital, at least one susceptible and some resistant to colistin. Finally, as seven patients carried the same clone, this could have influenced the results of risk-factor analysis if there had been a clone–host relationship, such as a propensity of the clone to infect the same type of tissue or patient, however we did not observe this in our study.This study has some limitations. The first is related to the retrospective nature of the study, which limited access to some clinical and laboratory data, such as the results from screening the patient for multidrug-resistant bacteria upon admission to hospital. The restrospective nature also impacted the availability of strains for sequencing from patients included in the study. Moreover as our initial aim was to molecularly characterise CRKP, no strains from patients in the CSKP control group for the case–control analysis were sequenced. The ST characterisation in this group might have provided more information on any relation between ST307 and the ‘colistin-resistant’ phenotype. The previous description of a carbapenemase-producing but colistin-susceptible In conclusion, this study concurs with previous ones, on risk factors for acquisition of CRKP and documents a CRKP ST307 epidemic clone in hospitals in Marseille, France, which is a country with an overall low prevalence of colistin resistance. Further work is warranted to understand why some clones have an ability to become increasingly resistant to antibiotics, by plasmid or chromosomal mechanism, as appears to be the case for CC258  and ST30


Constipation is a common problem among advanced cancer patients; however, many of them find limited effective from current therapies. Thus, we aimed to test the effect of a traditional Chinese herbal formula, modified MaZiRenWan (MZRW), by comparing with placebo among palliative cancer patients with constipation.This is a randomized, double-blind, placebo-controlled trial. Participants aged over 18 were recruited and randomized to MZRW or placebo group in addition to current prescriptions (including ongoing laxatives treatment) for two weeks. Exclusion criteria included cognitive impairment, presence of a colostomy or gastrointestinal obstruction and estimated life expectancy of less than one month. Individualized modification of MZRW was allowed according to the traditional Chinese medicine (TCM) pattern of patient. The primary outcome was the global assessment of improvement, which reflected whether the constipation had improved, remained the same or worsened.Sixty patients, with mean age 75.2 years (range 47–95 years), were randomized to MZRW or placebo group. Among the MZRW group, 59.3% (16/27) had improvement in the global assessment score, as compared with 28.6% (8/28) of the placebo group . Besides, the MZRW group had significant increase in stool frequency, and reduction in constipation severity and straining of defecation . No serious adverse event was reported due to the research medication.This pilot trial suggests modified MZRW is well-tolerated and effective for relief of constipation in patients with advance cancer. It could be considered as a potential treatment option for constipation in palliative care.Trial registration: The trial had been registered in ClinicalTrials.gov with identifier number NCT02795390 [https://clinicaltrials.gov/ct2/show/NCT02795390] on June 10, 2016.The online version contains supplementary material available at 10.1186/s13020-022-00580-0. Constipation is a common problem in advanced cancer patients with prevalence between 40 and 90% . It is aTraditional Chinese medicine (TCM) focuses on correcting maladjustments and restoring the self-regulatory ability of the human body , 7, whicFor better making use of TCM in the palliative care, further support with scientific proof is essential , 14. We https://clinicaltrials.gov/ct2/show/NCT02795390] on June 10, 2016. All participants were able to give voluntary, written and informed consent on their own before entering the trial. The consent form was provided in Additional file This was a multi-centre, double-blind, randomized, placebo-controlled trial conducted at the palliative care units of three public hospitals in Hong Kong, namely Caritas Medical Centre (CMC), Our Lady of Maryknoll Hospital (OLMH) and Hong Kong Buddhist Hospital (HKBH). The study protocol had been approved by the Kowloon West Cluster [KW/FR-16-077(98-20(TCM)] and Kowloon Central/Kowloon East Cluster [KC/KE-17-0116/ER-3] of Hospital Authority Research Ethics Committees, and the Hong Kong Baptist University [HASC/15-16/0316]. It had also been registered in ClinicalTrials.gov with identifier number NCT02795390 [Patients were age 18 years or above with a diagnosis of advanced cancer at the palliative care out-patient clinics of the three hospitals. They were eligible for this study if they had severity of constipation ≥ 3points  , PalliatAs it was a pilot study, 60 patients (30 patients per arm) were first enrolled. The results were important to calculate a sample size at a significance level of 0.025  for a larger scale clinical trial in future.Yin or Yang, while another two investigators (Cheng CW & Lam PY) were responsible to deliver the medication according to the codes and contact with patients. Chinese medicine practitioners (Mok HF & Kang YC) and patients were blinded in the whole study, and treatment assignments were not revealed until the termination of the whole study.All eligible subjects of both groups were randomly assigned to receive modified MZRW treatment or placebo control. For randomization, simple, complete, non-sequential random numbers were generated in advance by a computer program in a block of four and kept in sealed opaque envelopes. Two investigators with registered Chinese medicine practitioner licenses (Mok HF & Kang YC) were responsible to differentiate the TCM pattern of every patient and modify the core prescription by adding the herbs for deficiency of blood, Qi stagnation and moistening the dryness caused by the blood (Yin) deficiency may be helpful for advance cancer patients with constipation [Radix Astragali (HuangQi) granules 4 g  were chosen as the core prescription granules. The combined action of MZRW, which was composed of Cannabis Fructus (HuoMaRen) 14.4 g, Rhei Radix et Rhizoma (DaHuang) 7.2 g, Paeoniae Radix Alba (BaiShao) 3.6 g, Armeniacae Semen Amarum (KuXingRen) 7.2 g, Aurantii Fructus Immaturus (ZhiShi) 3.6 g and Magnoliae Officinalis Cortex (HouPo) 4.3 g, and Radix Astragali (HuangQi) 20 g could nourish Qi, moisten the intestine, promote the movement of Qi, and unblock the bowel [Rehmanniae Radix Praeparata (ShuDiHuang) 15 g and Angelicae Sinensis Radix (DangGui) 10 g for Deficiency of Blood, Ophiopogonis Radix (MaiDong) 15 g and Rehmanniae Radix (ShengDiHuang) 10 g for Deficiency of Yin, and Cistanches Herba (RouCongRong) 15 g and Achyranthis Bidentatae Radix (NiuXi) 10 g for Deficiency of Yang [MZRW group (with modification): From our previous study, a TCM prescription with the functions on replenishing deficiency, redirecting the flow of tipation . Therefohe bowel . Further of Yang . AdjustmYin and Yang) are made from dextrin (76.03%), tea essence (23.61%), gardenin (0.02%), and caramel (0.34%) to achieve packing, color, smell, taste, and texture comparable to the herbal granules [The placebos of core prescription and modifications on patterns . The severity of constipation was evaluated by an 8-point scale (0 = none to 7 = most severe). Besides, individual assessment of constipation related symptoms  were recorded using a 7-point ordinal scale  , 11.All efficacy and safety analyses were conducted according to the intention-to-treat (ITT) principle. Missing values were imputed by the last-observation-carried-forward method. The statistical analysis was performed using the Statistical Packages of Social Sciences (SPSS) for Windows version 16.0. The statistical significance was defined as two-sided p-value of < 0.05. Baseline characteristics were reported as mean (SD). Baseline differences between the groups were assessed by the Student’s t-test for normally distributed continuous variables and by non-parametric Mann–Whitney U test for non-normally distributed. For categorical variables, chi-squared test or Fisher’s exact test were used. Comparisons between groups were conducted by using an analysis of covariance (ANCOVA) with baseline as covariate.From November 2016 to August 2018, 733 patients were screened. 673 patients were excluded for the following reasons: PPS < 60%, constipation severity below 3/7, colostomy done, unable to communicate, significant impairment of liver and renal function, etc. Six subjects, three from each treatment group, withdrew from the study. Five of them withdrew after randomization without receiving any research medicine. One subject had taken one dose of research medication and withdrew due to progress of her own illness. A flow diagram was shown in Fig. For analysing those subjects with taking at least one time of research medication, 16 subjects in the MZRW group and eight subjects in the placebo group, in total, reported improvement of their symptoms. By contrast, 10 subjects in the MZRW group and 19 subjects in the placebo group claimed that symptoms remained the same. One subject from each group reported worsening of symptoms. Hence, the global assessment of improvement was 59.3% 16/27) for the MZRW group and 28.6% (8/28) in the placebo group . (Table 7 for theThe mean stool frequency of the MZRW group increased by 2.46 times/week while that of placebo group increased by 0.45 times/week (p = 0.04). Further, the MZRW group had greater improvement on the score of severity of constipation and sensation of straining when comparing with that of placebo group . There were, however, no significant differences on the changes on stool type, and sensations of incomplete evacuation, bloating, abdominal pain/cramping, nausea and passing of gas within two groups.  reported one or more adverse effects during the study. They included upper gastrointestinal symptoms, poor taste of research medications and changes of urination. All adverse effects were mild and subsided spontaneously. Table  One subjRadix Astragali (HuangQi). Several individual modifications for blood, Yin or Yang deficiency were also provided according to participants’ TCM patterns. In this pilot study, near 60% of the subjects reported global improvement in constipation after two-week treatment, and no serious adverse events was found. Moreover, modified MZRW is effective in increasing the bowel movements, and reducing the severity of constipation and the straining of defecation  among palliative cancer patients. Modified MZRW is a potential treatment option for relief of constipation in advanced cancer patients. Its efficacy and acceptance among advanced cancer patients as compared with current laxatives deserve further investigation.Constipation is a common clinical problem in patients with advanced cancer but with inadequate managed. An effective medication to relieve can greatly enhance their quality of life. MZRW and its modifications is the most frequently used formulae for clinical trials on constipation based on the systematic reviews our group conducted previously , 18. HowOpioid medications cause decrease in small and large bowel motility, increase tone in ileocaecal valve and anal sphincter. At the same time, opioid decreases electrolyte and water secretion in small bowel, and increase absorption in large bowel . TherefoPalliative care becomes a global health priority with increasing demand over the next 20 to 30 years , 24. It This pilot study was reported strictly according to the Chinese herbal medicine formula extensions of CONSORT  Statement . CertainMZRW is effective in bringing global improvement in constipation for 59.3% of patients (vs 28.6% in placebo group) with no serious adverse effects. It is more effective than placebo in increasing the number of bowel movements, and reducing the severity of constipation and the straining of defecation. MZRW is a potential treatment option for constipation in advanced cancer patients. Its long-term effectiveness and acceptance among advanced cancer patients could be further investigated in large-scale clinical trial in future.Additional file 1. Consent form.Additional file 2. Quality control reports of research medications.


Preoperative clinical factors, serum tumor markers, and PET, and CT radiomic features were analyzed. Prediction models were developed using the least absolute shrinkage and selection operator (LASSO) regression analysis. The performance of the models was evaluated and compared by the area under receiver-operator characteristic (ROC) curve (AUC) and DeLong test. The clinical utility of the models was determined via decision curve analysis (DCA). Then, a nomogram was developed based on the model with the best predictive efficiency and clinical utility and was validated using the calibration plots.A total of 315 NSCLC patients confirmed by postoperative pathology between January 2017 and June 2019 were retrospectively analyzed and randomly divided into the training (p < 0.05). A quantitative nomogram was subsequently constructed using the independently risk factors from the Combined Model. The calibration curves indicated a good consistency between the actual observations and nomogram predictions.In total, 122 SCC and 193 ADC patients were enrolled in this study. Four independent prediction models were separately developed to differentiate SCC from ADC using clinical factors-tumor markers, PET radiomics, CT radiomics, and their combination. The DeLong test and DCA showed that the Combined Model, consisting of 2 clinical factors, 2 tumor markers, 7 PET radiomics, and 3 CT radiomic parameters, held the highest predictive efficiency and clinical utility in predicting the NSCLC subtypes compared with the use of these parameters alone in both the training and validation sets (AUCs (95% CIs) = 0.932 (0.900–0.964), 0.901 (0.840–0.957), respectively)  contains supplementary material, which is available to authorized users. Non-small cell lung cancer (NSCLC) accounts for approximately 85% of lung cancer that is the most common cause of cancer-related mortality worldwide, with an estimated 1.4 million deaths each year . DiffereAn invasive biopsy for histological confirmation is commonly used in clinical practice . However18F-fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT)–based radiomics have been shown to have potential in differentiating ADC from SCC [Radiomics based on conventional medical images has been used to quantitatively assess tumor heterogeneity in more detail than visual analysis by analyzing the distribution and relationship of pixel or voxel gray levels in the lesion area , 13. 18Ffrom SCC , 15. Furfrom SCC , 17. HowHence, the aim of this study was to develop and validate a prediction model, integrating the clinical characteristics, tumor marker levels , and radWe conducted a retrospective analysis of records from patients with NSCLC who were diagnosed by curative surgical resection between January 2017 and June 2019. This retrospective study was approved by the ethics committee of Shanghai Proton and Heavy Ion Center, and the requirement for informed consent was waived. The inclusion criteria included the following: (1) ADC or SCC that was confirmed by postoperative pathology according to the 2015 World Health Organization (WHO) classification [n = 220) and validation set (n = 95). Baseline data pertaining to clinical characteristics, including gender, age, height, weight, smoking status , symptom , family history, the size, and location of lesion and the levels of serum tumor markers, including ferritin (FERR), squamous cell carcinoma antigen (SCCA), carbohydrate antigen 199 (CA 199), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cytokeratin 19 fragment antigen (CYFRA21-1), and neuron specific enolase (NSE) of each patient, were reviewed and recorded.Finally, totally 315 consecutive NSCLC patients were enrolled in this study, comprising 200 males and 115 females , as summarized in Table 18F-FDG PET/CT scans were performed on a Biograph 16 PET/CT scanner  according to standard clinical scanning protocols. All patients fasted for at least 6 h before the scan, and none had a blood glucose levels > 8.7 mmol/L. A whole-body scan was acquired approximately 1 h after intravenous administering of 5.18 MBq/kg of 18F-FDG. The CT scans were performed first  using a slice thickness of 3.0 mm and reconstructed to a 512 × 512 matrix (voxel size: 0.98 × 0.98 × 3.0 mm3). Then, PET scans were performed with 2 min in each bed, a TrueX algorithm  without filtering and smoothing was used to reconstruct the PET images. For all PET reconstructions, the matrix size was 200 × 200, resulting in anisotropic voxels of 4.07 × 4.07 × 3.0 mm3. The PET images were converted into SUV units by normalizing the activity concentration to the dosage of injected 18F-FDG and patient body weight.The max without knowing the pathology determined by consensus [18F-FDG uptake within the regions of interest, a joint reading of both the CT and PET scans was performed side by side.Tumor segmentation was performed using Inveon Research Workplace  software. Two experienced nuclear medicine physicians drew boundaries in the axial, coronal, and sagittal PET scans that were large enough to include the primary tumor to delineate the VOIs using a threshold of 40% of SUVonsensus , 22. To The radiomic features were extracted using a voxel-based methodology. First, the SUV values contained within the VOIs were relatively resampled to 64 different values to yield a limited range of values, with the goal of reducing the noise and normalizing the images . Then, thttp://www.r-project.org) software was used for the statistical analysis. A comparison between the groups was performed using an independent t test or a Mann-Whitney U test for continuous variables and Fisher’s exact test or χ2 test for categorical variables. A two-sided p < 0.05 indicated statistical significance. Intra- and inter-class correlation coefficients (ICCs) were used to evaluate the consistency and reproducibility of the intra- and inter-observer agreements of the radiomic feature extractions. An ICC > 0.75 indicated good consistency.The R  binary logistic regression with 10-fold cross-validation, which was used to select the most useful factors , 26. TheThe performance of the models was evaluated by the receiver-operator characteristic curve (ROC) analysis and compared by the DeLong test. The AUC with 95% confidence interval (CI), sensitivity, specificity, and accuracy were calculated for each model. The clinical application value of the prediction models was determined and compared through the decision curve analysis (DCA) by quantifying the net benefit to the patient under different threshold probabilities in the queue.To provide a visually quantitative tool to predict the histologic subtypes for NSCLC patients, we developed a nomogram on the basis of the prediction model with the highest AUC and clinical utility in the training set . Calibrap < 0.05). The levels of FERR, SCCA, CYFRA21-1, and NSE in SCC patients were higher than those in ADC patients (p < 0.05). There were no significant differences in patient’s weight, family history, lesion location, and levels of CA199, AFP, and CEA between the SCC and ADC groups according to the univariate analysis (p > 0.05).In total, 315 NSCLC patients comprising 122 SCC patients and 193 ADC patients were eventually enrolled in this study. The patients’ clinical characteristics and tumor markers of training set are summarized and compared in Table 18F-FDG uptake were manually segmented, and 212 radiomic features were separately extracted by the two physicians. The agreement between the two physicians was excellent . Thus, the mean measurement values of the two physicians were used for further analysis.A total of 315 regions with an increased For differentiating SCC from ADC, 4 independent prediction models  Model, PET- Radiomic (Rad) Model, CT-Rad Model, and Combined Model) were built separately on the basis of selected clinical factors-tumor markers, PET radiomic parameters, CT radiomic parameters, and the combination of above features by LASSO regression in the training set Fig. . The Prep < 0.001)  Figs.  and 4. T1) Figs. .Fig. 3ViThe performance of these 4 prediction models to discriminate SCC from ADC is shown in Fig. p < 0.05) (Table 3).The DeLong test showed that the Combined Model, which consisted of 2 clinical factors, 2 tumor markers, 7 PET radiomic parameters, and 3 CT radiomic parameters, presented the optimal discrimination and best predictive sensitivity, specificity, and accuracy among the 4 models in both the training set (AUC (95% CI) = 0.932 (0.900–0.964), sensitivity = 96.25%, specificity = 95.00%, accuracy = 84.09%) and validation set (AUC (95% CI) = 0.901 (0.840–0.957), sensitivity = 93.55%, specificity = 81.25%, accuracy = 85.95%) . The high ability to reproduce and validate radiomic studies is vital to generating sufficient and convincing scientific evidence for translating potential applications into clinical practice [In addition, since radiomic extracts information from the tumor, an appropriate tumor segmentation algorithm is important for measuring tumor image parameters . IdeallyT images . They fopractice , 36.This study also explored whether the prediction performance based on radiomics could be further improved by combining with clinical factors and tumor marker levels. The Combined Model established in the present study not only significantly improved the prediction efficiency for subtype compared to these factors alone in both the training and validation sets (AUCs = 0.932 (training set), 0.901 , respectively) but also had higher performance than previous researches –17. ThisIn addition, we generated an integrated nomogram on the basis of the Combined Model for facilitating its use in clinical practice. Clinical factors such as patient’s sex and age are recorded routinely at hospital admission. Moreover, we strongly recommend that serum tumor marker levels should be evaluated in patients who are highly suspected of having NSCLC or initially diagnosed with NSCLC, especially SCCA, CYFRA21-1. Both physicians and patients could perform a preoperative individualized prediction of the risk of ADC with this easy-to-use scoring tool, which can provide a noninvasive and accurate approach for patients who are unwilling or unable to undergo biopsy to develop more reasonable and effective treatment plans, especially the need of targeted therapy . The DCA18F-FDG uptake or small tumor size to ensure the quality of images and textural data. Small lesions are easier to be discovered in the early stage with the increasing use of imaging screening for lung cancer. Thus, a more sensitive tool that adaptively detects small tumors will be an important direction for future work. Finally, the patients with non-primary lung lesions were also excluded due to the purpose of this study. Noticeably that both primary and metastatic pulmonary nodules are very important for patients and clinical settings in the cancer center. The prediction model that widely used for lung lesions will be continually explored in future studies.Although the results were encouraging, the present study had several limitations. Firstly, the sample selection was biased in this single-center retrospective study, and a new multicenter prospective study is still necessary to be designed for the further evaluation and verification of the generalization ability of the models. Secondly, some NSCLC patients, especially ADC patients, were excluded from the radiomic analysis due to the faint In conclusion, an integrated nomogram was constructed and validated in our study, which could provide a relatively accurate, convenient, and noninvasive method for the individualized discrimination between ADC and SCC in NSCLC patients, assisting in clinical decision making for precision treatment.ESM 1(DOCX 790 kb)


Alzheimer’s disease (AD), frontotemporal dementia (FTD), and dementia with Lewy bodies (DLB) are the three major neurodegenerative dementias. In this study, we provide evidence that an alteration in extracellular vesicles (EVs) release is common across the three most common neurodegenerative dementias, AD, DLB, and FTD. Specifically, we analyzed plasma EVs in three groups of patients affected by AD, DLB, and FTD, and we found a significant reduction in EVs concentration and larger EVs size in all patient groups. We then investigated whether the loss of neurotrophic factors is also a common pathogenic mechanism among FTD, DLB, and AD, and if levels of neurotrophic factors might affect EVs release. Plasma levels of progranulin and cystatin C (CysC) were partially altered; however, taking together all variables significantly associated with the diagnostic groups only EVs size and concentration were able to distinguish patients from controls. The diagnostic performance of these two EVs parameters together (ratio) was high, with a sensitivity of 83.3% and a specificity of 86.7%, able to distinguish patients from controls but not to differentiate the different forms of dementias. Among the candidate neurotrophic factors, only CysC levels were associated with EVs concentration. Our study suggests that an alteration in the intercellular communication mediated by EVs might be a common molecular pathway underlying neurodegenerative dementias. The identification of shared disease mechanisms is of pivotal importance to develop treatments to delay disease progression. To this aim, further studies investigating plasma EVs size and concentration as early biomarkers of dementia are required. Brain abnormal protein accumulation and inclusions characterize all these neurodegenerative diseases : AD is cgression . Conversgression . In addigression . FTD-caugression ; we demogression . Brain-dgression . Moreovegression . A systegression .During progressive neurodegeneration and neuronal loss, exosomes could become the key player for neuronal communication and the crossroads of proteins: accordingly, survival of neurons could be easily affected by factors modulating exosome release and/or composition . We hypoTo test our hypothesis, herein we analyzed human plasma EVs and NTF  in dementia patients , investigating also the link between NTF and EVs.n = 30 AD, n = 30 DLB, n = 30 FTD sporadic patients and from n = 30 elderly subjects with normal cognitive function, as control group (CTRL), were analyzed. Patients were enrolled at the MAC Memory Clinic IRCCS Fatebenefratelli, Brescia, and at the Neurology 5/Neuropathology Unit, IRCCS Besta, Milan. Clinical diagnosis for probable AD, DLB, and FTD was made according to international guidelines  following the manufacturer’s protocol. Briefly, 125 μl of plasma was centrifuged at 2,000 × g for 20′ and then at 10,000 × g for 20′; supernatants were then transferred into new tubes, mixed with 62.5 μl (0.5 × plasma volume) of 0.2 μm filtered 1× phosphate-buffered saline (PBS), and then incubated with 37.5 μl  of Exosome Precipitation Reagent for 10′ at room temperature; after incubation, samples were then centrifuged at 10,000 × g for 5′. EVs pellets were resuspended in 100 μl of 0.2 μm filtered 1× PBS and stored at + 4°C or −20°C until nanoparticle tracking analysis (NTA). As a negative control, an aliquot of 125 μl of 1× PBS was processed as described above.EVs isolation was performed with Total Exosome Isolation Kit from plasma . Samples were diluted with 0.2 μm filtered 1× PBS in order to obtain an optimal range of 20–150 particles/frame. For each sample, 5 videos of 60″ duration were recorded and data were processed using NanoSight NTA Software 3.2. Post-acquisition settings were kept constant between samples. Data obtained were: particle concentration (particles/ml), average size (nm), and particle size distribution . Raw concentration data (particles/ml) obtained from NTA were normalized to obtain EVs concentrations in the human plasma sample.® Assays  following the manufacturer’s protocol. CysC plasma concentration was measured with Human Cystatin C Quantikine® ELISA kit  following the manufacturer’s protocol. All analyses were performed in duplicate. Measurements were carried out at the same study site on consecutive days, and researchers were unaware of whether the sample belonged to cases or to controls.GDNF, BDNF, and PGRN plasma concentrations were measured with Human Premixed Multiplex—Magnetic Luminexpost hoc tests, was used for the comparison across the four subject groups of the normally distributed continuous variables. Kruskal–Wallis test with Dunn’s post hoc tests was used for the group comparisons of non-normally distributed variables. Chi-square test was used to assess the association between demographic characteristic  of subjects with the four groups. A classification tree (CT)   was appln = 30, DLB n = 30, and FTD n = 30) and cognitively healthy controls (CTRL n = 30) were analyzed . Thus, the EVs preparations were enriched in exosomes. The negative control was below the detection limit, thus not interfering with the sample analysis (data not shown).EVs isolated from plasma samples of dementia patients . No significant differences were shown between patients’ groups (AD vs. DLB vs. FTD) in EVs concentration. Conversely, EVs size was significantly increased in AD, DLB, and FTD samples as compared to CTRL samples . No significant differences were shown between patients’ groups (AD vs. DLB vs. FTD) in EVs size.NTA showed a significant decrease in plasma EVs concentration in AD, DLB, and FTD samples compared to CTRL samples  . CysC levels were increased in DLB compared to CTRL . Plasma GDNF was not detectable in all samples.NTF analysis revealed that plasma levels of BDNF were not different among the four groups ; levels  to CTRL   four groups of outcome variable  and on (ii) dichotomized group variables  in order to detect the predictors that best classify subjects into CTRL or patients. The best classification was obtained using dichotomized group variables in which the average EVs size and average EVs concentration resulted to be the best predictors, i.e., the variables able to better classify the subject into CTRL or PTS. Although we considered all the socio-demographic and NTF in the classification tree, the best discriminant performance was found for EVs variables. Specifically, the EVs size alone  could classify the majority of dementia patients from controls (91.7% vs. 8.3%). Moreover, among the subjects, the ones with an EVs concentration lower than 2.3 × 10e 96.8%, . The inc9. As expected, ROC curves for AD, DLB, and FTD had a similar AUC .To estimate the diagnostic performance of these two EVs parameters (concentration and size), we performed a ROC analysis to measure the ability of the EVs concentration/size ratio to distinguish dementia patients from CTRL : an AUC p = 0.007) .Finally, in order to evaluate whether EVs size and concentration might be influenced by the NTF, we performed a linear regression analysis between these variables by adding the interaction with dichotomized (CTRL vs. PTS) groups. We found that only levels of CysC were associated with EVs concentration and in a different way between groups (group × CysC effect: = 0.007) : in CTRLExtracellular vesicles represent a new concept in the biomarker field, serving as transfer vehicles between cells of membrane and cytosolic proteins, lipids, DNA, and RNA with a wide range of regulatory functions .In this study, we provide evidence that an alteration in EVs release is common across the three most common neurodegenerative dementias, AD, DLB, and FTD and that plasma EVs dosage and size characterization might be a promising marker for dementias. The differential diagnosis between dementias was not supported by biomarkers for all patients: this could be a limitation of the study (reducing the likelihood of finding differences).9 of the EVs concentration/size ratio). As expected, this ratio could accurately distinguish patients from controls but was not able to differentiate different forms of dementias. A reduction of circulating EVs in dementia is in accordance with our previous studies on FTD and AD cellular models, demonstrating a loss of exosome/exosome-associated CysC in the genetic forms of these diseases  was high, with a sensitivity of 83.3% and a specificity of 86.7% (with a cut off point of 1.49 × 10diseases . These din vitro (primary neurons from CysC knockout mice) and in vivo (CysC transgenic mice) that CysC enhances brain-EVs secretion, resulting in a protective effect .The datasets of raw data generated for this study can be found in the Mendeley Data Repository (doi: The studies involving human participants were reviewed and approved by the Comitato Etico IRCCS San Giovanni di Dio Fatebenefratelli Brescia. The patients/participants provided their written informed consent to participate in this study.AL, RN, SB, and CS: data generation. AL, CF, LB, and RG: data analysis. RG: supervision and funding acquisition. GB and GF: clinical samples and phenotyping. AL and LB: original draft preparation for the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.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.


P = 0.000) or NDPN group (P = 0.043), while serum LCN2 levels in DPN group were higher than NDPN group (P = 0.001). Moreover, serum LCN2 levels positively correlated to TCNS scores, which reflects neuropathy severity . Multivariate stepwise regression analysis showed that BMI, triglycerides, and diastolic pressure were independently associated with serum LCN2 in DPN. Additionally, logistic regression analysis demonstrated that LCN2 (odds ratio (OR) = 1.009) and diabetes duration (OR = 1.058) were independently associated with the occurrence of DPN in T2D. Our report reveals the association of serum LCN2 with DPN in T2D. LCN2 might be used to evaluate DPN severity and serve a role in the pathogenesis of DPN.A variety of studies have demonstrated the role of lipocalin 2 (LCN2) in both diabetes and neurological disorders. Nevertheless, the relationship between LCN2 and diabetic peripheral neuropathy (DPN) needs to be elucidated in humans. Therefore, this study aimed to investigate the association of LCN2 with DPN in type 2 diabetes (T2D). A total of 207 participants with T2D and 40 participants with normal glucose tolerance (NGT) were included in this study. All participants were classified into DPN group and non-DPN (NDPN) group based on the Toronto Clinical Neuropathy Scoring (TCNS). Demographic and biochemical parameters were measured. Serum LCN2 levels were determined using an ELISA technique. Serum LCN2 levels in NGT group were lower than those in either DPN group ( Type 2 diabetes (T2D), the most common form of diabetes, has fallen into the leading causes of disability and death worldwide. The harms of T2D arise from its complications such as neuropathy, nephropathy, retinopathy, and cardiovascular disease. Diabetic peripheral neuropathy (DPN), the most prevalent and troublesome complication of T2D, affects up to 50% individuals , 2. DPN,et al. indicated that glial-derived LCN2 played an important role in the pathogenesis of DPN via PDK2-lactic acid axis in the dorsal root ganglion (DRG) in mice model , also known as 24p3 or neutrophil gelatinase-associated lipocalin, is a secreted glycoprotein in response to numerous physiological and pathological stimuli , 7, 8, 9ce model . NeverthA total of 207 participants with T2D from the Department of Endocrinology, the First Affiliated Hospital of University of Science and Technology of China (USTC) were included in this study from September 2018 to February 2021. As the control, 40 participants with normal glucose tolerance (NGT) from the Health Examination Center, the First Affiliated Hospital of USTC, were recruited from October 2018 to May 2019. NGT and T2D were diagnosed according to the 1999 World Health Organization criteria. All participants with T2D were classified into DPN group and non-DPN (NDPN) group. The diagnosis of DPN was based on the Toronto Clinical Neuropathy Scoring (TCNS) . A totalThe study was approved by the Ethics Committee of the First Affiliated Hospital of USTC and complied with the Declaration of Helsinki. Informed consents were obtained from all participants before inclusion in this study. This study was registered on the Chinese Clinical Trial Registry (ChiCTR2100046905).2. BP was tested in triplicate after at least 30 min of rest, and the average of three recordings was recorded.All participants were questioned and physical examination was done by trained doctors and nurses to obtain the information on age, sex, weight, height, blood pressure (BP), illness, and medical therapy history. The BMI was calculated as the weight/heightVenous blood samples were collected from all participants after an overnight fast of 10–12 h. Fasting blood glucose (FBG), total cholesterol (TC), triglycerides (TG), creatinine (in urine or serum) were assayed by an automatic biochemistry analyzer . Hemoglobin A1c (HbA1c) was measured by affinity chromatography with an HbA1c radiometer (Bio-Rad Laboratory Inc.). Urinary albumin was assayed using immune turbidimetry kits purchased from Northern Biotechnology Research Institute . Urine albumin creatinine ratio (UACR) was calculated as the urine albumin/urine creatinine. Fasting C-peptide (FCP) was assayed by electrochemiluminescence immunoassay (Roche Diagnostics GmbH). The estimated glomerular filtration rate was calculated by the Chronic Kidney Disease Epidemiology Collaboration equation.Serum LCN2 levels were assayed using an ELISA kit . Procedures were according to the manual instructions by kit provider.DPN was diagnosed using TCNS based on neuropathic symptoms, signs, and the presence of abnormal nerve conduction . All pars.d., skewed variables as medians with interquartile ranges, and categorical variables as frequencies. All variables were tested for normality using Kolmogorov–Smirnov test. Skewed distributed variables including TG and UACR were logarithm transformed to normality for further analyses. To compare the differences between two groups, independent t-test was performed for the normally distributed variables, and 2Χ-test for categorical variables. To compare the differences in the three groups, ANOVA was used followed by Bonferroni method. The correlation between LCN2 and TCNS score was performed by Spearman’s correlation analysis. The Pearson’s correlation analysis was used to examine the correlation between serum LCN2 and clinical parameters in DPN. Multivariate stepwise regression analysis was further performed to assess the association between serum LCN2 and clinical parameters after adjusting for potential confounders. Multiple logistic regression analysis was performed using the occurrence of DPN as a dependent variable. The confounders included the variables that had been reported to be associated with DPN. The P value less than 0.05 was considered to be statistically significant.Statistical analyses were conducted using SPSS software . Continuous variables with normal distribution were expressed as means ± P > 0.05). When compared with the NDPN group, patients in DPN group had longer diabetes durations (P = 0.008). For the other variables that might affect serum LCN2 levels including HbA1c, FBG, FCP, TC, TG, UACR, eGFR, and BP, no statistically significant differences were observed between the two groups .As shown in F = 14.864, P = 0.000; P = 0.000; NGT vs NDPN group, P = 0.043; NDPN vs DPN group, P = 0.001). The age, sex, and BMI in the three groups were matched .There were significant differences in serum LCN2 levels among NGT group (96.191 ± 28.322 ng/mL), DPN group (142.851 ± 51.195 ng/mL), and NDPN group  and TG , respectively, and negatively correlated to DBP .To examine the association of clinical parameters with serum LCN2, Pearson’s correlation analysis was performed in DPN group. Serum LCN2 was shown to be positively correlated to BMI , BMI , TG , and DBP  were shown to be independently associated with serum LCN2 levels in DPN.To further determine independent clinical parameters affecting serum LCN2, multivariate stepwise regression analysis was performed using sex, age, BMI, HbAMultiple logistic regression analysis was performed using the occurrence of DPN as a dependent variable in all participants with T2D. As shown in ob/ob mice , Local Scientific and Technological Development Project Guided by Central Government of China (grant number 2017070802D147), and Integrated Technology Application Research in Public Welfare of Anhui Province (grant number 1704f0804012).This work was funded by W W conceived and designed this study, wrote this manuscript, and participated in the data analysis. Z X, X R S, and W Z contributed to data collection and analysis. Y X and M Y X contributed to laboratory test and helped in editing the manuscript. S D Y contributed to the conception of this study, and edited the manuscript. S W X, J P W, and Z Z contributed to the study design and critical revision of the manuscript for important intellectual content. All authors read and approved the final version of the manuscript.


Endometriosis is a chronic and estrogen-dependent pelvic inflammatory disease, which may have various causes, such as oxidative stress. Dysmenorrhea, dyspareunia, and pelvic pain are well-known symptoms of endometriosis. The present clinical trial assessed the role of supplementation with antioxidant vitamins on the indices of oxidative stress as well as the severity of pain in women with endometriosis. n = 30), given vitamin C  and vitamin E  combination, or group B (n = 30), given placebo pills daily for 8 weeks.  We enrolled 60 reproductive-aged (15–45 years) women with pelvic pain in this triple-blind clinical trial. They had 1–3 stages of laparoscopic-proven endometriosis. The participants were randomized to group A , dysmenorrhea , and dyspareunia  significantly decreased in the treatment group after 8 weeks of supplementation.  Following treatment with vitamin C and vitamin E, we found a significant reduction in MDA and ROS compared with the placebo group. There was no significant decline in total antioxidant capacity after treatment. However, the severity of pelvic pain ( The present findings support the potential role of antioxidants in the management of endometriosis. The intake of vitamin C and vitamin E supplements effectively reduced dysmenorrhea severity and improved dyspareunia and severity of pelvic pain. Endometriosis (EMS) is a chronic and estrogen-dependent pelvic inflammatory disease that arises from ectopic endometrial implantation and growth outside the uterus cavity . EMS affThe cells to compensate for the ROS effects and to limit ROS production have the antioxidant system. This can balance between antioxidant defense and ROS production to inactivate ROS-induced cellular damage and repair them . Hence, Recently, the beneficial effects of antioxidant supplementation on EMS treatment have been noticed by researchers, but still, there is no convincing evidence in this regard . Data onSince adjuvant therapy by antioxidants such as vitamin C and vitamin E in EMS requires additional human clinical trials , this stIRCT201106296917N1).This randomized placebo-controlled clinical trial was carried out on 60 reproductive-aged (15–45 years) women with pelvic pain and 1–3 stages of laparoscopic-proven endometriosis referred to Sarem Hospital in northwest of Tehran city, Capital of Iran, from June to November 2017. The Iran University of Medical Sciences Ethical Committee approved this research protocol (code: IR.IUMS.REC.1394.25836), and all women signed an informed consent form, after they informed completely about study goals and methods. The trial protocol was registered in the Iranian Registry of Clinical trials (n = 30) were given a combination of vitamin C  and vitamin E  or group B (n = 30) given placebo pills  daily for 8 weeks. In this way, the name of groups (A or B) was written on two similar one-sided sheets, and then, the papers folded equally. After that, the participating women were allowed to choose one of the papers. The randomization process was performed by independent members who were not involved in the study. The vitamins and placebo tablets were not exactly similar, but were packaged in the similar boxes (each box contains 56 tablets) and then assigned special codes by an independent person who was not involved in the study. The participants, researchers, and statistician were blind about the groups. Vitamin C helps to incorporate vitamin E radical back to vitamin E recycling; it is recommended that vitamin C be included along with vitamin E supplementation [The inclusion criteria were no history of pelvic inflammatory disease, no supplement consumption during past 6 months, and no history of chronic autoimmune or metabolic/endocrine disease, cigarette smoking or tobacco and alcohol, and lack of special diet such as vegetarians. The exclusion criteria included any major side effects related to vitamins or placebo such as severe vomiting, abdominal cramps, and diarrhea with no other causes, dizziness, headache, or skin rash. The participants were randomly assigned to two groups by the simple randomization method: group A , ROS, and TAC (as primary outcomes) were measured by the enzyme-linked immunosorbent assay (ELISA) method. The participants were followed-up every two weeks by phone to ensure that the study protocol runs appropriately. Chronic pelvic pain, dysmenorrhea, and dyspareunia (as secondary outcomes) were assessed by VAS at baseline and 2, 4, 6,  and 8 weeks of intervention.t-test, paired t-test, and analysis of covariance (ANCOVA) were done. Dichotomous variables were compared by the chi-square test. SPSS ver. 16 was used to perform statistical analyses, considering p < 0.05 as statistically significant. Furthermore, intergroup comparisons were carried out by the repeated measures analysis of variance, and the Bonferroni test was used to test multiple comparisons.After 8 weeks, blood samples were drawn and processed again, as described above. Data are presented as mean ± SD or frequency and percentage. For comparing, the quantitative data-independent 2, respectively. The groups were not statistically significant differences regarding the variables mentioned above. The two groups were comparable with respect to most of their demographic parameters (µM) and ROS (5.46 ± 0.57 vs. 6.08 ± 0.38 µM) than the A group before intervention (p < 0.01), the ANCOVA test was performed for comparing MDA and ROS between two groups after intervention. The results of primary outcomes are given in p=0.002) and ROS (p < 0.001) compared to placebo intake, but, it did not change in TAC levels.The mean ± SD of age in groups A and B was 35.7 ± 5.71 and 38.03 ± 6.47 years, respectively. Also, body mass index (BMI) of women in groups A and B was 25.12 ± 4.02 and 25.54 ± 3.86 kg/mrameters . Since wp value <0.001), dyspareunia , and chronic pelvic pain  significantly decreased from baseline in group A in at least of the measurements.As secondary outcomes indicated in p value <0.001) and dyspareunia   was increased.Also, in group B, there was a significant reduction of dysmenorrhea  . In contThe intergroup comparison showed a higher significant reduction of dysmenorrhea, dyspareunia, and chronic pelvic pain VAS score in group A than group B .The role of OS in the pathophysiology of endometriosis is well demonstrated in studies suggesting that the reduction OS is a treatment option . VitaminIn this present study, we observed a significant reduction in peripheral MDA and ROS but not in TAC levels, due to a combination of vitamin C and vitamin E consumption. These vitamins did not affect TAC. Our data are consistent with previous studies. Mier-Cabrera et al. (2009) also reported a significant decrease in MDA and lipid hydroperoxides levels in women with EMS after 4 and 6 months of treatment with vitamin C and vitamin E . AnotherAlso, in the current study, a significant reduction of pain was observed on the VAS scale after 8 weeks of treatment with vitamin C and vitamin E compared with placebo. It has been suggested that the decrease in inflammation can suppress pain generating molecules.β-endorphin-like substances were increased after vitamin E injection resulting in pain relief in dysmenorrhea [Previous studies confirmed that enorrhea . Furtherenorrhea , 34. Theenorrhea .Considering the safety of vitamin E/C combination, vitamin E/C can be suggested for patients with endometriosis. Consistent with the present findings, Santanam et al. showed chronic pain alleviation using antioxidant supplementation with vitamin E/C in women suffering from endometriosis .Our result suggested that supplementation with vitamin C and vitamin E effectively reduces systemic oxidative stress indices in women with endometriosis. The present findings further support the potential role of antioxidants in the management of EMS. Hence, future large-scale trials with long follow-up durations are warranted to confirm the role of antioxidant supplementation in improving oxidative stress state and chronic pelvic pain, dysmenorrhea, and dyspareunia in EMS.


Neisseria meningitidis is the etiologic agent of meningococcal meningitis and sepsis. Initial colonization of meningococci in the upper respiratory tract epithelium is crucial for disease development. The colonization occurs in several steps and expression of type IV pili (Tfp) is essential for both attachment and microcolony formation of encapsulated bacteria. Previously, we have shown that host-derived lactate induces synchronized dispersal of meningococcal microcolonies. In this study, we demonstrated that lactate-induced dispersal is dependent on bacterial concentration but not on the quorum-sensing system autoinducer-2 or the two-component systems NarP/NarQ, PilR/PilS, NtrY/NtrX, and MisR/MisS. Further, there were no changes in expression of genes related to assembly, elongation, retraction, and modification of Tfp throughout the time course of lactate induction. By using pilT and pptB mutants, however, we found that lactate-induced dispersal was dependent on PilT retraction but not on phosphoglycerol modification of Tfp even though the PptB activity was important for preventing reaggregation postdispersal. Furthermore, protein synthesis was required for lactate-induced dispersal. Finally, we found that at a lower temperature, lactate-induced dispersal was delayed and unsynchronized, and bacteria reformed microcolonies. We conclude that lactate-induced microcolony dispersal is dependent on bacterial concentration, PilT-dependent Tfp retraction, and protein synthesis and is influenced by environmental temperature. Neisseria meningitidis is a human-restricted pathogen that is well adapted to survival in the upper respiratory tract. The nasopharyngeal epithelium is the natural reservoir for meningococci; however, bacteria can penetrate the cell barrier and cause sepsis and/or meningitis  by adding phosphoglycerol to PilE, which blocks the bundle formation and leads to dispersal   plays an important role in bacterial behavior in a density-dependent manner. The role of QS in 2 (AI-2) , 12. How to AI-2 , 13. Delhenotype .Neisseria is known to encode only four TCS . Pathogenic four TCS . The Misfour TCS –18. The our TCS ––22. Finailiation . A systeiliation .N. meningitidis can experience fluctuations in temperature during its colonization. It is transmitted through aerosol droplets to the human nasopharynx with an environmental temperature that can alternate between 32 and 34°C  with 2 mM lactate, it took 2 h to form microcolonies and, after an additional 30 min, the microcolonies dispersed in a synchronized way (luxS-deficient mutant (ΔluxS). However, the ΔluxS mutant showed no difference in timing of dispersal from that of the wild-type strain , ΔpilR (regulator of PilR/PilS), and ΔntrX (regulator of NtrY/NtrX) deletion mutants in strain FAM20 and confirmed them by PCR. Despite several attempts, we were unable to create a FAM20 ΔmisR (regulator of MisR/MisS) deletion mutant, indicating heterogeneity among strains and a possible essential role of MisR in FAM20. Therefore, we used an already existing ΔmisR mutant in the serogroup C isolate N. meningitidis L91543  aggregadisperse . In summpilE, pilD, and pilQ), elongation (pilF), retraction (pilT), and modification (pptB) of Tfp throughout the time course of lactate induction. However, no significant changes in gene expression were detected at 1, 5, 10, 20, 30, or 60 min after lactate addition .Pilus-associated factors are well known to affect clumping and aggregation of the bacteria. We next investigated the transcripts of genes related to assembly . In contrast, at 32°C, bacteria started to disperse around 1 h postinduction and then reformed microcolonies (l-lactate lanes), where the bacteria started to disperse after 1 h and after some time re-entered the aggregation phase. Similar results were found using 2 mM lactate (data not shown). Although bacteria formed aggregates at 32°C and responded to lactate, the dispersal was delayed and microcolonies reformed again after 2 to 3 h. These results indicate that not only the lactate concentrations but also the environmental temperature can influence microcolony dispersal in meningococci.Reduced temperature has been previously shown to affect protein expression of several virulence factors that enhance microcolony formation . Since tcolonies . Two reppilT-mediated retraction and protein synthesis. Our results showed that the activity of PptB was not responsible for lactate-induced dispersal, but it prevented reaggregation. Finally, we suggest that environmental temperature can regulate microcolony formation and dispersal.Understanding the mechanism behind microcolony dispersal is a major challenge in the field of meningococcal pathogenesis. Recently, we discovered that host-derived lactate induced the dispersal of meningococcal microcolonies . The ansN. meningitidis QS; the bacteria are known to produce functional QS signaling molecule AI-2, and its production is dependent on the luxS gene , NMB1030, and adhesion complex protein (ACP), involved in meningococcal aggregation, adhesion, and biofilm formation . It is tN. meningitidis depends on PilT-mediated retraction and protein synthesis for lactate-induced microcolony dispersal. To remain in a dispersed state, meningococci require the PptB activity and an optimal environmental temperature. These findings contribute to the understanding of the complex actions of N. meningitidis during microcolony dispersal. Further research may provide new targets for disease prevention.Collectively, our results suggest a hypothetical model . MeningoNeisseria meningitidis serogroup C strain FAM20 and its isogenic pilT mutant have been described previously  deletion mutant has been described  containing 1% heat-inactivated fetal bovine serum  was used in all experiments. Kanamycin was used at 50 μg/ml for the selection of mutants.The eviously . The misescribed . Prior tN. meningitidis FAM20 using homologous allelic replacement with spot transformation. High-Fidelity Phusion DNA polymerase (Thermo Fisher Scientific) was used for all PCRs. The FAM20 ΔpptB mutant was created using homologous allelic replacement after spot transformation with ΔpptB mutant chromosomal DNA  in the presence of 2 mM l-lactate . Microcolony dispersal was observed using time-lapse microscopy . Three positions were chosen per well, and images were acquired every 10 min for 6 h using a 40× objective. The images were independently evaluated by visual inspection. The aggregation phase was defined as the time from the start of incubation until dispersal of bacteria starts . The dispersal phase was defined as the time from initiation of bacterial dispersal until microcolonies were no longer visible in the images. The planktonic phase was described as dispersed single bacteria. For wild-type strain and 5 mM lactate, the dispersal was rapid and obvious and occurred from one image to another, i.e., within a 10-min time frame.7 CFU/ml. The bacterial solutions were transferred to 24-well glass-bottom plates (MatTek) and allowed to grow and form microcolonies for 3 h at 32°C or 37°C in a 5% CO2 environment in a live-cell observer . Prewarmed DMEM supplemented with sodium l-lactate  was added to microcolonies at a 1:1 ratio at final concentrations of 10 mM. During treatment with spectinomycin, the antibiotic was added at a final concentration of 100 μg/ml 30 min before the induction. Three positions were chosen per well, and images were acquired every 5 to 10 min for 4 or 5 h using a 40× objective. The aggregation phase is described as the start of incubation until the dispersal of bacteria starts. The dispersal phase is described as the initiation of bacterial dispersal until no aggregates are observed. The planktonic phase is described as dispersed single bacteria.The induction assays have been described previously . Brieflyl-lactate. At 60 min postinduction, bacteria were stained with a Live/Dead BacLight bacterial viability kit  according to the manufacturer’s protocol. The samples were analyzed by flow cytometry using an LSRFortessa (BD Biosciences) with a PI channel for dead cells and FITC for live cells. For each sample, 15,000 bacterial cells were counted, and data are presented as the percentages of dead or alive bacteria within this population. Data were analyzed with the FlowJo software.Bacteria were treated with 100 μg/ml of spectinomycin 30 min before induction with l-lactate and control DMEM were performed on FAM20 wild type as indicated above in 1-ml volumes in 2.0-ml Eppendorf tubes. Samples were collected at 1, 5, 10, 20, 30, and 60 min immediately after the addition of l-lactate or DMEM and treated with RNAprotect bacterial reagent (Qiagen). Total RNA was purified using an RNeasy plus minikit (Qiagen) according to the manufacturer’s protocol. Reverse transcription was performed with random hexamers using a SuperScript VILO Master Mix (Thermo Fisher). The resulting cDNA was amplified using LightCycler 480 SYBR green I Master mix (Roche) in a LightCycler 480 real-time PCR system. The PCR program was adapted from the manufacturer using an annealing temperature of 55°C. The 30S ribosomal protein rpsJ was used as a reference gene. The target mRNA levels in the samples were normalized to the reference gene and then compared to the value of the noninduced DMEM control sample.All of the primers used for quantitative real-time PCR analysis are listed in t tests were used to compare differences between two groups. In GraphPad Prism (version 5), analysis of variance with Bonferroni’s post hoc test was used to compare differences between multiple groups. P values below 0.05 were accepted as statistically significant.For statistical analysis in induction assays, the differences between the lengths of the dispersal phase were analyzed. In Excel, two-tailed and unpaired Student


Growing evidence suggests the relationship between obstructive sleep apnea (OSA) and venous thromboembolism (VTE). Few studies focused on VTE recurrence risk associated with OSA after anticoagulation cessation.In a prospective cohort study, patients with documented VTE, were followed for an indefinite length of time and VTE recurrence were documented and adjudicated. The primary outcome was recurrent VTE after anticoagulation discontinuation. Secondary outcomes included all-cause mortality and the clinical presentation of VTE. Univariable and multivariable analyses were performed to identify risk factors for recurrence and mortality.Among the 2109 patients with documented VTE included, 74 patients had moderate to severe OSA diagnosis confirmed by home sleep test or polysomnography. During a median follow-up of 4.8 (interquartile range 2.5–8.0) years recurrent VTE occurred in 252 patients (9 with OSA and 243 without OSA). The recurrence risk in the univariable and multivariable analysis was not increased in patients with OSA, regardless of the time of diagnosis (before or after index VTE or pooled). VTE phenotype was significantly more often PE with or without associated deep vein thrombosis in the first event and recurrence for OSA patients compared to non-OSA patients. The risk of death was not increased in the OSA population compared to non-OSA patients in multivariable analysis.In patients with OSA and VTE, the risk of all-cause mortality and VTE recurrence after anticoagulation discontinuation was not increased compared to non-OSA patients.The online version contains supplementary material available at 10.1186/s12959-021-00358-8. Venous thromboembolism (VTE), including pulmonary embolism (PE) and deep vein thrombosis (DVT), is a major health issue. PE is the most common cardiovascular cause of death after myocardial infarction and stroke  with an Obstructive sleep apnea (OSA), characterized by periodic narrowing and obstruction of the pharyngeal airway during sleep , is also, in a case-control study including 107 patients with PE and 102 patients without VTE, showed that OSA was more frequent in patients with PE and for every 10-unit rise in apnea-hypopnea index (AHI), the PE risk increased by 45% ) and median (interquartile range [IQR]); categorical variables were expressed as numbers and percentages. The student t-test was used to compare means between groups in case of normal distribution and the Mann-Whitney test in non-normal distribution. A Chi-square test or exact Fisher test was used to compare proportions as appropriate.p < 0.05) between the two groups. The second multivariable model was constructed by including (i) variables associated with increased risk of recurrence or death in univariable analysis, with p-value < 0.15 and a frequency > 3%; and (ii) variables whose distribution was significantly different between the two groups (p < 0.05). In multivariable models, risk factors were considered independent for a p-value < 0.05. All tests were two-sided. Statistical analyses were performed using R software Version 1.0.153 –© 2009–2017 RStudio, Inc.The rates of recurrent VTE were estimated using the Kaplan Meier method. An univariable Cox proportional hazard model analysis was performed to identify risk factors of recurrent VTE and death. Hazard ratio (HR) with 95% confident interval (CI) were provided. Multivariable analyses were performed using cause specific Cox models in two stages for the risk of recurrent VTE as well as for the risk of death (competitive risk) in association with OSA. The first multivariable model was constructed by including variables whose distribution was statistically different . Written informed consent was obtained from all participants before inclusion.n = 1908), a recurrent VTE (n = 352), age under 18 years old (n = 19), and without follow up (n = 64) . Demographic and clinical characteristics at inclusion are presented in Table Overall, 74 patients had OSA objectively confirmed by the home sleep test of polysomnography. Patients with OSA were older, predominantly obese or overweight, with history of cardiovascular and pulmonary disease. Sixty-three of them (85%) had a dedicated device (NIV or CPAP) for OSA. VTE was generally unprovoked, and clinical presentation was more often PE rather than DVT in OSA patients. Concomitant treatment  and anticoagulant therapy length were not significantly different between the two groups. The mean (standard deviation SD) follow-up of the overall population was 5.6 (4.2) years, 5.6 (4.2) years in the non-OSA group, and 5.4 (4.2) years in the OSA group (p = 0.38) between patients with: (i) OSA history ; (ii) OSA occurrence ); (iii) and patients without OSA ).The 5-year cumulative incidence rates of recurrent VTE were not statistically different (p = 0.33), OSA diagnosed after index VTE ; p = 0.328), AHI and nocturnal desaturation were not associated with an increased risk of recurrence ; p = 0.46) or OSA occurrence ; p = 0.24) as compared to other patients ; Recurrent VTE occurred in 252 patients during the follow-up after anticoagulation discontinuation; nine of these recurrences occurred in OSA patients and 243 in non-OSA patients (Table p = 0.023), 120 (49.4%) patients without OSA and seven patients (77.8%) patients with OSA had a recurrent VTE as PE with or without DVT (p = 0.09).The clinical presentation of index VTE and recurrent VTE were statistically different: 1117 (55%) patients without OSA and 51 (68.9%) patients with OSA had an index PE with or without DVT (p = 0.295). In univariable analysis, there was no increased risk of death in patients with a history of OSA compared to non-OSA patients ; p = 0.70) (eTable p = 0.09)  patients: 2 of the 74 OSA patients (2.7%) and 153 of 2035 non-OSA patients (7.5%)  was not significantly associated with an increased risk of VTE recurrence, nor with increased overall mortality, as well as AHI and nocturnal desaturation.Although OSA could be considered as a chronic syndrome with exposition to intermittent hypoxemia over the years before treatment and diagnosis, we considered two groups of patients, one with a history of OSA before the index VTE and one with an occurrence of OSA after the index VTE to evaluate its effect over the time. We found that OSA’s occurrence tended to increase the recurrence risk compared to OSA’s history. The effect of OSA pooled, as one variable was similar.p = 0.026). Firstly, OSA was diagnosed with HST after PE, while in the present study, most OSA diagnoses were made before index VTE. However, there was no difference statistically significant in the risk of recurrent VTE between OSA diagnosed before and after index VTE. Secondly, AHI ≥ 10/h was identified as an independent risk factor, but AHI ≥ 30/h was not. In the present study, OSA diagnosis was mainly severe OSA: the mean (SD) AHI (h-1) was higher (38.88 ± 18.4 vs 21.1 ± 20.5), the mean (SD) desaturation index (h-1) was higher (41.62 ± 35.15 vs 18.3 ± 19.7), and CPAP treatment was more frequent in OSA patients (63 of 74 (85.1%) vs 31 of 71 (43.7%)). The present study was not powered enough to evaluate CPAP effect on VTE recurrence risk but may have led to reducing the risk of recurrence as the majority of patients were treated. Some experimental studies suggested an endothelial dysfunction improvement with this treatment in OSA patients [p = 0.047). Nevertheless, recurrent VTE risk factors were not collected, and OSA was less severe, with only 12.5% of OSA patients with CPAP.Alonso-Fernández et al. evaluated the association between OSA and the risk of recurrent PE after anticoagulation discontinuation in 120 patients with a first episode of PE . OSA waspatients , 35. Xiepatients , in a prThe results of the present study are consistent with the “RIETE registry”: pre-existing OSA was not associated with recurrent VTE . The stuThe clinical presentation of index VTE was significantly different between groups; there was more index PE with or without DVT in OSA patients. Recurrence as PE with or without DVT tends to be more frequent in OSA patients without reaching statistical significance. In the general population, the proportion of PE with or without DVT and DVT alone are similar , and DVTConsistent with previous studies evaluating the risk of VTE recurrence, unprovoked VTE, older age, cancer, and male gender were associated with a higher risk of VTE recurrence , 37–39.The strengths of our study are related to (i) the prospective patient’s recruitment with documented VTE as PE, with or without DVT, or isolated DVT, that were followed-up for an extended period with a median up to 4.8 (2.5–8.0) years, with scheduled clinical reevaluation every 6 or 12 months, (ii) OSA assessment in all patients based on HST or polysomnography results including AHI, nocturnal desaturation and the characteristics of apneas, (iii) the use of predefined, validated and objective criteria for all cases of recurrent VTE and predefined criteria for assessing the cause of deaths, which were adjudicated by physicians who were not involved in patient care, (iv) the multicenter design.Several limitations should be considered. Obesity can be a confounding factor associated with VTE and OSA and might interact with its effect on recurrence. This is an observational study with data collected during a considerable period, with a random occurrence of OSA diagnosis and different sleep monitoring devices over the years. Moreover, many OSA patients included had CPAP treatment without random assignation, limiting additional evaluation of its influence and evaluating severe OSA on VTE recurrence. Sixty-four patients with OSA were excluded because on indefinite anticoagulation, the number of patients with OSA included in this study was therefore restricted. Lastly, only patients referred to the cohort centers were included in the study, they might have had more comorbidities and an index VTE with more severe clinical features than the general population with VTE. They also might have had a better prognosis due to a follow up by experienced teams with expertise in VTE, and we cannot exclude that clinical presentation have been influenced by ambulatory care of DVT.In this study, we failed to find an association between the risk of recurrent VTE, death and moderate to severe OSA after anticoagulation discontinuation. Nevertheless, larger studies are needed to evaluate OSA’s effect on VTE recurrence, given the previous evidence suggesting an association between OSA and the risk of recurrent VTE and considering the pro-coagulant state induced by intermittent hypoxemia. The effectiveness of CPAP and/or extended anticoagulation remains unknown and call for additional studies in patients with OSA and VTE.Additional file 1: eTable 1. Risk factor for recurrent VTE with OSA pooled.Additional file 2: eTable 2. Risk factors associated with mortality.Additional file 3: eTable 3. Risk factors associated with mortality with OSA pooled.


To evaluate the prognostic value of the aspartate transaminase/alanine transaminase (AST/ALT) ratio in primary non-muscle-invasive bladder cancer (NMIBC) using propensity score matching (PSM) analysis.We retrospectively collected the clinical and pathological data from 314 patients with primary NMIBC who underwent transurethral resection of bladder tumor. The full cohorts were divided into a low AST/ALT ratio group and a high AST/ALT ratio group according to the optimal cut-off value which was obtained based on the analysis of the receiver operating characteristic curve for the 3-year recurrence-free survival (RFS). After 1:1 PSM, the correlation between preoperative AST/ALT ratio and survival prognosis was evaluated by Kaplan–Meier analysis with log-rank tests. The independent prognostic factors for RFS and progression-free survival (PFS) were also analyzed.P = 0.007) and the worse pathological T stage (P < 0.001). After PSM, patients with a high AST/ALT ratio had poorer RFS and PFS than patients with a low AST/ALT ratio . In addition, multivariate Cox regression analysis indicated that preoperative AST/ALT ratio was considered as an independent prognostic factor of RFS  and PFS  in patients with primary NMIBC.The optimum cutoff value of the preoperative AST/ALT ratio was 1.40. Before PSM, a high AST/ALT ratio was correlated with the larger proportion of age > 60 years (The high AST/ALT ratio group tended to have poorer RFS and PFS than the low AST/ALT ratio group. Our results also indicated that the elevated preoperative AST/ALT ratio could be seen as a useful prognostic biomarker for predicting early disease recurrence and progression in patients with primary NMIBC. Bladder cancer is one of the most prevalent cancers in the urinary tract and the seventh most common tumor in the male population worldwide, while it drops to the 11th if both genders are considered . ApproxiAspartate transaminase (AST) and alanine transaminase (ALT) are found in the liver, heart, skeletal muscle, brain, kidney, and red blood cells, which are widely used to reflect hepatocellular damage in clinical practice. In 1957, the ratio of the serum activities of AST to ALT was first described as an important indicator for differentiating the etiology of acute hepatitis by De Ritis et al., which was also called the De Ritis ratio . The ASTThe purpose of this study was to evaluate the prognostic predictive value of the AST/ALT ratio on the survival prognosis of patients with primary NMIBC. A propensity score-matching (PSM) methodology was performed to minimize the interference of other confounding factors.We retrospectively reviewed the relevant medical records and collected clinical and pathological data of 314 patients with primary NMIBC who underwent TURBT at the Department of Urology of the First Affiliated Hospital of Nanchang University from January 2013 to May 2017. Patient inclusion criteria as following: (1) biopsy of cystoscopy and postoperative pathological outcomes were consistent in confirming non-muscle-invasive bladder urothelial carcinoma, (2) laboratory data of AST and ALT were obtained within 5 days before the first surgical treatment, and (3) adequate follow-up information is available for prognostic analysis. Exclusion Criteria: (1) any concomitant malignancy and pelvic radiation, (2) present hematological disorders and chronic liver disease within the last 3 months, (3) perioperative death. All patients obtained and signed informed consent. This study has been approved by the Institutional Ethical Review Board and the study protocol was carried out following the guidelines of the Helsinki declaration.To evaluate the optimum thresholds for the AST/ALT ratio, the receiver operating characteristic curve for 3-year recurrence-free survival (RFS) was analyzed. The optimum threshold for the AST/ALT ratio was obtained when there was a maximum Youden index value. This threshold transforms the AST/ALT rate into a dichotomous variable that divides all patients into a high AST/ALT group and a low AST/ALT group. To balance the differences of the covariates of baseline data in both the group, PSM analysis was done using a multivariable logistic regression model based on age, gender, the history of smoking, tumor size, tumor number, restaging transurethral resection of the bladder tumor (Re-TURBT), pathological stage, pathological grade, concomitant carcinoma in situ, type of intravesical therapy. Pairs of patients were derived using 1:1 greedy nearest neighbor matching within a caliper size of 0.1. Further, the clinicopathological data of the two paired groups were analyzed and the predictive value of the AST/ALT ratio in primary NMIBC was evaluated.TURBT was performed by skilled urologists at our center. Indications for Re-TURBT in patients with NMIBC remain controversial . Thus, RP < 0.05 was considered to be statistically significant.All categorical data were presented as a percentage or an absolute number, while continuous variables were presented as the median and interquartile range (IQR). The difference in clinicopathologic factors between the two groups was taken by the chi-square test or Fisher’s exact test before PSM, while the McNemar test was performed after PSM. The difference in survival was evaluated by Kaplan–Meier analysis with log-rank tests. The univariate and multivariate analyses were performed by a Cox proportional hazard model to identify the potential predictors of prognosis. PSM was performed using the MatchIt package in R software; all statistical analyses were performed using SPSS 26.0 Statistics software. A total of 314 patients newly diagnosed with NMIBC who underwent TURBT and were included in this study. The study population included 250 male and 64 female patients with a median age of 65 years (IQR 58–73 years), 137of whom had a history of smoking. 86 patients had a history of Re-TURBT. The tumor size was ≤ 3 cm in 247 (78.7%) patients, and > 3 cm in 67 (21.3%) patients. Multifocal tumors were found in 164 (52.2%) patients. Pathological T stage was Ta in 161 (51.3%) and T1 in 153 (48.7%) patients. Tumor grade was papillary urothelial neoplasms of low malignant potential in 13 (4.1%), low grade in157 (50.0%), and high grade in 144 (45.9%) patients. 29 (9.2%) patients had concomitant carcinoma in situ (CIS). 34 (10.8%) patients underwent early single instillation only and 48 (15.3%) patients were treated with postoperative adjuvant chemotherapy or BCG only, while 232 (73.9%) patients underwent combined therapy. Furthermore, 171 (54.5%) patients experienced recurrence, and 94 (29.9%) patients experienced progression after initial TURBT. The median follow-up time was 48 months (IQR 22.3–72 months).P < 0.001) , 22.0 U/L (IQR 19.0–27.0 U/L), and 1.4 (IQR 1.2–1.8), respectively. Patients who relapsed within 3 years after initial TURBT was more tend to have a high AST/ALT ratio (P ≥ 0.05). However, the pathological T stage of the high AST/ALT ratio group was worse than those of the low AST/ALT ratio group (P < 0.001), and the proportion of those aged > 60 years was also higher (P = 0.007) , and type of intravesical therapy  , tumor size (P = 0.008), Re-TURBT (P = 0.001), pathological stage (P = 0.011) and the AST/ALT ratio (P < 0.001) were related to the RFS of patients. In multivariate Cox analyses, these related prognostic factors including tumor size , tumor number , Re-TURBT , pathological stage , and the elevated AST/ALT ratio  continued independent association with RFS in multivariate Cox analysis  Fig. . The uniP < 0.001) , pathological stage (P = 0.047), pathological grade (P = 0.023), and the AST/ALT ratio (P < 0.001). According to Cox multivariate analysis, the independent prognostic indicators of RFS in primary NMIBC involved Re-TURBT , pathological grade , and the AST/ALT ratio   Fig. . In univ P = 0.02, and theThe serum levels of AST and ALT are biochemical markers widely used in clinical treatment to determine the liver function and to identify liver diseases such as viral hepatitis and alcoholic hepatitis. The AST is widely expressed in several organs, but ALT is s considered more liver-specific and enriched . Thus, pIn the present study, we find that the high AST/ALT ratio has independent predicting early disease relapse and progression value in primary NIMBC after PSM. In fact, several recent studies about the AST/ALT ratio all demonstrated that it could be considered as a significant prognostic biomarker in renal cell carcinoma, lung cancer, pancreatic cancer, and colorectal cancer. –15. Lee + as well as the glucose transporter. [This study describes the AST/ALT ratio as a novel significant biomarker for predicting recurrence and progression in patients with primary NMIBC. However, the mechanism behind the relationship between NMIBC and AST/ALT ratio remains obscure and requires further research. Several hypotheses have been presented to explain this mechanism. The most famous of these hypotheses is the “Warburg effect.” In tumor cell metabolism, it produces enough adenosine triphosphatase (ATP) by increasing glucose uptake and anaerobic glycolysis to promote the multiplying of cancer cells . Meanwhisporter. . It is ksporter. , 27. Whysporter. . TherefoDespite the current study showed novel findings, it has some limitations. First, this study was a retrospective analysis. Although all included patients received alike treatment, there were inevitably various inherent biases. Second, the AST/ALT rate was performed only once on the patient before TURBT and no comparative analysis was conducted at the interval of follow-up. Finally, it has the disadvantage of a single-center study and a small sample size. The results of this study need prospective larger studies to further consolidate, but this finding also would help clinicians in clinical decision making.This study has shown that the high AST/ALT ratio group tended to have poorer RFS and PFS than the low AST/ALT ratio group. Meanwhile, our results also indicated that the elevated preoperative AST/ALT ratio could be regarded as a significant prognostic biomarker for predicting early disease recurrence and progression of patients with primary NMIBC after TURBT. The findings would further help clinicians in making clinical decisions.