Our findings provide evidence indicating that psychological resilience plays a significant positive role in the SRH in our study sample. Further research is required to extend the growing knowledge regarding the application of psychological resilience on KOA.This study aimed to evaluate whether psychological resilience is an independent factor of self-rated health (SRH) among patients with knee osteoarthritis (KOA). A cross-sectional study with convenience sampling was designed. Patients with doctor-diagnosed KOA were recruited from the orthopedic outpatient departments of a hospital in southern Taiwan. Psychological resilience was measured by the 10-item Connor–Davidson Resilience Scale (CD–RISC-10), and SRH was measured by three items, including the current SRH, the preceding year-related SRH, and age-related SRH. The three-item SRH scale was categorized as “high” and “low–moderate” groups by terciles. Covariates included KOA history, site of knee pain, joint-specific symptoms measured by the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), comorbidity measured by Charlson Comorbidity Index, and demographic variables . A multiple logistic regression was used to detect the independent variables with significant odds ratios that can predict “high” SRH among participants. Results: In total, 98 patients with KOA (66 women and 32 men) with a mean age (±SD) of 68.3 ± 8.5 years were enrolled and were analyzed. A total of 38.8% (n = 38) of participants were categorized as “high SRH”, while 61.2% (n = 60) were categorized as “low–moderate SRH”. Multiple logistic regression showed that CD–RISC-10 had an increased odds ratio (OR) for high SRH (OR [95% CI] = 1.061 [1.003–1.122];  An aging society and the prevalence of chronic diseases highlight people’s need for good health. In order to improve the health of people, healthcare providers are always committed to seeking corresponding solutions. In addition to medications and lifestyle changes, healthcare providers are increasingly aware of the potential contribution of positive psychological factors to chronic disease management. Psychological resilience is an important topic that is discussed in the literature. The concept of resilience is originally derived from technical sciences and refers to the ability of a material to recover or rebound to its original shape after bending or compression. Psychological resilience has gradually become prevalent in the fields of psychology and psychiatry and has recently been garnering attention in the field of chronic illness.Psychological resilience has two distinct meanings. On the one hand, resilience can be narrowly described as a trait or a personality resource, an individual’s ability to resist being damaged or deformed by traumas or destructive forces. It enables individuals to adjust and modify habitual expression patterns of self-discipline and self-control so that they can face and adapt to the situation in the present and future. On the other hand, resilience broadly means readily “bouncing back” or recovering from those traumas or destructive forces. That is, when an individual is facing adversity, they can go further than merely coping by finding meaning in the trauma-inducing events, and they can further utilize this meaning to enhance their well-being to achieve the status of post-traumatic growth ,2. As an integrated concept of resilience, the American Psychological Association Dictionary of Psychology’s advanced definition defines resilience as “the process and outcome of successfully adapting to difficult or challenging life experiences, especially through mental, emotional, and behavioral flexibility and adjustment to external and internal demands” . This meIn recent decades, increasing attention is being paid to resilience in response to chronic illness. In this context, psychological resilience has been described as the flexibility and ability to cope, recover, or adapt when facing internal and external pressures brought on by a chronic illness ,7. As a KOA is a prevalent chronic illness and an age-related degenerative joint disease. KOA cannot be completely healed using medicine and usually requires a long period of supervision, observation, and management . KOA symSelf-rated health (SRH) is the result of a cognitive assessment of the integrated biological, mental, social, and functional aspects of an individual ,18. The With a special interest in this positive psychological trait, we hypothesized that patients exhibiting greater psychological resilience would report a better SRH. A cross-sectional study with convenience sampling was adopted, and participants were recruited from the orthopedic outpatient departments of a hospital in southern Taiwan from July to August 2020. Subjects were eligible for the study if they met the following criteria: (1) patients with doctor-diagnosed KOA, (2) those who agreed to participate in the study and provided informed consent, and (3) those with clear consciousness, no cognitive impairment, or other severe diseases that would limit their ability to complete a survey. Patients with a knee joint replacement or who were diagnosed with rheumatologic diseases were excluded. A total of 98 KOA patients participated in this study.According to the recommendations of Concato et al. and Peduzzi et al., the minimum required sample size should be based on the rule of event per variable (EPV) and the concept of an EPV of 10 is acceptable for logistic regression ,30. TherResearchers and two trained research assistants met the participants before or after their physician appointments at the orthopedic outpatient departments of a hospital in southern Taiwan. After the interviewers informed them of the study’s purpose and obtained written informed consent, face-to-face interviews were conducted. All participants were able to withdraw from the study and withdraw their consent at any time. Ethical approval was obtained from the institutional review committee of the hospital. The questionnaire included psychological resilience measured by the 10-item Connor–Davidson Resilience Scale (CD–RISC-10), SRH, and covariates . The Chinese version of the CD–RISC-10 is widely used to assess psychological resilience ,32, specSRH is a measure of how people perceive their health . In the In the present study, the content validity index of clarity (panel reviewed by experts) for this 3-item SRH scale was 1.00. The Cronbach’s alpha was 0.620. The reliability of the test is relatively low; however, it can be accepted . The SRHBased on previous studies on SRH, the following covariates were considered in this study. The sociodemographic characteristics included age, gender, education attainment , living arrangements (with/without a spouse), self-reported KOA history , site of knee pain , KOA symptoms, depression, and comorbidity. KOA symptoms were assessed using the Western Ontario and McMaster Universities Osteoarthritis (WOMAC) Index, which was developed in 1982 at the Western Ontario and McMaster Universities. The WOMAC Index includes 3 dimensions: stiffness (2 items), pain (5 items), and physical function (17 items). All items are scored on a scale of 0 to 4, corresponding to “none”, “mild”, “moderate”, “severe”, and “extreme”, respectively. Higher scores indicate greater severity of symptoms . WOMAC ip-value less than 0.05 was considered statistically significant.Descriptive statistics are reported as a percentage for categorical variables and as a mean ± standard deviation (SD) for continuous variables. Because most of the data did not follow a normal distribution, nonparametric analyses (Chi-square tests and Mann–Whitney U tests) were used to compare the demographic and clinical characteristics, as well as psychological resilience between the two levels of SRH (low–moderate and high SRH). If there were significant variables that were found between the two levels of SRH, the univariate logistical regression was conducted to assess the relationship between psychological resilience and SRH without considering covariates (univariate model). Multiple logistical regression with the conditional method was then used, controlling for all covariates (adjusted model). The results are reported as odds ratios (ORs) or adjusted odds ratio (AOR) with 95% confidence intervals (CIs). In terms of internal consistency, Cronbach’s alpha coefficients were calculated for all the scales. According to Guilford’s (1965) criteria, if Cronbach’s α value is greater than 0.7, then it is considered a good reliability value, while values between 0.7 and 0.35 are considered acceptable reliability values. Values less than 0.35 are rejected reliability values . The maiA total of 98 people with KOA, consisting of 32 men and 66 women, were enrolled in this study. The mean age (SD) was 68.3 ± 8.5 years, with a range of 51–90 years. In SRH, the median SRH score was 8 (range: 3–14). Using the upper tercile of the total SRH score (score = 9) as the cut-off point, 38 (38.8%) patients were dichotomized into “high SRH” (score ≥ 9) group, while 60 (61.2%) participants were dichotomized into “low–moderate” SRH (score < 9) group. p < 0.05 for all comparisons) of a long history of KOA (≥5 years), bilateral knee pain , and higher comorbidities (CCI < 1). In addition, participants in the low–moderate SRH group had higher scores in the total WOMAC index and its subscales (severe KOA symptoms), and lower scores in CD–RISC-10 (p < 0.05). However, there were no significant differences found in the proportion of the other variables between both SRH groups . p = 0.038). This result implies that KOA-related symptoms are negatively associated with a high SRH, and that psychological resilience was a positive predictor of SRH. p-values < 0.05, indicating that these four variables are significant predictors of SRH in people with KOA. The regression model had a high goodness-of-fit, with Omnibus test of model coefficients χ2 = 26.304, df = 4 (p < 0.001), indicating that this predictive model was able to distinguish between participants who did and did not report a high SRH. In terms of the strength of association, the model explained approximately 23.5–31.9% of the variance in SRH (range between Cox and Snell R2 and Nagelkerke R2).All tests on the Wald values for bilateral pain, WOMAC stiffness, WOMAC physical limitation, and the CD–RISC-10 score, resulted in The present study showed that psychological resilience was a key determinant of a high SRH among patients with KOA. After controlling the other significant variables, the higher the CD–RISC-10 scores, the higher the chance of reporting a high SRH in our study sample . ConversTo our knowledge, this is the first study to determine the association of psychological resilience with a high SRH among patients with KOA. This finding is congruent with the results reported in similar studies. A cross-sectional study surveyed 163 patients following a primary total knee arthroplasty and reported that psychological resilience measured by CD–RISC-10 was identified as a significant predictor of functional outcome . In addiIn the literature, psychological resilience has been considered as a kind of cognitive–affective state and can modify or alleviate perceptual chronic pain through the activation and inflammation of the immune system . Low psyOur significant finding represents opportunities for healthcare providers to give resilience-based interventions or strategies for patients with KOA. Specifically, healthcare providers can enable KOA patients to enhance their psychological resilience through the development or cultivation of resilience constructs or protective factors, such as a sense of self-efficacy, the ability to regulate emotions, coping mechanisms, or through creating a supportive environment . Once paIn the present study, we measured health status using a 3-item SRH scale, which was derived from the previous study, and investigated the association with psychological resilience. Given that the SRH is a simple and generally feasible method with acceptable validity, we used it as a measurement for the assessment of health status. The upper tercile of the total SRH score was used as a cut-off threshold (score = 9) for high SRH. Though high SRH does not guarantee health, it is indeed significantly associated with objective health measures and consequent morbidity . Due to As expected, the KOA-related symptoms  showed a negative relationship with high SRH. Physical health or illness-related factors, such as existing medical conditions, pain severity, functional problems, or disabilities, have also been reported to be strong predictors of poor SRH ,27. GiveThe present study sheds light on the relationship of some variables with SRH, especially the positive effects of psychological resilience, but it is still subject to some limitations. Firstly, due to the cross-sectional design of our study, the causality between relationships should be carefully interpreted. Advanced research to employ experimental and longitudinal designs may be required to determine if and how psychological resilience impacts SRH and to examine the interaction between KOA symptoms and SRH. Secondly, the relatively small sample size of this study (n = 98) not only limits the generalization scope but also makes Cronbach’s alpha of SRH and GDS-5 relatively low  . AccordiOur findings contribute to the growing body of research regarding the specific role of psychological resilience in high SRH among patients with KOA. Healthcare providers can devise and tailor preventive interventions that target psychological resilience to promote better health among patients with KOA. Future studies utilizing experimental designs may also be needed to elucidate the effectiveness of resilience on a person’s health.


We read with interest Yu and Zheng’s article, “Human Papillomavirus Type 16 Circular RNA Is Barely Detectable for the Claimed Biological Activity” . Yu and in vitro growth conditions can influence circE7 levels  is prone to underestimation bias , 6. RT-dIn contrast to RT-PCR, Northern blot assays are more reliable and not liable to detection artifacts. In our study, we quantitated Northern blots of total RNA from CaSki, SiHa, and SCC154 cells using an E7 probe generated by random-hexamer-primed labeling of a 450-bp fragment of E7. This fragment was amplified by PCR from a plasmid containing the full-length HPV16 genome. An RNase R-resistant E7 band was identified in multiple independent experiments. circE7 consistently accounted for ~1 to 3% of E7-containing transcripts, or ~7 to 21 copies/cell . Our Nor10–The abundance of circE7 is further bolstered by data from RNA sequencing, which is also less sensitive to artifacts than RT-dPCR. Publicly available Sequence Read Archive (SRA) data and The Cancer Genome Atlas (TCGA) sequencing data demonstrate that circE7 is present in both cervical cancer and head and neck squamous cell carcinoma (HNSCC). More recent analyses have demonstrated circE7 to be present in anal squamous cell cancer  and vulvAnother point raised by the authors was related to the specificity of the RNA interference (RNAi) used to assess circE7’s function. Using siRNAs, Yu and Zheng were unsuccessful in their attempts to knock down circE7 without also depleting E6*I RNA. In contrast, we employed a lentiviral, doxycycline-inducible short hairpin RNA (shRNA) vector to knock down circE7 and were successful in knocking down circE7 without significantly affecting E6*I levels as assessed by both RT-PCR and Northern blotting . CompareAn important control for the specificity of our shRNA knockdown experiments was the rescue of the shRNA knockdown through the expression of an shRNA-resistant circE7 . Lentivi6-methyladenosine modified, and associated with polysomes. In addition, circE7-mediated E7 expression is induced by heat shock. These biochemical features are consistent with overexpression and knockdown studies that demonstrate that circE7 functions as a translated circRNA. Additional studies will be required to determine whether linear and circular E7 isoforms interact cooperatively or independently to promote E7 production in viral replication and tumorigenesis.Finally, the authors inaccurately state that we conclude that “E6*I is nuclear and unable to encode E7” . This is19–


In this update, RM2Target encompasses 1 619 653 WER–target associations for nine RNA modifications in human and mouse, including m6A, m6Am, m5C, m5U, m1A, m7G, pseudouridine, 2′-O-Me and A-to-I. Extensive annotations of target genes are available in RM2Target, including but not limited to basic gene information, RNA modifications, RNA–RNA/RNA–protein interactions and related diseases. Altogether, we expect that RM2Target will facilitate further downstream functional and mechanistic studies in the field of RNA modification research.RNA modification is a dynamic and reversible process regulated by a series of writers, erasers and readers (WERs). Abnormal changes of WERs will disrupt the RNA modification homeostasis of their target genes, leading to the dysregulation of RNA metabolisms such as RNA stability and translation, and consequently to diseases such as cancer. A public repository hosting the regulatory relationships between WERs and their target genes will help in understanding the roles of RNA modifications in various physiological and pathological conditions. Previously, we developed a database named ‘m6A2Target’ to host targets of WERs in m6A, one of the most prevalent RNA modifications in eukaryotic cells. To host all RNA modification (RM)-related WER–target associations, we hereby present an updated database, named ‘RM2Target’ ( RNA modifications have been known to play critical roles in modulating RNA metabolisms, such as gene transcription, RNA stability, RNA splicing, nuclear localization and translation . RNA modBenefitting from the development of experimental and high-throughput sequencing technologies, more evidence could be provided to explore the relationship between WER and target genes. For instance, immunoprecipitation  and certhttp://rm2target.canceromics.org/) to host WER–target associations for 63 WERs from two organisms and nine types of RNA modifications, including N6-methyladenosine (m6A), N6,2′-O-dimethyladenosine (m6Am), N1-methyladenosine (m1A), pseudouridine (ψ), 5-methyluridine (m5U), 5-methylcytosine (m5C), 7-methylguanosine (m7G), 2′-O-methylation and A-to-I RNA editing. Compared to m6A2Target, RM2Target is far superior in all aspects , and 36 WERs for mouse  . All relFor all datasets, Genome Reference Consortium Human Build 38 (hg38) and Genome Reference Consortium Mouse Build 39 (mm39) were used as the reference genomes for human and mouse, respectively. fastp  was emplTo obtain potential targets that have protein–protein interactions with WERs, mass spectrometry results were summarized from the supplementary data of relevant literature.To obtain potential targets that have protein–DNA interactions with WERs, preprocessed ChIP-seq data were aligned to the reference genome using bowtie2 , and peaTo obtain potential targets that have protein–RNA interactions with WERs, RIP-seq and various types of CLIP-seq data were collected. For RIP-seq, STAR  was usedTo obtain potential targets with perturbation evidence, differential analyses were performed between control group versus perturbed group at five levels: RNA expression, modification, translation, alternative splicing and stability. Group information for all analyses can be found in the P-value < 0.05, fold change > 1.5). For modification enrichment, MeRIPseqPipe . For m5C BS-seq, information about the modification sites and quantification results were downloaded from relevant GEO datasets. For RNA stability, half-life of mRNA was calculated according to a previously published paper (2(FPKM of perturbed group/FPKM of control group), with a cutoff of 1.5 for the fold change. For alternative splicing, rMATS  were used and displayed in the web page.To identify the consensus motifs of target genes for each WER, we use the top 200 most reliable targets determined by confidence score to perform motif analysis with HOMER . Only taBasic information of WERs and target genes, such as official gene symbol, gene ID, gene type and genome location, were preferentially extracted from GENCODE  annotatiTo better illustrate the WER–target associations, modification sites in RMVar , RMBase https://genome.ucsc.edu/) , Cascading Style Sheets (CSS) and JavaScript (JS). All the interactive diagrams were generated by ECharts to visualize the analysis results. Furthermore, RM2Target implemented a genome browser to present genomic annotations using UCSC Genome Browser (sc.edu/) .in vivo or in vitro experiments using western blot, RT-qPCR, RNA stability assay, RIP assay, luciferase reporter assay, etc. methods, respectively. (ii) ‘Binding’: 461 746 and 61 395 WER–target associations with binding evidence were predicted by high-throughput analyses for human and mouse, respectively. Among them, there were 451 016 WER–target associations with protein–RNA interactions for human and 55 004 for mouse. A total of 3100 WER–target associations with protein–protein interactions were recorded in human. Growing studies have demonstrated that WERs may localize to the different region of chromatin and subsequently affect the deposition of RNA modifications co-transcriptionally   regulates cancer growth and progression . MoreovesnoRNPs) , also shThe deposition of RNA modifications is usually considered as a co-transcriptional process . More reRecent studies showed that some known WERs of RNA modification have modification-independent functions. For instance, the m6A writer METTL3 could promote the translation of target gene PAPBC1 without m6A modification . The m6ACompared to m6ATarget, the web interface of RM2Target has been redesigned to allow users to browse, search, and download all available RNA modification WER and target gene information more conveniently.Browse and search, in the browse page . We noted that ASNS was associated with cancer and malignant neoplasm of liver according to the disease annotations in RM2Target (Figure The m6A writer METTL3 has been proved to be associated with cancer progression in a variety of cancer types . Here, wt Figure . The expt Figure , and thet Figure . The expt Figure , and thet Figure . Additiot Figure . Taken tDetermining the regulatory network of WERs will be of great significance in the understanding of the molecular mechanisms underlying the regulation of RNA modifications and exploration of the potential application value of RNA modifications. To our knowledge, RM2Target is the first comprehensive and specific resources focusing on the targets of different WERs across various RNA modifications.Our previous version m6A2Target received much attention and facilitated a lot of m6A related research. For examples, the researchers use m6A2Target to explore the tumorigenic mechanisms of m6A WERs in various cancer types ,69. CompIn conclusion, we expect that RM2Target will become a powerful resource for the field of RNA modification research. As a long-term goal, we would like to continually update RM2Target.http://rm2target.canceromics.org.RM2Target is a comprehensive online database available at gkac945_Supplemental_FilesClick here for additional data file.


RNA‐binding Motif Protein39 (RBM39) is identified as a splicing factor and transcription coactivator. Despite mounting evidence that RBM39 plays a critical role in the development of specific malignancies, no systematic pan‐cancer investigation of RBM39 has been conducted. As a result, we set out to investigate RBM39’s prognostic significance and putative immunological activities in 33 different cancers. Based on TCGA and CCLE, GTEx, cBioportal and HPA, we used a series of bioinformatics approaches to explore the potential oncogenic role of RBM39, including analysis of the expression of the pan‐cancer species RBM39, the prognostic relationship between RBM39 expression and overall survival (OS), disease‐specific survival (DSS) and progression‐free interval (PFI), the relationship between RBM39 expression and clinical phenotype, analysis of the relationship between RBM39 expression and tumour mutational burden (TMB), microsatellite instability (MSI), DNA methylation and immune cell infiltration. Our results showed that RBM39 is overexpressed in most cancers. RBM39 was positively or negatively correlated with the prognosis of different tumours. RBM39 expression was associated with TMB and MSI in 9 and 12 cancer types. In addition, RBM39 expression was associated with DNA methylation in almost all tumours. There are eight tumours were screened for further study, including BRCA, COAD, HNSC, LIHC, LUSC, SKCM, STAD, UCEC. In the screed tumours, RBM39 was found to be negatively correlated with the infiltration of most immune cells. In addition, the correlation with RBM39 expression varied by immune cell subtype. Based on RBM39’s role in tumorigenesis and tumour immunity, we suggest it can serve as a surrogate prognostic marker. By 2020, it is estimated that there will be nearly 10 million cancer deaths and 19.3 million new cancer cases worldwide.RNA‐binding motif (RBM) proteins are members of the family of RNA‐binding proteins (RBPs), which are present in all organisms and can bind single‐ or double‐stranded RNA and play important roles as post‐transcriptional regulators in regulating RNA metabolism and gene expression.So far, most studies on the role of RBM39 in tumours have been limited to one specific type of cancer. There are no pan‐cancer studies on the relationship between RBM39 and various cancers. Therefore, we analysed the expression levels of RBM39 in different types of malignancies and its relationship with prognosis using multiple databases, including TCGA, Cancer Cell Line Encyclopedia (CCLE), Genotype‐Tissue Expression (GTEx), cBioPortal and Human Protein Atlas (HPA). In addition, we explored the relationship of RBM39 expression with DNA methylation and immune infiltration. We introduced co‐expression analysis and enrichment analysis of genes to investigate the biological functions of RBM39 in tumours. Our results suggest that RBM39 may play an important role in tumours and serve as a prognostic factor. Our study provides a theoretical basis to gain insight into the role of RBM39 in tumour immunotherapy.22.1https://portals.broadinstitute.org/). EdgeR software was used to compare RBM39 expression levels in tumour and normal tissues. Using the Kruskal–Wallis test, we determined the levels of RBM39 expression in several normal tissues and tumour cell lines.RNA sequencing, somatic mutations and clinical data were gathered from the TCGA and GTEx databases. Each tumour cell line's CCLE‐based expression data was obtained from the Broad Institute's Portal (2.2https://www.proteinatlas.org/) to compare differences in RBM39 protein expression between tumour tissues and normal tissues, including breast invasive carcinoma (BRCA), liver hepatocellular cancer (LIHC), lung adenocarcinoma (LUAD), lung squamous carcinoma (LUSC) and colon adenocarcinoma (COAD).IHC results of RBM39 protein expression were collected from the HPA database (2.3p < 0.05) survival analysis was used. Age, tumour stage and clinical phenotypes were analysed to see whether they were associated with RBM39 expression (p < 0.05).TCGA data on each sample's survival and clinical features were obtained. Three indicators, OS, DSS and PFI, were considered when analysing the correlation between RBM39 and survival. Kaplan–Meier  occurs when an insertion or deletion of repetitive units results in a change in microsatellite length as compared with normal tissues. Spearman rank correlation coefficient was used to analyse the correlation between RBM39 expression and MSI.2.5Mismatch repair is an intracellular process that allows mismatches to be repaired. DNA replication errors are not repaired when this mechanism fails, resulting in an increase in somatic mutations. The relationship between five of the MMRS genes  and RBM39 expression was studied using the TCGA data. The chemical modification of DNA known as DNA methylation can affect epigenetics and can influence gene expression without changing the DNA sequence. We used this study to examine whether RBM39 expression correlated with that of the four methyltransferases.2.6Tumour‐infiltrating lymphocytes are independently correlated with the status of primary lymph nodes and survival, and tumour immune cell scores are determined using immune scores and interstitial scores. We used software estimation to analyse the correlation between gene expression and immune cell score.2.7A neoantigen is encoded by a modified gene in tumour cells as a result of biological processes like point mutations, removal mutations and mutation fusions. Scanner calculates its binding affinity score based on antigenic epitopes with a length of 8–11 amino acids, whereas neoantigens have a score less than 500 nm. The Scanner software was used to quantify the number of neoantigens in each tumour specimen independently, as well as to examine RBM39 expression in relation to the number of antigens. In addition, 47 frequently studied immune checkpoint genes were isolated independently from RBM39 expression, and correlations with RBM39 expression were calculated.p < 0.05 was considered significant).We used CIBERSORT, a metagene tool that predicts immune cell phenotypes, to assess the correlations between RBM39 levels and immune cell infiltration in cancer in 33 tumours. , immune activation, immunosuppression, chemokine and chemokine receptor proteins. Gene Set Enrichment Analysis of RBM39 across cancers. The biological activities of RBM39 in malignancies were investigated using Gene Set Enrichment Analysis (GSEA).33.1We analysed the expression levels of the physiological RBM39 gene across tissues through the GTEx database Figure . RBM39 wFurthermore, to assess RBM39 protein expression, we matched IHC results from the HPA database to RBM39 gene expression data from the TCGA database. The results showed that normal breast, lung, liver and colon tissues showed low levels of RBM39 staining, whereas the corresponding breast, lung squamous, liver and colon malignancies had moderate to high expression, which was consistent with the expression pattern of RBM39 in the TCGA database for the five tumours Figure A–E.3.2p = 0.029), ACC , SARC , UCS , PRAD , PCPG . These results suggested that RBM39 was a high‐risk gene in LIHC, ACC, SARC, PRAD and PCPG, while it was a low‐risk gene in UCS. A Kaplan–Meier survival study revealed that patients with low RBM39 expression in LIHC, ACC, SARC, PRAD and PCPG had a longer OS, whereas UCS patients with low RBM39 expression had a shorter overall survival  and SARC . Kaplan–Meier survival analysis showed that patients with low RBM39 expression in both tumours had longer disease‐specific survival , ACC , UCEC , PRAD . However, high expression of RBM39 was associated with high PFI in SKCM   Figure A. KM sur) Figure B–F.3.3p < 0.01), KIRP (p < 0.01) and SARC (p = 0.01) had lower RBM39 expression, while UCEC (p = 0.02) patients <60 years had higher RBM39 expression  and minor insertions/deletions (INDELs). TMB is a quantifiable biomarker that indicates the number of mutations discovered in tumour cells. As shown in Figure 3.5With the exception of CHOL, practically all MMRs genes were related with RBM39 expression across cancers, suggesting that RBM39 may protect tumour cell survival by promoting DNA mismatch repair‐related genes expression Figure A. DNA me3.6R = –0.253, p < 0.001) and LUSC , and the top three tumours whose RBM39 expression was most significantly correlated with mesenchymal scores were LUSC , BLCA  and SARC ; the top three tumours with the highest correlation with the assessed immune scores were LUSC , UCEC  and SARC  , and in BRCA, the expression of RBM39 was negatively correlated with the number of neoantigens .We next examined the correlation between RBM39 with more than 40 immune checkpoint genes across cancers. RBM39 expression was shown to be favourably linked with immune checkpoint gene expression levels in a variety of tumour types, including KICH, LIHC, PAAD and UVM Figure . The fin3.8Next, we investigated the association of RBM39 expression with the level of infiltration of 24 immune‐related cells. Our data showed that the level of immune cell infiltration was substantially linked with RBM39 expression in the majority of malignancies Table . We screIn addition, there was a differential relationship between RBM39 expression and different subpopulations of T cells. The results showed that RBM39 was negatively correlated with T cells and Tregs, whereas positively correlated with Th cells and Tcm. Tumours with the strongest correlation coefficients between the degree of infiltration of each immune cell and RBM39 expression was shown in Figure We investigated the relationship between RBM39 expression and immune‐related genes in 33 malignancies using gene co‐expression analysis. Genes encoded MHC, immune activation, immune suppression, chemokine and chemokine receptor proteins were analysed. Heat maps showed positive correlations of RBM39 with immune‐related genes in KIRC, LIHC, PRAD and UVM Figure .3.9We used GSEA enrichment analysis to investigate the biological importance of RBM39 expression in various tumour tissues. The data showed that in nine tumours, including BRCA, GBM, CHOL, OV, SKCM, THCA, THYM, UCEC and UVM, RBM39 expression negatively correlates with immune‐related functions such as neutrophil degranulation and chemokine transduction Figure . While i4An increasing amount of evidence reveals that RBM39 is substantially linked to cancer growth in a range of malignancies. As an RNA‐binding protein, RBM39 is extensively involved in selective splicing of RNA.Using TCGA data, we performed a Kaplan–Meier survival analysis and discovered that greater RBM39 expression was associated with a poor prognosis for OS, DSS, and PFI in LIHC. RBM39 was discovered as an autoantigen in a cirrhotic patient who later acquired hepatocellular cancer.Tumour mutational burden is an intriguing predictive biomarker with the potential to usher immuno‐oncology into the era of precision medicine.Our results suggest that RBM39 plays an important role in cancer immunity. The tumour microenvironment (TME) not only influences tumorigenesis, development and metastasis, but also has a profound impact on therapeutic results.Tumour‐infiltrating immune cells have an important impact on tumorigenesis and progression and can antagonize or promote tumorigenesis and progression.Our study further elucidates the broader tumorigenic applicability of RBM39 and confirms that RBM39 expression is closely associated with the biological processes of immune cells and immune‐related molecules in most tumours. In addition, our study revealed that RBM39 is co‐expressed with genes encoding MHC, immune activation, immune suppression, chemokine and chemokine receptor proteins. These results suggest that RBM39 expression is closely associated with immune infiltration of tumour cells, affecting patient prognosis and providing a new target for immunosuppressant development. Furthermore, our enrichment analysis suggests that RBM39 may influence the aetiology or pathogenesis of cancer by acting in immune cell infiltration, immune chemokine transduction and immune anti‐infection pathways.In conclusion, our first pan‐cancer RBM39 analysis revealed that it is differentially expressed in tumour and normal tissues and that there is a link between RBM39 expression and clinical prognosis. Our findings imply that RBM39 can function as an independent prognostic biomarker in a wide range of malignancies. RBM39 expression leads to different prognostic outcomes for different tumours, which requires further investigation of the specific role of RBM39 in each tumour. In addition, RBM39 expression is associated with the tumour microenvironment and immune cell infiltration in different cancer types. Its effect on tumour immunity also varies by tumour type. These findings may help to elucidate the role of RBM39 in tumour development and provide a reference for achieving more precise and personalized immunotherapy in the future.Rui Zhang: Conceptualization ; data curation ; formal analysis ; investigation ; methodology ; writing – original draft ; writing – review and editing . Wei Wang: Data curation ; investigation ; writing – review and editing . Xueting Chen: Formal analysis (supporting); writing – review and editing (supporting). Nie Zhang: Formal analysis (supporting); writing – review and editing (supporting). Wanming Liu: Formal analysis (supporting); writing – review and editing (supporting). Longzhen Zhang: Funding acquisition (supporting); project administration (supporting); writing – review and editing (lead). Nianli Liu: Conceptualization ; funding acquisition (lead); project administration (lead); supervision (lead); writing – review and editing (lead).The authors report no conflicts of interest in this work.Figure S1Click here for additional data file.Figure S2Click here for additional data file.Figure S3Click here for additional data file.Figure S4Click here for additional data file.Figure S5Click here for additional data file.Figure S6Click here for additional data file.Table S1Click here for additional data file.


Legionella pneumophila DNA uptake regulator, RocC, bound to the transcriptional terminator of its primary partner, the sRNA RocR. The structure reveals specific recognition of the 3’ nucleotide of the terminator by a conserved pocket involving a β-turn-α-helix motif, while the hairpin portion of the terminator is recognized by a conserved α-helical N-cap motif. Structure-guided mutagenesis reveals key RNA contact residues that are critical for RocC/RocR to repress the uptake of environmental DNA in L. pneumophila. Structural analysis and RNA binding studies reveal that other ProQ/FinO domains also recognize related transcriptional terminators with different specificities for the length of the 3’ ssRNA tail.The ProQ/FinO family of RNA binding proteins mediate sRNA-directed gene regulation throughout gram-negative bacteria. Here, we investigate the structural basis for RNA recognition by ProQ/FinO proteins, through the crystal structure of the ProQ/FinO domain of the  In this work, the authors determine the crystal structure of a ProQ/FinO RNA chaperone bound to its RNA target. This provides insight into how this family of bacterial proteins recognize transcriptional terminator structures. RNA chaperones, such as the well-studied Hfq4, play roles in either increasing rates of sRNA–mRNA hybridization, affecting the stability of target mRNA, or rearranging target RNA folding impacting ribosome accessibility4.Small RNAs (sRNAs) control a variety of physiological responses across bacterial specieshttps://www.ebi.ac.uk/interpro/entry/pfam/PF04352/] constitute a large family of RNA chaperones that are widely distributed throughout the bacterial taxa8. This family is defined by a conserved ProQ/FinO domain, which is a largely α-helical fold that is often flanked by flexible N- or C-terminal regions9. The eponymous FinO protein was discovered as a regulator of F plasmid conjugation nearly 50 years ago, and acts to bind a single partner sRNA called FinP to stabilize FinP and facilitate its interactions with its antisense partner, the mRNA encoding the major F plasmid transcription factor, TraJ5. More recently, the application of RNA-seq technologies has enabled the elucidation of the biological partners and targets of several ProQ/FinO family proteins. These approaches have verified the recognition of FinP by FinO, but have also revealed that FinO, in certain instances, can also interact with related sRNAs from other co-resident plasmids10. Another plasmid-encoded ProQ/FinO family member, FopA, has also been shown to interact with a single antisense RNA11. Likewise, the ProQ/FinO domain-containing protein RocC of Legionella pneumophila interacts with only one trans-acting sRNA (RocR) to repress post-transcriptionally multiple mRNA targets6. While these studies have built the case that certain ProQ/FinO family members bind to only a very limited number of partners in a highly specific manner, other family members appear to have multiple biological sRNA targets. Probably best studied is ProQ, which binds hundreds of sRNAs and likely acts as a general regulator of gene expression with an impact similar to Hfq14. Similarly, a minimal ProQ/FinO domain protein, NMB1681, has been shown to bind a range of structured RNAs in Neisseria meningitidis15.The proteins containing a ProQ/FinO domain [16, and further work showed that the base of the hairpin and 3’ tail are both strongly protected from ribonuclease degradation by FinO17. Proteolytic mapping and protein deletion analysis revealed that the ProQ/FinO domain itself is responsible for transcriptional terminator binding19, and site-specific protein–RNA cross-linking identified key RNA contact surfaces on the core ProQ/FinO domain20. Further studies showed that the ProQ/FinO domains of ProQ and NMB1681, can also specifically recognize transcriptional termination structures22.Insight into how these proteins recognize their cognate RNAs initiated with FinO. Early studies showed that FinO specifically binds the 3’ transcriptional terminator structure of FinP in a manner that critically relies on both the GC-rich hairpin and 3’ polypyrimidine tail that define the terminator23. Interestingly, ProQ proteins also share another folded domain C-terminal to the ProQ/FinO domain that adopts a Tudor fold, and hydrogen-deuterium exchange experiments suggest that this region may also play a role in RNA binding24. Detailed recent work however confirms the key role of the ProQ/FinO domain of ProQ in the recognition of transcription terminators25. An analysis of the effects of ProQ point mutants on RNA binding in cells using a bacterial 3-hybrid approach indicated that a conserved concave surface on the ProQ/FinO domain could be the primary terminator binding surface26. Indeed, the same surface on the ProQ/FinO domain of FinO was found to interact with the FinP terminator, suggesting a common mechanism of interaction in the family20.More recent studies probing the mechanism of binding of ProQ/FinO proteins to biologically validated RNA targets have opened the possibility for more complex modes of RNA recognition. To date, ProQ has been most studied in this regard. RNA footprinting has suggested that while transcriptional terminators do appear to be protected, other regions far from the 3’ ends of the transcript can also be protected17 and ProQ24 with RNA, the molecular mechanisms underlying these interactions remain unclear. To obtain high-resolution structural information, we chose to study one of the most selective of the ProQ/FinO domain protein family, the L. pneumophila protein RocC and its primary sRNA partner, RocR. The RocC/RocR system regulates competence, a specialized physiological state involving the coordinated expression of multiple genes which allows the bacteria to uptake environmental DNA and integrate it into its chromosome 6. RocC binds and stabilizes RocR, which in turn binds and represses a series of mRNA targets that encode components of the DNA uptake system. Previous work showed that the ProQ/FinO domain of RocC specifically recognizes the transcriptional terminator  of RocR and that mutation of the ProQ/FinO domain abrogates RocC-mediated repression6.While molecular modeling integrating small-angle X-ray scattering with biochemical and biophysical data has yielded low-resolution models for interactions of FinOHere, we have determined the crystal structure of the ProQ/FinO domain of RocC bound to a minimal terminator derived from RocR. The structure reveals that the 3’ single-stranded RNA tail adopts a hook-like structure that docks the 3’ terminal nucleotide into a highly conserved pocket in the ProQ/FinO domain, while the hairpin portion of the RNA is recognized by a conserved α-helical N-cap structure that recognizes multiple contiguous phosphate groups on a single strand. The importance of the interactions visualized in the structure is supported by structure-guided site-directed mutagenesis with RNA binding and in vivo measures of competence. The structure also explains previous results from site-directed mutation studies on FinO and ProQ, suggesting a common mechanism of binding across the ProQ/FinO domain family. We further present evidence that different ProQ/FinO domain proteins selectively bind terminators with different lengths of single-stranded tails.1-126) could bind SL3 of RocR  at 3.2 Å  is confirmed by analysis of 1H NMR spectra of the free and RocC-bound RocR9bp-tet  adopts a hook-like structure. U25 maintains an A-form geometry, however U26 and C27 bend away, unstacking from U25 and instead stacking upon each other.We established a method to obtain a homogenous protein–RNA complex of various RocC/RocR mutant combinations using gel filtration chromatography, and SEC-MALS confirmed the formation of a one-to-one complex . The most similar example we found was from the 40 S subunit of the eukaryotic ribosome where a β-turn-α-helix motif contacts a nucleotide within the 18 S rRNA in the same orientation with the same hydrogen bonding arrangement  assay  led to a modest but statistically significant (~threefold) reduction in binding affinity, as well as a small reduction in transformation repression. Arg is commonly observed at this position in ProQ/FinO domains, however, Ser is the most conserved residue at this position , which forms the base of the 3’ nucleotide-binding pocket, resulted in an >200-fold reduction in binding affinity as well as a dramatic reduction in transformation repression similar to that observed in the Δols Fig. . We mutaMutation of residues in the N-cap RNA-binding motif also led to significant reductions in binding and biological activity. The most dramatic effect was observed for R75D which led to a >100-fold reduction in binding affinity, as well as a complete loss of the mutant protein’s ability to repress transformation. K71D and K73D also displayed significant reductions in binding affinity (~12-fold) as well as an almost complete loss in transformation repression. S70A resulted in a less pronounced defect in binding and little if any effect on transformation repression, however mutation of both Ser70 and Ser72 to Ala resulted in stronger defects in binding and repression.1-137). This construct had the same binding affinity as 1–126, indicating that this additional region does not play a significant role in RNA binding  showed lower levels of protein compared to WT . In agreement with the EMSA results, RocC showed a dramatic specificity for the 5-nucleotide tail, and only interacted weakly with the 3- or 8-nucleotide tails. Similarly, the ProQ/FinO domain of ProQ showed a strong binding to the 5-nucleotide tail RNA, and less binding to the other RNAs. In contrast, the ProQ/FinO domain of FinO showed less difference in binding specificity between the different RNAs, with only a weak preference for the 5- and 8-nucleotide tail lengths compared to the 3-nucleotide tail.ProQ/FinO family members exhibit profound differences in their ability to recognize different RNAs in vivo. Certain proteins, such as RocC and FinO, only have one biological partner whereas other family members, such as ProQ and NMB1681, can bind a range of RNAs. The structures of the ProQ/FinO domains of these proteins, in particular, their 3’ nucleotide-binding pockets and N-cap motifs, are well-conserved between these proteins. Previous work demonstrated that FinO, ProQ, and NMB1681, like RocC, all can bind transcription terminator structuresnce Fig.  and stru FP Fig. . All theSL3 complex presented here reveals a mechanism where RocC specifically binds its natural partner RocR by interacting with its terminator, i.e., the hairpin of SL3 and its 3’ single-stranded polypyrimidine tail. The relevance of this structure to RocC/RocR binding in solution and in vivo is supported by extensive mutagenesis data. This structure also provides a model to understand RNA recognition by other ProQ/FinO domain proteins. The pocket that binds the 3’ nucleotide of the terminator is particularly well-conserved in the ProQ/FinO family. The position of the N-cap motif is absolutely conserved in the ProQ/FinO family, and Arg75 is almost completely conserved (80% identity) with the major substitution being lysine. The structure explains previous biochemical and mutagenesis data on FinO as well as ProQ  all bind transcriptional terminator structures but with varying degrees of specificity. While FinO and RocC bind to just one or two physiological partners, ProQ and NMB1681 bind to dozens of different sRNAs. Our structure of the RocC/RocRxyl Fig. . In this25. Biochemically, we find that the isolated ProQ/FinO domains of RocC, FinO, and ProQ all have differing tail length specificities. RocC binds very specifically to its 5-nucleotide-tailed RNA target. ProQ also preferentially bound to the 5-nucleotide-tailed RocR compared to versions with either 3- or 8-nucleotide tails, while FinO showed little preference for the different tail lengths. Our structure would predict a minimal ssRNA tail length of three nucleotides, corresponding to the portion of the tail which must contour into the nucleotide-binding pocket . These strains , a single colony of BL21-Gold (DE3) bearing one of the pGEX-6P-1_RocC* plasmid was inoculated into 25 mL LB with 100 μg mL−1 ampicillin and 35 μg mL−1 kanamycin and incubated at 37 °C for 18 h with shaking. This preculture was then inoculated into 1 L LB with 100 μg mL−1 ampicillin and 35 μg mL−1 kanamycin and grown to O.D. 0.6–0.8 at 37 °C. GST-PP-RocC* expression was induced with 0.3 mM IPTG and incubation was continued at 18 °C for 20 h. The cell pellet was collected by centrifugation, flash frozen and stored at −80 °C. The cell pellet was resuspended in lysis buffer  and lysed using an Emulsiflex-C3 high-pressure homogenizer (Avestin). The lysate was incubated with 10 mL glutathione beads for 1 h at 4 °C. Incubated beads were washed with 150 mL of lysis buffer and the GST tag was removed by digestion with 3 C protease for 18 h at 4 °C. The digested protein was collected and purified by Superdex 75 16/60 gel filtration chromatography in different buffers depending on the purpose . FinO (45–186)19 and ProQ (1–130)21 were purified in the same way as RocC*.For each RocC*  at 16 °C. Protein/reservoir solution ratios of 1:1 or 1:2 yielded crystals. 32 mg mL−1 of RocC1-126 in 25 mM Tris:HCl pH 8.0, 30 mM NaCl, 1 mM DTT was crystallized by hanging drop vapor diffusion with a reservoir solution consisting of 0.2 M ammonium sulfate, 0.1 M HEPES:NaOH, pH 7.3, 25% PEG 3350 at 16 °C in either ratio 1:1 or 1:2 of protein solution:resevoir solution. The quality of RocC1-126 crystals was improved using additive screens  at 4 °C. In all, 2 μL of 32 mg mL−1 RocC1-126 was mixed with 1.6 μL of reservoir solution  and 0.4 μL of the additive screen was mixed to make a 4 μL drop. Various additives produced high quality of crystals: multivalent ions  chloride hexahydrate, 0.1 M chromium (III) chloride hexahydrate), Linker (0.3 M glycyl-glycyl-glycine), polymer (10% w/v polyethylene glycol 3350), carbohydrate , organic, non-volatile solvent , organic, volatile solvents . Homogenous RocC14-126: RocR9bp-tet complex in 10 mM HEPES-KOH pH 7.5, 100 mM KCl, 5 mM MgCl2, 1 mM TCEP was purified from excess free components using gel filtration chromatography and was crystallized by sitting drop vapor diffusion against a reservoir buffer containing 0.2 M lithium sulfate 0.1 M Tris: HCl, pH 8.5 30% (w/v) PEG 4000 . Crystals grew at 1:1 protein:precipitant ratio at room temperature.In all, 30 mg mL24-126 were collected on a RIGAKU MICROMAX-007 HF with a DECTRIS PILATUS3 R 200K-A detector to a final resolution of 2.10 Å. Data for RocC1-126 were collected at CLS (Canadian Light Source) BEAMLINE 08ID-1 with DECTRIS PILATUS3 S 6 M detector to a resolution of 2.02 Å. Crystals of the RocC14-126/RocR9bp-tet complex were collected at ALS (Advanced Light Source) BEAMLINE 8.2.2 with ADSC QUANTUM 315r detector and data was obtained to a resolution of 3.20 Å. All data was integrated with HKL-200036.Data for RocC24-126 was determined by molecular replacement (MR) using Phaser37. A pruned model of FinO85-158 generated using sculptor38 was used as a search model. Two protomers were found in the asymmetric unit. Non-crystallographic symmetry (NCS) was used in the early stages of refinement using PHENIX39. Missing parts of the model were manually built in Coot40 and refined using the PHENIX to a Rwork of 16.1% and an Rfree of 23.8% to a final resolution of 2.10 Å.The structure of RocC1-126 was solved by MR, using RocC24-126 as a search model with Phaser37. Nine protomers were placed in the asymmetric unit. NCS was used throughout the refinement and revealed α-helical density for the N-terminus of RocC for two of the protomers. Manual building and refinement were carried out with Coot40 and PHENIX39 with the assistance of ninefold NCS to a final Rwork of 19.3% and an Rfree of 21.3% to a resolution 2.02 Å.The structure of RocC14-126: RocR9bp-tet complex was phased by molecular replacement using the high-resolution structure of RocC1-126 as a search model. Ten protomers were placed in the asymmetric unit and NCS was used throughout the refinement. Refinement of ten protomers showed different densities indicative of RNA bound to four of the ten protomers. This difference density map was improved through fourfold NCS averaging  using T4 polynucleotide kinase (Invitrogen) and purified using denaturing PAGE as described34. Labeled RNAs were incubated 30 min on ice with proteins from 0 to 32 μM concentration in a final volume of 5 μL EMSA reaction buffer , and 12 U RNaseOUT (ThermoFisher)). The reactions were mixed with 5x native gel loading dye  and separated on 10% native gels run in 1x Tris-Glycine buffer at 4 °C. Gels were dried and imaged with phosphor imaging screens (Molecular Dynamics). Band intensities were quantified using Molecular Dynamics ImageQuaNT TL software . Prism5 (GraphPad) was used for graph fitting and equilibrium dissociation constant (KD) calculation with one site-specific binding with Hill slope:max is the maximum specific binding, and h is the Hill slope. The input data are X and Y, and Bmax, h, and KD are obtained from the fitting procedure. All EMSA results were repeated at least three times.In vitro transcribed RNAs were 5’ radiolabeled with ATP, [γ-14-126 was prepared as described in the protein expression and purification section. All components were dialyzed in ITC buffer . In all, 30–50 μM of protein in the sample cell was titrated with RNA titrand at concentrations between 350 and 400 μM. In total, 2 μL (except for first injection) of concentrated titrand were injected 19 times every 240 s. Data were analyzed using MicroCal PEAQ-ITC Analysis Software .ITC was performed using MicroCal PEAQ-ITC  at 25 °C. Ten nucleotide RNA (5’-GGCCCUUUCU-3’) was purchased from IDT and SL3-9bp-tet was chemically synthesized. RocCSL3 was purchased from IDT and 5’ FAM-labeled RocR3nt, RocR8nt were chemically synthesized. 5 μL of 80 nM RNA in FP reaction buffer ) was mixed with 15 μL of point mutated RocC14-126 to a final protein concentration of between 320 μM and 2.4 nM in a 20 μL reaction. Reactions were transferred to a 384-well plate and were incubated for 1.5 h at 25 °C. FP experiments were conducted using an Wallace EnVision manager (PerkinElmer) using 485 nm excitation and recorded at 538 nm. All experiments were repeated three times and the dissociation constant was calculated by fitting results to a sigmoidal curve with a 4-parameter logistic (4PL) equation:x is the protein concentration, y is the percent bound, a and d are the estimated minimum and maximum values. b is the slope factor and c is the protein concentration that gives 50% binding. Under these conditions (protein concentration >> RNA concentration), c ≅ KD. Input data is x and y and a, b, c, and d are all obtained from the 4PL fitting.5’ FAM-labeled RocRL. pneumophila strains was assessed as previously described using conditions in which the WT strain is not transformable but transformation is highly efficient in hypercompetent mutant 6.Transformability of 600 = 1 (≈109 cells mL−1) in 3 mL AYE. Two times 1 mL of cell suspension were centrifuged for 3 min at 5000 × g in a table-top microcentrifuge, and each pellet was resuspended in 50 µL of AYE with or without transforming DNA. Each suspension was spotted on a new CYE plate and let to dry. The plate was incubated overnight at 37 °C. Each spot was resuspended in 200 µL AYE. Ten-fold serial dilutions were then plated on non-selective medium and selective medium. Plates were incubated for 72 h at 37 °C and colony-forming units (CFU) counting was performed. Transformation frequency is the ratio of the number of CFUs counted on selective medium divided by the number of CFUs counted on non-selective medium. To test the transformability of the RocC punctual mutants (stains KanR), the DNA used was 2 µg (for 109 cells) of a 4-kb PCR fragment centered on a mutated allele of the rpsL gene. Transformants were thus selected on CYE + Streptomycin. To test the transformability of the RocCΔN mutants, the DNA used was either the same rpsLR PCR as above or 2 µg (for 109 cells) of the pGEM-ihfB::Kan, a plasmid containing a kanamycin-resistance cassette (KanR) inserted in the ihfB gene of L. pneumophila. As this plasmid is non-replicative in L. pneumophila, the internalized molecules recombine with the chromosome via a double crossover allowing the integration of the KanR cassette in the ihfB locus. Transformants were thus selected on CYE + Streptomycin or CYE + Kanamycin. For each tested mutants, transformation assays were performed three to seven times.The strains were streaked on CYE solid medium from the −80 °C frozen stock culture and incubated 3 days at 37 °C. The strains were then re-streaked on a new CYE plate and incubated overnight at 37 °C to obtain exponentially growing cells. Bacteria were resuspended to an ODFurther information on research design is available in the SUPPLEMENTARY FIGURESPeer Review FileReporting Summary


Recombinant human granulocyte colony-stimulating factor (rhG-CSF) reduces neutropenia events and is widely used in cancer patients receiving chemotherapy. However, the effects of rhG-CSF on distant organ metastasis (DOM) in non-small-cell lung cancer (NSCLC) patients following postoperative chemotherapy are not clear.A retrospective cohort study was performed on NSCLC patients who underwent complete surgical resection and postoperative systemic chemotherapy at The First Affiliated Hospital of Nanchang University between 1 January 2012 and 31 December 2017. The effect of rhG-CSF on DOM was assessed with other confounding factors using Cox regression analyses.n = 246 in the rhG-CSF group, n = 61 in the No rhG-CSF group). The incidence of DOM in postoperative NSCLC patients with rhG-CSF treatment was observably higher than in patients without rhG-CSF treatment . Univariate regression analysis revealed that rhG-CSF and pathological stage were independent risk factors for metastasis-free survival (MFS) (p < 0.05). RhG-CSF users had a higher risk of DOM  than nonusers of rhG-CSF. The association between rhG-CSF and the risk of DOM was significant only in patients presenting with myelosuppression  and not in patients without myelosuppression . The risk increased with higher dose density of rhG-CSF compared to rhG-CSF versus no users (p for trend< 0.001).We identified 307 NSCLC patients who received postoperative systemic chemotherapy (These analyses indicate that rhG-CSF use is related to DOM following postoperative chemotherapy in NSCLC. Non-small-cell lung cancer (NSCLC) is one of the most common cancers and the leading cause of cancer death worldwide . SurgeryRecombinant human granulocyte colony-stimulating factor (rhG-CSF) is commonly used for cancer patients following myelosuppressive cytotoxic chemotherapy, and it decreases the risk of potentially fatal infections and hospitalisation associated with febrile neutropenia (FN) and ensures that the dose intensity of chemotherapy improves the overall survival (OS) . HoweverHowever, both of these effects were eliminated in the presence of G-CSF inhibitors. However, clinical data rarely indicate that rhG-CSF use in cancer patients is related to a high DOM risk. The impact of rhG-CSF treatment in postoperative chemotherapy patients with myelosuppression on cancer metastasis, especially in NSCLC, is not clear. Therefore, we examined the pro-metastatic character of rhG-CSF in a retrospective cohort of NSCLC patients who received chemotherapy after surgery. These data provide experimental evidence to reduce the occurrence of cancer metastasis by pursuing G-CSF.The Ethics Committee of the First Affiliated Hospital of Nanchang University, China approved this retrospective cohort study, which abided by the rules of the Declaration of Helsinki. We reviewed the digital medical records of 307 consecutive NSCLC patients for pathological stage IB-IIIB (AJCC system 8th edition) who underwent complete surgical resection via lobectomy or greater and systematic lymph node dissection (lymph node dissection was performed depending on tumour location) and received postoperative system chemotherapy at The First Affiliated Hospital of Nanchang University between January 1, 2012 and December 31, 2017. Complete resection was defined as the macroscopic and microscopic absence of residual cancer (R0). Patients were excluded when they had been diagnosed with cancer other than NSCLC before surgery or when they underwent limited resection (segmentectomy or wedge resection), incomplete resection, neoadjuvant chemotherapy/radiotherapy, metastasis occurring before surgery or before receiving the first postoperative chemotherapy, or a follow-up time of fewer than 30 days after surgery. All patient records were anonymised before analyses. Follow-up duration was counted from the day of surgery to the day of DOM diagnosis, last follow-up, or loss to follow-up. The last follow-up date was December 31, 2020.One research group blinded to the outcome status collected clinical records data of the potential cases, and another research group blinded to the exposure status collected the outcome and follow-up of the case. The first and the corresponding authors were responsible for all data analyses and interpretation.Adjuvant chemotherapy was administered after surgery for NSCLC. Adjuvant chemotherapy dose intensity was applied according to the NCCN guidelines. During postoperative treatment, chemotherapy quickly induces myelosuppression, and rhG-CSFs were given to relieve or prevent myelosuppression. rhG-CSFs were administered 24 h after adjuvant chemotherapy. Patients were divided into two groups based on the use or non-use of rhG-CSF. All rhG-CSF users had at least one rhG-CSF prescription. The effects of the dose of rhG-CSF exposure on the postoperative metastasis risk of NSCLC were also analysed. Four subgroups  were divided based on the cumulative total rhG-CSF dose. Because of the impact of dose use over time, dose density of rhG-CSF on metastasis was used to evaluate the effects of density of rhG-CSF treatment on the outcome variable metastasis. Length of rhG-CSF exposure time (in days) was measured from the first prescription to the last to calculate the dose density of rhG-CSF treatment.We reviewed medical records to extract data on demographics, clinicopathological characteristics, and treatment histories: age, weight, sex, smoking habit (never smoked or smoked), anamnesis, histological type, grade, and pathological stage. During the postoperative chemotherapy and follow-up period, regimen and number of chemotherapies, the severity of myelosuppression (Common Terminology Criteria for Adverse Events (CTCAE), version 4.03), fever , and antibiotics were collected.All selected NSCLC patients achieved disease-free status after surgery. We reviewed clinical notes and checked all routine imaging records of enrolled patients after adjuvant chemotherapy, including ultrasound (US), thoracic radiography, computed tomography (CT), magnetic resonance imaging (MRI), and single-photon emission computed tomography (SPECT) at the time of diagnosis or during follow-ups, to assess their metastatic status. Metastasis-free survival (MFS) was defined as the duration from the day of chest surgery to investigator-assessed radiographic organ or node metastasis. MFS was censored on the last follow-up date if patients were alive and without any evidence of metastasis. Patient status of metastasis 1, 2, and 3 years after surgery was also measured. Patients without documented clinical or radiographic disease date of metastasis were censored at the last follow-up.χ2 tests for categorical data were used to analyse each covariate’s required data distribution between the rhG-CSF and No rhG-CSF groups. We used the Kaplan-Meier method to calculate overall MFS rates, and the differences in survival for univariate comparisons were calculated using the log-rank test. Forest plots were used to describe the underlying effect modification by each covariate. Interaction analysis was performed by calculating the respective categorical variable product terms individually in the model. Whether rhG-CSF and other covariates had an independent effect on NSCLC patients’ metastasis following postoperative chemotherapy separately was evaluated using univariate logistic regression and multivariate logistic regression models. The level of significance was established at p = 0.05. The statistical software packages R  and EmpowerStats  were used to perform all statistical analyses.Continuous variables are shown as the means ± SDs, and categorical variables are shown as frequencies (percentages). The t-test  or Kruskal-Wallis rank-sum test  for continuous variables and p < 0.001). Most patients with myelosuppression after chemotherapy tend to use rhG-CSF in clinical practice. A total of 107 patients received rhG-CSF as primary prophylaxis, and 139 patients received rhG-CSF as secondary prophylaxis. Essential characteristics were comparable between the No rhG-CSF and rhG-CSF groups. Patients in the two groups were similar in age, weight, sex, smoking, histology, differentiation, pathological stage, chemotherapy, fever, antibiotics, and complications. We excluded patients with a poor general physical condition before surgery and chemotherapy treatment. Therefore, the Eastern Cooperative Oncology Group performance status (ECOG PS) level of all postoperative NSCLC patients was 0–1, and there was no noticeable difference between the rhG-CSF and No rhG-CSF groups.Of the 307 postoperative NSCLC patients included in the study, 246 80.1%) patients who received rhG-CSF treatment during chemotherapy were used as the study group (rhG-CSF group), and 61 (19.9%) patients who did not receive rhG-CSF were included in the control group (No rhG-CSF group). The clinical characteristics and baseline parameters of the No rhG-CSF and rhG-CSF groups are presented in Table % patientp < 0.05). RhG-CSF treatment significantly correlated with metastasis in univariate Cox regression analysis . We found that the different doses and density gradients of rhG-CSF likely played an essential role in distant organ metastases , which is consistent with previous studies [A total of 136 of the 307 postoperative NSCLC patients (44.30%) had distant organ metastases following the chest surgery over the median follow-up period of 33.63 months. A total of 119 (48.3%) patients were in the rhG-CSF group, and 17 27.9%) patients were in the No rhG-CSF group, which suggests that the DOM rate was significantly higher in the study group than the control group (7.9% pati studies .Table 2Ep = 0.007), 2 years  and 3 years  of postoperative follow-up  and 1.33 times in the adjusted II model . We performed similar analyses for postoperative follow-up at 1, 2, and 3 years, and rhG-CSF treatment was associated with an increased risk of metastasis in the postoperative NSCLC patients  except for myelosuppression . Among patients who presented without myelosuppression after chemotherapy, rhG-CSF treatment resulted in a significantly high risk of metastasis . In contrast, rhG-CSF treatment reduced the risk of metastasis in patients who presented myelosuppression . Compared to the patients who had myelosuppression after chemotherapy, patients who presented without myelosuppression had a higher risk correlation of rhG-CSF use with metastasis , and at postoperative follow-up 1 year , 2 years  and 3 years  , in the non-adjusted and adjusted models (Table p for trend> 0.05). When the factor of time was considered, the dose density of rhG-CSF, which is dose of rhG-CSF used in the length of rhG-CSF exposure time, a positive relationship between the dose density of rhG-CSF treatment and risk of metastasis was observed for all models. Statistically significant results of trend tests were obtained in the total sample (p for trend< 0.001) (Table p for trend< 0.05)  Table . The ana5) Table .Distant organ metastasis is always associated with poor prognosis and shorter survival in NSCLC patients . The speThe influence of tumour staging on patient metastasis has been generally recognized . For patCSF was first discovered by Don Metcalf et al. . Mice trn = 125) group during cycle 1 of chemotherapy in NSCLC patients [n = 51) of patients with NSCLC who received chemotherapy without rhG-CSF treatment exhibited FN. There was a relatively low occurrence of FN in NSCLC patients even without rhG-CSF. It was evident that a large proportion of patients who received mild chemotherapy could also safely survive the risk period after chemotherapy in the absence of rhG-CSF. Guidelines pay close attention to whether the use of rhG-CSF reduces the occurrence of FN and its complications, the risk of infection and death, the duration of severe neutropenia, the duration of antibiotic use, the length of hospital stay, bone pain, and other indicators. However, it may ignore the dark side of rhG-CSF supplements, such as promoting cancer metastasis. The risk of metastasis with excessive use of rhG-CSF was increased remarkably in most of these patients, which led us to consider whether certain precautions, such as avoiding some infection factors and increasing nutrition after chemotherapy, could take the place of rhG-CSF use to lower the risk. Although patients with breast cancer benefitted from rhG-CSF use in extending DFS and OS, any damage from rhG-CSF use could be whitewashed by the profit supported by dose-intensity chemotherapy regimens [Current guidelines for rhG-CSF of National Comprehensive Cancer Network (NCCN) treatment recommend prophylaxis with rhG-CSF when the risk of FN is approximately 20% or higher or the risk of FN ranges from 10 to 20% combined with one or more risk factors, such as age and recent history of surgery . The purpatients  found thregimens . Howeverregimens .A developing number of studies –30 identNeutrophil extracellular traps (NETs) are a network structure consisting of modified chromatin structures and decorated with given cytoplasmic and granular proteins, which likely also played a crucial part in the enhanced risk of DOM with rhG-CSF use . NETs, fThe analyses of this research offer proof of the effect of rhG-CSF on the development of DOM in NSCLC patients with postoperative chemotherapy. Therefore, a physician in clinical practice should administer rhG-CSF to NSCLC patients based on chemotherapy regimens and the patient’s own risk factors using rhG-CSF once myelosuppression exists. The recommended guidelines for rhG-CSF use should formulate more comprehensive standards. Populations that are suitable to use rhG-CSF are also worthy of further exploration.Our study has the following limitations. First, due to loss to follow-up, we did not dissect the OS of participants in this study, which should be shown in subsequent research. Second, our study only included NSCLC patients who received chemotherapy postoperatively. Future studies should consider more tumour types. DOM was primarily judged using imaging examination and not biopsy, which likely caused false-positive results, despite the diagnosis estimated by two radiologists. Pathological discoveries may be used to supply evidence microenvironment variations in the metastatic organs. Lastly, this study was a retrospective study, and some selection biases may be present. Prospective research is also needed to verify these conclusion.Among NSCLS patients following postoperative chemotherapy, we found that rhG-CSF use was related to a high risk of metastasis, and this risk may increase significantly with the increase in dose density of rhG-CSF. The study suggests that patients without myelosuppression have more disadvantages from rhG-CSF. Our findings, if affirmed, lead to far-reaching clinical and public health influence because of the highest mortality rate of NSCLC globally. This result suggests that identifying patients with or without myelosuppression after chemotherapy could help guide the rational drug use of rhG-CSF and avoid its risks in clinical use.


Quantification of gene expression such as RNA-Seq is a popular approach to study various biological phenomena. Despite the development of RNA-Seq library preparation methods and sequencing platforms in the last decade, RNA extraction remains the most laborious and costly step in RNA-Seq of tissue samples of various organisms. Thus, it is still difficult to examine gene expression in thousands of samples.Arabidopsis thaliana.Here, we developed Direct-RT buffer in which homogenization of tissue samples and direct-lysate reverse transcription can be conducted without RNA purification. The DTT concentration in Direct-RT buffer prevented RNA degradation but not RT in the lysates of several plant tissues, yeast, and zebrafish larvae. Direct reverse transcription on these lysates in Direct-RT buffer produced comparable amounts of cDNA to those synthesized from purified RNA. To maximize the advantage of the Direct-RT buffer, we integrated Direct-RT and targeted RNA-Seq to develop a cost-effective, high-throughput quantification method for the expressions of hundreds of genes: DeLTa-Seq (Direct-Lysate reverse transcription and Targeted RNA-Seq). The DeLTa-Seq method could drastically improve the efficiency and accuracy of gene expression analysis. DeLTa-Seq analysis of 1056 samples revealed the temperature-dependent effects of jasmonic acid and salicylic acid in The DeLTa-Seq method can realize large-scale studies using thousands of animal, plant, and microorganism samples, such as chemical screening, field experiments, and studies focusing on individual variability. In addition, Direct-RT is also beneficial for gene expression analysis in small tissues from which it is difficult to purify enough RNA for the experiments.The online version contains supplementary material available at 10.1186/s13007-022-00930-x. Quantification of gene expression is a popular approach to study various biological phenomena –8. RecenThere is a trade-off between comprehensive gene quantification and the required amount of sequence data . For exaJasmonic acid (JA) and salicylic acid (SA) are majorly involved in defense and stress responses in plants . AlthougHere, we showed that a high concentration of DTT can inhibit intrinsic RNase in plant and animal crude tissue lysates, but does not inhibit reverse transcriptase. Using Direct-RT buffer containing DTT, we succeeded in direct-lysate RT from plant, yeast, and animal samples and RNA-Seq using the lysate. Finally, we combined direct-lysate RT and targeted RNA-Seq into a new method called DeLTa-Seq, which can drastically reduce the cost and labor of RNA-Seq, whilst maintaining accuracy in quantification. DeLTa-Seq will pave the way for large-scale research.Arabidopsis thaliana seedlings were homogenized in these reagents, followed by a 1 h incubation at 22 °C. RNA was purified from the lysates and checked by electrophoresis. Frozen rice (Oryza sativa) leaves were also homogenized with zirconia beads and dissolved in the reagents. Incubation and RNA purification were conducted as described above. In both cases, no peaks of 18S or 28S rRNA were detected, but short fragments of degraded RNAs were observed   . Here, we focused on the 100 marker genes used in a previous study  [A maximum of 9 seedlings of  mM DTT]  or CellAO. sativa was frozen in liquid nitrogen and stored at -80 °C until homogenization. Each frozen sample was homogenized with zirconia beads YTZ-4 using a TissueLyser II. Then, 400 µL of the same buffers used for A. thaliana seedlings were added, and immediately, the samples were mixed well by vortexing. According to the SuperPrep manufacturer’s instructions, 76 µL Stop Solution and 4 µL RNase inhibitor were added following incubation for 5 min at 22 °C.Each youngest fully expanded leaf of T. aestivum was frozen in liquid nitrogen and stored at − 80 °C until homogenization. Each frozen sample was homogenized with zirconia beads YTZ-4 using a TissueLyser II. Then, 400 µL of Direct-RT buffer was added, and immediately, the samples were mixed well by vortexing.Each coleoptile with the first leaf of S. cerevisiae in the exponential phase was homogenized with zirconia beads YTZ-1 and MS-100R  in 350 µL of 90 mM Tris–HCl (pH 7.6) containing 100 mM DTT.D. rerio larvae were homogenized in 50 µL of the same buffers used for A. thaliana seedlings with a Biomasher II . According to the SuperPrep manufacturer’s instructions, 9.5 µL Stop Solution and 0.5 µL RNase inhibitor were added following incubation for 5 min at 22 °C.Three The concentration of RNA in each lysate was measured using the Quant-iT RNA Assay Kit  and the Infinite M1000 PRO plate reader .The lysate was incubated for 1 h at 22 °C. Then, a 2.5 × volume of AMpure XP beads  was added to the lysate and the mixture was purified according to the manufacturer’s instructions. The RNA was eluted with an equal volume of RNase-free water to the lysate. One microliter of the purified RNA solution was electrophoresed using a Bioanalyzer 2100 with Agilent RNA nano or pico kit  to check for quality.RT reactions were conducted with 1 μL of 2 μM RT primer (CAGAAGACGGCATACGAGATGCGTCTACGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCNNNNNNTTTTTTTTTTTTTTTTTTV), 0.4 μL of 25 mM dNTP , 4.0 μL of 5 × SSIV Buffer (Thermo Fisher Scientific), 0.1 μL of SuperScript IV reverse transcriptase , lysate, and nuclease-free water to make a volume of 20 μL. In order to suppress production of cDNA from rRNA , reverseA. thaliana and O. sativa) or 140 ng (T. aestivum) of total RNA in the lysate. Then, the A. thaliana and O. sativa reaction mix was diluted 20 times with nuclease-free water. The T. aestivum reaction mix was used for subsequent experiments without dilution. The cDNA amount of AT3G18780, Os03g0836000, or HP620998.1 was measured by qPCR analysis. The composition of the qPCR mixture is described below: 2 µL diluted cDNA solution, gene-specific primer  [For Fig. 20998.1) , P7 primhttps://sites.google.com/view/lasy-seq/). RT reactions were conducted with 1 μL of 2 μM RT primer .We designed gene-specific primers for  Primer3 , with thFor Fig. https://sites.google.com/view/delta-seq/).An update of the DeLTa-Seq protocol is available at our website ’ section.For Fig. A. thaliana seedlings 7 days after sowing. RT reactions, pooling, and purification were conducted as described in the ‘Non-targeted RNA-Seq for Fig. For Fig. For non-targeted RNA-Seq, 20 μL of pooled cDNA was used. The following steps were conducted as described in the ‘Non-targeted RNA-Seq for Fig. For targeted RNA-Seq, the following experiments were conducted as described in the ‘Direct-lysate targeted RNA-Seq (DeLTa-Seq) for Fig. Equal amounts of non-targeted and targeted RNA-Seq libraries were mixed based on qPCR-based quantification with Kapa Library Quantification Kit (KAPA BIOSYSTEMS). Sequencing of 150-bp paired-ends was performed using a HiSeq.https://github.com/lh3/seqtk) to normalize total reads. The seed parameter ‘-s’ was specified differently in each subsampling.Subsampling of RNA-Seq reads was conducted with ‘seqtk’   using thanscript , 57, rRNnscripts , the S. ensemble , or D. rensemble  with RSEensemble , using Bensemble  with defWhen using UMI in the analysis, all obtained paired reads were processed using dynacomkobe/biodocker_rnaseq_pipeline:ver.0.2.0 with the default parameters, with quality trimming using Trimmomatic, and quantification with RSEM and Bowtie.https://github.com/naganolab/DeLTa-Seq.git. Briefly, read counts were normalized by calculating read per million (RPM) using the total read count of all transcripts as denominators. Differentially quantified genes (FDR = 0.05) were detected as described by Sun et al. [The expected counts of each gene in the RSEM outputs were used with R (version 3.4.2)  in the fn et al. .A. thaliana was sowed in 100 µL of 0.5 × Murashige and Skoog medium in each well of a 96-well plate and grown at 20 °C under continuous light for 7 days after vernalization treatment. Each of the 11 96-well plates was incubated at a different temperature  for 18 h. Then, each 96-well plate was divided into three parts of 32 wells each, and each part was filled with 50% EtOH, or 16.5 mM of JA or SA stock solution, forming a final concentration of 1%, 0.33 mM, and 0.33 mM, respectively; the plates were incubated for an additional 6 h. After incubation, medium was immediately discarded using 96 channel pipette ), and A. thaliana seedlings were homogenized twice in 90 s with 200 µL of 90 mM Tris–HCl (pH 7.6) containing 100 mM DTT using TissueLyser II . DeLTa-seq libraries were created as described above with minor modifications. RT products were obtained from 5 µL of lysate as the template. Pooled RT products were divided into two aliquots and each sample was allowed to react with either of the two gene specific primer mixes, Tradict targeting genes  .Tukey's test was conducted with multcomp R package (version 1.4-19) with the default parametors .Additional file 1: Data 1 Expressions of gene set 1 in EtOH-, jasmonic-acid-, and salicylic-acid-treated A. thaliana seedlings. Magenta lines represent trend curves of each target log2 RPM + 1 with the ambient temperatures.Additional file 2: Data 2 Expressions of gene set 2 in EtOH-, jasmonic-acid-, and salicylic-acid-treated A. thaliana seedlings. Magenta lines represent trend curves of each target log2 RPM + 1 with the ambient temperatures.Additional file 3:Table S1 List of the RT primers harboring 1st indexes. Table S2 List of the gene-specific primers for gene set 1 (Tradict target gene set). Table S3. List of the PCR primers harboring 2nd indexes. Table S4. List of the gene-specific primers for gene set 2 (JA- and SA-related genes).Additional file 4: Fig. S1. Performance of previously reported buffers for mammalian cultured cells in direct-lysate reverse transcription of A. thaliana, O. sativa, and D. rerio. Fig. S2. Comparison of RNA-Seq results of purified RNA and lysate of S. cerevisiae and D. rerio. Fig. S3. UMI conversion efficiencies of 96 technical replicates of non-targeted and targeted RNA-Seq.


N (average = 0.87), (Pr/Tb)N (average = 0.80), and (Tb/Yb)N (average = 1.09). Moreover, REY patterns of deposits of the Jiwozhai section differ markedly from those of modern seawater. The described geochemical aspects of the Jiwozhai section and the positive correlation of REY and Th contents displayed by the section point to a terrestrial siliciclastic contribution contemporaneous with reef-building. In contrast, REY patterns of the Upper Buzhai reef section samples are similar to those of modern seawater characterized by light rare earth element (LREE) depletion (average (Pr/Yb)N = 0.76), negative Ce anomalies (average Ce/Ce* = 0.88), and average super-chondritic Y/Ho ratios (average = 45.4)). Slightly positive Eu anomalies  of the Upper Buzhai reef section samples are attributed to the negligible effect of hydrothermal fluids. Middle REE (MREE) enrichment (average (Tb/Yb)N = 1.48) of Buzhai section carbonate samples and positive correlation of REY and Th suggest a riverine input. Combined with siliciclastic mineralogy, oxygen isotope values, and reef-building biota morphology of the studied two sections, we suggest that terrestrial nutrients delivered by rivers far outweighed upwelling as a source of nutrients supplied to the Givetian reef ecosystem of South China. Coral and stromatoporoid in tropic oceans thrived in turbid water containing abundant terrestrial sediment and the nutrient-laden water helped expand reef-builder habitats during the Givetian time.The Givetian Age witnessed the greatest expansion of stromatoporoid-coral reefs from low to higher latitudes of the Phanerozoic. Multi-proxy seawater surface temperature reconstruction suggests the establishment of a super-greenhouse climate as a major reason for reef expansion, yet many questions remain. This article presents the results of a rare earth element and yttrium  geochemical study as well as mineralogy and oxygen isotope values of two well-documented Middle Givetian reefal carbonate sections (Jiwozhai and Buzhai) of the Jiwozhai Formation of South China. The nearshore Jiwozhai patch reef succession displays greater biodiversity and more abundant coral than the marginal platform Upper Buzhai reef. Reefal and micritic carbonates of the Jiwozhai section are characterized by shale-like post-Archean Australian Shale (PAAS)-normalized REY patterns, by very weak negative Ce anomaly values , slightly elevated Y/Ho values , and near-unity values of (Pr/Yb) The Devonian Age experienced the greatest expansion of reefs of the Phanerozoic Eon, especially stromatoporoid-coral reefs during Givetian time . Reef deThe great expansion of metazoan reefs during the Devonian time was initially attributed to the establishment of super-greenhouse climatic conditions . HoweverModern reef-building corals are known to flourish in oligotrophic waters. Moreover, the combination of increasing nutrient levels and rising seawater temperature is known to be responsible for the decline of coral ecology in recent years . Terrest2+, is incorporated into inorganic and biogenic carbonate minerals  and seawater make REY concentration and distribution in carbonate rocks resistant to the effects of diagenesis and dolomitization (Particle reactive rare earth elements and yttrium (REY), nitrate, phosphate, and silica abundances of modern seawater display similar vertical distribution profiles . Furtherminerals . The lartization . Consequtization . The factization  validatetization .e.g., The Middle Devonian Givetian reef tract of the Dianqiangui Basin of South China extends from slope to near-shore environments for more than 1,700 km . The sedThe Dianqiangui Basin of the South China Block was located near the equator in the eastern part of the Palaeo-Tethys Ocean during the Givetian Age ; Fig. 1AStringocephalus burtini-Undispirifer undiderus and rugose coral assemblage Endophyllum guizhouense-Sunophyllum elegantum confirm a Givetian age of the Jiwozhai Formation  in the nearshore and abundant fringing reefs  on the platform margin comprise the dominant reef types of the Jiwozhai Formation. The enhanced biodiversity displayed by the Jiwozhai reef has been thoroughly described by Gray to dark-gray medium- to thick-bedded interlayered micritic limestone and marl comprise the primary lithology of the Jiwozhai Formation with scattered reef limestone deposits in the lower part of the unit. Variations in thickness and lithology of the Jiwozhai Formation between the studied sections likely reflect differences in paleogeographic locations of deposition. Patch reefs  is located in Dongyao village along a country road . The reeThe Jiwozhai patch reef section  is located in Dahekou Geopark in Dushan County and comprises three reef and bioclastic wackestone and packstone intervals . The JiwWe measured the Upper Buzhai reef and Jiwozhai patch reef sections of the Jiwozhai Formation and collected 34 fresh rock samples for mineralogy, lithology, trace element and oxygen isotopic analyses. Veining and weathered carbonate samples were avoided from field outcrops. One part of each specimen was used for the preparation of thin sections for petrographic observation. The other part was micro-drilled from stromatoporoids  or micrite (wackestone and packstone) with a tungsten carbide bit to obtain powder for mineralogy, trace element, and oxygen isotope analyses, and again the veining and weathered parts were avoided. Samples are numbered in order from bottom to top as BZ-1 to BZ-20 for the Buzhai section and JWZ-1 to JWZ-14 for the Jiwozhai section . Powders3-HF (1:4) mixture. The dissolution was maintained in an oven at 185 °C for 3 days. The solutions were then dried down to evaporate HF. The sample residues were re-dissolved with double distilled concentrated HNO3 followed by 1:1 HNO3 and dried again. Then, the samples were dissolved in a final 3ml 2N HNO3 stock solution. Finally, the sample solution was diluted to 4,000 times with 2 percent HNO3 and added with 6 ppb Rh, In, Re, and Bi internal spikes. USGS standard W-2a was used as reference standard and crossed checked with BHVO-2 and other reference materials. Instrument drift mass bios were corrected with internal spikes and external monitors. The ICP-MS procedure for trace element analysis follows the protocol of Trace element analyses were performed at Guizhou Tongwei Analytical Technology Co., Ltd. on a Thermo Fisher iCAP RQ ICP-MS equipped with a Cetac ASX-560 AutoSampler. Approximately 50 mg of each rock powder was dissolved in a Teflon bomb with a double-distilled concentrated HNOθ range of 5°–110° with a step size of 0.05°. The reference intensity ratio method is applied to estimate the relative contents of calcite and dolomite in the samples.Mineralogical analyses were performed by X’Pert Powder XRD analyzer (operating at 40 kV and 40 mA) using Cu Kα radiation in Guizhou Key Laboratory of Comprehensive Utilization of Non-metallic Mineral Resources of Guizhou University. Powder XRD patterns were collected in the 22 at 70 °C for 1 h. Finally, the released CO2 was sent by helium gas from Gasbench to Delta V Advantage for isotope analysis. The isotope data are reported as δ18O relative to VPDB standards. Reference material NBS18 was used for quality control. The analytical precision is ±0.20 ‰.Carbonate oxygen isotopic values were performed in the Key Laboratory of Karst Geo-resources and Environment of Guizhou University with Thermo Fisher Delta V Advantage stable isotope mass spectrometry. The brief analytical procedure is as follows. About 100 µg of carbonate powder was introduced into the reaction bottle and then sealed. The reaction bottle was blown with helium for 330 s. Phosphoric acid was then mixed with the carbonate powder to release CON, (Pr/Tb)N, and (Tb/Yb)N were calculated to define the degree of fractionation between light REE (LREE) and heavy REE (HREE), light REE and middle REE (MREE), and middle REE and heavy REE, respectively. Y/Ho ratios were calculated without normalization. Some rare earth element anomalies were calculated on a linear scale as the following N, and (Tb/Yb)N ratios of analyzed carbonate samples are 0.87, 0.80, and 1.09, respectively, are similar to those of the siliciclastic rock sample . Carbonate samples are characterized by Y/Ho ratios of 28.9 to 39.1 (average = 34.1), slightly greater than the 29.9 Y/Ho value of the siliciclastic rock sample. However, one reefal limestone sample with high carbonate contents (JWZ-6) captured oceanic signals characterized by the lowest (Pr/Yb)N ratio (0.60) and the highest Y/Ho ratio (39). Mn/Sr values of analyzed limestone samples range from 0.23 to 0.86 (average = 0.53), and one dolostone sample (JWZ-14) has the highest value of 3.76.Carbonates of the Jiwozhai reef section have total REY (TREY) concentrations ranging from 2.31 to 82.38 ppm (average = 32.65 ppm). The average concentration of REY of analyzed reefal limestone samples is 37.28 ppm, greater than the 16.22 ppm average of analyzed bioclastic packstone samples. One quartz sandstone sample (JWZ-1) contains the greatest TREY concentration of 155.2 ppm whereas the muddy dolostone sample (JWZ-2) is characterized by a slightly greater TREY concentration of 82.38 ppm. All analyzed samples regardless of lithology display flat  PAAS-normalized REY patterns defined by minor negative Ce anomalies and weak fractionation among LREE, MREE, and HREE . Ce anomr2 = 0.964, N = 13; r2 = 0.961, n = 14). These results are consistent with the mineralogy of the samples with the higher content of quartz and illite corresponding to the higher level of TREY. Both the reefal limestone and packstone samples have varied content of detrital minerals (quartz and illite) with quartz being predominant in the Jiwozhai section  of the section have the lowest δ18Ocarb values of −18.72 ‰ and −19.44 ‰, respectively.The N = 0.76), slightly negative Ce anomalies (average Ce/Ce* = 0.88), and super-chondrite Y/Ho ratios (average = 45.4). However, unlike the modern seawater REY pattern, 13 of 15 analyzed carbonate samples of the Upper Buzhai reef section display positive Eu anomalies  and MREE enrichment ((Tb/Yb)N = 1.26–2.42; average = 1.48).PAAS-normalized REY patterns for the Upper Buzhai reef section sample suite are presented in Five analyzed clastic sedimentary samples are characterized by Th and Zr concentrations  greater than those of carbonate samples . Among carbonate samples, reefal limestone samples are characterized by less Zr, Th, REY, and greater Y/Ho than are bioclastic packstone samples. Like the Jiwozhai reef section, samples of the Buzhai reef section display positive correlations of Th and Zr and TREY and Th  and 8B. δ18Ocarb trend to the Jiwozhai section with the higher values (−4.25 ‰∼−7.10 ‰) in the middle and lower part and lower values (−7.15 ‰ to −16.12 ‰) in the upper part of the section.The Upper Buzhai reef section presents a similar e.g., Th and Zr) and TREY concentrations and siliciclastic minerals, elevated Y/Ho ratios, and δ18Ocarb values relative to the Jiwozhai section. Carbonate deposits of the Buzhai section display seawater-like PAAS-normalized REY patterns whereas carbonate samples of the Jiwozhai section are characterized by shale-type PAAS-normalized REY patterns. Reefal limestone samples of the Upper Buzhai and Jiwozhai sections are variably dissimilar to the PAAS-normalized modern seawater REY pattern.The Upper Buzhai section carbonate sample suite is characterized by low immobile elements N for carbonate samples with Th < 1 ppm  and elevated (Tb/Yb)N ratios ranging from 1.26 to 1.72 (average = 1.47). Low Y/Ho ratios and MREE enrichment displayed by samples characterized by low Th content are attributed to the mixing of riverine water with seawater. The addition proportion of riverine water is below 1% as estimated by cross-plot of (Y/Ho) vs. (Sm/Nd)N for carbonate samples with Th < 1 ppm . Three bioclastic packstone samples present slightly lower Y/Ho values . Although terrestrial contamination cannot be ruled out, a Y/Ho ratio of 40 and Th content of 0.024 ppm of one reefal limestone sample (BZ-12) suggests some degree of freshwater contamination. Moreover, four samples  contain < 0.1 ppm Th, low (Pr/Tb) < 1 ppm , which iThe greater content of REY in shale than carbonate necessitates consideration of the possible role of terrestrial clastic contamination of the studied Jiwozhai Formation carbonate samples. High field strength elements such as Th and Zr are rarely susceptible to chemical weathering and diagenesis . This suElevated Th contents (average = 1.97 ppm) and shale-like REY patterns of Jiwozhai section samples compared to those of the Buzhai section suggest that the depositional site of the Jiwozhai section experienced a greater terrestrial input than did the depositional site of the Upper Buzhai section, an argument supported by the paleo-geographic location and fossil assemblages of the Jiwozhai and the Upper Buzhai sections. That is, the Jiwozhai patch reef was located much closer to the Givetian shoreline than was the Buzhai platform margin reef . As descModern oxic seawater is characterized by significantly negative Ce anomalies in PAAS-normalized REY patterns that reflect the lower solubility of tetravalent Ce than its neighboring La and Pr in seawater . Howevern = 15). Positive Eu anomalies (denoted as Eu/Eu*) are commonly cited as evidence of hydrothermal input -laden modern deep seawater is known to be an important source of nutrients for some reef ecosystems  in the reefal limestone and a greater biodiversity than the Buzhai reef  REE geochemistry of carbonate samples of two Devonian reef sections of South China suggest that shale-type PAAS normalized REY patterns of nearshore water in which the Givetian Jiwozhai Formation accumulated differed significantly different from marine water masses on the marginal platform of the Dianqiangui Basin. Together with a considerable amount of siliciclastic minerals and the elevated immobile elements contents in carbonate samples from the Jiwozhai near-shore reef section point to the terrestrial runoff effect during the reef development.(2) The nutrient source that sustained the reef ecosystem that encompassed the studied Jiwozhai and Buzhai sections appear to have been dominated by terrestrial runoff. Upwelling of nutrient-rich deep water played a minimal role in maintaining the Givetian reef ecosystem. Enhanced terrestrial sediment input associated with Givetian Jiwozhai coral-stromatoporoid reef development demonstrates that the coral ecosystem thrived in turbid waters.(3) Results of the present study suggest that tropic coral and stromatoporoid adaptation to turbid water played an important role in the Middle Devonian (Givetian) expansion of coral-stromatoporoid reef complexes during the global green-house climate.10.7717/peerj.13663/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.13663/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.13663/supp-3Supplemental Information 3Click here for additional data file.