Our findings showed that a rich reservoir of divergent AR-like genes  were distributed across the 13 CPR superphyla. These AR-like genes encode 89 different enzymes that are associated with 14 different chemical classes of antimicrobials. Most hits found (93.6%) were linked to glycopeptide, beta-lactam, macrolide-lincosamide-streptogramin (MLS), tetracycline, and aminoglycoside resistance. Moreover, two AR profiles were discerned for the Microgenomates group and “Candidatus Parcubacteria,” which were distinct between them and differed from all other CPR superphyla. CPR cells seem to be active players during microbial competitive interactions; they are well equipped for microbial combat in different habitats, which ensures their natural survival and continued existence.Microbes belonging to Candidate Phyla Radiation (CPR) have joined the tree of life as a new branch, thanks to the intensive application of metagenomics and sequencing technologies. CPR have been eventually identified by 16S rRNA analysis, and they represent more than 26% of microbial diversity. Despite their ultrasmall size, reduced genome, and metabolic pathways which mainly depend on exosymbiotic or exoparasitic relationships with the bacterial host, CPR microbes were found to be abundant in almost all environments. They can be considered survivors in highly competitive circumstances within microbial communities. However, their defense mechanisms and phenotypic characteristic remain poorly explored. Here, we conducted a thorough IMPORTANCE To our knowledge, this study is one of the few studies that characterize the defense systems in the CPR group and describes the first repertoire of antibiotic resistance (AR) genes. The use of a BLAST approach with lenient criteria followed by a careful examination of the functional domains has yielded a variety of enzymes that mainly give three different mechanisms of action of resistance. Our genome analysis showed the existence of a rich reservoir of CPR resistome, which is associated with different antibiotic families. Moreover, this analysis revealed the hidden face of the reduced-genome CPR, particularly their weaponry with AR genes. These data suggest that CPR are competitive players in the microbial war, and they can be distinguished by specific AR profiles. The increased use of exploring tools in the 21st century, such as high-throughput sequencing and its wide application in metagenomics, has led to broadening access to genomic data of uncultured microorganisms . Among tCPR are a group of highly distinct and abundant ultrasmall microbes , 7, whicPatescibacteria (the superphylum that contains most CPR genomes), members of this superphylum can escape bacteriophage attacks (attachment) by the natural suppression of common phage membrane receptors  was cocultured in 2015 based on its streptomycin resistance selection  have been reported in different human microbiomes   , 18–22. etc.) 18––28. TheiMoreover, according to metagenomic analyses of ancient DNA, CPR microbes have been reported in ancient samples of Neanderthal calcified dental plaque  dated thousands of years ago . In parain silico analysis using BLAST and search for functional domains. We found that CPR members are also players in this microbial “infinity war.”Given that CPR members (i) are widely spread in different ecological niches and microbiomes, (ii) have never been isolated and grown in pure culture, and (iii) have a high number of unknown biosynthetic activities within their genomes, few if any studies have investigated the defense mechanisms and competing behavior of CPR cells. In fact, survival strategies, which are, namely, AR gene components expressed by CPR members against other microbes in different hostile/competitive environments, have not yet been explored. For this purpose, we describe for the first time the first repertoire of AR genes in CPR genomes by Patescibacteria was divided into 4 groups or phyla: Parcubacteria, Gracilibacteria, Microgenomates, and unclassified Patescibacteria. The simple BLASTp search of the 3,654,820 CPR protein sequences predicted using the RAST server, against a total of 12,033 AR protein sequences, resulted in 320,121 matches , 1.8% phenicol, 1.96% fosfomycin, 0.62% rifamycin, 0.78% quinolone, and 0.5% other antibiotic families   and S3. In addition, we found AR hits in almost all studied CPR genomes across different superphyla; thus, 4,052 genomes were positive through our analysis out of 4,062 genomes tested (99.75%). The prevalence of the AR content was fairly diversified between the CPR superphyla, as the number of their available genomes was not homogeneous  and S3. d-alanine:d-alanine into d-alanine:d-lactate or d-alanine:d-serine   . Of thesclusters . More an10.1128/mSystems.00898-21.1FIG S1d-Alanine:d-Lactate and d-Alanine:d-Serine with the presence of the three essential genes in the CPR superphyla tested. Forty-eight CPR genomes have a total of 52 potential vancomycin clusters including four genomes each with two types of clusters. Download FIG S1, PDF file, 0.1 MB.Heat map of different vancomycin clusters Copyright © 2021 Maatouk et al.2021Maatouk et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Given that CPR members have very small genomes in comparison with other microorganisms , 4, it ierm  and cfr [n = 648 hits])  with MLS-inactivating enzyme activity  and gna  , tetBP (n = 778), and tetW (n = 564) (Although macrolide-lincosamide-streptogramin (MLS) antibiotics are chemically distinct, but because of their similar mechanism of action, they are classified in the same group . MLS ant8 hits])  and S3 dactivity  and S3. activity . The majn = 749)  and S3. n = 564)  and S3.Parcubacteria phylum including most CPR genomes and “Candidatus Wirthbacteria” with only two genomes   and acetyltransferase (gna) activity against aminoglycosides, phosphorylation of fosfomycin (fomA), and a remarkable number of class A and D beta-lactamases (Microgenomates possesses the greatest number of cat (chloramphenicol acetyltransferase) enzyme-encoding genes, streptogramin acetyltransferase (vat) genes, and rifampin phosphotransferase (rph) genes detected among all CPR genomes. In contrast, taking Saccharibacteria as an example of the group of other CPR superphyla, members of this superphylum have a high number of streptogramin lyases (vgb) and erythromycin esterases (erm) compared to other CPR groups   and S3. ctively) . The firctamases  and S3. R groups  and S3.10.1128/mSystems.00898-21.2FIG S2Candidatus Saccharibacteria,” unclassified Patescibacteria group, Candidate division WWE3 (Katanobacteria), “Candidatus Peregrinibacteria,” “Candidatus Berkelbacteria,” “Candidatus Dojkabacteria,” “Candidatus Doudnabacteria,” “Candidatus Gracilibacteria,” “Candidatus Absconditabacteria,” Candidate division Kazan-3B-28, and “Candidatus Wirthbacteria” together as the other CPR group. Download FIG S2, PDF file, 0.5 MB.Multi-informative heat map  with theCopyright © 2021 Maatouk et al.2021Maatouk et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Parcubacteria group, where bah (the amidohydrolase enzyme that inactivates bacitracin), icr (intrinsic colistin resistance enzyme), and fus (fusidic acid resistance enzyme) were found only in this CPR group  than in those found in the environment (80 hits over 187 genomes) . Interestingly, genomes from the three human-associated CPR superphyla showed a higher average of AR-like genes per genome than others detected from the environment. This was found specifically in the Absconditabacteria superphylum, with 9.8 AR-like genes/genome for those detected in humans and 2.75 for others found in the environment  . It is aironment .10.1128/mSystems.00898-21.3FIG S3Saccharibacteria superphylum. (B) Absconditabacteria superphylum. (C) Gracilibacteria phylum. Download FIG S3, PDF file, 0.1 MB.Network analysis of antibiotic resistance-like gene distribution between CPR genomes detected in humans (red) and the environment (green). (A) Copyright © 2021 Maatouk et al.2021Maatouk et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.00898-21.8TABLE S4Table S4, XLSX file, 0.01 MB.Number of antibiotic resistance hits according to the 3 CPR superphyla found in human and environmental samples. Download Copyright © 2021 Maatouk et al.2021Maatouk et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the To sum up, these results are suggestive of the influence of the CPR environment on its phenotypic characteristics and suggest a link between CPR members, other microbes, and their environment. Together, the high presence of AR-like genes in all CPR genomes may suggest that they have other vital functions for CPR cells and that therefore they are not strictly related to antibiotic resistance. They are likely to be functionally linked to other metabolic pathways and, subsequently, to participate in the survival of these microorganisms.There are significant knowledge gaps in our understanding of the physiological and biological processes of CPR, as well as of their interactions with host bacteria and their potential associations with human pathologies. Thus, it is essential to expand our research on these living microorganisms, which represent a new branch in the tree of life . This stin silico screening for AR in all CPR genomes available on NCBI, regardless of their quality and assembling methods, to detect the maximum of AR genes. The superphylum assignments were taken from NCBI and were not independently confirmed in our study, since our main aim is not reclassifying CPR but reporting AR genes in all their available genomes. Our analysis was based on a thoughtful strategy for these new microorganisms, using computational methods with adapted criteria. We revealed a rich repertoire of AR genes contained in almost all tested CPR genomes. We allocated the AR-like genes into families/groups to visualize the prevalence of AR genes in different CPR superphyla and, potentially, to find a correlation between genes encoding resistance to a particular antibiotic family and the superphylum of interest.Here, we conducted a thorough in silico analysis for the moment. AR screening of CPR genomes by analyzing nucleotide sequences against a database of bacterial resistance genes    resulted10.1128/mSystems.00898-21.9TABLE S5Table S5, XLSX file, 0.01 MB.Number of antibiotic resistance (AR) hits according to all CPR genomes tested, using the default parameters of ABRicate and ARG-ANNOT as AR database. Download Copyright © 2021 Maatouk et al.2021Maatouk et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the It was critical to establish an adapted strategy for AR screening in CPR genomes, as they have different nucleotide and protein sequences from bacterial ones . BecauseStreptomyces coelicolor A3(2) and Streptomyces hygroscopicus]  can be now reannotated as potential AR genes instead of being simple hypothetical proteins. The divergence observed in these sequences suggests that they have other functions involved in different metabolic pathways rather than resistance to antibiotics. Further studies are needed to confirm their functions.Concerning the resistance to the glycopeptide family, we could detect 20 different types of vancomycin resistance, in the 4,062 CPR genomes, which covers the high diversity that has previously been described for these genes . Howeverd-alanine:d-alanine peptidoglycan precursor  (data not shown). These genomes may naturally produce the d-Ala:d-Lac or d-Ala:d-Ser peptidoglycan precursors rather than the natural precursor d-Ala:d-Ala in bacteria. This supports the intelligent way in which these microorganisms survive with a limited number of genes, that is, an incomplete but functional cluster . This supports the idea that the CPR genome is simple but efficient. Further analysis should be carried out to verify the AR conferred by the absence or modification of the antibiotic’s targets, in addition to that conferred by the presence of active enzymes determined as part of this study.However, as described previously, the membrane of CPR cells is very similar to that of Gram-positive bacteria , which drecursor . CPR micOur results also show that there is almost one beta-lactam resistance gene per CPR genome; 77% of the tested genomes have at least one gene which codes for beta-lactamases . These genes may play a role in the degradation of substances used in metabolic pathways, including beta-lactams. Several studies have shown that beta-lactamase genes are multifunctional genes which play several roles including, but not limited to, endonuclease, exonuclease, RNase, and hydrolase . FurtherCandidatus Saccharibacteria”) with its host species bacterium, Schaalia odontolytica strain XH001  detected in humans and the environment can be linked to the source of isolation of CPR genomes tested and/or to antibiotic consumption in humans.It is worth noting that the number of AR-like genes was not found to be correlated with genome size. Thus, the CPR that are found in the environment have a larger genome size but a smaller average of AR-like genes than the CPR detected in human microbiomes. This reversed tendency in the three CPR superphyla  to survive in the environment (which secrete antibiotic without having the convenable resistance gene) or against other microbes which produce antibiotics. Otherwise, CPR could use their AR profile to protect their bacterial host against a given antibiotic, since they cannot survive without a viable one.in silico screening are expected to be confirmed in upcoming in vitro experiments. A specific database for AR gene screening in CPR genomes needs to be created to collect these new results for further studies.Finally, the AR-like genes detected in CPR genomes in our To conclude, this work contributes toward a new way of deciphering this new branch of the tree of life. We explicitly explored the CPR resistome by establishing an adapted AR screening strategy for these fastidious microorganisms. We found a gigantic reservoir of provisional AR, representing the first report of resistance genes in CPR genomes. These highly abundant microbes could be an interesting paradigm which constitutes an endless natural source of emerging resistances. Our findings represent a substantial opportunity for future scientific discoveries. If, as expected, the AR-like genes detected in CPR are involved in different metabolic pathways, further studies may lead to the successful growth of CPR cells in pure culture.https://www.ncbi.nlm.nih.gov) were selected and downloaded from the NCBI GenBank database , 126 “Candidatus Peregrinibacteria,” 55 “Candidatus Berkelbacteria,” 53 “Candidatus Dojkabacteria,” 39 “Candidatus Doudnabacteria,” 33 “Candidatus Gracilibacteria,” 13 “Candidatus Absconditabacteria,” 11 Candidate division Kazan-3B-28, and two “Candidatus Wirthbacteria” (under reserve of NCBI superphylum assignments) (Streptomyces spp. [Streptomyces coelicolor A3(2) with GenBank ID CP042324.1 and Streptomyces hygroscopicus with GenBank ID CP003275.1] were selected and downloaded from the NCBI GenBank database.For this study, all nucleotide sequences of CPR genomes available on 12 September 2020 on the NCBI website   . Only 35Genome annotation was generated using the Rapid Annotation using Subsystem Technology tool kit (RASTtk) as implemented in the PATRIC v3.6.8 annotation web service  Fig. 1)Fig. 1).For antimicrobial resistance profiling, we carried out an in-house BLAST search against the protein databases from ARG-ANNOT (Antibiotic Resistance Gene-ANNOTation) , BLDB B, and NDAPredicted AR genes in each CPR genome were individually compared to proteins in each AR database by reciprocal BLASTP . The numhttps://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi)  (psb.cgi) . The prepsb.cgi) .www.displayr.com), to show the distribution of different AR-like genes on CPR superphyla and their mechanisms of AR.AR-like genes detected in CPR tested genomes are represented using Cytoscape v.3.8.2 to highlight the link between different antibiotic families and distinct CPR superphyla. These genes are also represented in a multi-informative heat map created by the Displayr online tool (


The epigenomic regulation of transcriptional programs in metastatic prostate cancer is poorly understood. We studied the epigenomic landscape of prostate cancer drivers using transcriptional profiling and ChIP‐seq in four clonal metastatic tumors derived from a single prostate cancer patient. Our epigenomic analyses focused on androgen receptor (AR), which is a key oncogenic driver in prostate cancer, the AR pioneer factor FOXA1, chromatin insulator CCCTC‐Binding Factor, as well as for modified histones H3K27ac and H3K27me3. The vast majority of AR binding sites were shared among healthy prostate, primary prostate cancer, and metastatic tumor samples, signifying core AR‐driven transcriptional regulation within the prostate cell lineage. Genes associated with core AR‐binding events were significantly enriched for essential genes in prostate cancer cell proliferation. Remarkably, the metastasis‐specific active AR binding sites showed no differential transcriptional output, indicating a robust transcriptional program across metastatic samples. Combined, our data reveal a core transcriptional program in clonal metastatic prostate cancer, despite epigenomic differences in the AR cistrome. Epigenomic and transcriptomic profiling of 4 clonal metastatic samples taken from a patient with prostate cancer during a single autopsy reveals sample‐shared/specific Androgen Receptor (AR) binding sites with enhancer activity. Sample‐shared AR sites are conserved across disease transition states, which are enriched in genes essential for prostate cancer proliferation. Remarkably, genes associated with active metastatic sample‐specific AR binding sites showed no differential transcriptional output. ARAndrogen ReceptorATRXATRX Chromatin RemodelerChIP‐seqChromatin Immunoprecipitation SequencingCTCFCCCTC‐Binding FactorESR1Estrogen Receptor 1ETSETS Transcription Factor ELK1EZH2Enhancer Of Zeste 2 Polycomb Repressive Complex 2 SubunitFFPEFormalin‐Fixed Paraffin‐EmbeddedFOSFos Proto‐Oncogene, AP‐1 Transcription Factor SubunitFOXA1Forkhead Box A1FOXA2Forkhead Box A2H3K27acHistone 3 lysine acetylationH3K27me3Histone 3 lysine trimethylationHDACHistone DeacetylaseJUNJun Proto‐Oncogene, AP‐1 Transcription Factor SubunitmCRPCmetastatic castration‐resistant prostate cancerNENeuroendocrineNR3C1 (GR)Nuclear Receptor Subfamily 3 Group C Member 1PDXPatient‐Derived XenograftPGRProgesterone ReceptorPSAProstate Serum AntigenPTENPhosphatase And Tensin HomologSPOPSpeckle Type BTB/POZ ProteinTP53Tumor Protein P531In metastatic castration‐resistant prostate cancer (mCRPC), under the selective pressure of low circulating testosterone levels, tumors typically respond by restoring the Androgen Receptor (AR) pathway by means of activating mutations or splice variants of the AR , 2, 3, 4et al. [Previously, Haffner et al.  describeIn order to comprehensively survey the epigenome, we generated ChIP‐seq data for AR, its pioneer factor FOXA1 , the chr22.1All samples were procured as part of the Johns Hopkins Prostate Cancer Rapid Autopsy Program with approval by the Johns Hopkins Institutional Research Board and written informed consent. The tissue procurement was conducted in accordance with the Declaration of Helsinki. The patient and the patient's next of kin provided consent. Clinicopathological information is described previously  and belo2.2−1 and underwent a radical prostatectomy. Five years after surgery, his PSA had risen to > 6 ng·mL−1, without any initial radiographic evidence of metastatic disease which prompted the treatment with an investigational prostate cancer vaccine (GVAX) [89Sr, mitoxantrone, abiraterone acetate, etoposide, cisplatin, and external beam radiation along with chemoembolization of the liver lesions. Seventeen years after the initial diagnosis, the patient died of progressive castration‐resistant prostate cancer.The detailed clinical history of the patient in this study has been reported previously . In briee (GVAX) . Over th2.3RNA was extracted from fresh‐frozen tissue samples using the RNeasy Mini kit . Purified RNA was then used to prepare a sequencing library and sequenced on Applied Biosystems SOLiD (V3). Sequencing reads were aligned to hg18 (NCBI36), and raw counts were generated using Bioscope. We used DESeq2 v1.22.2  to norma2.4ChIP‐seq for AR, FOXA1, CTCF, H3K27ac, and H3K27me3 were performed using 5 μg of antibody and 50 μL of Protein A/G magnetic beads  per sample. Antibodies applied here were AR ; FOXA1 ; CTCF ; H3K27ac ; H3K27me3 . As control, input of each sample was generated separately. Immunoprecipitated DNA was processed for sequencing using standard protocols and sequenced on an Illumina Hi‐seq 2500 with 65 bp single end reads.2.5http://broadinstitute.github.io/picard/). We called peaks in all samples for AR, FOXA1, CTCF and H3K27ac against input DNA using two peak callers, macs2 (v2.1.1) [k‐means function [Raw sequencing data were aligned using BWA v0.5.20 to hg19 and filtered for mapping quality > MQ20 with samtools version 1.8 . Duplica(v2.1.1)  and DFil(v2.1.1) . The int(v2.1.1) . Count p(v2.1.1) . To prod(v2.1.1) . DNA reg(v2.1.1) , 35. To function . The topfunction  with thefunction , 38. Ing33.1PTEN, SPOP, and TP53, copy number alterations including a high‐level copy number gain of the AR locus and structural rearrangements such as an inversion of the ATRX locus in all metastases . Due to technical challenges in calling broad peaks, H3K27me3 data were included only as raw tracks in further analyses.Minimal intra‐individual genetic heterogeneity allowed us to assess whether diverse metastatic environments would associate with changes in the epigenetic landscape. To investigate the constellation of epigenome and transcriptome changes in each metastatic site, we generated RNA‐seq data and ChIP‐seq data for AR, FOXA1, H3K27ac, and H3K27me3 Fig. . For ChI samples . We exclThe AR is a major driver of progression in mCRPC , 40. Betn = 41 630)  Fig. . For AR,30) Fig.  with sim30) Fig. . As expe30) Fig. . These f30) Fig. . Of note30) Fig. . Cumulat3.2P < 0.005), albeit significantly lower than in sample‐shared regions . Moreover, we observed the same pattern for FOXA1, further supporting the hypothesis that absent AR sites are not devoid of enhancer activity . Although the number of reads is of sufficient depth for calling quality peaks (average mean depth > 100), the number of reads in AR sample‐specific regions for ChIP‐seq datasets of AR, H3K27ac, and FOXA1 is consistently lower than sample‐shared regions  with the other datastreams for each sample. This allowed us to uncover epigenetic patterns in both sample‐shared and sample‐specific regions. In sample‐shared AR regions, we found high H3K27ac signal, medium CTCF, low H3K27me3 signal, and high FOXA1 signal, suggesting that these highly conserved AR‐bound regions are found in active enhancers/promoters .The shared AR binding sites (sample‐shared) are very highly bound by H3K27ac and FOXA1. Based on these findings, we hypothesized that the shared AR binding sites represent core binding events of a prostate lineage‐specific epigenetic program that is established during organogenesis and maintained throughout cancer progression. To test this, we investigated the distribution of AR sites in previously published ChIP‐seq datasets from benign prostate tissues, primary prostate cancers  and cellP = 0.03, Fig. To formally test the hypothesis that sample‐shared AR regions are associated with prostate cell survival, we evaluated the enrichment of essential genes (LNCaP) in the gene list that was associated with H3K27ac‐positive (active) sample‐shared AR binding sites . EssentiTo better understand the relationship between all factors and sample types, we examined the correlation of samples using peak occupancy (reads in peaks) and unsupervised clustering. We found sample clustering to be driven by factor, with CTCF and H3K27ac data clustering separately from AR/FOXA1 data, regardless of sample type Fig. . This re3.3k‐means clustering  using ing Fig. . Subsequing Fig. . Genes iing Fig. . These sing Fig. A. Very fing Fig. B. Ingenuing Fig. . Surprising Fig. . We alsoing Fig. C. This iMuch to our surprise, homogeneity was found overall; very few differential pathways were found associated with the sample‐specific differentially bound active AR sites. Therefore, we next investigated the correlation of normalized gene expression for all genes across samples. Also, in this analysis, despite the observed selectivity of AR sites between metastatic sites, gene expression for all samples was highly significantly correlated Fig. . To furt4Tumor‐stromal interactions are widely accepted to contribute to the tumorigenic and metastatic process, but a direct connection between the tumor metastatic niche and tumor epigenome remains thus far elusive. Here, we have profiled clonal metastases from four distinct metastatic sites of a patient who died of mCRPC. We applied a comprehensive integrated approach to capture both transcriptomic and epigenomic alterations, in an effort to better understand the heterogeneity of the epigenome and cistromic changes between different metastatic sites. The unique strength of this study lies in the assessment of multiple mCRPC metastatic lesions from different sites that were derived from a single patient, which show a very high level of genomic homogeneity. This design allowed us to interrogate epigenomic differences in the background of a shared driver gene alterations.cis gene expression [While the vast majority of AR chromatin binding sites were conserved among all metastatic sites, we identified a subset of AR‐bound regions that were sample/metastatic site‐specific. These regions were similar, both in genomic region as well as in DNA sequence motif preference, with the exception of one sample, M45_HLN that shows focal areas of necrosis that may explain cistromic differences. FOXA1 facilitates but does not have transactivating potential alone. We hypothesize differential FOXA1 does not necessarily translate to differences in downstream transcriptomics depending on the transcription factor, co‐factor binding and regulatory effects. In a subset analysis of differential AR‐bound regions marked by H3K27ac, we found that there were very few differences in gene expression programs that are associated with differentially bound regions. This indicates that although there were differences in active AR‐bound regulatory elements, these changes did not translate into differential transcriptional outputs. Importantly, we did not observe sample‐selectivity in H3K27ac occupancy at the AR‐selective sites but did still observe H3K27ac binding, indicating the absence of AR binding does not impact the activity of the corresponding enhancer region. In addition, we did not observe an increase in H3K27me3 levels when AR is absent from a specific site, supporting the conclusion that enhancer activity remains in the absence of AR. However, as both FOXA1 and H3K27ac were present in all samples at specific regions where AR was sample‐specific, we hypothesize that possible other yet‐to‐be classified transcription factors may compensate for AR at these regions. It is also possible that at the sample‐specific AR sites, the AR dynamics in that particular sample at that site were less transient. Interestingly, we found binding at core AR regions is stronger in all sample types: normal, primary tumors, cell lines, and PDX samples, indicating an early epigenetic programming event. Of note, we also found zero overlap of core AR regions with metastasis‐specific AR sites  further pression .The patient in this study was first diagnosed with distant metastatic disease  around 13 years after radical prostatectomy. In the following years, up until death, he developed additional metastases with extensive involvement of the liver, lung and lymph nodes . Over thN = 1’ studies is increasingly recognized [The biological implications of these findings should be interpreted with caution, since there are several limitations to this study. The study is based on the in‐depth analysis of a single case, which raises concerns about the generalizability of the findings. However, the value of ‘cognized , since iIt is important to note that all samples analyzed here were procured at an autopsy and are therefore representative of metastatic tumor cell populations that have evolved in this case over a period of over 17 years and have developed resistance to numerous therapies. It is therefore possible that the tumor burden at autopsy appears from a genetic and epigenetic standpoint more homogeneous, since tumor cells had to pass through numerous treatment‐induced clonal bottle necks. It remains to be explored if samples collected in a longitudinal fashion at different stages of tumor progression show a similarly conserved core epigenome program. Although additional longitudinally collected samples were available for this case, they comprise > 25‐year‐old Formalin‐Fixed Paraffin‐Embedded (FFPE) material, which is not compatible with the ChIP‐seq protocols used in this study. This highlights the need for thoughtful prospective sample collection and for the development of robust protocols to assess cistromic characterization in FFPE materials.This case is characterized by high expression of prostate specific markers and no evidence of divergent differentiation to neuroendocrine (NE) or AR negative prostate cancer , 51, 52.5In summary, this study represents the first in field evaluation of cistromic and epigenomic heterogeneity in any type of metastatic cancer and sets the stage for further studies in this field which will likely yield major new insights into the biology of advanced prostate cancer.LW has received funding from Genmab, BV. WGN is on the Scientific Advisory Board of Cepheid, Inc. WZ has received research funding and speaker’s compensation from Astellas Pharma.YZ and SY performed the experiments. TMS performed computational analyses. AMD‐M, TMS, MCH, and WZ conceived study design. LW, AMB, MCH, and WZ performed study supervision. TJ, AMD‐M, and YZ performed sample procurement and processing. SY and WGN provided infrastructural support. TMS, MCH, and WZ wrote manuscript, with critical input from MLF and JWS.https://publons.com/publon/10.1002/1878‐0261.12923.The peer review history for this article is available at Fig. S1. ChIP‐seq quality measures of anatomically distinct metastases. (A) Boxplots of millions of mapped reads > MQ20 for AR (pink), FOXA1 (turquoise), CTCF (gray) and H3K27ac (dark blue). AR, FOXA1 and H3K27ac have n = 4. CTCF has n = 3. The one outlier H3K27ac sample (outlined in red) indicates the data are less than the 25th percentile (Q1) ‐ 1.5*(interquartile range). (B) Boxplots of number of peaks of AR (pink), FOXA1 (turquoise), CTCF (gray) and H3K27ac (dark blue). Number of samples is the same as left plot.Click here for additional data file.Fig. S2. Shared and unique chromatin binding sites of AR and FOXA1. (A) Venn diagrams depicting the number of shared and unique AR (left), FOXA1 (center) and CTCF (right) binding sites in 3 ‐ 4 anatomically distinct metastases.Click here for additional data file.Fig. S3. Overlap of LNCaP essential genes and genes associated with active sample‐shared AR sites. (A) UpSetR diagram depicting number of shared (red) and unique (gray) genes in LNCaP essential genes [al genes  and geneClick here for additional data file.Fig. S4. Percentage and overlap of genes in significantly enriched Hallmarks gene sets and gene set expression correlation. (A) Stacked barplots indicating the percentage of genes which were in sample‐specific lists (orange) in significantly enriched gene sets (gray). (B) UpSetR diagram depicting the shared and unique genes in Hallmarks gene sets that were sample‐specifically significantly enriched. Red box indicates overlaps of genes in gene sets with the Androgen Response gene set. (C) Correlation matrix of normalized log‐transformed gene expression data for remaining Hallmark gene sets for all samples. Pearson correlation and significance are indicated in the top right (*** indicates P < 0.0001).Click here for additional data file.Table S1. SeqPos DNA motif enrichment at AR sites (shared and sample‐specific).Click here for additional data file.Table S2. Genes associated with H3K27ac‐positive sample selective AR sites and their respective Ingenuity Pathway Analysis Top Canonical Pathways and Upstream Regulators.Click here for additional data file.


AR) copy number (CN).Cabazitaxel improves overall survival (OS) in metastatic castration-resistant prostate cancer (mCRPC) patients progressing after docetaxel. In this prospective study, we evaluated the prognostic role of CTC gene expression on cabazitaxel-treated patients and its association with plasma androgen receptor  or odds ratio (OR) and 95% CI.Patients receiving cabazitaxel 20 or 25 mg/sqm for mCRPC were enrolled. Digital PCR was performed to assess plasma p=0.011), AKR1C3 , AR , EPCAM , PSMA , MDK , and HPRT1  was significantly associated with OS. ALDH1 , AR , EPCAM , PSMA , MDK , and HPRT1  expression was associated with early PD. AR CN status was significantly correlated with AR-V7 (p=0.05), EPCAM (p=0.02), and MDK (p=0.002) expression. In multivariable model, EPCAM and HPRT1 CTC expression, plasma AR CN gain, ECOG PS=2, and liver metastases and PSA were independently associated with poorer OS. In patients treated with cabazitaxel 20 mg/sqm, median OS was shorter in AR-V7 positive than negative patients .Seventy-four patients were fully evaluable. CTC expression of AR-V7  dose was associated with poorer outcomes in AR-V7 positive patients compared to AR-V7 negative patients in a post hoc subgroup analysis.Clinicaltrials.govNCT03381326. Retrospectively registered on 18 December 2017.The online version contains supplementary material available at 10.1186/s12916-022-02244-0. AR mutations, AR amplification, constitutively active AR variants, intracrine steroid synthesis, and AR bypassing seem to be the mechanisms involved in CRPC evolution [Prostate cancer (PCa) is one of the leading causes of cancer death in men worldwide . Androgevolution . As the volution , 4, chemvolution . Howevervolution . Taxanesvolution –9. This volution , 11. CurAR copy number (CN) have already been demonstrated to be useful liquid biopsy-based approaches, found to be associated with worse outcomes to AR-targeted therapies abiraterone and enzalutamide [Cell-free DNA (cfDNA) biomarkers, such as lutamide –14.AR CN status.Circulating tumor cells (CTC) are released into the bloodstream from the primary tumor and/or metastasis. So, CTC analysis can be considered a non-invasive liquid biopsy test that takes into account the heterogeneity typical of PCa. Specific genomic alterations found in CTC are associated with clonal evolution or selection of cells contributing to treatment resistance in mCRPC . SeveralThis prospective study (NCT03381326) was approved by the Institutional Review Board of IRCCS Istituto Romagnolo per lo Studio dei Tumori (IRST) “Dino Amadori,” Meldola, Italy (protocol code: IRST B030). Inclusion criteria were: histology of prostate adenocarcinoma without neuroendocrine differentiation, a progressive disease despite “castration levels” of serum testosterone (< 50 ng/dL), ongoing LHRH analog treatment or prior surgical castration, and evidence of PD during or after treatment with docetaxel. Additional eligibility criteria included an Eastern Cooperative Oncology Group (ECOG) performance status 0–2, adequate cardiac, renal, hepatic, and bone marrow function. PD was defined as either biochemical progression  . Serum Pg for 15 min and plasma aliquots were stored at -80 °C. Two 5-ml peripheral blood samples from each patient were retrieved at different time points in AdnaCollectTM tubes (Qiagen) to enrich and isolate CTC.We collected blood samples from 100 patients at different time points: pre-treatment, after one cycle treatment , at first radiological evaluation, at the end of treatment/at PD. Peripheral blood samples were collected pre-treatment (baseline) in cfDNA BCT tubes , centrifuged at 1800×Plasma DNA was extracted with the QIAamp Circulating Nucleic Acid Kit  according to the manufacturer’s instructions, using 1 mL of plasma. Total extracted DNA was quantified by a spectrophotometer  using 2 μl of DNA.AR copy number (CN) analyses were performed by QuantStudio3D digital Polymerase Chain Reaction (dPCR) System (Thermo Fisher Scientific) in a duplex assay using FAM and VIC fluorescent probes. AR CN was evaluated with two assays  and two reference genes were selected as control genes: RNaseP, TaqMan Copy Number Reference Assay, and AGO1 (Hs02320401), modified with a VIC-labeled probe. DNA samples from three healthy male donors were pooled and used as calibrator. Data were analyzed using QUANT STUDIO 3D ANALYSIS SUITE CLOUD Software (Thermo Fisher Scientific). The average number of copies per reaction microlitres was determined using Poisson distribution. A ratio of target copies and reference copies was measured for each sample, then a ratio between sample and calibrator was calculated. AR CN status is defined as the mean value of the result of the two assays. AR gain status was defined using a cut-point value > 2.01, as previously described [escribed .EMT-2/StemCell Select™, targeting EPCAM, EGFR, and ERBB2. This method permits the characterization of CTC and the assessment of potential biomarkers in CTC, even though it does not allow CTC enumeration or morphology.CTC were isolated by the immunomagnetic-based AdnaTest EMT-2/StemCell Detect™ (both Qiagen) according to manufacturer’s instructions. Oligo (dT)25-coated beads allow mRNA isolation from the lysate of pre-enriched CTC. cDNA was obtained from reverse transcription using Sensiscript Reverse Transcriptase Kit™ (Qiagen), as previously described [The cell lysates derived were stored at − 20 °C no longer than 2 weeks, then proceeding with mRNA-isolation by AdnaTest escribed .We selected 17 cancer-related assays from the TATAA GrandPerformance CTC Assay Panel, a customizable gene panel that allows single-cell expression profiling that was run at TATAA Biocenter. Biomarkers’ choice was based on their role in prostate cancer progression and metastasis, steroid synthesis and signaling, stemness, EMT process, neuroendocrine differentiation. All samples were profiled in duplicates for expression of AR-V7, AKR1C3, AKT2, ALDH1, AR, EPCAM, PSMA, MDK, PARP, MRP1, PI3KCA, POU5F1, PSCA, TUBB3, VIM, ACTB, and HPRT1. This gene expression panel is composed of 17 genes, of which ACTB was used as a control gene. The limit of quantification (LoQ) for the assays in the panel ranged from 20 to 200 copies/reaction. LoQ was calculated as the concentration of the last standard point before the RSDr (relative standard deviation of replicates) is > 35% based on copy number. Regarding specificity, capillary gel electrophoresis was performed during assays validation in order to identify that the PCR product had the correct length in bp (+/− 10 bp) as expected, indicating that the specific target was amplified.CTC positivity for the examined patient cohort was defined as the expression of at least 1 of the following 12 biomarkers: AR-V7, AKR1C3, AKT2, ALDH1, AR, EPCAM, PSMA, MDK, PIK3CA, PSCA, TUBB3, VIM. These biomarkers were selected based on literature data supporting their capacity to identify CTC as positive in prostate cancer or confirming their role in prostate cancer or in EMT –30.The samples were preamplified using TATAA PreAmp Primer Mix (TATAA Biocenter AB) and TATAA PreAmp GrandMaster® Mix . Preamplification was performed in a thermocycler . No template controls (preAmp NTC) and human genomic DNA samples  were included.ACTB control gene for all the testing genes due to its stability and abundance in most of the samples analyzed. The Cq values were converted to relative quantities and transformed to log base 2 scale.The preamplified samples were 8× diluted according to the recommendation for the TATAA PreAmp GrandMaster® Mix. The diluted preamplified samples were analyzed for the 17 genes selected from the GrandPerformance CTC Assay Panel (TATAA Biocenter AB) and the ValidPrime™ assay (TATAA Biocenter AB). The qPCR analysis was performed using TATAA Probe GrandMaster® Mix (TATAA Biocenter AB). No template control and preAmp NTC were included, as described elsewhere . Cycle oBiomarker values evaluated at baseline and over time were summarized among all patients for whom at least one blood sample was available using descriptive statistics .Relationship among clinical characteristics and the value of each biomarker at baseline was investigated using chi-square test or Fisher’s test, as appropriate.OS was calculated from the date of the start of cabazitaxel to death or last follow-up. Progression-free survival (PFS) was calculated as the time between the start of cabazitaxel and the first date of progression or death, whichever comes first, or last tumor evaluation.Time-to-event outcomes (i.e. PFS and OS) were evaluated using the Kaplan-Meier method. Univariate and multivariable Cox regression models were used to estimate the hazard ratio (HR) and relative 95% confidence intervals (95% CI). To identify prognostic factors for OS and PFS, multivariable Cox regression model with stepwise backward elimination method were performed.The effect of the interaction between AR-V7 expression and initial cabazitaxel dose on OS was evaluated using Cox regression model including AR-V7 expression, initial cabazitaxel dose, and initial dose-by-AR-V7 expression.The impact of change in biomarkers at various landmark times on survival outcomes was assessed by landmark analyses. Patients with early PD/death before the landmark times were excluded. For these analyses, PFS and OS times were measured from the landmark times to these survival outcomes. The landmark times were at 1 month and 3 months of treatment.Logistic regression analysis was performed to estimate odds ratio (OR) and 95% CI of the association between biomarkers and PSA response and early PD, defined as PD occurring within the first three months of treatment.p< 0.05 was considered significant. Statistical analysis was conducted using SAS Statistical Software release 9.4 .All tests were two sided, and AR CN analyses  patients, whereas AR CN gain in 34 (46%). Median follow-up was 37 months (range 1–61). Median PFS and OS were 6.9 (95% CI 5.2–8.5) and 14.1 (95% CI 11.1–18.7) months, respectively. Univariate analysis reported that ECOG PS (p = 0.043 and p = 0.005), liver metastases (p = 0.005 and p = 0.0001), PSA (p = 0.0002 and p < 0.0001) and AR CN status (p = 0.0003 and p = 0.003) were significantly associated with PFS and OS, respectively. Gleason score (p = 0.01) and presence of pain (p = 0.028) were significantly associated only with PFS or OS, respectively.Between December 2014 and December 2018, 100 patients were recruited in the IRST B030 protocol. Among 100 patients enrolled, 74 were considered fully evaluable for CTC and At baseline, 67 of 74 (91%) samples were CTC positive (CTC+) and 7 out of 74 (9%) samples were classified as CTC negative (CTC−). After the first month of therapy, 20 out of 20 (100%) samples were CTC+. After the first radiological evaluation, 51 out of 56 (91%) samples were CTC+ and 5 out of 56 (9%) samples were CTC-. At the end of treatment, CTC were detected in 38 out of 43 samples (88%). Twenty-seven patients remained CTC+ over the whole observation period . On the other hand, none of the patients remained CTC− at all the 3 time points evaluated. No association between CTC+/CTC− and clinical characteristics or outcome was found.Correlation analysis of the biomarkers at baseline is shown in Additional file p = 0.05), AR , EPCAM , PSMA , MDK , and HPRT1  CTC expression and early PD  compared to AR-V7 negative patients.As an exploratory analysis, we investigated the impact of AR-V7 expression on treatment outcome in 44 patients (59%) treated with an initial reduced dose (20 mg/sqm) of cabazitaxel. Among these patients, 8 (18%) had AR-V7 CTC expression. Regarding OS and PFS, no difference was observed between AR-V7 positive  and negative patients treated with full-dose cabazitaxel. However, in the initial reduced dose subgroup, AR-V7 positive patients had a worse median OS .The interaction test involving AR-V7 expression effect in the initial reduced and full doses groups suggested that the correlation between AR-V7 expressions and worse outcome was significantly associated with the initial reduced dose of cabazitaxel for OS . AR CN status was significantly associated with AR-V7 expression , EPCAM , and MDK .Of the 74 patients evaluated, 40 had AR CN status and CTC expression of AR-V7, AKR1C3, AR, EPCAM, PSMA, MDK, and HPRT1) and four clinical prognostic factors such as ECOG PS, liver metastases, PSA, and presence of pain as covariates were constructed and analyzed in our cohort. The clinical prognostic factors selected have been demonstrated to be independent predictors of OS in univariate analysis in this cohort. Using Cox multivariable model with stepwise backward elimination method, EPCAM and HPRT1 CTC expression, plasma AR gain, ECOG PS = 2, presence of liver metastases and PSA were all independently associated with shorter OS . However, it has to be noted that this result is referred to a patient only. Fifty-six patients had available CTC analysis at baseline and after 3 months of treatment, we observed that OS was associated with AKT2 increased expression , considering a variation cut-off of 10% for the gene expression.We then evaluated the modulation of CTC biomarkers from baseline to one and three cycles after therapy, correlating it with patients’ prognosis. Twenty patients had available CTC analysis at baseline and after the first month of treatment; we found that an increased expression of AR-V7 was significantly correlated with PFS and OS  with median PFS and OS of 5.2 (95% CI 0.7-10.6) and 10.6 (95% CI 1.3-16.7) months, respectively. Three of these patients expressed AR-V7 at baseline and during treatment: one patient remained positive in each the time points analyzed  and had PFS and OS of 2.5 and 8.6 months, respectively; another patient remained positive from baseline to the sample collected after 3 cycles of treatment corresponding to PD sample (PFS and OS of 2.1 and 12.3 months); the last patient was positive at baseline, became negative after 3 cycles treatment and then returned positive at the end of treatment (PFS and OS of 16.7 months). Additional file The molecular characterization of CTC from mCRPC patients treated with cabazitaxel provided through a panel of specific biomarkers, including genes related to PCa and taxanes chemotherapy, with a promising applicability as “liquid biopsy,” has been studied here. Many CTC subpopulations with different phenotypes circulate in the blood stream of metastatic patients, so the detection of multiple genes by molecular assays is crucial.Our study, performed on a prospectively collected cohort of patients with mCRPC undergoing treatment with cabazitaxel, showed that the expression of specific CTC biomarkers is able to identify patients with worse prognosis and/or experiencing early PD.AR CN gain, ECOG PS = 2, the presence of liver metastases, and PSA were all independently associated with poorer OS. EPCAM is a transmembrane glycoprotein that is consistently expressed by epithelial-derived tumor cells and is associated with poor clinical outcome [A prognostic multivariable model was designed to evaluate the association between OS and the most significant clinical and biological markers found in our analysis. EPCAM and HPRT1 CTC expression, plasma  outcome . EMT is  outcome , 34. Sur outcome . Moreove outcome .AR CN gain, confirming the previous results [AR CN gain [Notably, AR-V7 expression that actually represents the most clinically relevant CTC biomarker , 38 in m results . AR-V7 a results , 40, som results –43. In t results , the num results , 45. Our results . We foun CN gain .We also performed a CTC expression analysis during cabazitaxel treatment in a subgroup of patients and we showed that AKT2 increased expression after 3 months of treatment was correlated with a better OS. Literature data reported that AKT2 overexpression resulted in a significant decrease in migration, whereas AKT2 knockdown promoted migration of PC3 and DU145 PCa cells . HoweverAR CN status, AR-V7 and AR expression, but other AR aberrations, such as AR mutations, were not included in the current analysis; thus a complete scenario regarding AR status and its clinical utility would be needed.We recognize some limitations of our study, such as the relatively modest sample size. For this reason, we had to exclude MRP1 from the analyses because it was expressed in one patient only. A technical limitation was represented by our CTC isolation protocol that allows for simple and fast samples processing, but does not allow for CTC enumeration; furthermore, no cell morphological assessment could be performed. As a result, gene expression is not adjusted for the number of CTC and may be influenced by CTC quantity. In addition, we considered In conclusion, our results suggest that CTC expression biomarkers, especially if evaluated before treatment initiation, could give important prognostic indications in mCRPC cabazitaxel-treated patients, and that AR-V7 positive patients could avoid an initial reduced dose. Moreover, this non-invasive, liquid-biopsy-based approach overcomes the need for tissue biopsies from metastases, still representing a major issue in PCa clinical management. The availability of predictive biomarkers could improve treatment selection, identifying the patients most likely to respond to therapy while avoiding to treat those with a low probability of response, reducing the potential treatment-related adverse events and the costs on the health care systems. Larger prospective multi-center clinical trials are warranted.Additional file 1: Table S1. Correlation analysis of gene expression biomarkers; we performed an analysis to evaluate the correlation of all the biomarkers considered.Additional file 2: Figure S1. AR-V7 expression trend from baseline to disease progression for all AR-V7 positive patients or for patients divided in: non responders and responders. The lines represented the AR-V7 expression trend for all patients. Globally, AR-V7 expression was stable from baseline to the first radiological evaluation (3 months) in our patients, whereas we found a modulation of AR-V7 expression from 3 months to PD (at progression) in non responders who became AR-V7 positive at progression. One responder patient changed AR-V7 expression from positive at baseline to negative at 3 months.Additional file 3. Raw data: Raw data biomarkers prostate cancer; the dataset used and analysed during the current study.


Methamphetamine use is a serious problem in China. Compulsory isolation detoxification is the main treatment measure for drug dependents, whereas psychological interventions in compulsory isolation detoxification centers are extremely inadequate. The current study aimed to examine the effects of mindfulness-based relapse prevention (MBRP) on methamphetamine dependence patients in Chinese compulsory isolation detoxification treatment institutions. Forty-one methamphetamine dependent patients received 16-sessions of MBRP in 8 weeks and assessments were conducted at the baseline, 4-, 8-week (after the whole intervention). Results of repeated measured ANOVAs showed there was no significant effect on emotions and cravings. Findings indicated that the effects of MBRP are still difficult to make firm conclusions due to the insignificant results. Future studies should modify the MBRP and ensure that it is suitable for compulsory isolation detoxification treatment institutions in China. Substance use is a serious public health problem and social problem. According to a report by the Chinese government, the number of drug users in China has reached 1.801 million in 2020, 57.2% of them abuse synthetic drugs such as methamphetamine and ketamine, and methamphetamine abuse remains a serious problem –5.In China, compulsory isolation detoxification is the main treatment measure for drug dependents. Compared with the previous treatment system, compulsory isolation detoxification addresses the importance of psychological intervention among the drug dependents since according to the “Drug Control Law of the People's Republic of China,” drug dependents have been defined as both offenders and patients, and psychological interventions should be delivered to help them to keep abstinence. To meet the requirement of “Drug Control Law,” we try to find new psychological intervention strategies which are eligible for compulsory isolation treatment sites.Mindfulness-based relapse prevention therapy (MBRP) developed by Witkiewitz et al.  is one oThe effect of MBRP on SUDs has received considerable empirical support . CompareThe pilot study aimed to examine the effectiveness of MBRP on methamphetamine dependent patients in Chinese compulsory isolation detoxification treatment institutes.Forty-one participants were recruited from the compulsory isolation detoxification treatment center in < city>Shanghai < /city>, China from April to May in 2019. The inclusion criteria were:  capable n = 21) and the control group (n = 20). The MBRP group received 16 sessions of 2-h MBRP interventions , which was developed based on the Mindfulness-based relapse prevention for addictive behaviors: a clinician guide written by Bowen et al. The intervention was led by a professional mindfulness trainer. The control group received the treatment as usual (TAU). Assessments were conducted at the baseline, after the 8th session (mid-test), and the end of the intervention (post-test). See This study was designed as a randomized controlled clinical trial and has been registered at ClinicalTrials.gov (ID: NCT03748875). Forty-one participants were randomly divided into the MBRP group (The Chinese version of the Beck Depression Inventory (BDI) was used to measure depression . This scParticipants' anxiety was measured using the Chinese version of the State-Trait Anxiety Inventory (STAI) . This scCraving was measured using the Visual Analog Scale (VAS). The 100 mm line was used to represent the craving degree from 0 (no craving) to 100 (very strong craving). A higher score indicates a higher level of craving.2 test and independent t-test were used to examine group differences at the baseline. Emotions and craving measured at the baseline, mid-test, and post-test were submitted to 3 (Time) × 2 (Group) repeated measures ANOVAs to examine the effects of the intervention. The significant level was set at 0.05.Data were processed with SPSS 21.0. χ2-test and independent t-tests indicated no significant difference between the two groups at the baseline except depression (ps > 0.05). The BDI scores in the MBRP group were significantly higher than that in the control group at the baseline . Thus, depression was regarded as a covariate in the following analyses. See The results of the χF = 4.890, p = 0.034, power = 0.575; F = 4.445, p =.043, power = 0.535]. The further pairwise comparison showed that the BDI scores in the MBRP group were significantly higher than that in the control group (p = 0.034), the score of state anxiety in the MBRP group was significantly lower than that in the control group (p = 0.043). Moreover, time showed a significant main effect on BDI . The BDI scores at the post-test were significantly lower than that at the baseline (p = 0.009) and the mid-test (p = 0.020). However, no significant interaction effects of time and groups were found (ps > 0.05). See more details in Five participants dropped out because of private reasons. The remaining data from 36 participants were included in further analyses. The results of repeated measured ANOVAs showed that the main effects of group on BDI and state anxiety were significant [This pilot study initially examined the effectiveness of MBRP on methamphetamine dependent patients in Chinese compulsory isolation detoxification treatment institutes. We focused on the effects of MBRP on patients' emotions and cravings in the present study.The main effect of time on BDI is significant, which indicated that the depression level of methamphetamine dependent patients declined over time. Previous studies found that in the rehabilitation period, the depression level of methamphetamine dependent patients improved during the time course, which was consistent with the current findings . Most noHowever, numerous studies have suggested that the MBRP intervention has no significant effects on the improvement of emotions and cravings among SUDs. Previous research observed no significant effects of MBRP on anxiety and craving . AccordiRegarding the current study, it was conducted in the compulsory isolation detoxification treatment institutes, in which patients' daily schedules were strictly arranged. Thus, the lacking meditation practice might reduce the intervention effect. Another impacting factor is the changing motivation. The participants in the compulsory isolation detoxification treatment institutes are relatively lacking in changing motivation, which might be an important reason for the insignificant effect of MBRP intervention in the current study . Other fEven the results were negative, the present study still provides preliminary evidence for the implementation of MBRP on methamphetamine patients. Given the specific settings for compulsory rehabilitation treatment in China, it might be necessary to modify MBRP and ensure it is suitable for the current situation.via self-report scales which might be impacted by social desirability bias, all the included methamphetamine dependent patients are male while gender might influence the vulnerability to methamphetamine toxicity, and lack of biological indicators as the biological improvements might not be reflected in behaviors , Science and Technology Commission of Shanghai Municipal (19411969200 and 21DZ2201000), Shanghai Municipal Science and Technology Major Project (2018SHZDZX05), Shanghai Clinical Research Center for Mental Health (19MC1911100), Shanghai Engineering Research Center of Intelligent Addiction Treatment and Rehabilitation (19DZ2255200), and Shanghai Key Laboratory of Psychotic Disorders (13DZ2260500).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. The reviewer CZ declared a shared affiliation, with no collaboration, with the authors JZ, YL, JS, DS, QR, MZ, and JD at the time of the review.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In this article, the range of works connected with the repair of a historical Maryan bell from 1639 are presented. The first attempts to repair damaged bells occurred in the 1930s in Poland. However, this process was stopped because of extensive technological difficulties. Welding and soldering-welding were the basic methods. There is one difference between these two methods—connecting surfaces are melted during the welding process but only heated until the melting temperature of the material added to the connection (that is the solder) during the soldering-welding process. It was important to heat the bell to the proper temperature during welding. Uneven heating causes the enlargement of existing cracks or the appearance of new ones, or even the complete destruction of the bell. Nowadays, a method of even heating using a special heating mat has been devised. Thanks to this method it is possible to control the heating and cooling process. The most important task during the whole operation of bell welding was obtaining the original sound. During this research, the chemical composition was examined to prepare a welding rod with a suitable chemical composition. After the repair process, an analysis of the sound of the bell was conducted. It was shown that the repair of bells is possible when correct thermal parameters are used. The most highly recommended technique for repairing bells is gas welding. There are a lot of churches with very old, historical bells in Poland, as it has historically been a Catholic country. Unfortunately, their strength is decreasing and cracks and scratches are have appeared. These defects make them useless, because not only does the sound become worse, but reacting to the damage too late can cause the complete destruction of the bell as well.The lifetime of the bell was determined to be 200–300 years  on the bThe repair of the bell is possible up to its complete destruction. Repairing the bell is an expensive and time-consuming process. However, attempts to repair these bells are not rare, because the bell is a precious item not only thanks to its material value but also its historical and artistic value as well. Despite the avoidance of the repair of cracked bells in Poland for a long time, this problem has been considered in many other countries ,3.It is necessary to examine the chemical composition of the alloy used to produce the bell to repair the crack. The oldest bells are made of gunmetal, while the younger are made of bronze. Copper at a concentration of about 80% and tin with a concentration between 19% and 21% are the main components in both alloys. Gunmetal also contains zinc, lead, carbon, and iron. Trace amounts of silver and gold are also possible to observe in both alloys because of the tradition to add these elements into the liquid metal to ennoble the material ,5,6.The melting temperature of bronze is about 850–950 °C. The alloys with high tin content are characterized by great strength, but concurrently low toughness—they are very brittle . This, iIn Poland, the first attempts to repair damaged bells were observed in the 1930s. Welding and soldering-welding were the base methods. There is one difference between these two methods: connecting surfaces are melted during welding process and only heated until the melting temperature of the material added to the connection (that is solder) during the soldering-welding process. Additionally, a welding rod with a chemical composition similar to indigenous metal was used during welding process, whilst sticks made of brass alloy (known as bronzite) were used for soldering-welding.First, it was desirable to estimate the size of the crack. A simple penetration study was conducted with the use of chalk and kerosene. Chalk was rubbed into the inner side of the bell and the external side was lubricated with kerosene. The greasy spot was used to show the range of the crack. After estimating the size of the damage, the place of repair should be properly prepared. In both cases, the method of action was similar. A groove with a v–shape was cut along the crack and metal was poured in. In this procedure the hole at the end of the crack must be remembered. Its task was to limit the increase of the crack during bell heating. An acetylene torch was used as the welding tool in both methods. Uniformity of the weld obtained was the main difference between welding and the soldering-welding process. The weld had nearly the same chemical composition as welded material during the welding process. The weld had different chemical composition to the repaired bell during the soldering-welding process, which caused worse sound.The position heated by charcoal (in the past it was often used as a fuel) was used to heat the bell before repair. This solution was connected with uneven bell heating and cooling. It may have caused new cracks or increased the old ones as a result.The company Rduch Bells & Clocks and Foundry Department collectively made a decision to repair a cracked Maryan bell from the 17th century at the request of urban restorer in Krosno–Marta Rymar. The bell hangs in a church tower. It is the smallest bell of three cast in 1639, weighing 580 kg. It is characterized by producing a “G#” sound. The bell was cast by two bell founders, Szepan Meutel and Jerzy Olivier, for a special order from the great philanthropist Robert Wojciech Portius. The Maryan bell is one of three treated bells. On the tower there are also the Urban bell and the Jan bell. The bells are tuned to a major scale. This means that they sound happy, merry, and concurrently noble. The lack of one bell or unclear sound will cause the whole set to lose its musical value. This is why it was so important not only to repair the crack but to do it in such a way as to avoid changes to the sound. The first step was to take a material sample to determine the averaged chemical composition of the alloy, which was analyzed with a glow–discharge spectrometer LECO GDS500A  ;In the next step a series of welding rods with the same chemical composition were prepared on the basis of these results, which were used during bell welding process;The next step was to determine the size and range of the crack, and penetration research was conducted;The next step was preparing the bell for the welding process by properly bevelling the sides of the bell;The next step was heating and keeping the bell at the proper temperature;The next step was obtaining the required temperature to conduct welding process;After welding slow cooling was conducted to avoid stresses;The last step was analysis of the sound of the repaired bell.The most important task during welding process was to obtain the original sound. Fortunately, in 2013, during the change of the clapper, acoustic measurements were conducted. Thanks to this it was possible to obtain the sound before and after the damage to the bell (the damage occurred in 2017—exact date is unknown). This was the base for further activities. The work was divided into a few steps:Accuracy in all of these activities allowed us to obtain the ideal sound from the repaired bell. The samples of material obtained were examined with the use of a spectrometer to determine averaged chemical composition  interval of 1.2 . The lower (tone lower than the prime about the octave) and upper (tone higher than the prime about the octave) octaves with prime, tierce and quint were found to be beautifully harmonious after the repair. There was no distortionary vibration and the bell sustained its note for a long time.The frequency spectrums of the Maryan bell before and after the crack are presented in Determined by the Wavanal program, values of frequency of basic side tones : the lower octave (hum), prime , minor tierce, quint, and upper octave  of the bells examined are presented in If the welding process is carried out with the correct parameters, especially with thermal ones, and with monitoring and control of the heating and cooling rates, the repair even of contaminated high tin bronze bells is possible;After repair by welding, the bell has a “better” sound, and this is probably due to a kind of heat treatment performed, including a cycle of slow heating and cooling which improves the properties of the material; andThe most highly recommended technique for repairing bells is gas welding, due to the relatively low temperature in the bonding area and the efficiency of the process.Based on the experience gained during the work and research carried out on the Maryan bell, the following conclusions can be drawn:


In the present study, we explored a novel accelerative effect of DEX on cardiac fibroblast (CF) differentiation mediated by LPS and clarified its potential mechanism. LPS apparently increased the expression of α-SMA and collagen I/III and the phosphorylation of p38 and Smad-3 in the CFs of mice. These effects were significantly enhanced by DEX through increasing α2A-AR expression in CFs after LPS stimulation. The CFs from α2A-AR knockout mice were markedly less sensitive to DEX treatment than those of wild-type mice. Inhibition of protein kinase C (PKC) abolished the enhanced effects of DEX on LPS-induced differentiation of CFs. We also found that the α-SMA level in the second-passage CFs was much higher than that in the nonpassage and first-passage CFs. However, after LPS stimulation, the TNF-α released from the nonpassage CFs was much higher than that in the first- and second-passage CFs. DEX had no effect on LPS-induced release of TNF-α and IL-6 from CFs. Further investigation indicated that DEX promoted cardiac fibrosis and collagen I/III synthesis in mice exposed to LPS for four weeks. Our results demonstrated that DEX effectively accelerated LPS-induced differentiation of CFs to myofibroblasts through the PKC-p38-Smad2/3 signaling pathway by activating α2A-AR.Dexmedetomidine (DEX), a selective α Myocardial fibrosis is characterized by net accumulation of extracellular matrix proteins in the cardiac interstitium and results in both systolic and diastolic dysfunctions . It is a2-AR agonist, has gained more popularity in the intensive care management of patients due to its sedative, analgesic, and anxiolytic properties [2-AR antagonists have an inhibitory effect on the progression of hepatic fibrosis [2-AR expression in kidneys effectively offsets interstitial fibrosis [Dexmedetomidine (DEX), a highly selective αoperties . In 1999operties . Shehabioperties . It has fibrosis . The dowfibrosis . However2-AR on cardiomyocytes [2A-AR [2-AR, thereby accelerating cardiac fibrosis and potentially causing increased mortality in septic patients with DEX treatment.Cardiac fibroblasts, which account for 60–70% of the cells in the heart, are a key source of the extracellular matrix (ECM) that regulates the structure of the heart and hence mechanical, chemical and electrical signals between the cellular and non-cellular components ,15. The myocytes , norepins [2A-AR . Thus, wCurrently, there is no information on the role of DEX in CFs. Thus, the purpose of this study was to characterize the effects of DEX on the differentiation of CFs into myofibroblasts induced by LPS and the mechanism underlying these effects.LPS is a potent inducer of fibroblast-to-myofibroblast differentiation, which is characterized by the high expression of α-SMA . To dete2-AR agonist on p38 phosphorylation was mediated by activation of PKC [Following acute myocardial injury, myofibroblasts secrete elevated levels of collagen I/III . To deten of PKC . To inve2-ARs have been divided into three subtypes: α2A-AR, α2B-AR and α2C-AR. Previous studies have reported that only α2A-AR mRNA, but not αB-AR or α2C-AR, was detected in cultured adventitial fibroblasts of rat aortae [2-AR subtypes in CFs of mice, the mRNA levels of α2A-AR, α2B-AR and α2C-AR in CFs with and without LPS stimulation for 2 h were measured by qPCR. The mRNA expression of both α2A-AR and α2B-AR was detected, but α2C-AR was undetectable. Compared with control CFs, the mRNA level of α2A-AR was increased in LPS-treated CFs  mice, then treated with DEX and/or LPS. The CFs of α2A-AR KO mice treated with LPS showed significantly increased expression of α-SMA and collagen I/III, which are major markers of myofibroblasts. However, unlike the CFs of wild-type mice, DEX had no effect on the LPS-induced upregulation of α-SMA and collagen I/III in the CFs of α2A-AR KO mice  antibodies were obtained from Abcam . Antibodies against vimentin (#5741), p38 (#9212S), p-p38 (#4511S), PKC (2683S) and GAPDH (#2118) were purchased from Cell Signaling Technology, Inc. . Antibodies recognizing Smad 2/3 (sc-133098) were purchased from Santa Cruz Biotechnology, Inc. . Antibodies against collagen I (WL0088), collagen III (WL03186), and p-Smad 2/3 (WL02305) were obtained from Wanleibio . Alexa Fluor 647-labeled goat antibody against mouse IgG (A21235), Alexa Fluor 488-labeled goat antibody against rabbit IgG (A21206), goat anti-rabbit IgG antibody (#31462) and goat anti-mouse IgG antibody (#31438) were obtained from Thermo Fisher Scientific . The enzyme-linked immunosorbent assay (ELISA) kits for the detection of mouse TNF-α (MTA00B), interleukin (IL)-6 (M6000B) and IL-10 (M1000B) were purchased from R&D Systems .LPS  and DEX (SML0956) were purchased from Sigma Aldrich . Go6983 (HY-13689) and SB203580 (HY-10256) were obtained from MedChemExpress . Anti-α-SMA (ab7817), p-PKC (ab63387) and anti-αg for 10 min at 4 °C, resuspended in DMEM supplemented with 10% FBS and plated in a 6-well plate (1.0 × 105 cells/well). After 48 h of culture, CFs were treated with vehicle and LPS at a concentration of 1 μg/mL for 15 min, followed by normal saline or DEX (0.1 μM) treatment. In separate experiments, CFs were preincubated with Go6983  or SB203580  for 30 min before treatment with LPS. Moreover, cell viability was measured using Cell Counting Kit-8 .Cardiac ventricles were excised from 6- to 8-week-old male C57Bl/6J mice, thoroughly minced with sterile fine scissors and digested in 10 mL HBSS containing 0.1% collagenase type 2  at 37 °C. During this incubation, the digesting tissue was triturated for one min with a sterile serological pipette every 5 min. The supernatant cell suspension containing liberated fibroblasts was then collected into a tube containing cold Dulbecco’s modified Eagle’s medium (DMEM). The undigested fraction was reconstituted with fresh digestion media, and the same digestion procedure was repeated till almost the entire tissue was dissociated into single cells. The cell fraction was collected after a final centrifugation at 500× 2A-AR knockout  mice were generated by Adra2atm1Bkk mice (Strain Name: B6.129-Adra2atm1Bkk/J), which were obtained from Jackson Laboratory . All animals were given free access to standard mouse chow and water and maintained at room temperature (24-C T 2-C) with a 12-h light-dark cycle. The mice were then randomly assigned to one of the following four groups: control, LPS, LPS + DEX and DEX. Mice were injected i.p. with LPS at a dose of 10 mg/kg once a week for four weeks. The control group received 0.3 mL of saline instead of LPS. DEX (80 μg/kg) was given i.p. after LPS treatment for 2 h every week. After 4 weeks, the mice were anaesthetized with diethyl ether and sacrificed. Then, the left ventricles of hearts were obtained for western blot and Masson’s staining analysis. All animal experiments were accomplished according to the guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health. Our study was approved by the Ethics Committee of Jinan University.Male C57Bl/6J mice (8–10 weeks old) weighing 25 to 28 g were obtained from Guangdong Experimental Animal Center. αAdult mouse cardiac fibroblasts with/without LPS or DEX stimulation were fixed with 4% formalin solution and then permeabilized with PBS containing 0.1% Triton X-100. After three washes with PBS, the cells were blocked in PBS with 1% BSA and 0.1% Triton X-100, followed by incubation with a 1:200 dilution of antibodies against α-SMA and vimentin at 4 °C overnight. The cells were then incubated with the secondary anti-rabbit Alexa Fluor 488 antibody for vimentin and secondary anti-mouse Alexa Fluor 647 antibody for α-SMA at room temperature for 1 h. The cells were then washed three times with PBS and incubated with a 1:200 dilution of 4′,6-diamidino-2-phenylindole (DAPI) for 15 min. The cells were observed with a Leica fluorescence microscope.Western blot experiments were performed as previously described . Cells a2A-AR, α2B-AR and α2C-AR in mouse CFs. Total RNA was isolated from cells using TRIzol reagent according to the manufacturer’s instructions. RNA samples were reverse-transcribed using random hexamer primers in the presence of RNase inhibitor . cDNA was amplified using ChemoHS qPCR mix (Monad), 0.2 μM of each primer and nuclease-free water. Amplified cDNA signals were detected and analyzed by CFX Maestro Software v1.1  using β-Actin as an endogenous control. The results are expressed as the fold increase over the control. The specific primers used in this study were as follows. α2A-AR: forward 5′-GTGACACTGACGCTGGTTTG-3′, reverse 5′-CCAGTAACCCATAACCTCGTTG-3′. α2B-AR: forward 5′-TCTTCACCATTTTCGGCAATGC-3′, reverse 5′-AGAGTAGCCACTAGGATGTCG-3′. α2C-AR: forward 5′-CTGTGGTGGGTTTCCTCATCG-3′, reverse 5′-ACTTGCCCGAAGTACCAGTAG-3′. α-SMA: forward 5′-TTCCTTCGTGACTACTGCCG-3′, reverse 5′-TATAGGTGGTTTCGTGGATGCC-3′. Collagen I: forward 5′-CCCTGAAGTCAGCTGCATACACAA-3′, reverse 5′-CCTACATCTTCTGAGTTTGGTGAT-3′. Collagen III: forward 5′-GAGATGTCTGGAAGCCAGAACCAT-3′, reverse 5′-GATCTCCCTTGGGGCCTTGAGGT-3′. β-actin: forward 5′-GGCTGTATTCCCCTCCATCG-3′, reverse 5′-CCAGTTGGTAACAATGCCATGT-3′.qPCR was used to measure the mRNA levels of αAdult mouse cardiac fibroblasts were treated with LPS for 24 h, and then, the culture supernatants were collected. The concentrations of TNF-α, IL-6 and IL-10 in the supernatant were measured by using a commercially available ELISA kit according to the manufacturer’s instructions.For evaluation of cardiac fibrosis, the left ventricles were embedded in 10% formalin and sliced at 5 μm. The tissue sections were stained with Masson’s trichrome according to the manufacturer’s instructions and then examined under light microscopy. After Masson staining, cardiomyocytes were stained red, whereas blue staining indicated positive staining for collagen. The fibrotic changes were assessed by a pathologist in 10 randomly selected high-power fields for each tissue slide. ImageJ software was used to quantify the fractional area of cardiac fibrosis .5 cells/well. After 24 h of incubation, the cell monolayer was scratched with a sterile pipette tip. Then, the cells were incubated with vehicle and LPS for 15 min, followed by normal saline or DEX (0.1 μM) treatment for 24 h. The scratch areas were photographed using an IX51 Inverted Phase Contrast Fluorescence Microscope  and quantified by ImageJ software. The relative migration ratio of the cells was calculated according to the formula: (size at 0 h–size at 24 h)/size at 0 h.An in vitro scratch assay was used to assess the migration capacity of cells. CFs were seeded into 6-well plates at a density of 5 × 10t-test or one-way ANOVA followed by the Student-Newman–Keuls test. Differences with p values < 0.05 were considered statistically significant. SPSS software was used to analyze the data.Mean densitometry values and all other quantitative data are presented as the mean ± standard error of the mean (SEM). Comparisons among groups were made using Student’s 2A-AR expression in CFs. Accordingly, the intensified effect of DEX on LPS-induced differentiation of CFs is mediated by accelerating the activation of the PKC-p38-Smad2/3 signaling pathway through increasing α2A-AR (In summary, this is the first study to demonstrate that LPS increased αg α2A-AR . Our stu


Long noncoding RNA nuclear enriched abundant transcript 1 (lnc‐NEAT1) and its target microRNA‐125a (miR‐125a) are reported to regulate immune and inflammation process in allergic rhinitis (AR). Hence, this study intended to investigate the correlation between lnc‐NEAT1 and miR‐125a expressions, as well as their clinical values in pediatric AR patients.+ T cells were analyzed by flow cytometry.Peripheral blood mononuclear cell samples from 80 pediatric AR patients, 40 disease controls (DCs), and 40 healthy controls (HCs) were collected to detect lnc‐NEAT1 and miR‐125a expressions by reverse transcription‐quantitative polymerase chain reaction. For pediatric AR patients only, serum interferon‐gamma (IFN‐γ) and interleukin (IL)‐10 were measured by enzyme linked immunosorbent assay; meanwhile, T helper (Th) 1 and Th2 cells in CD4p < 0.001). Moreover, lnc‐NEAT1 expression negatively correlated with miR‐125a expression in pediatric AR patients (p = 0.002), but not in DCs (p = 0.226) or HCs (p = 0.237). Furthermore, in pediatric AR patients, lnc‐NEAT1 expression positively associated with TNSS (p < 0.001), sneezing score (p = 0.006), and congestion score (p = 0.008); miR‐125a expression was negatively related to TNSS (p < 0.001), itching score (p = 0.040), and sneezing score (p = 0.005). Additionally, lnc‐NEAT1 expression positively, while miR‐125a expression negatively correlated with Th2 cells and IL‐10 , but they were not correlated with Th1 cells or IFN‐γ in pediatric AR patients.Lnc‐NEAT1 was overexpressed, while miR‐125a downregulated in pediatric AR patients compared to DCs and HCs (all Circulating lnc‐NEAT1 and miR‐125a are aberrantly expressed and linked with Th2 cells and symptom severity in pediatric allergic rhinitis. + T cells were analyzed by flow cytometry. Intriguingly, lnc‐NEAT1 was overexpressed while miR‐125a downregulated in pediatric AR patients than in DCs and HCs. Moreover, lnc‐NEAT1 negatively correlated with miR‐125a in pediatric AR patients. Furthermore, in pediatric AR patients, lnc‐NEAT1 positively associated with TNSS, sneezing score and congestion score; miR‐125a was negatively related to TNSS, itching score and sneezing score. Additionally, lnc‐NEAT1 positively, while miR‐125a negatively correlated with Th2 cells and IL‐10 in pediatric AR patients.This study enrolled 80 pediatric AR patients, 40 DCs, and 40 HCs to detect their blood lnc‐NEAT1 and miR‐125a expression by RT‐qPCR. For pediatric AR patients only, serum IFN‐γ and IL‐10 were measured by ELISA; meanwhile Th1 and Th2 cells in CD4 The enrollment criteria were as follows: (1) diagnosed as AR according to Allergic Rhinitis and its Impact on Asthma (ARIA) guidelines, 2016 revision2.2After enrollment, the demographics of all subjects were recorded, and the serum IgE level of all subjects was examined. For pediatric AR patients, the disease severity was evaluated by the total nasal symptom score (TNSS) in accordance with a previous study,2.3® qPCR Mix (TOYOBO). The relative expressions of lnc‐NEAT1 and miR‐125a were calculated using the 2−ΔΔCt method. GAPDH was used as the internal reference for lnc‐NEAT1, and U6 as the internal reference for miR‐125a. Besides, qPCR primers were designed referring to previous studies.The lnc‐NEAT1 and miR‐125a expressions in PBMC of all subjects were detected using the reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR) assay. Total RNA was extracted by RNeasy Protect Mini Kit (Qiagen), then reserve transcription was completed using PrimeScript™ RT reagent Kit (Takara). After that, qPCR was achieved by KOD SYBR2.4+ T cells of pediatric AR patients were analyzed by the multicolor flow cytometric analysis using commercial Human Cell Differentiation Kit (Bio‐Techne China Co. Ltd.). The procedure of the experiment was performed strictly according to the kit instruction. In brief, after stimulation, T helper cells were stained using specific fluorescent antibodies, then the cells were counted by a fluorescence activated cell sorting (FACS) flow cytometer using anti‐human interferon‐gamma (IFN‐γ) antibody and anti‐human interleukin 4 antibody (Bio‐Techne China Co. Ltd.). Sequentially, the proportions of Th1 and Th2 cells in CD4+ T cells were calculated.The proportions of Th1 and Th2 cells in CD42.5IFN‐γ (Th1 cell cytokine) and IL‐10 in serum of pediatric AR patients were detected by enzyme linked immunosorbent assay (ELISA), which was implemented using commercial Human ELISA Kit (Bio‐Techne China Co. Ltd.). The experiment was performed referring to the specification provided by the manufacturer.2.6P value <0.05 was indicated in the above analysis.SPSS 24.0 (IBM Corp.) and GraphPad Prism 6.01 (GraphPad Software Inc.) were used for statistical analysis and graph construction, respectively. Comparisons of clinical characteristics among groups were analyzed by one‐way analysis of variance (ANOVA) test, chi‐square test, and Kruskal‐Wallis H rank sum test. Comparisons of lnc‐NEAT1 and miR‐125a expressions among groups were determined by Kruskal‐Wallis H rank‐sum test and adjusted by the Bonferroni test. The receiver operating characteristic (ROC) curve analysis and the area under the curve (AUC) were used to evaluate the performance of lnc‐NEAT1 and miR‐125a expressions, and lnc‐NEAT1/miR‐125a axis in identifying different subjects. Correlations of two continuous variables were assessed using Spearman's rank correlation test. Statistical significance was concluded when 33.1p = 0.582), gender (p = 0.432), height (p = 0.675), and weight (p = 0.846), except the IgE level, which was varied (p < 0.001) among all subjects. Moreover, the detailed clinical characteristics are listed in Table The mean ages of pediatric AR patients, DCs, and HCs were 6.2 ± 2.7 years, 6.7 ± 2.5 years, and 6.5 ± 3.1 years, respectively (Table 3.2p < 0.001) and miR‐125a (p < 0.001) were both differed among pediatric AR patients, DCs, and HCs : 0.876–0.967) and pediatric AR patients from DCs  Figure . Likewisp < 0.001, Figure As to the lnc‐NEAT1/miR‐125a axis, it also differed among pediatric AR patients, DCs, and HCs , while no correlation was found in the lnc‐NEAT1 expression with the miR‐125a expression in DCs  or in HCs , the sneezing score , and the congestion score , while no association was observed in the lnc‐NEAT1 expression with the nasal rhinorrhea score  and the itching score , the itching score , and the sneezing score ; nevertheless, there was no correlation of the miR‐125a expression with the nasal rhinorrhea score  or the congestion score  and IL‐10 , but not correlated with Th1 cells  or IFN‐γ  and IL‐10 , but there was no relationship between the miR‐125a expression and Th1 cells ; besides, the miR‐125a expression was positively correlated with IFN‐γ   Lnc‐NEAT1 was overexpressed, while its target miR‐125a was downregulated in pediatric AR patients compared to DCs and HCs. Additionally, both the lnc‐NEAT1 expression and the miR‐125a expression suggested good potential in distinguishing pediatric AR patients from DCs and HCs. (2) The lnc‐NEAT1 expression was negatively correlated with the miR‐125a expression in pediatric AR patients. (3) In pediatric AR patients, the lnc‐NEAT1 expression was positively related to disease severity, while the elevated miR‐125a expression was linked with alleviative disease severity. (4) In pediatric AR patients, the lnc‐NEAT1 expression was positively related to Th2 cells and IL‐10, but not correlated with Th1 cells or IFN‐γ, which caused Th1/Th2 imbalance.Lately, some studies have explored that lnc‐NEAT1 and miR‐125a are dysregulated in some inflammatory disorders, such as bronchial asthma and AR.Apart from exploring the dysregulation of lnc‐NEAT1 and miR‐125a, various studies also note that they are inter‐correlated in sepsis, coronary artery disease, etc.Recent evidence indicates that the lnc‐NEAT1 expression is positively associated with disease severity in asthma patients and other allergic diseases.+ T cells; moreover, STAT6 signaling pathway induced the differentiation of T cells toward Th2 cells.AR is also exacerbated due to the imbalance of Th1/Th2 differentiation, and its imbalance arouses the binding of IgE with eosinophil and mast cells, which is a promoting factor of allergic inflammation.There were some limitations in this study. Firstly, the sample size was relatively small, which might cause a weak statistical power, and a further study with a larger sample size was necessary. Secondly, the lnc‐NEAT1 and miR‐125a expressions were detected at a single time point in this current study; therefore, their longitudinal change for the continuous monitoring of the disease progression needed to be explored. Thirdly, we hypothesized that the lncRNA NEAT1 expression involved in the allergic inflammation response of AR via targeting miR‐125a, whereas its detailed molecular mechanisms needed to be answered in the future study. Fourthly, according to the type of allergen, AR could be divided into seasonal AR and perennial AR; and according to the frequency of symptoms, AR could be classified into intermittent AR and persistent AR.In conclusion, circulating lnc‐NEAT1 and miR‐125a are dysregulated and correlated with the disease severity and Th1/Th2 imbalance in the pediatric AR patients, which makes it possible to distinguish the risk stratification and consequently help the clinicians provide individual treatment to each pediatric AR patient. Hence, they may serve as potential biomarkers for the management of the pediatric AR patients.The authors declare that they have no competing interests.Fig S1Click here for additional data file.