AR gene on chromosome X, often reported with shorter survival and higher prevalence of GBM among males. Copy number (CN) and mRNA expression of AR were tested with droplet digital PCR in 91 fresh‐frozen GBM samples and 170 formalin‐fixed, paraffin‐embedded samples collected at Linköping University Hospital. The fresh‐frozen cohort was also subjected to pyrosequencing methylation analysis of 17 CpG sites in the AR promoter. Additionally, the gene expression of AR was analysed in the fresh‐frozen cohort and The Cancer Genome Atlas (TCGA) cohort of isocitrate dehydrogenase wild‐type primary GBM . The association of AR expression and overall survival (OS) was tested with Kaplan–Meier log rank analysis after dichotomisation by maximally selected rank statistics. We found that AR CN alterations were more common in female GBM. AR gene expression correlated with methylation levels of different CpG sites in males and females but there was no difference in expression between sexes. Survival analysis of TCGA cohort revealed the opposite effect of AR overexpression on OS of males and females, with high AR expression correlating with shorter OS in females and longer OS in males. Additional gene set enrichment analysis showed that AR expression correlated with DNA repair response, especially in the male group. In summary, we found that high AR gene expression in GBM exhibits sex‐dependent effects on patient survival, which, for males, is linked to DNA repair response.We sought to analyse the androgen receptor (AR) in glioblastoma (GBM) due to the location of the  Glioblastoma (GBM) is more common among males; however, the reason for sex differences remains largely unknown. Through molecular analyses of androgen receptor (AR) in GBM samples, we found that AR is commonly expressed in tumours of both sexes, but copy number alterations of the gene coding for AR are more frequent in females. Additionally, high AR expression correlates with worse survival among females but better survival among males. Aktprotein kinase BARandrogen receptorATRXalpha thalassemia/mental retardation syndrome X‐LinkedCNcopy numberDDX3XDEAD‐box helicase 3 X‐linkedDHTdihydrotestosteroneEGFRepithelial growth factor receptorFDRfalse discovery rateFFPEformalin‐fixed, paraffin‐embeddedGBMglioblastomaGSEAgene set enrichment analysisIDHisocitrate dehydrogenaseMGMTO‐6‐methylguanine‐DNA methyltransferasemiRmicroRNAmTORmechanistic target of rapamycin kinaseOSoverall survivalPI3Kphosphoinositide 3‐kinaseSVZsubventricular zoneTCGAThe Cancer Genome AtlasTCPAThe Cancer Proteome AtlasTGFβtransforming growth factor β1Glioblastoma (GBM) remains a nearly incurable brain tumour with a poor prognosis  . GBM belSex chromosomes are often purposefully omitted in analyses, due to the challenges they present. The gene coding for the androgen receptor (AR) is located on chromosome X, Xq12. The AR is a ligand‐dependent nuclear transcription factor and a member of a superfamily of steroid hormone nuclear receptors. Androgens, testosterone and dihydrotestosterone (DHT), mediate their effects via AR, and when bound to cytoplasmic AR cause the release of heat shock proteins . The ligin vitro, e.g., enzalutamide, led to inhibition of proliferation of GBM cells [A recent pan‐cancer study revealed that AR is overexpressed in GBM , and fewBM cells , 12, 13.BM cells . Hence, There are a limited number of studies exploring AR antagonists as possible treatment for GBM, and not much is known about AR genetic and epigenetic characteristics in GBM, which could influence the receptor's activity. Mutation frequency in AR in GBM is very low, only about 1%, as found in cBioPortal v3.7.5 based on The Cancer Genome Atlas (TCGA) data , 16. Hen22.1All patients included in this study underwent surgery at Linköping University Hospital, were at least 18 years old, and provided written informed consent. The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Regional Ethics Committee of Linköping University, Sweden . All patient samples, as well as control samples were collected in South‐East Sweden. Table The second cohort consisted of 91 fresh‐frozen samples, partially overlapping with the FFPE cohort  and aligned using blast to confirm correct amplification and enable counting of CAG repeats. Eight samples with a different number of repeats were selected and used as controls and markers in the following fragment analysis. DNA extracted from blood of 799 healthy controls and 167 GBM patients was amplified using the same set of primers as for sequencing but with the forward primer labelled with FAM fluorochrome at the 5′ end. Samples were then diluted 1 : 12 with water, and 1 μL was mixed with 0.3 μL of GeneScan 600 LIZ dye Size Standard (Applied Biosystems) and 15 μL of Hi‐Di Formamide , and separated in the 3500 Genetic Analyzer. Fragments were analysed with the genemapper v.4.1 (Applied Biosystems) and the number of CAG repeats was determined by comparison of fragment sizes to control samples included in every run.For all samples included in the CAG repeat analysis, DNA was extracted from blood using Maxwell 16 Blood DNA Purification Kit  on the Maxwell 16 Instrument (Promega). A fragment encompassing the variable region was amplified with primers: forward‐ACCGAGGAGCTTTCCAGAAT, reverse‐ GCTGCCTGGGGCTAGTCTCTT, and sequenced for 20 healthy males from the control cohort. Amplification of exon 1 of AR was done with MyTaq DNA Polymerase , and a touchdown protocol was applied. The first three cycles were performed with annealing temperature 63 °C, followed by three cycles with annealing temperature 62 °C and 29 cycles with annealing temperature 61 °C. Sequencing was performed according to the BigDye Terminator v3.1  protocol with fragment separation on 3500 Genetic Analyzer . Electropheograms were visualised in the 2.3psq assay design 2.0.1.15 (Qiagen), are given in the Table pyroq‐cpg 1.0.9 software .Methylation analysis of 17 CpG sites spanning 750 bp of the promoter region of AR in 91 fresh‐frozen tumour samples  was done by pyrosequencing. EZ DNA Methylation Kit  was used for bisulfite conversion of 350 ng of DNA, out of which 20 ng was used for each of the subsequent amplification reactions done with the HotStarTaq Master Mix Kit (Qiagen). The promoter region was divided into seven amplicons and all used primers adapted from Hornig et al. [2.4quantasoft v1.7.4 (BioRad).The AR CN analysis was performed on 91 fresh‐frozen GBM and 83 IDH‐mutated gliomas, as well as 170 FFPE GBM samples using droplet digital PCR. The same technique was used for assessment of mRNA expression, both for total AR as well as the AR‐V7 variant. In all instances, droplet generation was done using the Automated Droplet Generator , and after PCR, droplet reading was performed on the QX200 Droplet Reader (BioRad). The results were analysed using the For CN analysis, DNA from fresh‐frozen tissue was subjected to enzymatic digestion . This step was omitted for DNA from FFPE tissue due to its pre‐existing fragmentation. Then, 20 ng of DNA was mixed with probes and ddPCR Supermix for Probes (no dUTP) (BioRad), and amplified according to the manufacturer's protocol with annealing temperature of 60 °C. The FAM‐labelled probe targeting AR  was used together with the reference HEX‐labelled probe AP3B1 . Deletion was reported for CN values below or equal to 1.7 for females and 0.7 for males, amplification for females was reported for CN values above 2.3 and 1.3 for males.Reverse transcription of 500 ng–1 mg RNA for expression analysis was done with Maxima First Strand cDNA Synthesis Kit for RT‐qPCR (ThermoFischer Scientific). We used primers and probes for AR gene expression, as reported by Ma et al.  and GUSB2.5gsea 4.1.0 software [gsea software default settings, high and low AR expressions were treated as categorical values, and phenotype labels for the two groups were created based on the survival analysis results. In the second analysis, the AR expression values were treated as continuous data, allowing us to look for enrichment of gene sets associated with the increasing AR expression within each sex group, without the influence of survival data. Here, the Euclidean distance as a metric for ranking genes was used. We also investigated the enrichment of microRNA (miR) targets gene sets associated with AR expression due to the involvement of AR in miR regulation. In all analyses, only gene sets with false discovery rate (FDR) < 25% and P < 0.01 were considered significant.The gene expression data from TCGA GBM samples were used in the gene set enrichment analysis (GSEA), keeping the analysis separate for males and females. The analyses included hallmark gene sets version 7.4 and was performed on software , 27. In 2.6P < 0.05. All statistical analyses were done in spss v.26  unless stated otherwise, and maximally selected rank statistics was done in rstudio v.1.4  with r version 4.0.3 using packages maxstat and survminer. Normality of distribution of CAG repeats, AR gene expression and methylation data were tested with the Shapiro–Wilk test before other tests were performed. The Kruskal–Wallis test with Bonferroni correction for multiple pairwise comparisons was used to compare AR gene expression from TCGA data and between fresh‐frozen GBM and IDH‐mutated gliomas, whilst the Mann–Whitney U test was used for 2‐group AR mRNA expression between females and males. The Pearson coefficient was used for correlation analysis between AR CN and gene expression. The CAG repeats were compared with Mann–Whitney U test. In females, two alleles were separated  before statistical analysis, but we also compared the biallelic mean values. Additionally, we dichotomised data based on the median value from the control group  and divided into a group with lower and equal or higher number of CAG repeats [2 test.Differences in OS were analysed with the Kaplan–Meier log rank method complemented by maximally selected rank statistics  used for repeats . Such gr33.1First, we analysed CN changes in FFPE and fresh‐frozen GBM samples, and detected amplifications and deletions of the gene. In both cohorts, combined CN alterations were more frequent in females than in males Table . More frP = 0.099) . In female GBM, no such relationship was found , however, in IDH‐mutated gliomas the correlation was the opposite, found in females but not in males (Table P = 2 × 10−8) and mesenchymal (P = 1 × 10−6) subtypes. Additionally, we divided samples by sex, and multiple pairwise comparison revealed significant differences between several subtypes and across sexes, but no differences were found between females and males within the same subtypes   Fig. . Correlaes Table . Howeverpes Fig. . Protein65) Fig. . Interes65) Fig. .P = 0.258)  Fig. .3.2We further investigated the influence of AR CN and gene expression on OS. First, we analysed the cohort of FFPE samples, with the advantage of all patients following an equal treatment regimen, and we compared survival between patients with AR amplification, deletion and normal CN with the log rank Kaplan–Meier analysis. Males and females were analysed in combination and separately, but no significant differences were found. There were also no survival differences in the analyses of amplification vs. remaining samples, in the sex‐combined and sex‐separated setting Fig. .2) cut‐off value estimated with maximally selected rank statistics ensuring the best separation of survival curves and allocation of at least 20% of samples in each group. In females, the cut‐off value was 4.8303log2, and in males it was estimated at 4.9594log2. Surprisingly, we recorded the opposite effect of AR expression on survival with high AR mRNA expression associating with better survival among males    Fig. , and wor35) Fig. . No sign35) Fig. .3.3Pyrosequencing allows for semi‐quantitative evaluation of methylation at specific CpG sites. We analysed the promoter region of AR, with the first CpG site located 750 bases upstream from the transcription start site Fig.  in the c3.4We performed GSEA for TCGA cohort, trying to resolve cellular mechanisms that are responsible for the sex difference linking AR expression with survival, however, no gene sets were identified. We then repeated GSEA, but instead of dividing samples into two groups based on their survival, we looked for gene sets associated with AR expression (AR‐positive samples). Surprisingly, only one set of genes, namely DNA repair genes, was associated with AR expression in males Fig. . In fema3.52, P = 0.075). We did not observe any significant differences between the CAG length distributions in either sex, even after treating data as binary qualitative and dividing samples based on the median CAG repeat value in the control group , and among control males 21.9 (±2.8), whereas for the female patients it was 20.1 (±2.2) for the shorter allele and 23.4 ±2.6) for the longer allele, and in female controls 20.4 (±2.3) and 23.6 (±2.4), respectively. The number of repeats ranged from 14 to 35 in males and from 10 to 31 in females. Among the samples tested, we found 62 (15.5%) homozygous females in the control group and 15 (24.6%) in the GBM group but the difference between groups was not significant  acts as a transcription factor and the main controller of cell cycle in response to DNA damage [O‐6‐methylguanine‐DNA methyltransferase (MGMT) [The results presented in this study complement the current knowledge about AR and its involvement in GBM, also indicating a sex‐specific influence of AR on patients' survival. The abundance of AR expression in GBM is encouraging as it may constitute a novel therapeutic target. In fact, enzalutamide, an oral AR antagonist used for prostate cancer treatment , was shoA damage . It has A damage . AdditioA damage . As repoA damage . In otheA damage , 39, 40.e (MGMT) . Silencie (MGMT) , 43. TheGlioblastoma presents to be a tumour dependent on other sex hormone receptors as well. Intriguingly, oestrogen receptor beta was shown to act as a tumour suppressor in GBM by downregulating DNA damage response , which cWe also report on the frequency of AR CN changes, which affect females more often than males but without a clear direction towards amplifications or deletions. In the previously published work by Zalcman et al. , AR amplAs reviewed by Bramble et al. , neural High AR expression in GBM has been previously reported by several groups , 31, 58.In the methylation analysis, we found a sex difference in the CpG sites, where methylation inversely correlated with AR expression. Two CpG sites were identified for males and three different for females. Hornig et al. , showed To our knowledge, this is the first study exploring CAG‐length polymorphism in AR in GBM patients. A decreasing number of CAG repeats encoding polyglutamine was shown to be associated with an increased risk of prostate cancer , and lon5In summary, sex differences are observed in the association of AR expression and survival as well as in the frequencies of AR CN alterations, but no association was found between CAG repeat number and GBM development. AR is commonly expressed in GBM of both sexes and the negligeable presence of the AR‐V7 transcript suggests the need for the ligand to trigger downstream signalling, which is more strongly linked to DNA damage response in males than in females. Hence, further research focusing on both mechanistic studies and discriminating sex differences associated with AR and other sex hormone receptors might translate into sex‐dependent treatment of GBM in the future.The authors declare no conflict of interest.MŁ and AM conceptualised the study, administered the project and performed data curation. MŁ designed the methodology, investigated and visualised the study, and wrote and prepared the original draft. MŁ and MT performed the formal analysis. MM, CB, MS, PM, MH and AM provided resources. MŁ, MT, MM, CB, MS, PM, MH and AM wrote, reviewed and edited the manuscript. AM supervised the study and acquired funding. All authors have read and agreed to the published version of the manuscript.Fig. S1. Relative expression of AR in the fresh‐frozen tumours collected in South‐East Sweden.Fig. S2. Analysis of AR CN changes influence on overall survival in the FFPE Linköping cohort (Kaplan‐Meier log‐rank method).Fig. S3. Survival analysis of IDH‐mutated tumours from TCGA.Table S1. Primers used for the methylation analysis of the promoter region of AR and amplicon specific annealing temperatures.Table S2. Mean methylation values for each analysed CpG site within the AR promoter region from the fresh‐frozen cohort.Table S3. Results of Spearman correlation analysis between methylation of 17 CpGs in the promoter of AR and AR gene expression.Table S4. Spearman's correlation of AR protein expression and 9 selected proteins; TCGA cohort .Click here for additional data file.


National big data pertaining to the status of common respiratory diseases is essential to devising appropriate policies to promote proper treatment and prevention of respiratory diseases amid the prolonged coronavirus disease 2019 (COVID‐19) pandemic. The aim of this study is to investigate the prevalence of common respiratory diseases and their association with sociodemographic characteristics, comorbidities, and medical history using 11 years (2008–2018) of the Korea National Health and Nutrition Examination Survey (KNHANES) data, ultimately to present foundational data for policy decision making and disease prevention measures.Among the participants of the KNHANES survey (2008–2018), 93 028 adults aged ≥40 years who underwent a lung function test were included in this cross‐sectional study. The participants were divided into four groups: Asthma, chronic obstructive pulmonary disease (COPD), asthma + COPD, and no respiratory disease. Their data were analyzed for demographic factors, health behavior, and disease‐related factors. Multiple logistic regression was used to calculate the odds ratio (OR) adjusted for sex, age, household income, educational level, occupation, body mass index (BMI), smoking status, alcohol consumption, physical activity, and comorbidities.Of all participants, 1.83%, 12.63%, and 1.27% had only asthma, only COPD, and asthma + COPD, respectively. With respect to the patients with asthma who also had asthma + COPD, the OR of asthma + COPD was 5.272 in underweight patients and 6.479 in patients aged ≥70 years. Meanwhile, a high association between COPD and asthma + COPD was found in female patients, whereas asthma was more highly associated with asthma + COPD in male patients.The study confirmed that old age, sex, smoking status, BMI, previous history of atopic dermatitis, and lung cancer were independent risk factors for asthma, COPD, and asthma + COPD. The present study demonstrated the need for a multidisciplinary integrative approach to respiratory diseases, and the findings could be used for developing policies for the treatment of COVID‐19 and respiratory diseases and the prevention of infectious diseases. The odds ratio (OR) of having asthma + COPD among patients with asthma was lower in females and older people. Regarding BMI, risk of asthma + COPD was higher in underweight patients with asthma as compared with those with normal BMI, whereas the association was lower in overweight subjects. In contrast to asthma patients, females, older ages, and overweight patients with COPD had higher risk of asthma + COPD. Global Burden of Disease Study 2020 reported that deaths associated with lower respiratory tract infections have decreased over the past 20 years, and yet many deaths are still caused by respiratory diseases.Asthma is a heterogeneous chronic inflammatory disease with several etiologies, and its symptoms include breathing difficulty and coughing.Chronic obstructive pulmonary disease (COPD) is a lung disease characterized by irreversible airflow limitation that has become a public health issue with a high prevalence and mortality rate. According to the World Health Organization (WHO), COPD will become the third leading cause of death by 2030. The socio‐economic burden associated with COPD is substantial in Korea. The direct medical cost per COPD patient was $2800 in 2009, which was slightly higher than that of Canada and the United States.The definition of asthma + COPD remains controversial, with no commonly accepted definition. In clinical practice, diagnosis of asthma or COPD is suggested if three or more of the symptoms corresponding to asthma or COPD are present. On the other hand, diagnosis of asthma + COPD should be considered when the patient presents symptoms of asthma and COPD alike.Previous studies have used the Korean National Health and Nutrition Examination Survey (KNHANES) data to investigate respiratory diseases. A study used data from the 2007–2012 KNHANES to divide patients with asthma + COPD into asthma‐dominant and COPD‐dominant groups to investigate their socio‐economic status and quality of life. The results showed that the asthma‐dominant asthma + COPD group had lower socio‐economic status and poorer quality of life than the COPD‐dominant asthma + COPD group.The COVID‐19 pandemic remains active since the index case in late 2019. COVID‐19 is known to have a greater repercussion on individuals with preexisting respiratory conditions, such as asthma and COPD.As described, typical respiratory diseases include asthma, COPD, and asthma + COPD, but each disease has various risk factors according to its own characteristics. Previous studies have focused on the associations of a single factor, but no study has investigated that specific factor, among the multitude of complex factors, which shows the strongest association. Moreover, while previous studies have investigated asthma, COPD, and asthma + COPD separately, and some have investigated two of these diseases, no study has compared all three diseases during the period between 2008 and 2018. Accordingly, the present study aims to analyze the risk factors that influence asthma, COPD, and asthma + COPD and compare the risk factors using data from Korean adults aged ≥40 years from the KNHANES 2008–2018.22.1KNHANES is a nationwide survey conducted by the Korea Centers for Disease Control and Prevention (KCDC), and the present study used data from the fourth to seventh KNHANES survey (KNHANES IV‐VII). Starting from 1998, KNHANES has been conducted in 3‐year waves. It is a nationwide health and nutrition survey with representativeness and reliability that has been conducted annually since 2007. The survey collects information on the general characteristics, health behavior, chronic disease status, and nutritional intake status of the Korean population. The findings are used as basic data for establishing health care policies.2.2The present study used data from the KNHANES IV‐VII, collected between January 2008 and December 2018. In the present study, 93 028 individuals were surveyed, of which 35 235 adults aged ≥40 years who took part in a lung function test were included in the final analysis. Of the 93 028 surveyed individuals, 42 619 individuals aged <40 years and 15 174 individuals with missing data regarding asthma and COPD were excluded. Consequently, the analysis of the present study included a total of 35 235 individuals  criteria.Sex, age, household income, educational level, and occupation were selected as demographic characteristics. Age was divided into 40–49, 50–59, 60–69, and ≥70 years, whereas the educational level was divided into elementary school graduate or lower, middle school graduate, high school graduate, and college graduate or higher for individuals aged ≥40 years. For household income, the household equivalent income quartiles from the KNHANES  were used. The occupation was divided into unemployed; professional; office work; sales and services; agriculture, forestry, and fishery; machine fitting and simple labor; and others.2 (m2), in accordance with the WHO standards.BMI, smoking status, alcohol consumption, physical activity, previous history, lung function test, and GOLD criteria were analyzed as health‐related characteristics. BMI was divided into underweight (BMI < 18.5), normal (18.5 ≤ BMI < 25), and overweight (25 ≥ BMI) based on anthropometric results to calculate body weight (kg)/height2.3.2The asthma group was defined as patients answering “Yes” to whether they had been diagnosed with asthma by a doctor.2.3.3The COPD group was defined based on the results of the lung function test. FVC  and FEV1  were determined and used to calculate FEV1/FVC (mean ± SE). Patients with FEV1/FVC < 0.7 were defined as having COPD.2.3.4Individuals with both asthma and COPD were defined as the asthma + COPD group. Hence, this group was comprised of those who responded “Yes” to the question of whether they had been diagnosed with asthma by a doctor and who had an FEV1/FVC < 0.7.2.4KNHANES is a nationwide sample survey that used complex sample analysis considering weights, stratification variables, and cluster variables. The distribution of the characteristics of the study participants was expressed as frequency and percentage, while continuous data were expressed as mean and 95% confidence interval (95% CI). To identify the risk factors for asthma + COPD among patients with respiratory diseases, we performed logistic regression analysis with asthma + COPD as a binary dependent variable. Additionally, the risk factors for each respiratory disease were also analyzed using each respiratory disease  as the dependent variable. Sex, age, household income, education, occupation, BMI, smoking status, alcohol consumption, physical activity, and type of comorbidity were included as covariates in the logistic regression analysis. Furthermore, the annual status of asthma, COPD, and asthma + COPD from 2008 to 2018 was investigated. The logistic regression results were presented as odds ratio (OR) and 95% CI. The data used in the present study were statistically analyzed using SAS V9.4  with the significance level set to <0.05 (two‐tailed).3Among all Korean adults aged ≥40 years who underwent a lung function test, 1.83%, 12.63%, and 1.27% had asthma, COPD, and asthma + COPD, respectively. In the asthma group, the prevalence was three times higher in women than in men, whereas the prevalence among men was 3.2 and 1.2 times higher than that in women in the COPD and asthma + COPD groups, respectively. Table Among the disease‐related factors, the lung function test results showed a decreasing mean FEV1/FVC value in the order of the NRD (0.80), asthma (0.78), COPD (0.64), and asthma +COPD (0.58) groups. With respect to the GOLD criteria, the NRD and asthma groups showed the highest prevalence in stage 1; the COPD group, in stages 1 and 2; and the asthma + COPD group, in stage 2.Table Table Among the demographic factors, the female sex in the asthma group showed a higher association than the male sex, with an OR of 2.550 (95% CI 1.764–3.687), whereas for females in the COPD group, the association was significantly lower, with an OR of having asthma of 0.294 (95% CI 0.255–0.338). In the asthma group, there was no significant difference according to age. In the COPD group, the OR of having COPD increased with age: 50–59 years , 60–69 years , and ≥70 years , relative to the 40–49 years age bracket. In the asthma + COPD group, the OR of having asthma + COPD also increased with age: 60–69 years  and ≥70 years  relative to the 40–49 years age bracket. For household income, there were no associations among household incomes in the asthma group, whereas the low group  had a higher risk for COPD than the high group. Likewise, the low group  had a higher risk for asthma + COPD than the high group.With respect to educational level, the association with asthma was higher among elementary school graduates  than among college graduates, whereas the association with COPD was higher among elementary school graduates , middle school graduates , and high school graduates  than among college graduates. In the asthma + COPD group, there was no statistically significant association. Based on occupation, machine fitting and simple labor  showed a lower association than being unemployed in the asthma + COPD group.Among health behavior and disease‐related factors, overweight subjects  showed a higher association than those with normal weight in the asthma group, whereas underweight subjects  showed a high association with COPD, and overweight subjects  showed a low association with COPD. In the asthma + COPD group, underweight subjects  showed a high association with asthma + COPD. In the asthma group, there was no significant association with smoking status, whereas, in the COPD group, former smokers  and current smokers  showed a higher association than nonsmokers. A similar pattern was also found in the asthma + COPD group . With respect to alcohol consumption, individuals who consume any alcohol showed significantly lower associations with asthma and COPD than those who do not drink at all. In the asthma + COPD group, individuals who consume ≤1 drink per month showed a lower association. Individuals with a previous history of atopic dermatitis showed a higher association with asthma  and asthma + COPD , whereas individuals with a previous history of lung cancer showed a higher association with COPD .4The present study used reliable and nationally representative data to analyze the demographic characteristics, health behavior, disease‐related factors, and risk factors associated with respiratory diseases among 35 235 Korean adults aged ≥40 years.In this study, women showed a high association with asthma than men. Such findings supported the results from a previous study reporting that 71% of adult asthma patients are women.Furthermore, women showed a lower association with COPD than men. Such findings support the fact that COPD is more frequent in men than women due to COPD being historically linked to smoking status and occupational exposure.Finally, individuals aged 60–69 and ≥70 years showed a higher association with asthma + COPD than those aged 40–49 years. These findings support the results from previous studies indicating that the prevalence of asthma + COPD increases significantly with age and that age is associated with asthma + COPD.Studies in the US have also used data from the KNHANES to investigate the status of respiratory diseases. The results revealed that the prevalence of asthma + COPD had increased from 0.96% to 1.05% between 2007 and 2012. The age‐standardized prevalence of asthma + COPD was higher among individuals with low socio‐economic status and previous history of myocardial infarction or stroke. Old age and smoking status showed even higher associations with the prevalence of asthma + COPD. Among the participants with COPD, a higher prevalence was correlated with being non‐Hispanic Black, being obese, and having a history of myocardial infarction or stroke. asthma + COPD is associated with the use of oxygen therapy for severe asthma and COPD. The participants with asthma + COPD showed a poorer FEV1 in the lung function test results than those with asthma or COPD alone.4.1The present study had some limitations. First, because it was a cross‐sectional study using KNHANES data, caution should be taken when interpreting the causal relationships between the variables. In other words, whether the associated factors are independent causative factors or epiphenomenon factors cannot be known by their causal relationships. Second, asthma has a characteristic higher incidence at younger ages, but because the study population included only adults aged ≥40 years, the identification of factors may have been influenced by the fact that more COPD cases than asthma cases were included. Third, asthma was diagnosed by a doctor and thus may have been subject to underdiagnosis, overdiagnosis, or poor accessibility to health care services.Despite these limitations, the present study also had the following strengths. First, it used data from surveys conducted by a Korean government agency. The surveys were nationwide and were conducted on sample populations from the same region using the same questions during the same survey period. Because the surveys targeted the general population, the obtained data have high reliability. Second, this was the first study to analyze the risk factors of three different respiratory diseases over multiple years. While previous studies have analyzed the risk factors of respiratory diseases, no study has analyzed such factors over multiple years. Additionally, the present study compared the associations of asthma‐asthma + COPD and COPD‐asthma + COPD to investigate which risk factors would increase the likelihood of asthma or COPD progressing to asthma + COPD. Finally, the present study measured the ORs of each respiratory disease relative to a group of healthy controls to identify the risk factors that influence specific respiratory diseases.This study analyzed the correlation between respiratory diseases and associated factors from 2008–2018. As a result of the global COVID‐19 pandemic, there may have been changes to the status of respiratory diseases since 2019. Subsequent studies can comparatively analyze the status of respiratory diseases prior to and after the advent of the COVID‐19 pandemic by examining respiratory diseases after 2019. Therefore, this study provides useful data shedding light on the characteristics of respiratory diseases in the Korean population prior to the advent of COVID‐19. In the future, studies should analyze respiratory diseases and their associated factors in Korea also using data since 2019.5The present study identified the associations between respiratory diseases and demographic factors, health behavior, and disease‐related factors. In particular, the independent risk factors influencing specific diseases were being a woman and having a previous history of atopic dermatitis for asthma; older age, being a current smoker, and having a previous history of lung cancer for COPD; and older age, being underweight, being a current smoker, and having a previous history of atopic dermatitis for asthma + COPD. Our findings could be used to developing policies to treatment of COVID‐19, respiratory diseases and the prevention of infectious diseases.The authors have no conflict of interests to declare.http://knhanes.cdc.go.kr). The present study was conducted after approval from the IRB (JASENG IRB File No. 2021‐05‐013).The KNHANES IV–VII were conducted by the Korea Centers for Disease Control and Prevention (KCDC) and written consent was obtained from all participants. All survey protocols were approved by the Institutional Review Board (IRB) of the KCDC . The data from KNHANES is available on the ‘Korea National Health and Nutrition Examination Survey’ website (Conceptualization: Yoon Jae Won, Sook‐Hyun Lee, In‐Hyuk Ha. Data curation: Yoon Jae Won, Yu‐cheol Lim, In‐Hyuk Ha. Formal analysis: Yu‐cheol Lim, In‐Hyuk Ha. Methodology: Yoon Jae Won, Sook‐Hyun Lee, In‐Hyuk Ha. Project administration: Sook‐Hyun Lee, Beom‐Joon Lee, In‐Hyuk Ha. Validation: Yoon Jae Lee, Maurits Van den Noort, Beom‐Joon Lee. Writing – original draft: Yoon Jae Won, Sook‐Hyun Lee, In‐Hyuk Ha. Writing – review & editing: Yoon Jae Won, Sook‐Hyun Lee, Yu‐cheol Lim, Yoon Jae Lee, Maurits van den Noort, Beom‐Joon Lee, In‐Hyuk Ha.Table S1. Association of risk factors for asthma+COPD among Korean participants with asthma or COPD in the KNHANES 2008–2018Click here for additional data file.


UMOD gene associate with kidney function, blood pressure (BP), and hypertension. Elevated BP is linked to kidney function and impaired cognitive as well as physical performance in later life. We investigated the association between UMOD rs4293393–A > G and kidney function, BP, cognitive and physical function in the Berlin Aging Study II (BASE–II). Data of 1556 older BASE–II participants (mean age 68.2 ± 3.7 years) were analyzed. BP was determined by standardized automated measurements, estimated glomerular filtration rate (eGFR) by CKD Epidemiology Collaboration creatinine equation. Cognitive function was assessed by Mini-Mental State Examination and Digit Symbol Substitution Test, while physical function by Handgrip Strength and Timed Up and Go-Test. Association analyses were performed by covariance and logistic regression models adjusting for sex. G–allele carriers at UMOD rs4293393 exhibited significantly higher eGFR values compared to non–carriers , and a lower risk of eGFR < 60 mL/min/1.73 m2 . However, UMOD rs4293393 genotypes were not associated with BP, diagnosis of hypertension or cognitive and physical function parameters. Our data corroborate previous findings on the association of UMOD rs4293393-G with better kidney function in older adults. However, no association between UMOD and BP or physical and cognitive parameters in these community-dwelling older adults was detected.In genome-wide association studies, genetic variants in the  Expression of the kidney–specific protein uromodulin, also known as Tamm–Horsfall protein, is restricted to the thick ascending loop of Henle (TAL) followed by proteolytic cleavage and secretion into the urine . AlthougUMOD promoter region such as rs12917707–G > T as well as other single–nucleotide polymorphisms (SNPs) in high linkage disequilibrium (LD) including rs4293393–A > G  have shown genome-wide significant association with estimated glomerular filtration rate (eGFR) and the incidence of chronic kidney disease (CKD) , where MAF in non-Finnish North-Western Europeans is listed with 18.6%  and the n = 56; AG, n = 451) had significantly lower SCr concentrations compared to individuals homozygous for the rs4293393 risk genotype  for the rs4293393 risk allele .Carriers of the protective rs4293393 G–allele . Further separate analysis for SBP and DBP by adjusting for the use of antihypertensive medication in treated individuals also revealed no significant associations (not shown).Analyses of covariance adjusted for sex showed no significant association of rs4293393 with mean SBP, DBP, or heart rate , HGS , or with morbidity index  or frailty .Concerning cognitive and physical function, no association of rs4293393 with cognitive function as determined by MMSE and DSST was detected  [UMOD variants in LD with SCr [UMOD variants play an important role in kidney function also in older adults. Yet, this did not translate into associations of rs4293393 with cognitive assessment results in our analysis. Of note, an association between kidney dysfunction based on eGFR and cognitive impairment was reported for eGFR values <60 ml/min/1.73 m2 [2 and those with eGFR of 60 to below 90 mL/min/1.73 m2 at baseline [2 but >60 ml/min/1.73 m2 did not associate with several tests of cognitive function, including the DSST and MMSE [2) and cognitive performance assessed by MMSE, among other tests [2 (9.1%) and being frail (0.8%). In general, the prevalence of diseases and age-related comorbidities such as hypertension, heart failure, and diabetes is rather low in our cohort


Lactobacillus spp. as a protective and therapeutic agent, we aimed to investigate the efficacy of Lactobacillus spp. in modulating JAK/STAT and nuclear factor kappa B (NF‐κB) inflammatory signaling pathways.Probiotics have a beneficial effect on inflammatory responses and immune regulation, via Janus kinase/signal transduction and activator of transcription (JAK/STAT) and NF‐κB signaling pathways. To evaluate the precise effects of TIRAP], Interleukin 1 Receptor Associated Kinase[IRAK4], Nuclear factor‐kappa B Essential Modulator [NEMO], and receptor interacting protein [RIP]) followed by treatment of the HT‐29 cell line with sonicated pathogens before, after, and simultaneously with Lactobacillus spp. A cytokine assay was also used to evaluate interleukin (IL)‐6 and IL‐1β production after treatment with Lactobacillus spp.A quantitative real‐time polymerase chain reaction (qPCR) assay was used to analyze the expression of JAK/STAT and inflammatory genes (TIR‑associated Protein [Lactobacillus spp. downregulated JAK and TIRAP, IRAK4, NEMO, and RIP genes in the NF‐κB pathway compared to sonicate‐treated cells. The expression of STAT genes was different after treatment with probiotics. The production of IL‐6 and IL‐1β decreased after probiotic treatment.Lactobacillus spp. cocktail showed anti‐inflammatory effects on HT‐29 cells by modulating JAK/STAT and NF‐κB signaling pathways in all three treatment variants. Therefore, Lactobacillus spp. as a dietary supplement can both prevent and reduce inflammation‐related diseases such as inflammatory bowel disease.Our   Lactobacillus plantarum was first isolated from saliva. Later, it was demonstrated that L. plantarum produces hydrogen peroxide and therefore has remarkable antimicrobial activity. Lactobacillus. rhamnosus has significant beneficial effects, such as a positive role in obesity and activities against resistant bacteria (vancomycin‐resistant enterococci). Lactobacillus. reuteri naturally colonizes the digestive tract, especially after consumption of dairy products. It is one of the Lactobacillus strains that can be transmitted to infants via breast milk. Lactobacillus brevis is another Lactobacillus strain with remarkable effects on intestinal homeostasis and inhibitory effects on intestinal inflammation.Probiotics play several important roles in improving health. Modulating the gut microbiota, regulating the immune system, showing anti‐inflammatory effects, and having activities against pathogens by regulating mucus secretion are some of the important functions of probiotics.As mentioned earlier, modulation of the immune system and anti‐inflammatory effects are among the beneficial effects of probiotics. These processes are critical for the management and control of inflammatory diseases, including inflammatory bowel disease (IBD). Both Th‐1 and Th‐2 activities have been observed in Crohn's disease (CD) and ulcerative colitis (UC), the two subtypes of IBD. Therefore, in IBD, there is often an overproduction of inflammatory cytokines leading to inflammation.Lactobacillus spp. in modulating JAK/STAT and NF‐κB signaling pathways to understand how probiotics might play an effective role in reducing inflammation before, during, and after the presence of an inflammatory state.Several signaling pathways, such as Janus kinase/signal transducer and activator of transcription (JAK/STAT) and nuclear factor kappa‐light‐chain‐enhancer of activated B cells NF‐κB, are involved in the production and function of cytokines and therefore play roles in the inhibition or progression of IBD.22.1Lactobacillus spp. including L. plantarum, L. rhamnosus, L. brevis, and L. reuteri were isolated from the natural gut microbiota of fecal samples from 53 healthy volunteers aged 1−36 years, and the probiotic and phenotypic characteristics of these strains were previously investigated.l‐cysteine and incubated at 37°C for 20 h. After centrifugation at 8000 rpm for 5 min, the pellets were diluted with the antibiotic‐free MRS medium containing 10% fetal bovine serum (FBS), and the optical density (OD) was adjusted to 0.08 (0.5 McFarland). To make the probiotic cocktail, an equal amount of Lactobacillus spp. was mixed and the final concentration was adjusted to 0.08 OD using a spectrophotometer at 600 nm. For treatments, the culture pellet was collected and diluted in RPMI‐1640 with 10% FBS without antibiotics to obtain the final OD concentration of 0.08 at 600 nm.In the current study, the in vitro assay was conducted to evaluate the effects of probiotics on the NF‐κB and JAK/STAT signaling pathways. Four Escherichia coli (ETEC) and Salmonella typhimurium (ST) were cultured in Luria−Bertani (LB) broth (Thermo Fisher Scientific). After that, heat‐killed cultures (100°C for 10 min) were sonicated , and cell debris was centrifuged . All methods were performed according to the relevant guidelines and regulations, and ethical approval for the previous study was obtained from the Pasteur Institute of Iran Committee (IR.PII. REC.1398.060). Signed informed consent was obtained from all participants.For induction of inflammation sonicated pathogen (SP) was used. For preparation, Enterotoxin‐producing 33.15 cells per well were seeded.The human colon adenocarcinoma cell line HT‐29 was purchased from the Cell Bank of the Pasteur Institute of Iran. For HT‐29 cell line, RPMI‐1640 (Thermo‐Gibco) supplemented with 10% fetal bovine serum (Biochrom) and 1% penicillin−streptomycin (Sigma‐Aldrich) was used. Cells were then separated with 0.25% trypsin‐EDTA (Gibco), washed twice with PBS, and counted. The cell suspension was centrifuged and the precipitate was diluted with RPMI‐1640 and 2 × 103.2Lactobacillus spp.  and SP. The components are as follows: sonicated enterotoxigenic E. coli (SP‐ETEC), sonicated Salmonella typhi (SP‐ST), and Lactobacillus spp. To study the effect of probiotics before, during, and after the inflammatory state, Lactobacillus spp. were added as follows: (a) first, Lactobacillus spp. was added to the HT‐29 cell line and, after 6 h, SP‐ETEC and SP‐ST were added to cause inflammation (LP). This treatment was used to observe the effect of Lactobacillus spp. before the inflammatory condition was triggered. (b) First, SP‐ETEC and SP‐ST were added to the HT‐29 cell line and, after 6 h, Lactobacillus spp. was added to determine the supposed effects (PL). This treatment was used to see the effect of Lactobacillus spp. after triggering the inflammation. (c) Lactobacillus spp. and SP (SP‐ETEC and SP‐ST) were simultaneously added to the HT‐29 cell line (P + L). This treatment was used to show the effect of Lactobacillus spp. while the inflammatory condition was induced. In the next step, each well was washed twice with PBS to remove the nonadherent bacteria. These treatments were performed in duplicate and the cell culture was maintained at 37°C and 5% CO2 for up to 48 h. Determination of MOI was performed as previously indicated.HT‐29 cells were exposed to 3.3http://pga.mgh.harvard.edu/primerbank) was used to select quantitative real‐time polymerase chain reaction (qPCR) primers (Table ‐ΔΔCt was used to obtain the relative gene expression in the comparative CT method.gapdh), was selected as the housekeeping gene to normalize the data. To evaluate the mRNA quantification of the studied genes, the ABI step one plus detection system (Applied Biosystems Corp.) and SYBR Green Master Mix (Amplicon Bio) were used.An RNA extraction kit (Roche) was used to extract total RNA according to the manufacturer's instructions. The amount and quality of purified RNA were determined using a NanoDrop1000 UV‐Vis spectrophotometer (measuring absorbance at 260/280 nm). The cDNA template was synthesized using the cDNA synthesis kit (Yekta Tajhiz) according to the manufacturer's instructions. The online website Primer Bank ‐6 and IL‐1β.To determine the phenotypic effects of probiotic treatment on inflammation reduction, cytokine production was assessed by ELISA assay. After SP and 3.5p < .05 were considered statistically significant. The results were presented as mean ± SD.Graphs and statistical analysis of the data were performed using SPSS (ver. 25) and GraphPad Prism software to compare variables of different groups. Statistical differences between multiple groups were determined using ordinary one‐way analysis of variance (ANOVA). 4Lactobacillus spp. The cocktail form with four different species was used to enhance the anti‐inflammatory effect of the probiotic strains. In addition, the SP was used to induce the inflammatory state because bacterial components, including lipopolysaccharide (LPS) and flagellin of gram‐negative bacteria, are released during the sonication process and these components could trigger inflammation via triggering inflammatory signaling pathways. Lactobacillus spp.‐treated HT‐29 cells were compared with C24 and C48 (negative controls) and SP24 and SP48 (positive controls) to evaluate the efficacy of Lactobacillus spp. in up or downregulating the studied genes. It should be noted that the concept of treatment stages (phases) refers to taking probiotics before, after, and during the induction of inflammation.In the present study, we investigated the anti‐inflammatory effect of live 4.1STAT gene expression are shown in Figure p < .05). However, both up and downregulation was observed for STAT genes after probiotic treatments compared to positive controls in a different state of inflammation.Data on STAT1, STAT2, and STAT4 gene expressions showed that Lactobacillus spp. treatment upregulated gene expression at most treatment stages. However, LP24 and PL24 downregulated the expression level in STAT1, STAT2, and STAT4, respectively (p < .001). In STAT5, again LP24 could significantly decrease the expression level (p < .05), while LP48 was able to significantly increase the expression level (p < .0001).Comparative analysis of STAT3 and STAT6, the general trend was downward in most treatment phases. In particular, P + L24 in both genes and PL48 in STAT3 had the greatest effect on reducing expression levels (p < .05).For 4.2JAK expressions are shown in Figure JAK gene expression between SP and negative controls showed that SP‐ETEC and SP‐ST could significantly increase gene expression.Data on JAK1 showed that Lactobacillus spp. could downregulate the expression level both before and after causing inflammation (p < .0001). However, when Lactobacillus spp. were added simultaneously with the SP, the expression level was significantly increased (p < .0001).Comparative analysis of JAK2, Lactobacillus spp. was able to downregulate the expression level before and after triggering inflammation compared to SP48 (p < .0001). When Lactobacillus spp. was added simultaneously with the sonicated pathogen, the result was not homogeneous. Lactobacillus spp. downregulated the expression level in the first 24 h of treatment, but gene expression was increased after 48 h. In JAK3, the general trend was downward in all treatment phases, except for PL24.For TYK)2 gene expression showed that Lactobacillus spp. treatment upregulated gene expression at most treatment stages. However, when Lactobacillus spp. was added to the HT‐29 cell line before inflammation induction, the expression level was decreased after 24 h of treatment (LP24) (p < .0001). Adding Lactobacillus spp. together with SP (during inflammation state) had the most effects on increasing the expression level of the TYK2 gene (P + L24) (p < .05).Comparative analysis of tyrosine kinase (4.3NEMO), TIR‑associated Protein (TIRAP), Interleukin 1 Receptor Associated Kinase (IRAK), and receptor interacting protein (RIP) between SP and negative controls showed that SP‐ETEC and SP‐ST could significantly increase gene expression, especially after 48 h. Lactobacillus spp. treatment had significant reducing effects at all treatment stages. In other words, it could reduce expression before, after, and during inflammation induction. It should be noted that Lactobacillus spp. treatments reduced the expression of TIRAP and RIP to zero.Inflammatory gene expression data are shown in Figure 4.4Lactobacillus spp. significantly decreased cytokine production before, after, and during inflammation. It appears that the addition of Lactobacillus spp. along with the SP (during inflammation) had the greatest effect on reducing inflammatory cytokines.The result of proinflammatory cytokines production are shown in Figure 5IBD as a chronic recurrent disease affects various aspects of the patient. The exact cause or pathogenesis of IBD is not yet fully understood, but dysbiosis of the gut microbiome along with dysregulation of the immune system could lead to inflammation, which is usually observed in patients with IBD. One of the biggest challenges in IBD patients is the fact that the probability of relapse within the next year is 70%−80% in patients with active disease. Thus, if therapeutic choices could prolong the period of remission in the patient, this inactive phase could persist for the next few years. Patients with CD usually suffer more severe symptoms than patients with UC, including hypovolemia, protein‐calorie malnutrition, and anemia. However, it takes 2 years after the initial diagnosis for the first relapse and surgical therapies are usually not used until 10 years after the initial diagnosisLactobacillus in an in vivo model.Lactobacillus spp. treatments in all versions of inflammation induction had significant reduction effects, especially when probiotics and sonicated pathogens were added simultaneously. These phenotypic results were confirmed by the molecular investigation. The results of NF‐κB expression levels were homogeneous. The addition of  sonicated pathogens could significantly increase the expression level. This evidence could show the probable role of pathogenic components to cause inflammation. However, treatment with Lactobacillus spp. had a significant reducing effect. Lactobacillus spp. had a reducing effect on the expression of inflammatory genes before, after, and during the initiation of inflammation. NF‐κB is considered one of the main mediators of inflammatory responses and can induce the expression of several proinflammatory genes.The results of the cytokine assay (reduction of anti‐inflammatory cytokines) in the present study are consistent with the results of our previous study that showed the anti‐inflammatory effect of Lactobacillus spp. had both decreasing and increasing effects on expression levels. However, these results are also consistent with the phenotypic results and the anti‐inflammatory properties of the probiotics. In the current study, the overall trend of expression of some STATs, including STAT3 and STAT6, was downward. In general, downregulation of this pathway has remarkable effects on reducing inflammation. Several studies have reported that reducing the expression of STAT3, for example, can lead to a reduction in IL‐6 and thus contribute to the reduction of the inflammatory state.−/− mice and that deficiency of STAT6 may be helpful in improving IBD.STATs, including STAT1, STAT2, STAT4, and STAT5, was approximately increased. These data could be confirmed by other reports. The overall function of JAK/STAT in controlling IBD is complicated. For example, Rauch et al.STAT1 expression might be elevated in CD, but this condition could not usually be observed in patients with UC. The association between STATs, including STAT1, and anti‐inflammatory cytokines, including the IL‐10 superfamily, may be one of the reasons supporting the beneficial effects of upregulating STAT1 in the treatment of inflammatory diseases such as IBD.STAT5. IL‐22 as one of the anti‐inflammatory cytokines could activate STAT1, STAT5, JAK1, and TYK2 and mediate its function.STATs genes.The results of the JAK/STAT pathway in the current study were different. In other words, JAK1, JAK2, and JAK3 was downward. Targeting JAKs to control IBD has been discussed in several studies. In other words, any agent that has an inhibitory effect on the activities of JAK could control the inflammatory state in patients with IBD.TYK2 seem to be the opposite. The general trend in the expression of TYK2 was increasing. TYK2, together with the STATs component, might be associated with several cytokines. IL‐10 and IFN‐λ are two of the cytokines with anti‐inflammatory properties associated with TYK2.The results of JAKs also showed remarkable data on the control of inflammation. The overall trend for Lactobacillus spp. on signaling pathways could provide clear insight into how probiotics modulate and reduce inflammation. One of the most important issues is understanding the anti‐inflammatory properties of probiotics and clarifying whether probiotics could be used as a preservative or as a therapeutic. Finding the easiest way to both prevent and relieve the symptoms of IBD could be of great importance to IBD patients. Since we use Lactobacillus spp. as pre, post, and cotreatment, and observation of its beneficial effects on all three variants, the results may suggest that these probiotic strains, in turn, could prevent and treat the severity of IBD.In conclusion, evaluating the precise molecular effects of Performed the experiments: Shadi Aghamohammad, Seyedeh Tina Miri, and Saeideh Najafi. Data analysis: Amin Sepehr, Mahdi Rohani, and Shadi Aghamohammad. Writing of the manuscript: Shadi Aghamohammad and Amin Sepehr. Revised manuscript: Mohammad R. Pourshafie and Mahdi Rohani. Conceived and designed the experiments: Mohammad R. Pourshafie and Mahdi Rohani.The authors declare no conflict of interest.The experimental protocols were established following the Declaration of Helsinki and approved by the ethics committee of Pasteur Institute of Iran (IR.PII.REC.1398.060). Signed informed consent was obtained from all participants.


Larix gmelinii; LG) compared with those in degraded forest ecosystems of four forest vegetation types  in the Heilongjiang Zhongyangzhan Black‐billed Capercaillie Nature Reserve in northern China, using Illumina MiSeq sequencing of 16 S rRNA genes. Soil physicochemical properties  differed significantly (p < .05) among the five forests. SOC, C/N, TP, AN, and AP were highest in QM, whereas SOC was lowest in LGQM. Soil pH was lowest in BD and highest in LGQM. α diversity was highest in LG and lowest in QM. The soil bacterial community composition in the climax forest was significantly different from that in the four degraded forests (p < .05). The dominant bacterial phyla were Acidobacteria, Proteobacteria, Verrucomicrobia, Bacteroidetes, Actinobacteria, Gemmatimonadetes, Firmicutes, Chloroflexi, and Rokubacteria. The highest relative abundances of these phyla were: 30.7% for Acidobacteria in LGQM, 42.6% for Proteobacteria in LG, 17.6% for Verrucomicrobia in BD, 5.5% for Firmicutes in BP, and 6.9% for Actinobacteria in QM. The dominant bacterial genera across the five forest vegetation types were Bryobacter and some poorly characterized taxa . Redundancy analysis indicated that SOC, C/N, TP, AN, and AP were the main soil physicochemical properties that shaped the bacterial communities. Our study revealed distinct bacterial diversity and composition in the climax forest compared with values in degraded forests, suggesting that the biotic and abiotic factors associated with climax ecosystems play an important role in shaping soil bacterial community structure and thus biogeochemical functions. The results of this study contribute to a deeper understanding and better predictions of the network among belowground systems and of the functions and services of degraded forests compared with climax ecosystems.Bacteria are a crucial component of forest soil biodiversity and play an important role in numerous ecosystem processes. Here, we studied the patterns of soil bacterial community diversity and structure in a climax forest ( Soil bacterial diversities of conifer forests were significantly higher than broad‐leaved forests as well as mixed coniferous and broad‐leaved forests. Soil organic, C/N, total phosphorus, available phosphorus and available nitrogen were the main drivers of soil diversity and community structure. We measured soil bacterial composition, diversity, and function using Illumina MiSeq sequencing of the 16 S rRNA genes (V3–V4). We hypothesized that: (1) the degraded forests would have better soil conditions than the climax L. gmelinii forest, owing to the occurrence of broadleaved trees in the degraded forests, and thus (2) the soil bacterial community in the degraded forests would have a higher diversity and a more complex structure than the climax forest. The overall aim of this study was to contribute to a deeper understanding and better predictions of the network among belowground systems and of the functions and services of climax ecosystems.bed Fox, , and thu22.1Larix gmelinii, LG) forest and the following four secondary forests were selected for our research: Betula dahurica forests (BD), Betula platyphylla forests (BP), Quercus mongolica forests (QM), and Larix gmelinii and Quercus mongolica mixed forests (LGQM). All forests are older than 40 years. The soil is deeper than 25 cm, developed from granite and classified as Borice Luvisols, according to the American Soil Taxonomy. Table The experimental site was located in the Heilongjiang Zhongyangzhan Black‐billed Capercaillie Nature Reserve  were established in each forest. Fifteen to 20 soil samples (0–20 cm) were obtained, using a soil auger  after removing the litter layer from each plot. Approximately 2.5 kg soil was collected from each plot. The soil samples were immediately placed in an icebox and kept at 4°C until processing in the laboratory. The soil samples were then pooled to form a single composite soil sample per plot and passed through a 2‐mm mesh to remove the roots and other debris. Each composite sample was then divided into two sub‐samples; one was stored at −80°C for DNA extraction (approximately 10 g soil), whereas the other was air‐dried for soil physicochemical analyses (approximately 1 kg soil).2.33.The methods used to measure soil pH, soil organic carbon (SOC), total nitrogen (TN), available nitrogen (AN), total phosphorous (TP), and available phosphorus (AP) were described by Sui et al. . Briefly2.4−1), 1 μl DNA template (50 ng·L−1), and enough ultrapure water (ddH2O) to reach a 25 μl reaction volume. The amplification conditions as follows: pre‐denaturation at 95°C for 5 min, 35 cycles of denaturation at 95°C for 30 s, annealing at 52°C for 45 s, extension at 60°C for 60 s, and finally an extension step at 72°C for 10 min. The PCR products were inspected using 2% agarose electrophoresis, and the PCR amplicons were purified with the AxyPrep DNA purification kit . Three independent PCR replicates were produced per sample then pooled in equal amounts; the pooled samples were then paired‐end sequenced on the Illumina MiSeq v3 platform (2 × 300 bp). The raw sequences were uploaded to the NCBI Sequence Read Archive database under accession number PRJNA691134.Soil total DNA was extracted using the Power Soil DNA isolation kit 12888 according to the manufacturer's instructions. Using 50 ng DNA as a template, the 338F (5′‐ACT CCT ACG GGA GGC AGC A‐3′) and 806R (5′‐GGA CTA CHV GGG TWT CTA AT‐3′) universal primers were used to amplify the V3–V4 regions . Exact barcode matching was implemented, which permitted a two‐nucleotide mismatch during primer matching. The obtained operational taxonomic units (OTUs) were clustered by CD–HIT at a similarity threshold of 97%, and the taxonomy was annotated to the SILVA database (v138.1). Non‐bacterial sequences  needed to be removed from OTU tables prior to statistical analyses, which were implemented using R v3.3.2 . OTU‐level α diversity indices, such as richness, Chao1 index, Abundance‐based coverage estimator (ACE), and Shannon index, were calculated using the OTU table in QIIME2. Venn diagrams, rarefaction curves, and heatmap representations of the relative abundances of bacterial OTUs among soil samples were generated using the “vegan” package in R Oksanen, . The cor33.1p < .05) in soil physicochemical properties among the five forests  of soil bacteria differed significantly with forest type . The bacterial community structure of the two Betula forests (BD and BP) was similar, whereas the community structure of QM was significantly different from that of the four other forests  associated with soil physicochemical properties. Among the top 15 OTUs with the strongest correlation with soil physicochemical properties, excluding the unclassified OTU, eight OTUs were significantly positively and seven OTUs negatively correlated with soil physicochemical properties , resulting in low soil bacterial diversity. However, our results are contrary to the intermediate disturbance hypothesis that disturbance increases diversity. This is because the original LG forest belongs to the climax community, with high‐ecosystem stability, resulting in a high diversity of soil bacteria. Secondary forests grow as part of secondary succession following disturbance by humans and soil habitat destruction, and soil bacteria are extremely sensitive to changes in habitat. Moreover, this study area is characterized by a cold temperate climate, meaning that the habitat is difficult to restore once the forest is destroyed, thus also leading to a decrease in soil bacterial diversity. This also shows that climax ecosystems in cold regions have high‐ecosystem functionality that is reduced and difficult to restore after “a persistent decrease”.The soil bacterial α diversity in the LG forest was higher than that in the broadleaved forests and mixed coniferous and broadleaved forest. This may be because the LG forest is a climax community with high‐ecosystem stability. During deforestation, the soil habitat is destroyed and the diversity of the soil bacteria decreases. Although secondary succession occurs, the soil ecosystem habitat does not recover, which may be responsible for the decreases in bacterial diversity. Our results were consistent with those from previous studies. For example, Jiang et al.  conducte, et al.  investig4.2Soil microorganisms play a critical role in the conversion of organic matter and in biogeochemical cycles  were the key environmental factors affecting the soil bacterial community composition Table . The soiIn our study the relative abundance of soil Actinobacteria in the different forests was related to SOC, TN, and AN, which is consistent with results from previous studies ; writing – review and editing . Beat Frey: Writing – review and editing . Mingyu Wang: Data curation . Xiaohong Weng: Investigation . Xin Wang: Methodology ; resources . Fuyuan Chen: Investigation . Xianda Li: Investigation . Zhong Du: Methodology ; visualization . Libin Yang: Investigation ; methodology ; visualization ; writing – review and editing . Xin Sui: Funding acquisition (lead); methodology (lead); software (lead); supervision (lead); validation (lead); visualization (lead); writing – original draft (lead). Mai‐He Li: Writing – review and editing .This work was funded by the Heilongjiang Province Postdoctoral Research Start‐up Fund Project (LBH‐Q21167); the Natural Sciences Foundation of Heilongjiang Province (LH2020C088); the Outstanding Youth Foundation of Heilongjiang University (JCL202006); and the China Scholarship Council Visiting Scholar Program (201908230401).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.


In this case–control study, our results suggest that the rs3746444 GG genotype increased ESCC risk , which remained consistent after Bonferroni correction. There was no association of rs11614913 and rs2910164 polymorphisms with ESCC. After adjusting by age, sex, smoking, and drinking status and body mass index (BMI), the multiple logistic analysis suggested that rs11614913 T → C variation reduced ESCC susceptibility in females and in the ≥63 years old subgroups, while rs2910164 C → G variation increased ESCC risk in both two BMI subgroups.MicroRNAs (miRNAs) are a group of small, non‐coding, and endogenous RNAs that regulate gene expression and over 50% of them are located at cancer‐related genomic regions or fragile sites. According to previous studies there is significant association of miRNA single nucleotide polymorphisms (SNPs) with tumorigenesis , however, the conclusions have been inconsistent. To investigate the relationship between  We genotyped and assayed the SNPs of rs11614913, rs2910164, and rs3746444 with propriety multiplex SNP genotyping system – SNPScan, which use the highly specific ligation reaction to discriminate alleles. Fluorescence signals are read out and genotypes are determined using the software, GeneMapper. We found that rs3746444 GG genotype increased ESCC risk. All the patients with dysphagia alone or with other symptoms such as chest pain and weight loss and were diagnosed on the basis of flexible upper endoscopy with biopsy.http://www.geneskybiotech.com/en/index.php/Index/fuwuer/id/29) and previously published articles,The blood samples were obtained from peripheral vessels and collected in tubes contained Ethylene Diamine Tetraacetic Acid (EDTA). We utilized the Promega kit  to extract genomic DNA from lymphocytes according to the instructions of the manufacturer, and then amplified sample DNA by polymerase chain reaction (PCR). Three loci, including rs11614913, rs2910164, and rs3746444, were assayed with a custom‐by‐design 2x48‐Plex SNPscan™ Kit  according to the manufacturer's manual , gender distribution, other selected variables, and the genotype frequency in ESCC patients and controls was evaluated with a χ2 test. The crude/adjusted odds ratios (ORs) and the corresponding 95% confidential intervals (CIs) were used to estimate the association between rs2910164, rs11614913, and rs3746444 and the risk of ESCC. Haplotype frequencies of microRNAs were tested through the website  and data analyzed using GraphPad Prism 5 software. The deviation of the Hardy–Weinberg equilibrium (HWE) in the control group was tested with an online χ2 test (http://ihg.gsf.de/cgi‐bin/hw/hwa1.pl). Differences were considered to have statistical significance when p‐values were less than 0.05.Statistics were performed using SAS software (version 9.4). Differences between cases and controls in age were assessed with Student's 33.1p = 0.305 and p = 0.331). There were significant differences in smoking, drinking status, and BMI between cases and controls (p < 0.001). The main data of the three polymorphic sites are summarized in Table miRNA‐499 rs3746444 AA, AG, GG were 71.75%, 26.07%, and 2.18% in controls compare to 72.46%, 23.33%, and 4.22% in patients with ESCC. The p‐value of HWE in controls were 0.578, 0.048, and 0.680, respectively  were recruited in our study (Table 3.2p = 0.009) when compared to the rs3746444 AA genotype, and the frequency of rs3746444 GG also had a close association with an increased risk of ESCC when the rs3746444 AA/AG genotype as reference . After adjustment for gender, age, tobacco using, BMI, and alcohol consumption, there was a significant connection between the rs3746444 GG genotype and increased risk of ESCC as well . For rs3.4http://analysis.bio‐x.cn/myAnalysis.php) (Table rs11614913Crs2910164Ars3746444 was used as a reference (because it was the haplotype with highest frequency), a relationship of seven other haplotypes frequencies to the risk of ESCC was not observed .We constructed eight haplotypes by using an expectation–maximization algorithm on website (p) Table  accordin3.5Association between miRNAs loci and lymph node metastases in ESCC cases fell short of statistical significance in our research Table .4miR‐146a rs2910164, miR‐196a2 rs11614913, and miR‐499 rs3746444 polymorphisms could increase the risk of various cancers, such as esophagogastric junction adenocarcinoma, breast cancer, lung cancer, cervical cancer, and non‐Hodgkin lymphoma.miR‐146a rs2910164, miR‐196a2 rs11614913, and miR‐499 rs3746444 polymorphisms to ESCC susceptibility. Our results showed that the rs3746444 G allele significantly increased ESCC risk, while rs2910164 and rs11614913 SNPs were not related to ESCC in overall result. Moreover, for subgroup analyses, multivariate regression analysis revealed that the rs3746444 GG genotype increased ESCC risk in male, female, age < 63 years, never smoked, never drink, and BMI ≥24 kg/m2 subgroups but decreased ESCC risk in the subgroup of alcohol consumption. The rs2910164 C > G polymorphism increased ESCC risk in the BMI <24 kg/m2, ≥24 kg/m2 subgroups, and the rs11614913 T > C SNP decreased ESCC risk in the female and age ≥ 63 years subgroups. However, we should interpret the results with more cautions, and follow‐up experiments with more specimens and gene–environment factors should be performed.Previous studies confirmed that MiRNA‐499 is located on chromosome 20, and rs3746444 is in the seed sequence of miR‐499a‐3p, which may reduce the expression of miRNA‐499a.SRY‐sex‐determining region box (SOX) is a target gene of miRNA‐499 that suppresses cell growth and is regarded as an antitumor factor.SOX‐6 overexpression could inhibit the proliferation induced by the rs3746444 A/G polymorphism, causing the activation of the Wnt/β pathway, which is involved in oncogenesis and progression. A polymorphism of rs3746444 in the miR‐499 gene has been implicated in tumorigenesis through regulating the expression level of the SOX gene. Numerous studies have reported that SOX‐6 expression was deregulated in ESCC, oral squamous cell cancer, and pancreatic cancer.miRNA‐146a precursor, decreasing mature miRNA‐146a and pre‐miRNA‐146a production and increasing reported gene expression; the downregulated expression of both miRNA‐146a precursor and mature miRNA‐146a could be reversed when C was mutated back to G.The rs2910264 polymorphism with G to C variation occurs at the first nucleotide of the MiRNA‐196 is located on the homeobox (HOX) genes.miRNA‐196a have been found so far, named miRNA‐196a‐1, miRNA‐196a‐2, and miRNA‐196b, respectively. MiRNA‐196a2 is located in the region between HOXC‐10 and HOXC‐9 on chromosome 12,miRNA‐196a and miRNA‐196a*), and rs11614913 is located on miRNA‐196a* mature sequence, not only influencing the mature miRNA‐196a2 level, but also affecting target gene expression.miRNA‐196a2 transfected with pre‐miRNA‐196a2‐C was higher than that transfected with pre‐miRNA‐196a2‐T. Previous studies reported that the homozygote genotype of rs11614913 could reduce the risk of ESCC.miR‐146a, miR‐149, and miR‐196a2, and found that Crs2910164Crs2292832Crs11614913 and Grs2910164Trs2292832Crs11614913 increased the risk of GC, while Crs2910164Trs2292832Trs11614913 and Crs2910164Crs2292832Trs11614913 reduced the GC risk. Mohan Damodaranrs73318382Grs57095329Grs2910164Crs11614913Grs41275794Trs12976445Grs10404453Grs1297533 reduced the risk of prostate cancer, indicating locus–locus interaction of SNPs and gene–gene interaction on cancer risk.Haplotype analysis has stronger statistic power than single SNP analysis,There were some limitations in our study. Primarily, other variants in miRNA genes were not estimated. Second, due to the insufficient sample size, we did not perform a replication of the study. Finally, recruiting controls from hospitals instead of from communities may not accurately represent the whole Chinese populations. Some potential influencing factors were not analyzed and this might reduce the reliability of results.miR‐146a gene, rs11614913 in the miR‐196a gene, and rs3746444 in the miR‐499 gene and susceptibility to ESCC.In summary, our study identified the association between miRNA loci and ESCC in a Chinese Han population. Well‐designed case–control studies including a larger sample size are required to verify these main results and validate the potential gene–gene interaction and gene–environment factors which is implicated in polymorphisms of the rs2910164 in the The authors declared that they have no conflict of interest to this work. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.Weifeng Tang: Conception and design of experiment, revising the manuscript. Chao Liu: Analysis of data, drafting the manuscript. Wenhui Gao: Collection of specimens, extraction of DNA. Lu Lv: Acquisition of data, extraction of DNA. Yijun Shi: Genotyping.The study was approved by the Ethics Committee of Zhenjiang First People's Hospital (ID: K‐20210064‐W) Zhenjiang, China. All patients provided written informed consent.Appendix S1Click here for additional data file.


To study the clinical signs and mechanisms  of myoendocarditis in the long‐term period after COronaVIrus Disease 2019 (COVID‐19).Fourteen patients  with biopsy proven post‐COVID myocarditis were observed. The diagnosis of COVID‐19 was confirmed by IgG seroconversion. The average time of admission after COVID‐19 was 5.5  months. An endomyocardial biopsy (EMB) of the right ventricle was obtained. The biopsy analysis included polymerase chain reaction diagnosis of viral infection, morphological, immunohistochemical (IHC) examination with antibodies to CD3, CD45, CD68, CD20, SARS‐Cov‐2 spike, and nucleocapsid antigens. Coronary atherosclerosis was ruled out in all patients over 40 years.The new cardiac symptoms  appeared 1–5 months following COVID‐19. Magnetic resonance imaging showed disseminated or focal subepicardial and intramyocardial late gadolinium enhancement, hyperemia, edema, and increased myocardial native T1 relaxation time. Antiheart antibodies levels were increased 3–4 times in 92.9% of patients. The mean left ventricular (LV) ejection fraction (EF) was 28% . Active lymphocytic myocarditis was diagnosed in 12 patients, eosinophilic myocarditis in two patients. SARS‐Cov‐2 RNA was detected in 12 cases (85.7%), in association with parvovirus B19 DNA—in one. Three patients had also endocarditis . As a result of steroid and chronic heart failure therapy, the EF increased to 47% .COVID‐19 can lead to long‐term severe post‐COVID myoendocarditis, that is characterized by prolonged persistence of coronavirus in cardiomyocytes, endothelium, and macrophages (up to 18 months) in combination with high immune activity. Corticosteroids and anticoagulants should be considered as a treatment option of post‐COVID myoendocarditis. The possibility of acute coronavirus‐induced myocarditis was demonstrated in a series of studies. The first detection of coronavirus particles by electron microscopy in myocardial biopsy of a patient with COVID‐19 and cardiogenic shock has been described,The maximum time since infection, in which RNA‐positive myocarditis has been detected, is only 4 weeks.1.1To study the clinical signs and mechanisms  of myoendocarditis in the long‐term period after acute COVID‐19.2Fourteen patients  with morphologically verified post‐COVID myocarditis were included in the study.Inclusion criteria were a history of serologically verified new coronavirus infection, appearance or marked progression of cardiac symptoms  after COVID‐19, presence of Dallas morphological and immunohistochemical criteria for active myocarditis according to ESC guidelines 2013.Exclusion criteria were previously verified by MRI and/or EMB myocarditis, immunosuppressive therapy, coronary artery stenoses over 50%, valvular heart diseases, hypertensive heart disease, diffuse connective tissue disease, systemic vasculitis, sarcoidosis.Peculiarities of acute COVID‐19. The diagnosis of COVID‐19 was confirmed by the appearance of SARS‐Cov‐2 IgG. PCR examination of nasopharyngeal smears was performed in 10 patients and was positive in four patients. Nonsevere bilateral viral pneumonia was diagnosed by chest CT scan in five patients; in eight patients chest CT was not performed. There were no cases of severe respiratory failure or evidence of acute cardiac injury during the acute COVID‐19. Treatment varied, but steroid therapy was administered in one patient only.2.12. Seven patients had a history of arterial hypertension, which was well controlled. One patient had a history of smoking, one—atopic bronchial asthma, one—diabetes mellitus, and one—bicuspid aortic valve without significant valvular dysfunction. Ten patients had no symptoms of heart disease before the COVID‐19. Other four patients had cardiac symptoms of unclear genesis before coronavirus infection  in two patients, and moderate left ventricular (LV) dysfunction in two patients]. The myocarditis was not previously diagnosed.The mean body mass index was 28.3 ± 3.5 kg/mAfter COVID‐19, all patients experienced prominent cardiac symptoms. The mean time to clinic admission after COVID‐19 was 5.5  months, ranging from 2 to 9 months; the time to onset of symptoms after acute coronavirus infection was 1–5 months. In two cases, symptoms of myocarditis appeared not after COVID‐19, but only after subsequent vaccination.2.2EMB of the right ventricle (RV) was obtained. The analysis of myocardial biopsy specimens included hematoxylin‐eosin and Van Gieson stains, immunohistochemical (IHC) examination with antibodies to CD3, CD45, CD68, CD20, SARS‐Cov‐2 spike, and nucleocapsid antigens. This study was supported by Russian Foundation for Basic Research grant no. 20‐315‐90021/20. The specialized cardiac pathologist evaluated the all EMBs.n = 6) were also performed.The myocardium was examined by polymerase chain reaction (PCR) for DNA of parvovirus B19, herpes group viruses, adenovirus, and SARS‐Cov‐2 RNA. Total RNA was extracted from myocardial fragments using the RNeasy Mini Kit . A QuantiTect single‐step PCR kit (Qiagen) was used to identify SARS‐CoV‐2. Primers selection was based on the publicly available data on gene DNA and mRNA sequences in the NCBI database, Primer‐BLAST software was used. The evaluation of antiheart antibodies (AHA) titers by indirect immunofluorescence method, echocardiography (EchoCG), 24‐h ECG monitoring, coronary angiography, and cardiac MRI  CHF. In 11 patients, it was biventricular . We observed the moderate dilatation of all heart chambers and decreased LV ejection fraction (EF) up to 20%–30% Figure . High puC‐reactive protein (CRP) elevation and leukocytosis remained in three patients. AHA titers were elevated 3–4 times (1:160‐1:320) in all except one patient. A typical ECG sign was a low QRS voltage (in 57.1%). Three patients developed persistent AF. Two‐thirds of the patients had PVCs and nonsustained ventricular tachycardia (VT). Two patients developed a left bundle branch block and another patient—AV block with pauses up to 5 s during AF.On cardiac MRI only a patient with IE had signs of myocardial edema Figure , the oth3.2A correlation of the clinical data of the patients with the myocardial morphological studies is presented in Table The diagnosis of active myocarditis was confirmed in all cases. The evidence of activity included signs of cardiomyocytes' death  and severe dystrophy, as well as interstitial edema in all patients, Figure IHC study confirmed the diagnosis of myocarditis in all cases Figure . In some3.3Real‐time PCR analysis of myocardium detected no adenovirus or herpetic virus genome. Parvovirus B19 DNA was found in one patient (see Table A pronounced positive expression to the SARS‐CoV‐2 nucleocapsid was detected in cardiomyocytes and infiltrate cells (mainly macrophages). Positive reaction to spike protein was observed in vascular endothelium and in infiltrate cells, including infiltrate in the endo‐ and pericardium, Figure 3.41.IE was diagnosed in a 39 y.o. patient with severe myocarditis . All patients were treated with corticosteroids, in one case of absence of coronavirus in the myocardium—in combination with mycophenolate mofetil 2 g/day. A good immediate response to treatment  was achieved  post‐COVID myocarditis. SARS‐CoV‐2 RNA was detected in the myocardium of 12 out of 14 patients. The longest follow‐up after acute COVID‐19 was 18 months. Maximal late myocarditis was diagnosed in a patient who was vaccinated 1 year after acute COVID‐19. Only two such patients were included in the study. This suggests a trigger (resolving) role of the vaccine administered already after a coronavirus infection. At the same time, in both patients coronavirus was detected in the myocardium, which does not allow us to talk about isolated postvaccine myocarditis.Our study presents, for the first time, The absence of virus in one case demonstrates the virus‐negative post‐COVID myocarditis and other mechanisms for its development. This is primarily due to AНA production, which we detected in 73.5% of inpatients with coronavirus pneumonia in acute COVID‐19.To date, corticosteroids remain the only group of drugs for which a positive effect on prognosis in COVID‐19. Steroids resulted in a clear improvement in all of our patients. The feasibility of using anticoagulants in patients with myocarditis, myocardial microvascular thrombosis, and especially thromboendocarditis should be discussed.5SARS‐Cov‐2 infection leads not only to acute but also to subacute/chronic myocarditis, with clinical manifestations occurring within 1 to 4–6 months after acute COVID‐19. The main mechanisms of post‐COVID myocarditis are long‐term persistence of coronavirus in myocardium  combined with high immune activity (high titers of AHA). In a pandemic, any unclear myocardial dysfunction requires serodiagnosis of coronavirus infection. Corticosteroids and anticoagulants should be considered in the treatment of post‐COVID myoendocarditis.


MYC/BCL2, and stratified the series into three groups, yielding hazard ratios from 0.15 to 5.49 for overall survival, and from 0.17 to 5.04 for progression‐free survival. Group differences were highly significant (p < 0.0001), and the scoring system was applicable to younger patients (<60 years of age) and patients with advanced or localized stages of the disease. Results were validated in an independent dataset from 166 DLBCL patients treated in two distinct clinical trials. This risk score combines clinical and biological data in a model that can be used to integrate biological variables into the prognostic models for DLBCL cases.Diffuse large B‐cell lymphoma (DLBCL), the most frequent non‐Hodgkin's lymphoma subtype, is characterized by strong biological, morphological, and clinical heterogeneity, but patients are treated with immunochemotherapy in a relatively homogeneous way. Here, we have used a customized NanoString platform to analyze a series of 197 homogeneously treated DLBCL cases. The platform includes the most relevant genes or signatures known to be useful for predicting response to R‐CHOP  in DLBCL cases. We generated a risk score that combines the International Prognostic Index with cell of origin and double expression of  It is a highly clinically, morphologically, and biologically heterogeneous group of aggressive lymphoproliferative disorders . Althoug the COO , 16–22. Other prognostic markers have also been proposed, including Epstein–Barr virus (EBV), P53, CD5, CD30, PD‐L1, and others , 28, 29.MYC/BCL2, and a selection of genes known to predict survival in R‐CHOP‐treated DLBCL can integrate all these sources of information to create a robust and reproducible prognostic score. These results were validated in an independent DLBCL cohort.Interactions between these markers are complex and, in many cases, have not been fully assessed. Here, we examine whether a customized NanoString panel including genes providing information about COO, 22.1A total of 197 DLBCL patients were retrospectively enrolled in the study, which was developed in collaboration with many Spanish hospitals working in adherence to the clinical protocols of the GELTAMO Group, under the supervision of the Fundación Jiménez Díaz Ethics Committee, and conducted in accordance with the Declaration of Helsinki. All participants signed an informed consent form at diagnosis. Samples were collected and clinical data were managed, following protocols guaranteeing the confidentiality of donor data. Material and data from other centers were anonymously transferred to the Lymphoma Research Laboratory  after obtaining approval from the corresponding ethics committees, in accordance with the relevant Spanish legislation . Diagnoses were made using whole sections. All cases were reviewed, and a consensus diagnosis was made by two expert hematopathologists  based on the World Health Organization Guidelines from 2017. Double Hit/Triple Hit (DH/TH) large B‐cell lymphoma were not excluded from this study. Samples and clinical data from patients were provided by the following departments and biobanks: Pathology Department of Bellvitge Hospital and HUB‐ICO‐IDIBELL Biobank, funded by Instituto of Salud Carlos III (PT17/0015/0024); Pathology Department of Clínico Universitario of Santiago Hospital‐CHUS and HULA Biobank‐Biobanco do Complexo Hospitalario Universitario of Santiago de Compostela (PT17/0015/0002); Biobank of Virgen del Rocío Hospital (PT17/0015/0041); Valdecilla Biobank (PT17/0015/0019); and MD Anderson Biobank (PT17/0015/0008).Tissue for biological studies was collected at the time of diagnosis before initiating an R‐CHOP/R‐CHOP‐like regimen. Clinical features ; Eastern Cooperative Oncology Group Performance Status; lactate dehydrogenase; number of extranodal sites) were also recorded at the time of the initial diagnosis . ClinicaThe validation series comprised the 166 DLBCL patients retrospectively enrolled in two multicenter clinical trials (RHDS0305 and DLCL04) and treated with R‐CHOP and R‐CHOP‐like regimen , 32, 33.2.2RNA was isolated from formalin‐fixed, paraffin‐embedded (FFPE) diagnostic tissue biopsies. Total RNA was extracted with an RNeasy FFPE kit  according to the manufacturer's instructions. RNA quality and quantity were assessed with an RNA 6000 Nano kit  using the Agilent 2100 Bioanalyzer System (Agilent Technologies).2.3MYC and BCL2 gene expression [We used the Research Use Only Version of the NanoString LST assay .Briefly, the probes were hybridized to 400 ng of the total RNA for 16 h at 65°C. Automated removal of excess probe and immobilization of probe–transcript complexes on a streptavidin‐coated cartridge were carried out in the nCounter Preparation Station. Gene expression values were normalized with respect to housekeeping genes. Normalized count data were logCOO was determined on paraffin‐embedded tissue using the Lymph2Cx NanoString assay, in which low and high linear predictor scores enable samples to be assigned to the GC and ABC groups, respectively. We classified each of the samples using the NanoString COO algorithm and the scores . Samples2.4Tissue microarrays (TMAs) were designed and constructed using two 0.6‐mm tissue cores per case, taken from archival FFPE patient tumor blocks, using reactive tonsils as control. Immunohistochemical staining was performed and assessed by an expert central pathology committee. All diagnostic immunostaining was done, following standardized protocols in whole sections. The panel of antibodies was chosen on the basis of their biological and clinical relevance to clinical classification and DLBCL pathogenesis. Positive tumor cells were scored in percentage classes of the following markers: CD10, BCL6, MUM1, P53, CD5, CD30, Ki67, PDL1, BCL2, MYC, EBV (EBER), CYCLIN D1, and CD20 , who considered either the cytoplasmic, membranous, or nuclear staining intensity, or the percentage of positive cells. The thresholds used were those recommended by the WHO classification of tumors of hematopoietic and lymphoid tissue criteria: CD10, MFluorescence in situ hybridization (FISH) was performed following the routine protocol at the Pathology Department of our hospital. Vysis LSI Break Apart FISH probes  were used to analyze BCL2 (18q21.33 region), BCL6 (3q27.3 region), MYC (8q24.21 region), and IGH (14q32.33 region), in accordance with the manufacturer's instructions.2.5Survival data were available for 197 patients with DLBCL from the discovery series and for 166 patients with DLBCL from the validation series. Follow‐up was calculated based on observation time by Kaplan–Meier curve analysis. Progression‐free survival (PFS) was calculated from the date of diagnosis to the date of disease progression. Overall survival (OS) was evaluated from the date of diagnosis to the date of death. The Kaplan–Meier method was used to estimate OS and PFS, and the log‐rank (Mantel–Cox) test was used to assess differences in OS and PFS between the patient groups.2.6https://www.R‐project.org, R Foundation for Statistical Computing, Vienna, Austria) and GraphPad PRISM version 8.4.0 software . Categorical variables are reported as percentages. Continuous variables are summarized as the median and range. For all analyses, values of p < 0.05 were considered statistically significant.All statistical analyses were performed using R 3.6.1  as cut‐off points, we generated three risk groups  with different prognoses.We performed a Cox proportional hazards analysis with the IPI, COO, and Finally, to reinforce the robustness of the conclusions, we validated the prediction model in the validation set (N = 166).33.1The clinical characteristics of the discovery series are summarized in Supporting information The median follow‐up for the whole series was 27 months (range: 1–211), and survival probability for the whole series was 76.84% at 27 months. Survival probability at 36 months was 70.16% and at 60 months it was 68.1%. The median follow‐up for the patients in the validation series was 63.8 months (range: 0.3–119). At the time of initial diagnosis, we observed that the median age of the series was 65 years (range: 17–91), and more than half were male (52.3%). At the time of initial diagnosis, this series consisted mostly of patients at advanced stages of the disease (stages III–IV: 63%).IHC with specific antibodies, listed in Supporting information Table 3.2MYC/BCL2 double expression, as defined in the NanoString data, was significantly associated with shorter OS in the Cox univariate analyses  as individual variables in our series by Kaplan–Meier survival analysis. The IPI predicted shorter OS . The full results of the univariate analyses for OS and PFS are shown in Supporting information Univariate and multivariate Cox proportional hazards analyses were used to identify risk factors. Clinical and biological risk factors were evaluated, and IPI, COO, and p < 0.05 in the multivariate Cox models. Data in Supporting information MYC/BCL2 were independent prognostic predictors of OS and PFS, respectively. A risk score was then calculated from the final multivariate Cox regression model by incorporating the three risk factors  {coxph ∼ IPI + MYC‐BCL2 + NANOSTRING, data = PFS)}, which were weighted by their Cox coefficients. For the discovery series, risk scores for each patient are indicated in Supporting information Table We considered risk factors with a significance of The risk score was further split into terciles for OS  and for PFS .p < 0.0001) .For OS, the HRs for the three groups determined by risk score were: low‐risk, HR = 0.15, 95% CI: 0.07–0.33; intermediate‐risk, HR = 2.63, 95% CI: 1.20–5.76; and high‐risk, HR = 5.49, 95% CI: 2.54–11.90 (p < 0.0001).Analysis of PFS gave the following HRs: low‐risk, HR = 0.17, 95% CI: 0.08–0.48; intermediate‐risk, HR = 3.94, 95% CI: 1.98–7.86; and high‐risk, HR = 5.04, 95% CI 2.49‐10.20 , since previous studies had shown that prognostic markers and the response to therapy differs among clinical stages . TherefoNext, we examined whether this risk model could be applied to localized clinical stages (stages I–II) (72 patients). For OS, the risk score was split into low‐risk (38 patients) and high‐risk (34 patients) groups, using the median as the cut‐point. For PFS, low‐risk (35 patients) and high‐risk (37 patients) groups were compared. Figure MYC/BCL2 double expression, determined by NanoString, yields a score that makes it possible to recognize substantive differences in survival probability. This can be used for young patients (for PFS) and patients at advanced (stages III–IV) and localized (stages I–II) clinical stages of the disease.Taken together, these findings indicate that the prognostic model, which combines IPI with both COO and 3.4To evaluate the performance of the predictive model, data from a retrospective validation series of FFPE samples from 166 DLBCL patients  were analyzed after they had been treated with immunochemotherapy , 32, 33.The characteristics of the validation set are summarized in Supporting information Table The risk model was then applied to the validation series. As a result, the risk score was further split into terciles for OS: low‐risk (58 patients), intermediate‐risk (58 patients), and high‐risk (50 patients) groups. For PFS, low‐risk (58 patients), intermediate‐risk (52 patients), and high‐risk (56 patients) groups were established.p < 0.005) ; and high‐risk, HR = 7.32, 95% CI: 2.4–21.8 (p = 0.000345).  Analysis of PFS identified the following HRs: low‐risk, HR = 0.4004, 95% CI: 0.2240–0.7155; intermediate‐risk, HR = 3.07, 95% CI: 1.30–7.24 (p = 0.0105); and high‐risk, HR = 5.63, 95% CI: 2.52–12.60 (p = 0.00002). Risk scores for each patient in the validation series are presented in Supporting information Table OS probabilities for the three groups were: low‐risk, HR = 0.2866, 95% CI: 0.1473–0.5575; intermediate‐risk, HR = 3.85, 95% CI: 1.28–11.60  or NF‐kB activity (Derenzini's model) [Our own group and others have published several studies, all of which have concluded that the IHC analysis of COO markers and MYC/BCL2 protein expression could be used to predict the response to R‐CHOP , 47–50, COO data  and are activity , and cons model) .This study is not comparable with those of Ennishi et al. ADDIN EN.CITE or Sha et al . The forMYC/BCL2 expression—in a risk model that produces a patient‐specific risk estimate based on a firmly established molecular approach that can be used with paraffin‐embedded tissues. The model is also applicable to early stages and younger patients, which is something that models based on IHC techniques have failed to achieve [This proposal combines a consolidated prognostic clinical model with the main sources of biological variation among the DLBCL cases—COO and  achieve .This study was performed using consecutive routine cases diagnosed with DLBCL, and thereby closely mimicked the routine composition of cases that receive this diagnosis.An obvious question that arises is whether this can not only stratify patients into different risk groups, but also help establish a different therapeutic approach. To obtain an answer will require specific clinical trials to be designed and executed, although here we have demonstrated that the risk score can be applied to patients of different ages and clinical stages, encouraging the pursuit of risk‐adjusted therapeutic schemes. Our study was not designed to identify specific therapeutic targets for these high‐risk patients, but other experimental studies have shown that MYC and BCL2 can be targeted using BET and BCL2 inhibitors .M.A.P. declares having received lecture fees and advisory board fees from Millennium/Takeda, Jansen, NanoString, Kyowa Kirin, Gilead, and Celgene.The authors declare that they have no significant relationships with, or financial interests, in any commercial companies pertaining to this article.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.