The association between alcohol consumption and blood lipids in Chinese children and adolescent: findings from the China Health and Nutrition Survey.

BACKGROUND: Alcohol consumption by children and adolescents is receiving increasing attention. It may cause dyslipidemia, a risk factor for cardiovascular disease. However, the association between alcohol consumption and blood lipids in children and adolescents is unclear, and so we aimed to characterize this association. METHODS: Data from the China Health and Nutrition Survey were extracted from children and adolescents aged 7-18 years for whom information was available on alcohol consumption. The population was divided into drinking and nondrinking groups. The χ2, Student's t, or Mann-Whitney U test was used to compare groups. Univariate and multivariate linear regression and propensity score matching (PSM) analysis were used to identify the association between alcohol consumption and blood lipids. RESULTS: This study included 408 children and adolescents with 35 drinkers and 373 nondrinkers. The drinkers had significantly lower values of total cholesterol (TC) (3.8 mmol/L for nondrinkers versus 3.5 mmol/L for drinkers, p = 0.002) and high-density lipoprotein cholesterol (HDL-C) (1.3 mmol/L for nondrinkers versus 1.2 mmol/L for drinkers, p = 0.007), but not for low-density lipoprotein cholesterol (LDL-C) (2.1 mmol/L for nondrinkers versus 2.0 mmol/L for drinkers, p = 0.092) or triglyceride (TG) (0.9 mmol/L for nondrinkers versus 0.8 mmol/L for drinkers, p = 0.21). The univariate and multivariate analyses led to the same conclusions. After PSM there was still a significant negative association between alcohol consumption and TC or HDL-C. CONCLUSION: Alcohol consumption in children and adolescents exhibited significant negative associated with TC and HDL-C, but not with LDL-C or TG. These findings need to be confirmed in future prospective research, and the health effects of blood lipid changes caused by drinking in children and adolescents need to be clarified.

Transcriptional regulation of the fidaxomicin gene cluster and cellular development in Actinoplanes deccanensis YP-1 by the pleiotropic regulator MtrA.

IMPORTANCE: Cascade regulation networks are almost present in various kinds of microorganisms, but locating and systematically elucidating specific pleiotropic regulators related to a certain gene cluster can be a tricky problem. Here, based on the promoter of the fidaxomicin pathway-specific regulator FadR1, we utilized a "DNA to Proteins" affinity purification method and captured a global regulator MtrA, which positively regulates fidaxomicin biosynthesis. In the mtrA overexpressed strain, the production of fidaxomicin was improved by 37% compared to the native strain. Then, we combined the "Protein to DNAs" affinity purification method (DAP-seq) with the results of RNA-seq and systematically elucidated the primary and secondary metabolic processes in which MtrA directly or indirectly participates. Thus, our work brought up a new way to improve fidaxomicin production from the perspective of global regulation and analyzed the regulatory mechanism of MtrA. Meanwhile, we provided a novel methodology for the research of cascade regulation networks and vital secondary metabolites.

Degradation mechanism of AtrA mediated by ClpXP and its application in daptomycin production in Streptomyces roseosporus.

The efficiency of drug biosynthesis depends on different transcriptional regulatory pathways in Streptomyces, and the protein degradation system adds another layer of complexity to the regulatory processes. AtrA, a transcriptional regulator in the A-factor regulatory cascade, stimulates the production of daptomycin by binding to the dptE promoter in Streptomyces roseosporus. Using pull-down assays, bacterial two-hybrid system and knockout verification, we demonstrated that AtrA is a substrate for ClpP protease. Furthermore, we showed that ClpX is necessary for AtrA recognition and subsequent degradation. Bioinformatics analysis, truncating mutation, and overexpression proved that the AAA motifs of AtrA were essential for initial recognition in the degradation process. Finally, overexpression of mutated atrA (AAA-QQQ) in S. roseosporus increased the yield of daptomycin by 225% in shake flask and by 164% in the 15 L bioreactor. Thus, improving the stability of key regulators is an effective method to promote the ability of antibiotic synthesis.

Global trends in the incidence rates of MDR and XDR tuberculosis: Findings from the global burden of disease study 2019.

Purpose: The study aimed to quantify the global trends of the incidence rates of multidrug-resistant (MDR) tuberculosis (MDR-TB) and extensively drug-resistant (XDR) tuberculosis (XDR-TB). Methods: Cases, age-standardized rates (ASRs), and incidence rates of MDR-TB and XDR-TB during 2010-2019 were obtained from the Global Burden of Disease Study 2019. The incidence trends of MDR-TB and XDR-TB were evaluated using the estimated annual percentage changes (EAPCs) in ASRs. The relationships among the ASRs of MDR-TB and XDR-TB, the MDR rate, the XDR rate, and socio-demographic index (SDI) were assessed using locally weighted regression and Pearson's correlation coefficient. Results: The global ASR of MDR-TB on average decreased by 1.36% (EAPC = -1.36, 95% confidence interval [CI] = -2.19 to -0.52) per year whereas that of XDR-TB was stable (EAPC = 0.69, 95% CI = -0.15-1.54) during 2010-2019. The incidence trends of MDR-TB in most regions and countries were decreasing, but those of XDR-TB were increasing. People aged 35-44 and 55-64 years had the highest incidence rates for MDR-TB and XDR-TB. The MDR and XDR rates both peaked in those aged 35-44 years. Areas with higher SDI tended to have lower ASRs of MDR-TB (p < 0.001, ρ = -0.43). Conclusion: The current achievements for the incidence trends of MDR-TB and XDR-TB are insufficient. More strategies and tools need to be developed to further curb MDR-TB and XDR-TB, especially in high-risk areas and age groups, and in low SDI regions.

Degradation Mechanism of AAA+ Proteases and Regulation of Streptomyces Metabolism.

Hundreds of proteins work together in microorganisms to coordinate and control normal activity in cells. Their degradation is not only the last step in the cell's lifespan but also the starting point for its recycling. In recent years, protein degradation has been extensively studied in both eukaryotic and prokaryotic organisms. Understanding the degradation process is essential for revealing the complex regulatory network in microorganisms, as well as further artificial reconstructions and applications. This review will discuss several studies on protein quality-control family members Lon, FtsH, ClpP, the proteasome in Streptomyces, and a few classical model organisms, mainly focusing on their structure, recognition mechanisms, and metabolic influences.

Analysis of the Global Disease Burden of Down Syndrome Using YLDs, YLLs, and DALYs Based on the Global Burden of Disease 2019 Data.

Purpose: This study aimed to determine Down syndrome (DS) burden using years lived with disability (YLDs), years of life lost (YLLs), disability-adjusted life years (DALYs), and the trends in these parameters. Methods: We obtained the annual YLDs, YLLs, DALYs, and age-standardized rates (ASRs) of DS from 2010 to 2019 using the Global Health Data Exchange tool. The estimated annual percentage changes (EAPCs) in ASR were used to quantify and evaluate DS burden trends. Gaussian-process regression and Pearson's correlation coefficient were used to assess the relationship between DS burden and socio-demographic index (SDI). Results: Global DALYs decreased by 2.68% from 2010 to 2019 but the ASR was stable, which was mostly explained by the stability in the ASR for YLLs. The ASR of YLDs showed an increasing trend (EAPC = 1.07, 95% CI = 0.45 to 1.69). There was notable regional imbalance, with most of the DALYs or ASRs in areas with relatively low SDI. The DALY rates of DS were mostly from the YLLs of children younger than 1 year. Lower SDI areas tended to have higher DS burdens (ρ = -0.3, p < 0.001). Conclusion: This systematic analysis of the global disease burden of DS from 2010 to 2019 revealed that although the global DS DALY and YLL rate is stable, the YLD rate is increasing. And the DS burden varies significantly differences among regions or countries. The present results suggest that future strategies should focus on DS-related deaths in children younger than 1 year and the DS burden in low-SDI regions or countries, since this may be effective in further reducing DS burden.

Improving the Yield and Quality of Daptomycin in Streptomyces roseosporus by Multilevel Metabolic Engineering.

Daptomycin is a cyclic lipopeptide antibiotic with a significant antibacterial action against antibiotic-resistant Gram-positive bacteria. Despite numerous attempts to enhance daptomycin yield throughout the years, the production remains unsatisfactory. This study reports the application of multilevel metabolic engineering strategies in Streptomyces roseosporus to reconstruct high-quality daptomycin overproducing strain L2797-VHb, including precursor engineering (i.e., refactoring kynurenine pathway), regulatory pathway reconstruction (i.e., knocking out negative regulatory genes arpA and phaR), byproduct engineering (i.e., removing pigment), multicopy biosynthetic gene cluster (BGC), and fermentation process engineering (i.e., enhancing O2 supply). The daptomycin titer of L2797-VHb arrived at 113 mg/l with 565% higher comparing the starting strain L2790 (17 mg/l) in shake flasks and was further increased to 786 mg/l in 15 L fermenter. This multilevel metabolic engineering method not only effectively increases daptomycin production, but can also be applied to enhance antibiotic production in other industrial strains.

A novel strategy of gene screen based on multi-omics in Streptomyces roseosporus.

Daptomycin is a new lipopeptide antibiotic for treatment of severe infection caused by multi-drug-resistant bacteria, but its production cost remains high currently. Thus, it is very important to improve the fermentation ability of the daptomycin producer Streptomyces roseosporus. Here, we found that the deletion of proteasome in S. roseosporus would result in the loss of ability to produce daptomycin. Therefore, transcriptome and 4D label-free proteome analyses of the proteasome mutant (Δprc) and wild type were carried out, showing 457 differential genes. Further, five genes were screened by integrated crotonylation omics analysis. Among them, two genes (orf04750/orf05959) could significantly promote the daptomycin synthesis by overexpression, and the fermentation yield in shake flask increased by 54% and 76.7%, respectively. By enhancing the crotonylation modification via lysine site mutation (K-Q), the daptomycin production in shake flask was finally increased by 98.8% and 206.3%, respectively. This result proved that the crotonylation modification of appropriate proteins could effectively modulate daptomycin biosynthesis. In summary, we established a novel strategy of gene screen for antibiotic biosynthesis process, which is more convenient than the previous screening method based on pathway-specific regulators. KEY POINTS: • Δprc strain has lost the ability of daptomycin production • Five genes were screened by multi-omics analysis • Two genes (orf04750/orf05959) could promote the daptomycin synthesis by overexpression.

Rational engineering strategies for achieving high-yield, high-quality and high-stability of natural product production in actinomycetes.

Actinomycetes are recognized as excellent producers of microbial natural products, which have a wide range of applications, especially in medicine, agriculture and stockbreeding. The three main indexes of industrialization (titer, purity and stability) must be taken into overall consideration in the manufacturing process of natural products. Over the past decades, synthetic biology techniques have expedited the development of industrially competitive strains with excellent performances. Here, we summarize various rational engineering strategies for upgrading the performance of industrial actinomycetes, which include enhancing the yield of natural products, eliminating the by-products and improving the genetic stability of engineered strains. Furthermore, the current challenges and future perspectives for optimizing the industrial strains more systematically through combinatorial engineering strategies are also discussed.

Genome-based rational engineering of Actinoplanes deccanensis for improving fidaxomicin production and genetic stability.

Microbial fermentation is currently still the major way to produce structural complicated clinical drugs. Yet, the low productivity and genetic instability of producing strains remain the bottlenecks in microbial pharmaceutical industry. Fidaxomicin is a microbial drug against the Clostridium difficile infection. Here, a genome-based combinatorial engineering strategy was established to improve both fidaxomicin production and the genetic stability of Actinoplanes deccanensis YP-1. Guided by genomic analysis, several genetic instability-associated elements were cumulatively deleted, generating a more genetically stable mutant. Further rational engineering approaches including elimination of a pigment pathway, duplication of the fidaxomicin gene cluster, overexpression of a positive regulator and optimization of the fermentation medium, led to an overall 27-folds improvement in fidaxomicin production. Taken together, the genome-based rational combinatorial engineering strategy was efficient to enhance the fidaxomicin production and ameliorate the genetic stability of YP-1, it can also be widely used in other industrial actinomycetes for strain improvement.

Trends of the incidence of drug use disorders from 1990 to 2017: an analysis based on the Global Burden of Disease 2017 data.

AIM: Drug use disorders are an important issue worldwide. Systematic attempts to estimate the global incidence of drug use disorders are rare. We aimed to determine the incidence of drug use disorders and their trends. METHODS: We obtained the annual incident cases and age-standardised incidence rate (ASR) of drug use disorders from 1990 to 2017 using the Global Health Data Exchange query tool. The estimated annual percentage changes of the ASR were used to quantify and evaluate the trends in the incidence rate. Gaussian process regression and the Pearson's correlation coefficient were used to assess the relationship between the ASR and socio-demographic index (SDI). RESULTS: The number of drug use disorders' cases increased by 33.5% from 1990 to 2017 globally, whereas the ASR exhibited a stable trend. The ASR was higher in men than in women. Most cases (53.1%) of drug use disorders involved opioid. A positive association (ρ=0.35, p < 0.001) was found between ASR and SDI. Teenagers aged 15-19 years had the highest incidence rate. CONCLUSIONS: The incident cases of drug use disorders were increasing, but the incidence rate did not change significantly from 1990 to 2017. Current preventive measures and policies for drug use disorders might have little effect. The present results suggest that future strategies should focus on men, teenagers and high-risk regions in order to improve the current status of drug use disorders.

The Gap Between Global Tuberculosis Incidence and the First Milestone of the WHO End Tuberculosis Strategy: An Analysis Based on the Global Burden of Disease 2017 Database.

OBJECTIVE: The first milestone (in 2020) of the End Tuberculosis (TB) Strategy of the World Health Organization was a 20% reduction in TB incidence rate compared with the 2015 baseline. This study aimed to determine the incidence rate of TB and how it has changed since 2015 at the global, regional, and country levels. METHODS: This study used the most recent data from the Global Burden of Disease study in 2017 to extract TB incidence rates at the global, regional, and country levels. The annual percentage change in the incidence rate (APCIR) of TB based on 2015 was calculated to evaluate the trend in the changes at various levels, including globally and at the regional and country levels. An APCIR of -4% from 2015 to 2020 is considered acceptable. RESULTS: The global APCIR was only -1.1% from 2015 to 2017. Only 2 of the 21 analyzed regions had APCIRs lower than -4%: Southern Sub-Saharan Africa and Eastern Europe. Worse still, six regions exhibited increasing TB incidence rates. At the country level, although 143 of 195 countries and territories showed reductions in TB incidence rates, the APCIR was lower than -4% in only 11 of them. CONCLUSION: This study suggests that it will be difficult to achieve the 2020 incidence rate milestone of the End Tuberculosis Strategy. This indicates the need to design and implement suitable strategies to address the current situation in order to achieve the next milestone and targets of the End Tuberculosis Strategy.

Comprehensive dissection of dispensable genomic regions in Streptomyces based on comparative analysis approach.

BACKGROUND: Large-scale genome reduction has been performed to significantly improve the performance of microbial chassis. Identification of the essential or dispensable genes is pivotal for genome reduction to avoid synthetic lethality. Here, taking Streptomyces as an example, we developed a combinatorial strategy for systematic identification of large and dispensable genomic regions in Streptomyces based on multi-omics approaches. RESULTS: Phylogenetic tree analysis revealed that the model strains including S. coelicolor A3(2), S. albus J1074 and S. avermitilis MA-4680 were preferred reference for comparative analysis of candidate genomes. Multiple genome alignment suggested that the Streptomyces genomes embodied highly conserved core region and variable sub-telomeric regions, and may present symmetric or asymmetric structure. Pan-genome and functional genome analyses showed that most conserved genes responsible for the fundamental functions of cell viability were concentrated in the core region and the vast majority of abundant genes were dispersed in the sub-telomeric regions. These results suggested that large-scale deletion can be performed in sub-telomeric regions to greatly streamline the Streptomyces genomes for developing versatile chassis. CONCLUSIONS: The integrative approach of comparative genomics, functional genomics and pan-genomics can not only be applied to perform a multi-tiered dissection for Streptomyces genomes, but also work as a universal method for systematic analysis of removable regions in other microbial hosts in order to generate more miscellaneous and versatile chassis with minimized genome for drug discovery.

Insurance status is related to overall survival in patients with small intestine adenocarcinoma: A population-based study.

BACKGROUND: Our goal was to investigate the effect of insurance status on the overall survival (OS) in cases of small intestine adenocarcinoma. METHODS: The SEER (Surveillance, Epidemiology, and End Results) database was used to identify 3822 patients who were diagnosed with small intestine adenocarcinoma between 2007 and 2015. The proportional hazard ASSUMPTION was evaluated by proportional-hazards assumption test and Schoenfeld residual test. The Kaplan-Meier method and Cox proportional-hazards regression analysis were performed to evaluate the association between insurance status and OS. RESULTS: We found that the insurance status at the time of diagnosis affected OS at the population level, both in those aged <65 and ≥65 years. Cox multivariate analysis of patients aged <65 years revealed that the hazard of death was greater in the Medicaid group (hazard ratio [HR] = 1.641, 95% confidence interval [CI] = 1.299-2.073, P < 0.001] and uninsured group (HR = 1.472, 95% CI = 1.095-1.979, P = 0.010) compared with the insured group, while the OS did not differ significantly between the Medicaid and uninsured groups. Similarly, the hazard of death among patients aged ≥65 years was higher in the Medicaid than the insured group (HR = 1.403, 95% CI = 1.136-1.733, P = 0.002). CONCLUSION: Our results suggest that patients with small intestine adenocarcinoma with insurance coverage have a significantly better OS than patients who have Medicaid or are uninsured, while the OS does not differ between Medicaid and uninsured patients.

Activation of anthrachamycin biosynthesis in Streptomyces chattanoogensis L10 by site-directed mutagenesis of rpoB.

Genome sequencing projects revealed massive cryptic gene clusters encoding the undiscovered secondary metabolites in Streptomyces. To investigate the metabolic products of silent gene clusters in Streptomyces chattanoogensis L10 (CGMCC 2644), we used site-directed mutagenesis to generate ten mutants with point mutations in the highly conserved region of rpsL (encoding the ribosomal protein S12) or rpoB (encoding the RNA polymerase ß-subunit). Among them, L10/RpoB (H437Y) accumulated a dark pigment on a yeast extract-malt extract-glucose (YMG) plate. This was absent in the wild type. After further investigation, a novel angucycline antibiotic named anthrachamycin was isolated and determined using nuclear magnetic resonance (NMR) spectroscopic techniques. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis and electrophoretic mobility shift assay (EMSA) were performed to investigate the mechanism underlying the activation effect on the anthrachamycin biosynthetic gene cluster. This work indicated that the rpoB-specific missense H437Y mutation had activated anthrachamycin biosynthesis in S. chattanoogensis L10. This may be helpful in the investigation of the pleiotropic regulation system in Streptomyces.

Development and validation of a nomogram for predicting cancer-specific survival in patients with Wilms' tumor.

Purpose: The objective of this study was to develop and validate a nomogram for predicting the cancer-specific survival (CSS) in patients with Wilms' tumor (WT). Methods: Patients with WT diagnosed between 2002 and 2015 in the Surveillance, Epidemiology, and End Results (SEER) database were divided randomly into training and validation cohorts in this study. Multivariate Cox regression analysis was used to screen variables. A Cox proportional-hazards regression model and a nomogram were constructed based on variables that significantly affected the CSS in the training cohort. The nomogram for distinguishing and predicting the CSS was evaluated using the concordance index (C-index), the area under the time-dependent receiver operating characteristic curve (AUC), and calibration plots. Results: In total, 1631 patients from the SEER database were enrolled, with 1141 categorized into the training cohort and 490 into the validation cohort. All significant variables associated with CSS-age, the number of examined lymph nodes, SEER stage, and tumor size-were included in the nomogram. The C-index values of the nomogram in the training and validation cohorts were 0.746 and 0.703, respectively. The 3-, 5-, and 10-year AUCs were 0.755, 0.749, and 0.724, respectively, in the training cohort, and 0.718, 0.707, and 0.718 in the validation cohort. The calibration plots indicated the nomogram could accurately predict the 3-, 5-, and 10-year CSS. Conclusions: We have developed and validated the first nomogram for predicting the survival of WT patients. The nomogram is a reliable tool for distinguishing and predicting the CSS in patients with WT. Information provided by the nomogram may help to improve the clinical practices related to WT.

Effect of marital status on duodenal adenocarcinoma survival: A Surveillance Epidemiology and End Results population analysis.

Numerous studies have shown that marital status may be a prognostic factor in various malignancies, but little is known about its effect on duodenal adenocarcinoma. The aim of the present study was to determine the association between marital status and survival in patients with duodenal adenocarcinoma. The Surveillance, Epidemiology and End Results database was utilized to analyze 2,018 patients who had been diagnosed with duodenal adenocarcinoma between January 2004 and December 2015. Kaplan-Meier and Cox regression analyses were also used to determine the impact of marital status on overall survival (OS) and cause-specific survival (CSS). The 5-year OS rate was higher in married patients (32.6%) compared with unmarried (26.8%) patients (P<0.001), as was the 5-year CSS rate (38.8 vs. 33.7%; P<0.001). Multivariate analysis demonstrated that marital status was an independent prognostic factor for duodenal adenocarcinoma, with married patients having improved OS (P<0.001) and CSS (P=0.001) compared with unmarried patients. Subgroup analysis showed that marital status played a role in the survival of patients at American Joint Committee on Cancer Tumor-Node-Metastasis stage I, but not of patients at stages II, III or IV. The survival outcomes for duodenal adenocarcinoma are improved in married patients compared with those in unmarried patients. Therefore, attention should be paid to the impact of social factors and socio-economic factors on unmarried patients, in order to improve their survival outcomes.

FadR1, a pathway-specific activator of fidaxomicin biosynthesis in Actinoplanes deccanensis Yp-1.

Fidaxomicin, an 18-membered macrolide antibiotic, is highly active against Clostridium difficile, the most common cause of diarrhea in hospitalized patients. Though the biosynthetic mechanism of fidaxomicin has been well studied, little is known about its regulatory mechanism. Here, we reported that FadR1, a LAL family transcriptional regulator in the fidaxomicin cluster of Actinoplanes deccanensis Yp-1, acts as an activator for fidaxomicin biosynthesis. The disruption of fadR1 abolished the ability to synthesize fidaxomicin, and production could be restored by reintegrating a single copy of fadR1. Overexpression of fadR1 resulted in an approximately 400 % improvement in fidaxomicin production. Electrophoretic mobility shift assays indicated that fidaxomicin biosynthesis is under the control of FadR1 through its binding to the promoter regions of fadM, fadA1-fadP2, fadS2-fadC, and fadE-fadF, respectively. And the conserved binding sites of FadR1 within the four promoter regions were determined by footprinting experiment. All results indicated that fadR1 encodes a pathway-specific positive regulator of fidaxomicin biosynthesis and upregulates the transcription levels of most of genes by binding to the four above intergenic regions. In summary, we not only clearly elucidate the regulatory mechanism of FadR1 but also provide strategies for the construction of industrial high-yield strain of fidaxomicin.

Determining the optimal cutoff point for lymph node density and its impact on overall survival in children with Wilms' tumor.

OBJECTIVE: Previous studies showed that the lymph node density (LND) was a predictor of survival in Wilms' tumor (WT). However, the optimal LND cutoff point is controversial due to methodological shortcomings of previous studies, and no studies have shown the effect of LND on survival in children with WT. The purpose of this study was to remedy this situation. METHODS: We identified 376 children with WT. LND cutoff point was determined using the median value, the X-tile program, the survival-tree algorithm, and the time-dependent ROC curve analysis. Survival functions were estimated by the Kaplan-Meier method. We used Cox regression analysis to determine the impact of LND on survival. Smooth curve fitting between relative mortality risk and LND was performed. RESULTS: The LND cutoff point was 0.44, 0.65, 0.65, and 0.64 according to the median value, the X-tile program, the survival-tree algorithm, and the time-dependent ROC curve analysis, respectively. The 5-, 10-, and 20-year overall survival rates were 86.9%, 86.9%, and 84.7%, respectively, in the <0.44 group and 81.3%, 80.3%, and 80.3%, respectively, in the ≥0.44 group. Survival did not differ significantly between the two groups (P=0.185). The 5-, 10-, and 20-year overall survival rates were 87.8%, 87.8%, and 86.0%, respectively, in the < 0.65 or < 0.64 group and 76.5%, 75.1%, and 75.1%, respectively, in the ≥ 0.65 or ≥ 0.64 group. Children with the high LND had a significantly worse survival (P=0.011) if 0.64 or 0.65 was used for the stratification. LND was a significant predictor for overall survival in the multivariate Cox regression analysis (HR =1.797; 95% CI, 1.043-3.097; P=0.035). Smooth curve fitting suggested that the risk of mortality tended to be ascending with the increase in LND in general. CONCLUSION: The three methods including the X-tile program, the survival-tree algorithm, and the time-dependent receiver operating characteristic (ROC) curve analysis are equivalent in their ability to stratify patients and clearly better than the median method. The results showed that the optimal LND cutoff point was around 0.65 and the LND was a reliable predictor of overall survival in children with WT.