A Common Rejection Module (CRM) consisting of 11 genes expressed in allograft biopsies was previously reported to serve as a biomarker for acute rejection (AR), correlate with the extent of graft injury, and predict future allograft damage. We investigated the use of this gene panel on the urine cell pellet of kidney transplant patients. Urinary cell sediments collected from patients with biopsy-confirmed acute rejection, borderline AR (bAR), BK virus nephropathy (BKVN), and stable kidney grafts with normal protocol biopsies (STA) were analyzed for expression of these 11 genes using quantitative polymerase chain reaction (qPCR). We assessed these 11 CRM genes for their abundance, autocorrelation, and individual expression levels. Expression of 10/11 genes were elevated in AR when compared to STA. Psmb9 and Cxcl10could classify AR versus STA as accurately as the 11-gene model . A uCRM score, based on the geometric mean of the expression levels, could distinguish AR from STA with high accuracy (AUC = 0.9886) and correlated specifically with histologic measures of tubulitis and interstitial inflammation rather than tubular atrophy, glomerulosclerosis, intimal proliferation, tubular vacuolization or acute glomerulitis. This urine gene expression-based score may enable the non-invasive and quantitative monitoring of AR. Kidney transplantation (KTx) is the preferred modality for treatment of end-stage renal disease (ESRD) by any cause . While tInterrogation of proteomic, RNA, and microRNA biomarkers in the urine of KTx patients has been demonstrated by our groups and others –17 to beIn this study, we apply the knowledge gained from harnessing a Common Rejection Module (CRM) of 11 genes , originaIn this study, we assess the CRM gene set for use on urine samples from KTx patients, paired with allograft biopsies with known histology, for the non-invasive diagnosis of AR and other immune mediated injuries. Further, we develop a urine CRM (uCRM) score that accurately discriminates between STA and AR patients. We evaluate the clinical potential of this score in detection of bAR by correlating this score with histology scores of tubulitis and interstitial inflammation.Biobanked urine samples (n = 1760) from KTx recipients enrolled at Stanford University in between 2000 and 2011 and UCSF Medical Center enrolled in between 2014 and 2016 were included in the study. The study was approved by the Institutional Review Board and Ethics Committee of the University of California San Francisco, CA. All patients provided written informed consent to participate in the research, in full adherence to the Declaration of Helsinki. The clinical and research activities being reported are consistent with the Principles of the Declaration of Istanbul as outlined in the Declaration of Istanbul on Organ Trafficking and Transplant Tourism. For urine samples utilized in establishing the uCRM threshold for AR, 178 urine samples were identified with paired kidney allograft biopsies with clearly defined pathologies of either Banff graded AR , 31 or n150 unique urine samples were assessed for the uCRM assay in 150 unique kidney transplant patients. Baseline clinical and demographic variables by AR, bAR, BKVN, or STA phenotype are shown in All kidney biopsies were blindly and centrally analyzed at each institution by staff pathologists (RS and ZL) and were graded by the Banff classification , 33 for ™ Master Mix . qPCR was performed on cDNA synthesized from 50 ng of total RNA, then 1.56 ng of cDNA was processed through specific target amplification and sample dilution with the pooled Taqman assays for the 11 uCRM genes in multiplex, with Taqman PreAmp Master mix (ABI) to 5 μl final volume, for 18 cycles in a thermal cycler, then diluted with DNA Suspension Buffer . Microfluidic qPCR was performed on the 96.96 dynamic array  using 2.25 μl of the diluted sample from specific target amplification, along with Taqman Assays (ABI) for each gene transcript  was collected from kidney transplant patients before biopsy procedure and prior to any treatment intensification for AR. RNA was extracted from urinary cell sediment following our previously reported protocol . In brieanscript , Taqman All qPCR assays were run in duplicates. All the data are presented as mean ± SEM. For comparisons of the CRM genes per phenotype, a mixed-effects model with the Geisser-Greenhouse correction was used, with multiple comparisons corrections performed using the two-stage linear-step procedure of Benjamini, Krieger, and Yekutieli. Pearson correlation and hierarchical clustering were performed in Morpheus (Broad Institute). For machine learning prediction models, the data was split into a training set (80%) and testing set (20%). A decision tree classification model, validated on the testing set, was used to determine the most accurate uCRM score cut-off. Variable Selection Using Random Forests (VSURF) was used to classify AR vs STA as well as to evaluate and rank individual gene importance. The Random Forest variable importance output is defined as the mean percentage decrease in accuracy of the model if the variable (gene) were excluded (randomly permuted) from the model. Unsupervised clustering to visualize phenotype separation was done using the t-distributed stochastic neighbor embedding algorithm (t-SNE) in Mathematica 11.3 . Network analysis of the CRM genes was performed using GeneMANIA . StatistThe study was approved by the ethics committees of both Stanford University Medical School and UCSF Medical Center. All adult patients and parents/guardians of non-adult patients provided written informed consent to participate in the research, in full adherence to the Declaration of Helsinki. The clinical and research activities being reported are consistent with the Principles of the Declaration of Istanbul as outlined in the Declaration of Istanbul on Organ Trafficking and Transplant Tourism.Baseline clinical and demographic variables for all 150 KTx recipients with AR, bAR, BKVN, and STA phenotypes are shown in To determine relative abundance of the CRM gene transcripts in the urine sediments, the cycle threshold (Ct) values were used as a metric of abundance. The lower the Ct value, the higher its abundance among the CRM genes. Among the 11 CRM genes, BASP1 was the most abundant transcript in the urinary cell sediment. BASP1 was followed by TAP1, PSMB9, and ISG20 as the 4 topmost abundant transcripts. LCK and CD6 were among the least abundant transcripts in the urine sediments in the CRM gene-set. Since the Ct values ranged from the lowest Ct value of 14 to the highest Ct value of 20, there was a 64-fold difference between the BASP1 and CD6 gene transcripts, with CD6 being the least abundant transcript. to very strong . Although CXCL9 and CXCL10 are in the same class of chemokines, the gene expression correlation between them was only moderate (r = 0.46). The graphical presentation of correlation matrix is presented in .Next, we evaluated gene expression of each of 11 CRM genes for their relative expression in AR, bAR, BKVN, and STA. A summary of results of the analysis is presented in Expression of CD6, CXCL10, CXCL9, LCK, NKG7, and PSMB9 were differentially regulated in urine samples with patients with BKVN when compared to the samples from STA patients. Out of the six genes with statistically different gene expression values between BKVN and STA samples, expression of only NKG7 was substantially lower in BKVN urine.Because expression of the CRM gene-set was not homogenous across the transplant phenotypes and there was substantial physiological cross-talk between the different genes , we usedTo explore the classification performance of the uCRM score on AR, borderline AR, and STA cases, a decision tree classifier was produced . The decWhen including the BKVN samples, all phenotypes were significantly different from one another after multiple comparisons correction except bAR and BKVN . When diNotably, the trend of increasing uCRM score from STA to bAR to AR suggested that the uCRM score could detect gradations of inflammation that were clinically relevant. As such, we evaluated whether the uCRM score was associated with the extent of histological AR lesions observed in matched biopsies from the same patient, collected simultaneously. As seen in There is an urgent need in transplant medicine for developing reliable and non-invasive monitoring tools that may help transplant clinicians predict the risk of allograft injury, preferentially before allograft damage has already been established. While a number of transcriptional biomarkers have been associated to AR, most of the studies have basically focused on a unique or single transcriptional factor and do not reflect the entire molecular complexity of the biological process of allograft rejection , 38. MorSeveral reports have shown the value of studying different biomarkers predicting AR in urine samples of kidney transplant recipients –22. IncrWhile many of the individual CRM genes and gene products have been assessed individually, this is the first report of the collective, noninvasive use of the CRM genes in the prediction of AR in KTx. For example, urinary CXCL9 mRNA and protein and CXCL10 mRNA had previously been evaluated in multicenter studies for the diagnosis of AR , 45, 46. limited sample size of the study, (ii) absence of other transplant injury phenotypes such as chronic allograft injury or drug toxicity, and (iii) lack of assessment of uCRM score in a longitudinal sample in a larger cohort size. These promising findings suggest that additional, prospective studies are needed to validate and fully assess the potential utility of the uCRM score in the clinical setting. In summary, we present a non-invasive, urine-based biomarker developed from a Common Rejection Module composed of 11 genes that can identify transplant injury and rejection in kidney transplant patients.S1 FigA network view of the CRM genes. Cross-hatched bubbles are the CRM genes while solid bubbles indicate partners identified through network analysis. Connections are color-coded based on the type of interaction. Red, physical interaction; purple, co-expression; orange, predicted; blue, co-localization; turquoise, pathway; green, genetic interactions; yellow, shared protein domains.(PDF)Click here for additional data file.S2 FigA. ROC curve of the uCRM score in discriminating between AR and the combination of bAR and STA phenotypes . B. Scatter dot plot of the uCRM score for AR, bAR, STA, and BKVN phenotypes. Significance was determined by nonparametric Kruskal-Wallis test with Dunn’s multiple comparisons correction. C. ROC curve of the uCRM score in discriminating between AR and the combination of bAR and STA phenotypes . * < 0.05. ** < 0.01. *** P < 0.001. **** P < 0.0001.(PDF)Click here for additional data file.S3 FigThe correlation between the uCRM score and various histological parameters was determined. Other than the tubulitis (t) and interstitial inflammation (ii) scores that were significantly correlated , none of(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file.


Adventitious roots (ARs) are roots that are generated from nonrooting tissues. ARs are usually produced both during normal development and in response to stress conditions, such as flooding, nutrient deprivation, heavy metal stress and wounding. The ability of plants to form ARs is a key trait that enables plant propagation, especially for most tree species.2O2 homeostasis showed enrichment in one cluster that was highly upregulated from DAE0 to DAE8. Pharmacological assay confirmed that an appropriate content of H2O2 in stem bases could accelerate the formation of ARs in poplar.Here, the kinetics of AR formation in a tissue culture of a hybrid variety of poplar were investigated. AR formation mainly occurred during the first 8 days and both pre- and newly- formed primordia contributed to AR formation in poplar by histological study. RNA-Seq-based transcriptome analysis was performed for stem bases collected at 0, 2, 4, 6 and 8 days after excision (DAE). Based on the data, the expression patterns of 8 phytohormone-related genes were investigated, and their influences on AR formation were considered. Subsequent gene expression cluster analysis showed a number of biological processes involved in AR formation. Among these biological pathways, genes involved in H2O2 on AR formation was also confirmed. These results enhance our understanding of the regulation of AR formation in tree species.Based on the results of this study, we were able to predict a regulatory network for 7 phytohormones that are involved in poplar AR formation. The influence of HThe online version of this article (10.1186/s12870-019-1700-7) contains supplementary material, which is available to authorized users. Arabidopsis thaliana, Oryza sativa and Petunia hybrids [Adventitious roots (ARs) are roots that are generated both in vitro and in field cultivation from nonrooting tissues such as stems, leaves and hypocotyls. ARs are usually produced both during normal development and in response to stress conditions, such as flooding, nutrient deprivation, heavy metal stress and wounding . The for hybrids , 3. Gene hybrids , 4. All  hybrids –7. Thus,Phytohormones, also known as plant hormones, play important roles in plant growth and environmental responses. Phytohormones act as the most important endogenous factors controlling the process of AR formation in plants. Among the diverse phytohormones, auxin has been confirmed to play a central role in AR formation in many studies . General2O2), one of the main types of ROS, has been predicted by RNA-Seq analysis to positively regulate AR formation by modulating a number of genes [2O2 has also been shown to increase the ability of marigold to form adventitious roots [Among the endogenous factors that affect AR formation, reactive oxygen species (ROS) also play important roles in regulating AR formation. Generally, ROS are considered signaling molecules and have been reported to regulate stomatal movements and biotic and abiotic stress responses as well as many aspects of plant development, including lateral root formation –20. Receof genes . On olivof genes . Exogenous roots , 24. ComPtRR13 was also found to act as a negative regulator of adventitious root development in Populus [2O2 pathways may contribute to poplar AR formation. Finally, pharmacological assay confirmed the influence of H2O2 on AR formation in poplar. Thus, in this study, we provide novel information on the regulation of AR formation in tree species.Poplar is a model tree species due to the small size of its genome, the early release of a high-quality draft genome and the availability of a highly efficient transgenic platform. Usually, poplar is considered as easy-rooting plant, however, different types of species still varied for this trait . Recentl Populus . Subsequ Populus . Several Populus , 30. HowTo investigate the kinetics of poplar AR formation, 30 microcuttings with 2–3 upper leaves and an apical bud (ca. 2–3 cm) of “NL895” were cultured in WPM in the absence of plant hormones and other growth regulators. The first visible AR was observed at 5 days after excision (DAE5), and > 80% of the microcuttings showed roots at DAE8. The number of ARs from DAE0 to DAE8 is shown in Fig. P = 0.05), significant difference among DAE0, DAE2 and DAE4; while difference between DAE6 and DAE8 didn’t reach significant level. The typical sections from DAE0 to DAE8 are illustrated in Fig. When using anatomical method to investigate poplar AR formation in WPM, we found there were different AR primordia rate among the 5 tested stages. The percent are DAE0 (30.4% ± 7.0%), DAE2 35.2% ± 13.7%), DAE4 (45.7% ± 17.9%), DAE6 (60.0% ± 12.3%) and DAE8 (55.3% ± 11.0%)  were collected for each time point. In total, 15 RNA-Seq libraries were created  using the collected samples. A total of approximately 284 million 150-bp paired-end (PE) reads were obtained  were calculated by setting expression at DAE0 equal to 1 Fig. . To compPhytohormones are regarded as key regulators of AR formation. During AR formation, different hormones interact with each other and form a large and complex network that regulates this dynamic biological process. To gain insight into the roles of phytohormones in AR formation, we investigated phytohormone-related genes in poplar with homology searching . In total, 807 genes were predicted to be involved in biosynthesis, transport, metabolism and reception of the 8 phytohormones , while the H2O2 content at these 3 time points was significantly lower than at DAE8 (P < 0.05). Therefore, we concluded that the H2O2 content showed three levels during AR formation: the H2O2 content was at a baseline level at the beginning time point; it increased and remained at a relatively high level from DAE2 to DAE6, and it then increased sharply at DAE8.To test the above hypothesis, we mainly focused on genes in the peroxidase pathway. A total of 7 differentially expressed genes encoding peroxidase were grouped in cluster 6, and most of these genes showed increased expression after DAE0 and peak expression at DAE6 Table . The genlts Fig. . After R2O2 during poplar AR formation, we performed hydroponic culture by using semi-hardwood cuttings of poplar “NL895” as plant material. In the preliminary experiment, five cuttings with uniform growth status were grown in hydroponic solution supplemented with 0, 500 or 1000 μM H2O2. Cuttings grown in 500 μM H2O2 produced markedly more ARs at DAE11 than cuttings grown in the other H2O2 concentrations , and the number of ARs for each cutting was recorded every day. The number of ARs was recorded after 11, 15, 19 and 23 days of growth in hydroponic solution supplemented with 0, 250, 500, 750 or 1000 μM H2O2. The results showed that the average number of ARs was significantly affected by the concentration of H2O2 , while the differences between the average AR numbers seemed to decline after day 19. Additionally, the average AR numbers of the cuttings grown in the solutions with 750 and 1000 μM H2O2 were lower than those of the cuttings grown in the untreated control solution (0 μM). Therefore, three findings were evident from these data. First, treatment with exogenous H2O2 could affect AR formation; second, AR developed faster in the 500 μM solution than in the other solutions; and third, higher concentrations of exogenous H2O2 slowed AR formation in poplar. All these results also suggest that appropriate levels of H2O2 production can accelerate AR formation in poplar. To validate the above results, poplar “NL895” cuttings were also grown in hydroponic solution with 0.5 mM DMTU, which is a chemical H2O2 scavenger. By day 8, the H2O2 content in the stem base of cuttings that were grown in DMTU solution was 142.7 ± 7.3 (n = 30), while that in the stem base of untreated controls was 367.7 ± 3.2 (n = 30). This revealed that DMTU reduced the accumulation of H2O2 . The average number of ARs in the cuttings grown in hydroponic solution with 0.5 mM DMTU was significantly lower than that in the cuttings grown in control solution at DAE12 . Although the average AR length was not significantly different between cuttings grown in solutions supplemented with 0 and 250 μM H2O2, the absolute number of ARs was lower in cuttings from the solution with 250 μM H2O2. This result might suggest that H2O2 negatively regulates AR elongation in poplar.To further understand the roles played by HPoplars is considered as easy-rooting species and there is still complex biological processes during AR formation . In prevPhytohormones are the most important factors affecting AR formation. Among diverse phytohormones, auxin has been confirmed to play a central role in AR formation in many studies –10. SincBesides phytohormones, other factors could be deduced to show influence on poplar AR formation according to the RNA-Seq results. According to the data listed in Additional file 2O2 on poplar AR formation by using pharmacological tests. The accumulation of H2O2 was increased after DAE0 . It suggested that there were 3 levels of H2O2 content from DAE0 to DAE8, with the lowest, moderate and highest at DAE0, DAE2 to DAE6 and DAE8, respectively. Since there were visible ARs at DAE8 and we couldn’t completely exclude the influence of newly formed ARs on H2O2 content assay, thus, the much higher H2O2 accumulation at DAE8 might be partially attributed to the newly formed ARs . However, the accumulation of H2O2 was still higher at DAE2 to DAE6 than at DAE0 (from 53.5 ± 2.0 to 110.2 ± 6.7 mmol/g.prot), it revealed that accumulation of H2O2 was indeed increased during the early stage of poplar AR formation. In previous studies, H2O2 was also found to be involved in AR or lateral root formation, such as in Mung bean [In this study, we investigated the influence of HAE0 Fig. . Howeverung bean , Marigolung bean , 24 and ung bean . Compari2O2 could accelerate the formation of AR, while higher H2O2 content could slow AR formation and reduce root elongation. H2O2 is a type of reactive oxygen species that has various roles in many biological processes, including responses to biotic and abiotic stresses and plant growth [2O2 and a number of phytohormones [2O2 during poplar AR formation. Since the accumulation of H2O2 increased steadily during the whole AR formation process and since two phytohormones, auxin and BRs, positively regulated AR formation, these results might suggest that there was direct or indirect regulation among H2O2, auxin and BRs. In A. thaliana, ROS act as downstream regulators of auxin in the promotion of root hair development [2O2 was regulated by auxin and BRs during AR formation. An appropriate H2O2 content promoted AR formation. However, there are 3 stages for poplar AR formation, primordium organization, primordium differentiation and elongating roots. The H2O2 concentrations at these 3 stages were significantly different (P < 0.05). Thus, we cannot conclude whether H2O2 plays a role at all 3 stages or only at a special stage. Therefore, more research should be performed to comprehensively determine the influence of H2O2 on AR formation in poplar.The data clearly revealed that an appropriate content of Ht growth . Howeverhormones . Here, welopment . Based o2O2 during poplar AR formation by using RNA-Seq and pharmacological testing. The results showed a large and complex regulatory network among diverse phytohormones and H2O2. The knowledge gained from this study will help us to understand the underlying mechanisms of AR formation.Here, we were able to provide a global view of the roles of phytohormones and HPopulus. euramericana), which forms ARs easily both in tissue culture and field cultivation, was used as plant material in this study. This variety was obtained from the tree germplasm repository of Huazhong Agricultural University (HZAU) under legal permission. For the investigation of the kinetics of poplar AR formation and for the preparation of RNA for high-throughput sequencing, tissue culture plantlets of “NL895” were used as plant material. Woody plants with different juvenile periods exhibit very different AR-forming abilities; therefore, two strategies were employed to avoid this influence. First, all microcuttings with undifferentiated ARs were obtained from apical buds that were generated from the first subculture of the differentiated poplar explants. Second, only microcuttings with 2–3 upper leaves and an apical bud (ca. 2–3 cm) were propagated in woody plant medium (WPM) for further analyses. The WPM was prepared according to previous report [A hybrid variety of poplar “NL895”  and then treated with protocol described in a previous study . Section2O2 content assay, we followed protocol described in Bio-101 with slightly modification [2O2 with Amplex Red hydrogen peroxide/peroxidase assay kit  according to its introduction. Six technique replications were set for each biological sample.For Hfication . BrieflyTotal RNA was isolated using an RNeasy Plant Mini Kit according to the manufacturer’s instructions  Co., Ltd., Beijing, China). The quality of all RNA samples was examined by performing agarose gel electrophoresis and examination of the A260/280 wavelength ratio. The RNA integrity number (RIN) of all samples were also tested by an Agilent Bioanalyzer 2100. All samples had sufficient quality were used for RNA-Seq library construction according to a previously described protocol . The conP. trichocarpa genome version 3.0 was used as the reference genome in this study [The raw RNA-Seq reads were quality trimmed and quality filtered using Trimmomatic software with the default parameter settings . The P. is study . Clean ris study , 32. Valis study , 32. A fA. thaliana were obtained from the Arabidopsis Hormone Database (http://ahd.cbi.pku.edu.cn/) [ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/). The predicted proteins from the P. trichocarpa genome version 3.0 were used as queries. BLAST hits with scores greater than 250  were kept. The biological processes of the retained poplar query proteins were annotated based on the functions of their top hit counterparts in the Arabidopsis Hormone Database.For the annotation of phytohormone-related genes in poplar, a homology searching strategy was employed. First, phytohormone-related genes in edu.cn/) , and genhttps://www.r-project.org/). The z-scores of all the differentially expressed genes were then used to form clusters with the “k-means” command in R software. The number of clusters was determined as previously described [http://structuralbiology.cau.edu.cn/PlantGSEA/) [To cluster the differentially expressed genes, the z-score  of each FPKM value was determined with the “scale” command in R software (escribed . FunctiontGSEA/) .–ΔΔCq method was used to calculate the relative gene expression based on the RT-qPCR data [Ten differentially expressed genes were selected for RT-qPCR assay. The primers for these 10 randomly selected differential expressed genes are listed in Additional file PCR data . The exphttps://www.r-project.org/). The function ‘aov’ was used for variance analysis and all multiple comparisons was performed under the significant level of P value equal to 0.05.All the Statistical analysis was conducted by using the R software Additional file 2:Figure S1. Dendrogram of RNA-Seq samples used for time course transcriptome analysis. (PPTX 125 kb)Additional file 3:Figure S2. Number of genes that are differentially expressed during poplar AR formation. (PPTX 9022 kb)Additional file 4:Table S3. Phytohormone-related genes in poplar (XLSX 30 kb)Additional file 5:Table S4. Normalized expression values of differentially expressed phytohormone-related genes (XLSX 28 kb)Additional file 6:Figure S3. Boxplot of FPKM values for differentially expressed genes related to phytohormone transport. FPKM represents fragments per kilobase of transcript per million fragments mapped. Each boxplot consists of the Q1 , Q3 , the median Q2 (the line inside into the box), Q3 + 1.5 IQR  and Q1–1.5 IQR (the lowest line outside the box). Values larger than Q3 + 1.5 IQR or less than Q1–1.5 IQR were considered as outliers and they were plotted as dots outside of the Q3 + 1.5 IQR or Q1–1.5 IQR. If Q3 + 1.5 IQR is less than Q3 or Q1–1.5 IQR is less than Q1, their corresponding lines will not be shown. (PPTX 38 kb)Additional file 7:Figure S4. Boxplot of FPKM values for differentially expressed genes related to phytohormone metabolism. The figure legends are similar to Additional file Additional file 8:Figure S5. Boxplot of FPKM values for differentially expressed genes related to phytohormone receptors. The figure legends are similar to Additional file Additional file 9:Table S5. Functional enrichment of genes in the 12 clusters. (XLSX 15 kb)Additional file 10:Figure S6. Hydroponic culture of poplar cuttings with different concentrations of supplemented H2O2 at DAE11. Panels a, b, and c represent poplar cuttings grown in 0, 500, and 1000 μM H2O2, respectively. Bar = 1 cm. (PPTX 579 kb)Additional file 11:Table S1. Primers used for RT-qPCR assay. (XLSX 9 kb)


Yet, residual aortic regurgitation (AR) secondary toparavalvular leaks (PVL) remains a procedural limitation.3,5-7Therefore, other methods for proper AR assessment have been evaluated in recent years,such as 3D echocardiography, hemodynamic AR index, aortography and even cardiovascularmagnetic resonance, each one with its specific advantages and disadvantages.1,5,7While Doppler echocardiography has been the most common method to assess AR followingTAVR, its accurate quantification is challenging since AR jets are often multiple andeccentric.8 investigate a quantitative angiographic assessment of AR byvideodensitometry (VD-AR) before and after BPD was performed. VD-AR was shown todecrease significantly from 24.0 [18.0-30.5] % to 12.0 [5.5-19.0] % (p < 0.001) afterBPD, with some degree of AR grade improvement for up to 70% of patients treated. Ofnote, significant AR (VD-AR > 17%) was observed in 47 patients (77%) before and in 19patients (31%) after BPD; moreover, in up to a quarter of these, pre-BPD VD-AR was below17%, indicating that this additional maneuver could have been avoided. The study has itsinherent limitations, e.g., the cohort was relatively small, with retrospective patientselection and imaging acquisition, and the decision whether or not to perform BPD wasleft to the discretion of the operators. Accordingly, the study only comprises caseswhere BPD was deemed necessary, and only aortograms with good quality imaging wereselected.In the current issue of the journal, Miyazaki et al.9,10 This technique provides an accurate assessment of theseverity of PVL, and a VD-AR index greater than 17% correlated with increased mortalityand with impaired cardiac reverse remodeling after TAVR.11,12 And while VD-AR measurements are performed offline only,real-time online assessment is underway so as to enable this method to help guiding TAVRin the near future. After all, BPD is currently performed in about 10% to 20% ofpatients following TAVR, and it reduces the severity of PVL by at least one grade inmore than two thirds of patients.13,14Nevertheless, BPD may be associated with an increased risk of cerebrovascular events andannular trauma, therefore judicious utilization of this procedure isrecommended.13,14Notably, the technique used to quantify VD-AR is a novel method that can accuratelydetermine the regurgitation fraction in aortograms performed during TAVR; it usesdedicated software and showed excellent reproducibility and accuracy.In conclusion, since PVL has a negative impact on clinical outcomes after TAVR, itsproper assessment through a multimodality, multiparametric, integrative approach isfundamental. Priority should be given to PVL prevention through accurate sizing ofaortic annulus by 3D imaging techniques, THV devices with improved sealing features, andoptimal THV sizing and positioning. Still, if PVL does occur after TAVR, theinterventional cardiologist can consider corrective procedures such as BPD,valve-in-valve, or leak closure. The novel VD-AR after TAVR also allows quantitativelyassessing post-TAVR regurgitation and may assist decision making on whether or not toperform BPD, as well as determining its efficacy. Future prospective studies arewarranted to further confirm the present results.


Androgen receptor (AR) plays important role in the development, progression, and metastasis of prostate cancer (PCa). Caffeic acid phenethyl ester (CAPE) is the main component of honey bee propolis. We determined if CAPE affects the signaling and stability of AR in PCa cells.Effects of CAPE on AR transcriptional activity and localization were determined by reporter gene assay and immunofluorescent microscopy. Western blotting, fluorescent polarization, computer simulation, and animal experiment were performed to investigate the molecular mechanism how CAPE reduces the stability of AR.CAPE treatment dose-dependently suppressed the transcriptional activity of AR as well as the protein levels of AR and its target gene PSA. Cyclohexamide treatment revealed that androgen stabilized AR protein, but AR stability was diminished by CAPE. Fluorescence microscopy demonstrated that androgen promoted the nucleus translocation of AR in PCa cells, while treatment with CAPE reduced protein level of AR in both nucleus and cytoplasm. CAPE treatment suppressed the phosphorylation of Ser81 and Ser213 on AR, which regulates the stability of AR. CDK1 and AKT are the kinases phosphorylating Ser81 and Ser213 on AR, respectively. CAPE treatment significantly reduced the protein level and activity of CDK1 and AKT in PCa cells. Overexpression of CDK1 or AKT rescued the AR protein level under CAPE treatment.Our results suggested that CAPE treatment reduced AR stability and AR transcriptional activity in PCa cells, implying the possibility of using CAPE as a treatment for advanced PCa.The online version of this article (10.1186/s12964-019-0404-9) contains supplementary material, which is available to authorized users. Androgen receptor (AR) is an androgen-activated transcription factor belongs to the nuclear receptor superfamily . BindingAR regulates male sexual maturation, maintenance of normal prostate function, prostate carcinogenesis, and prostate cancer (PCa) progression , 5. AR pCaffeic acid phenethyl ester (CAPE), a strong antioxidant, is the major bioactive component in honeybee hive propolis , 18. CAPAR. PC-3AR cells were maintained in DMEM (Gibco/Invitrogen) supplemented with 10% charcoal-stripped fetal bovine serum (CS-FBS) , penicillin (100 U/ml), and streptomycin (100 μg/ml). Caffeic acid phenethyl ester (CAPE) was purchased from Sigma Aldrich. Cyclohexamide was purchased from Calbiochem/Merck Millipore .LNCaP 104-S and LNCaP 104-R1 cells were generated from ATCC FGC clone (ATCC CRL-1740) as described in previous publication . LNCaP C5 cells per well. After 24 h, pRL-TK  and p3xARE-∆56-c-Fos-GL3 (reporter gene vector) were co-transfected by using PolyJet in vitro DNA transfection reagent (SigmaGen Laboratories) for 5 h, and substituted medium containing DHT or/and CAPE for 48 h. Cell lysates were lysed in 100 μl 1X passive lysis buffer (Promega). Dual-luciferase reporter assay kit (Promega) was used to measure transcriptional activity by Turner Biosystems 20/20n Luminometer.Cells were seeded in 12-well plates at the density of 2.5 × 10Cell lysate was lysed and Western blot was performed as previously described . AntibodCell lysate were prepared for RNA extraction using RNeasy mini kit . Two micrograms of total RNA of each samples was used as templates for synthesis of complementary DNA (cDNA) by RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Scientific). qPCR analysis was performed by Maxima SYBR Green/ROX qPCR Master Mix (2X) (Fermentas/Thermo Fisher Scientific). The mRNA expression was analyzed by ABI PRISM 7500 . The following sequences were used as qPCR primers: AR-Fw: CTGAAACTACAGGAGGAAGG, AR-Rv: TGCAGAGGAGTAGTGCAGAG; PSA-Fw: CATCAGGAACAAAAGCGTGAT, PSA-Rv: AGCTGTGGCTGACCTGAAATA; CDK1-Fw: CTGGGGTCAGCTC GTTACTC, and CDK1-Rv: TCCACTTCTGGCCACACTTC. GAPDH-Fw: ACAGT CAGCCGCATCTTCTT and GAPDH-Rv: ACGACCAAATCCGTTGACTC.Cells were seeded in 35 mm imaging dish (ibidi), and fixed with 4% formaldehyde on ice for 15 min and permeabilized in 0.3% Triton X-100 (in PBS) for 10 min. Blocking for an hour and cells stained with indicated antibody for 16 h at 4 °C. Alexa Fluor 488 dye (Thermo Fisher Scientific) was used as secondary antibody for green-fluorescent dye. The cell nuclei were stained by DAPI. The image of fluorescence was taken by Leica TCS SP5 AOBS Confocal Spectral Microscopy using a 63x oil-immersion objective len and a 10x eyepiece. A scale bar showing 25 μm was arranged at the lower right part of each image.2, 0.4% NP-40, pH 7.5) and centrifuged for 2 min at 3000 rpm at 4 °C. Removing the supernatant and re-suspending the pellet by lysis buffer. Centrifuging for 2 min at 3000 rpm and collecting the supernatant in new eppendorf (cytosol fraction). The pellet was re-suspended for 15 min on ice in nuclear extraction buffer  and nuclear fraction was collected by centrifugation for 10 min at 13000 rpm at 4 °C.Cells were lysed in lysis buffer  was used to evaluate the statistical significance of results from proliferation assay experiments.AR cells (AR-negative PC-3 cells being overexpressed of wild type AR)  293 cells, which is AR-negative. Luciferase reporter gene assay indicated that dihydrotestosterone (DHT) stimulated AR transcriptional activity dose-dependently while CAPE treatment suppressed AR transcriptional activity in HEK293-AR cells Fig. a. TreatmAR) Fig. b and LNCAR) Fig. c. LNCaP PSA is a target gene of AR. As CAPE treatment reduces AR transcriptional activity, we predict that CAPE treatment can suppress PSA expression in human PCa cells. We determined the mRNA level of PSA in androgen-dependent AR-positive LNCaP 104-S cells and androgen-independent AR-rich LNCaP 104-R1 cells in the presence or absence of androgen. Our qRT-PCR data revealed that while androgen stimulated the expression of PSA mRNA, CAPE treatment suppressed the androgen-induction of PSA mRNA in both LNCaP 104-S , in the presence of cycloheximide (CHX) for 48 h. DHT stabilized AR protein, while CAPE promoted the degradation of AR protein Fig. a and LNCAR protein stability is also regulated by phosphorylation on Ser213, which is regulated by PI3K-AKT signaling. Treatment with DHT induced AR phosphorylation on Ser213 in both LNCaP 104-S Fig. a and LNCWe performed animal study to determine if CAPE treatment reduces protein expression level of AR and CDK1 in vivo. Compared to tumors in control nude mice, CAPE treatment  significantly reduced AR protein expression level  Tumor tissue was lysed and determined by western-blotting. (B) Protein level of AR, CDK1 and AKT was quantitated by ImageJ software. (TIFF 4670 kb)Additional file 2:Figure S2. The hydrophobicity of three compounds  was analyzed by Druglikeness software. The logP value revealed drug diffusion permeability. Molinspiration druglikeness software is an on-line service software which can be used to compare various molecule properties and structure features which determine whether a particular molecule is similar to the known drugs (https://www.molinspiration.com/docu/miscreen/druglikeness.html). We used this software to compare the hydrophobicity of DHT, CAPE and bicalutamide. (TIF 3862 kb)Additional file 3:Figure S3. The nuclear receptor ligand score of three compounds  was analyzed by sophisticated Bayesian statistics. Molinspiration druglikeness software was used (with sophisticated Bayesian statistics) to compare the ability of DHT, CAPE, and bicalutamide to bind AR. This software compare the structures of representative ligands active on the particular target with structures of inactive molecules and to identify substructure features typical for active molecules (https://www.molinspiration.com/docu/miscreen/druglikeness.html). The values revealed the ability to bind with AR. (TIF 5090 kb)Additional file 4:Figure S4. The binding ability of AR-ligand binding domain with DHT, CAPE and Bicalutamide was determined by AR competitor assay. AR competitor assay was performed with the PolarScreen AR Competitor Assay kit (Thermo Fisher Scientific) following the manufacturer’s protocol. Reaction plate was incubated for 6 h. Fluorescence polarization was measured by SpectraMax Paradigm Reader and the data was analyzed by Graphpad software. The fluorescence polarization was measured to predict the IC50. (TIFF 1466 kb)Additional file 5:Supplemental Material and Methods. (DOCX 16 kb)


D value of 0.71 μM. Importantly, and in contrast to ILT4, NRP1 interacted with CSPs that were covalently bound to target surfaces in the course of complement activation, therefore representing a classical complement receptor. The binding site of CSPs was mapped to the b1 domain of the coagulation factor V/VIII homology domain of NRP1. Taken together, our results demonstrate a novel role for NRP1 as a receptor for CSPs deposited on surfaces during complement activation. Further work is required to elucidate the functional consequences of the NRP1-CSP interactions in immunity.Complement split products (CSPs), such as the fragments C4d and C3d, which are generated as a consequence of complement regulatory processes, are established markers for disease activity in autoimmunity or antibody-mediated graft rejection. Since immunoglobulin-like transcript 4 (ILT4) was previously shown to interact with soluble CSPs, but not with CSPs covalently-bound to target surfaces following classical complement activation, the present study aimed to identify novel cellular receptors interacting with covalently-deposited CSPs. By applying an unbiased screening approach using a cDNA mammalian expression library generated from human monocyte-derived dendritic cells and probed with recombinant human C4d, we identified neuropilin-1 (NRP1) as a novel receptor for C4d, C3d, and iC3b. NRP1, a highly conserved type 1 transmembrane protein, plays important roles in the development of the nervous and cardiovascular system as well as in tumorigenesis through interaction with its established binding partners, such as vascular endothelial growth factor (VEGF) and semaphorin 3A (Sema3A). NRP1 is also expressed on immune cells and serves as a marker for murine Tregs. Although NRP1 contains domains homologous to ones found in some complement proteins, it has not been linked to the complement system. We demonstrate that binding of C4d to NRP1 expressing cells was dose-dependent and saturable, and had a K The complement system mediates robust first line defense mechanisms against invading pathogens and participates in the elimination of cellular and humoral debris . It funcHost protection is mediated by a combination of cellular and humoral complement regulatory proteins . They acIn the present study, we screened a cell pool expressing a retroviral cDNA library generated from human monocyte-derived dendritic cells (moDCs) and identified neuropilin-1 (NRP1), also known as CD304 and blood dendritic cell antigen-4 (BDCA4), as a novel cellular complement receptor for C4d, C3d, and iC3b. NRP1, a well-described receptor for vascular endothelial growth factor (VEGF) and semaphorins, has been implicated to be involved in various biological processes including angiogenesis, neuronal development, cell survival, migration, and tumor-invasion –16. We pMurine thymoma BW5147 (referred to as BW throughout this work) cell line and Jurkat cell line (JE6.1) were cultured as previously described , 17. ForTM or LSRFortessaTM . Data was analyzed using FlowJo software . Sorting of high surface molecule expressing cells was carried out with a Cell Sorter SH800 .Flow cytometric data acquisition was performed using FACSCaliburRh-C4Ad, rh-C4Bd, and rh-C3d were expressed and purified as previously described , 20. Ih-Purification from human plasma and thioester carbonyl-specific-biotinylation via amine-PEG2-biotin reagent of ih-C4d was carried out as previously described . IodoaceTM cell sorter . Single cell clones were established from the resulting cell pool by limiting dilution culturing. Genomic DNA was isolated from a C4d-reactive single cell clone and PCR-amplification of the integrated DNA was performed as previously described  and human serum  for 2 h at 37°C.Natural complement deposition of C4d was performed as previously described . In brieBW cells were retrovirally transduced to express either the wildtype NRP1 or one of the following NRP1 deletion variants , all of which express the MAM/dimerization domain, the transmembrane segment and the cytoplasmic tail. In addition, BW cells expressing murine NRP1 (mNRP1) and ILT4 were generated. For activation of moDCs, Jurkat cells were retrovirally transduced to express CD40L. Wildtype NRP1, NRP1 deletion variants, mNRP1, ILT4, and CD40L were cloned into the retroviral expression vector pCJK2  and cellPeripheral blood mononuclear cells (PBMCs) were isolated from heparinized whole blood of healthy volunteer donors purchased from the Austrian Red Cross by standard density-gradient centrifugation with Lymphoprep . Donors gave their informed consent and approval was obtained from the ethics committee of the Medical University of Vienna (ECS1183/2016). Monocytes were isolated and differentiated into monocyte-derived dendritic cells (moDCs) as previously described , 23. MoDex vivo  . Additioex vivo .Since complement receptors commonly bind several ligands, we assessed whether NRP1 would also bind to additional C3- and C4-derived CSPs. We generated BW cells expressing high levels of NRP1 and probed them with recombinant human or isolated human C4Ad, C4Bd, C3d, C4b, C3b, and iC3b. These experiments showed that NRP1, in addition to human C4d of both isotypes, strongly bound rh-C3d and ih-iC3b, whereas only weak binding was detected for ih-C4b and ih-C3b . These iD value of 0.71 μM (±0.09 μM) was calculated from the binding curve  domains (a1/a2), two coagulation Factor V/VIII homology domains (b1/b2) and a MAM  (c) domain . In ordeAlthough NRP1 and ILT4 belong to different molecular families and do not share structural similarities, they both interact with C4d and other CSPs. Since ILT4 is an established receptor for MHC class I molecules , we asseNRP1 is a highly conserved type 1 transmembrane protein that was originally described as a neuronal adhesion molecule –30. The in vitro assays evaluating the function of this receptor, makes it challenging to understand the functional role of NRP1 in human immunity. In the nervous and vascular system, NRP1 mediates its functions in a co-receptor and ligand-dependent manner. Currently, it is not known whether in the immune system, NRP1 functions independently or also as part of a receptor complex. NRP1-NRP1 interactions between T cells and DC in the immunological synapse have been proposed to mediate the initiation of primary human T cell responses  . These cHuman NRP1 has been implicated to play an important role in T-cell-DC interactions , but prein vitro assays and a defined signaling pathway for this receptor. However, since we found murine NRP1 to interact with CSPs, it might be possible to assess a potential immunomodulatory effect of naturally deposited CSPs and murine NRP1 in vivo.Further research will be necessary to reveal a potential functional significance of the CSP-NRP1 interaction, which at present is hampered by the lack of both reliable The datasets generated for this study are available on request to the corresponding author.Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.CB, JH, PS, and WP: conceptualization and design of experiments. CB, JH, WP, AD, and JL: acquisition of experiments. CB, JH, WP, AD, JL, and PS: analysis and interpretation of data. JH, PS, WP, GZ, MW, and DI: resources. CB, JH, and PS: wrote the manuscript. CB, AD, WP, JL, MW, DI, GZ, PS, and JH: manuscript review, read, and approved.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The androgen receptor (AR) is an emerging prognostic marker and therapeutic target in breast cancer. AR is expressed in 60–80% of breast cancers, with higher prevalence among estrogen receptor-positive (ER+) tumors. Androgen treatment inhibits ER signaling in ER+/AR+ breast cancer cell lines, and AR expression is associated with improved survival for this subtype in epidemiologic studies. However, whether AR expression modifies the efficacy of selective ER modulators or aromatase inhibitors for ER+ cancers remains unclear.We evaluated the prognostic and predictive value of AR expression among 3021 postmenopausal ER+ breast cancer patients in the Breast International Group (BIG) trial 1–98. The BIG 1–98 study was a four-armed, double-blind, phase III randomized clinical trial that compared 5 years of tamoxifen or letrozole monotherapy, or sequences of 2 years and 3 years treatment with one drug and then the other. AR expression was measured by immunohistochemistry and the percentage of AR-positive nuclei was quantified. The association between AR expression and prognosis was evaluated using Cox proportional hazards models. Continuous AR-by-treatment interactions were assessed using Subpopulation Treatment Effect Pattern Plots (STEPP).p = 0.12, log-rank test). In multivariable-adjusted models, AR expression was not associated with BCFI . The letrozole versus tamoxifen monotherapy treatment effect did not significantly differ for AR+ tumors  and AR− tumors  (p-heterogeneity = 0.16). STEPP analysis also suggested no heterogeneity of the treatment effect across the continuum of AR expression.Eighty-two percent of patients had AR+ (≥ 1%) tumors. Patients with AR+ cancers were more likely to have smaller, lower-grade tumors, with higher expression of ER and PR. AR expression was not associated with breast cancer-free interval (BCFI) (415 events) over a median 8.0 years of follow-up  contains supplementary material, which is available to authorized users. Sex steroid hormone receptor signaling is vital in the development and progression of breast cancers. The role of the estrogen receptor (ER) in breast cancer has been well elucidated, and estrogen blockade using selective estrogen receptor modulators (SERMs) and aromatase inhibitors is fundamental in breast cancer therapeutics , 2. The Preclinical and clinical data support differential effects of AR signaling in breast cancers, dependent on tumor ER expression –10. In EThe Breast International Group (BIG) trial 1–98 was a phase III randomized clinical trial that evaluated letrozole (an aromatase inhibitor) versus tamoxifen (a SERM) as an adjuvant therapy for early stage hormone receptor-positive breast cancer among 8010 postmenopausal women . The iniThere is conflicting evidence surrounding AR as a predictive marker of tamoxifen response, with prior studies indicating that high AR expression is associated with improved response and that a high AR:ER ratio is associated with tamoxifen resistance , 22. No We leveraged the resources of the BIG 1–98 trial to evaluate AR protein expression as a marker of prognosis among postmenopausal women with early stage ER+ breast cancer. Additionally, we also assessed AR expression as a predictor of differential efficacy of letrozole and tamoxifen adjuvant therapy in this population.ClinicalTrials.gov (identifier NCT00004205).BIG 1–98 was an international, multicenter, phase III, double-blind, four-arm randomized trial conducted among postmenopausal women with early stage hormone receptor-positive breast cancer. The design of the trial has been described in detail previously –17. FromTumor tissue biospecimens were collected retrospectively in accordance with institutional guidelines and national laws, with the financial support of Novartis. Tissue blocks and/or slides were obtained for 6549 participants at the International Breast Cancer Study Group (IBCSG) Central Pathology Office in Milan, Italy. For 3784 participants, two 1-mm-diameter cores per participant were obtained from their formalin-fixed paraffin-embedded tumor specimens and assembled into tissue microarrays (TMAs) in Italy.n = 42 individuals). Expression of ER, PR, and Ki67 was previously evaluated on whole sections by immunohistochemistry (IHC) at the IBCSG Central Pathology Office, as was the expression of HER2 by IHC and fluorescent in situ hybridization [AR was centrally evaluated by immunohistochemistry using the Dako AR441 antibody (1:200 dilution) at the Dana-Farber/Harvard Cancer Center Specialized Histopathology Services Core, Boston, MA, USA (TMA sectioning and antibody staining) and Beth Israel Deaconess Medical Center, Boston, MA, USA (scoring). The percentage of positive epithelial nuclei was estimated using Definiens Tissue Studio automated imaging analysis software version 4.4.2 . The average of AR expression across the two cores was calculated for each participant weighted by the number of detected nuclei. A cutoff of 1% expression or greater was used to define AR positivity in primary analyses. The Spearman correlation between AR expression measured by Definiens and three-tier manual pathologist scoring  was 0.61 in a test TMA , which was defined in concordance with STEEP criteria as the time from randomization until the earliest of invasive local-regional or distant recurrence or new invasive breast cancer in the contralateral breast . Diseasen = 3021), while predictive analyses were limited to the monotherapy population (n = 1753).A comparison of trial participants with and without assessable AR expression is shown in Additional file The associations between AR expression and patient and tumor characteristics were evaluated using chi-square tests for categorical variables and Wilcoxon rank sum tests for continuous variables. The distribution of BCFI and DFS were estimated using the Kaplan-Meier method. The association between AR expression and BCFI and DFS was evaluated both by the log-rank test and using Cox proportional hazards models to estimate hazard ratios (HRs) and 95% confidence intervals (95% CIs). Models for this association were stratified by randomized treatment assignment and receipt of chemotherapy, and were adjusted for participant, tumor, and local treatment covariates. AR expression was evaluated both as a dichotomous variable (≥1% versus < 1%) and as a continuous variable. Restricted cubic splines were used to evaluate potential non-linearity of the association between AR expression and BCFI and DFS, though tests for non-linearity were highly non-significant, and AR expression was therefore modeled linearly as a continuous variable . ER, PR,p < 0.05 was considered statistically significant.AR was assessed as a predictive biomarker using Cox proportional hazards models. In these analyses, participants who selectively crossed over from tamoxifen monotherapy to letrozole monotherapy were censored at the time of crossover. Heterogeneity of the treatment effect by dichotomous AR expression was assessed using a likelihood ratio test for an AR-by-treatment interaction. Subpopulation Treatment Effect Pattern Plot (STEPP) analysis was used to evaluate heterogeneity of the treatment effect across the continuum of AR expression , 29. Theα = 0.05 for a two-sided test, there was 80% power to detect an HR of 0.69 or lower for BCFI [For evaluation of AR as a prognostic marker, with a sample size of 3021 women, 415 events, 82% AR+ tumors, and setting for BCFI .p < 0.001) and lower histologic grade tumors (p < 0.001). Correspondingly, women with AR+ cancers were more likely to have received breast conserving surgery (p < 0.001), but receipt of chemotherapy did not differ by AR status (p = 0.21). AR+ tumors had higher ER and PR expression than AR− tumors (both p < 0.001), but were less likely to overexpress HER2 (p = 0.04). Ki67 expression did not significantly differ between AR+ and AR− tumors (p = 0.07).Distributions of participant, tumor, and treatment variables are shown in Table p = 0.12, log-rank test)   (Table p = 0.27). When evaluating AR expression continuously, each successive 10% increase in AR expression was not associated with BCFI .Over a median of 8.0 years follow-up (IQR 7.0–9.0 years) after randomization, a total of 415 BCFI events  and 676 DFS events 22%) occurred. Five-year BCFI was 93.1% (95% CI 92.1–94.2%) among women with AR+ tumors and 90.5% (95% CI 88.1–93.0%) among women with AR− tumors, though differences in BCFI were not significant (2% occurrn = 1753), BCFI was better for those assigned to letrozole than tamoxifen, regardless of tumor AR expression  , as was found in the total trial population (Table p = 0.002) than among AR+ breast cancers , though this difference was not statistically significant (p-heterogeneity = 0.16). Among those assigned to letrozole monotherapy, AR expression was associated with non-significantly worse BCFI , while among those assigned to tamoxifen, AR expression was not associated with BCFI . Similar trends were observed for DFS.In multivariable-adjusted Cox models, participants assigned to letrozole monotherapy experienced significantly better BCFI compared to participants assigned to tamoxifen monotherapy  and when evaluating the continuum of AR expression.n = 5648 patients) finding tumor AR expression to be associated with improved DFS in the multivariate analysis  [n = 1571 patients) [The androgen receptor is an emerging prognostic marker for breast cancer, with a recent meta-analysis by Bozovic-Spasojevic et al. of 13 studies 37–0.58) . This as . This a37–0.58) . In vitr37–0.58) , 32. How37–0.58) –35, bene37–0.58) , 37, and37–0.58) , 38, 39 37–0.58) . Epidemiatients) . Prior matients) –43, thouatients) . Treatmeatients) . Howeveratients) . Cistromatients) .n = 938), AR expression was associated with improved disease-specific survival for women receiving endocrine therapy and chemotherapy, but not endocrine therapy alone [In contrast to our own study, several prior observational studies have found AR to be a predictive marker for endocrine therapy response in ER+ breast cancers. However, this evidence is conflicting and primarily relies on univariate analyses. In the largest study to date  a table comparing trial participants for whom AR expression was and was not assessable; 2) a Kaplan-Meier curve comparing disease-free survival by tumor AR expression; 3) a Kaplan-Meier curve comparing disease-free survival by cross-classified tumor AR expression and treatment assignment. Table S1 Hypothesis testing for differences in distributions between tumors with and without assessable AR used chi-square, Wilcoxon rank sum and log-rank tests for categorical, continuous and time-to-event endpoints, respectively. Design variables (two- or four-arm randomization period and treatment assignment) were not compared. Individuals with missing data were excluded when performing hypothesis tests for continuous variables. (DOCX 53 kb)


The genome of Bacillus subtilis 168 contains SPβ, an active prophage, skin, a defective prophage, and ICEBs1, an integrative conjugative element. Each of these MGEs harbors the classic SSR unit attL-int-rdf-attR. Here, we demonstrate that these SSR units are all compatible and can substitute for one another. Specifically, when SPβ is turned into a defective prophage by deletion of its SSR unit, introduction of the SSR unit of skin or ICE converts it back to an active prophage. We also identified closely related prophages with distinct SSR units that control developmentally regulated gene rearrangements of kamA . These results suggest that SSR units are interchangeable components of MGEs.Site-specific recombination (SSR) systems are employed for transfer of mobile genetic elements (MGEs), such as lysogenic phages and integrative conjugative elements (ICEs). SSR between   •Lysogenic phage-derived SSR unit is sufficient to drive SSR of ICE and vice versa•Defective prophage-derived SSR unit can drive the excision of the active lysogenic phage•Closely related prophages with distinct SSR units control each gene rearrangements•Correspondence between MGEs and their cognate SSR units is not absolute Genetics; Molecular Genetics; Microbiology; Microbial Genetics For example, the bacteriophage λ genome, which carries an attP site, integrates in the genome of its host Escherichia coli at the attB site located between the bio and gal operons  infect bacterial host cells by injecting their genetic material. Phage virions consist of a protein coat that protects a DNA or RNA genome. Lytic (virulent) phages cause host cells to lyse after production of viral particles. Lysogenic (temperate) phages can switch between the dormant state (prophage), where the phage genome is integrated into the host chromosome, and the productive state, following excision of the phage genome from the chromosome. The loci where lysogenic phages integrate into the host genome are known as attachment ( operons . Similarynthesis .attL-int-rdf-attR (SSR unit) (attL (left) and attR (right), are hybrids of the attP and attB sites. Attachment sites are recognized by an integrase, which either catalyzes phage integration (by recombination of attP and attB) or, conversely, excision of the DNA comprised between attL and attR. In addition, excision reactions require phage-encoded small proteins, known as recombination directionality factors (RDFs)  belongs to the LSR-type family. Each excision reaction requires an RDF, known as Xis for λ and SprB for SPβ  and an RDF (XisBs1ICE), respectively (The basic genetic unit of site-specific recombination (SSR) systems in the genome of an integrated mobile genetic element (MGE) is SR unit) . The phas (RDFs) . Integra for SPβ . Integra for SPβ . In the ectively .B. subtilis strain 168, these SSR units can be carried simultaneously by active prophages, ICEs, defective prophages, or non-prophage-like elements. Inactive prophages are often observed in genomes of spore-forming bacteria. Many of these are found in sporulation-specific genes  generated after asymmetric division of the sporulating cell. Importantly, both cell types receive identical copies of the bacterial genome after division, but because the mother cell eventually lyses and the forespore matures into a spore, the spore genome can be viewed as the germ cell genome that will retain the inactive prophage. Several other genes specifically expressed in the mother cell of spore forming bacteria are known to be interrupted by MGEs. In B. weihenstephanensis, these include spoVFB (dipicolinate synthase subunit B gene) and spoVR (involved in spore cortex formation) (gerE (encoding a mother cell-specific transcription factor) is interrupted in B. cereus  . Similar. cereus .Bacillus species are usually produced in response to nutrient deprivation. Their dormant state is sustained until environmental conditions become favorable again for growth. Sporulation is an elaborate developmental process with well-defined temporal stages in the differentiation of the mother cell and forespore. Each cell engages in specific gene expression programs governed by a cascade of cell-specific sigma factors (σF and σG in the forespore and σE and σK in the mother cell) that control the expression of sporulation genes temporally and spatially  interrupted by the defective prophage skin. To reconstitute a functional sigK, excision of skin is necessary (skin (SpoIVCA), which promotes excision  are split by an element that carry their cognate SSR unit. Although some rdf genes have not yet been identified, excision processes are likely mediated by an individual integrase and its cognate RDF. It was recently discovered that the sporulation gene spsM was interrupted by an active prophage, SPβ, carrying the SSR unit attL-sprB (rdf)-sprA (int)-attR. The timing of phage excision is controlled by sprB, whose expression is dependent on stress-inducible and mother cell-specific promoters   . There aB. subtilis SSR units are compatible among lysogenic phages and ICEs. Our experiments also show that SSR units from a defective prophage and from an ICE can rescue a phage rendered inactive by deletion of its endogenous SSR unit. In addition, we found in other B. subtilis strains and related species closely related prophages with distinct SSR units, suggesting that transfer between MGEs of independent SSR units is possible.Accordingly, SSR units could be transferred as independent units between lysogenic phages and ICEs. Here, we demonstrate that B. subtilis 168 genome, the 134-kb SPβ prophage  carry the SSR unit attLSPβ-sprB-sprA-attRSPβ. Among the components of this unit, the integrase gene, sprA, encodes an LSR and has a σA-dependent promoter (PV), whereas its cognate RDF gene, sprB, has distinct promoters, i.e., a stress -inducible σA-dependent promoter (PSt) and a sporulation-specific σE/K-dependent promoter (PE/K) (Bs1 and ICEBs1cat (a version carrying a chloramphenicol resistance cassette) harbor the SSR unit attLBs1ICE-intBs1ICE-xisBs1ICE-attRBs1ICE  and ImmR (immunity repressor)  with the ICEBs1-derived SSR unit (attLBs1ICE-intBs1ICE-xisBs1ICE-attRBs1ICE)  was induced by MMC (0.5 μg/mL) and the phage lysate was used to infect a SPβ-, ICEBs1-, and skin-cured strain (Δ3)  and quantitative polymerase chain reaction (qPCR)-based analyses. These assays showed that, although the timing of SPβBs1ICE excision was slower than that of SPβkan because of transcriptional regulation of SSR unit in SPβBs1ICE, excision rates were significantly increased in response to MMC-mediated induction for both the SPβkan and chimeric SPβBs1ICE lysogens . Phage titers for SPβBs1ICE were found to be slightly lower than those of the positive controls, i.e., wild-type SPβ or SPβkan (Bs1ICE was approximately 30-fold lower than that of SPβkan (probably due to non-native SSR units), site-specific integration at attBBs1ICE was 100% accurate in lysogens carrying SPβBs1ICE . In total, these data indicate that the ICEBs1-derived SSR units are sufficient for integration/excision of SPβ at the attBBs1ICE site. These results further imply that SSR units can be repurposed to control the life cycle of unrelated MGEs, including that of lysogenic phages.In the prophage  integratly 45.2°  (Kunst ely 45.2° . SPβ andr (PE/K) B (Abe etr (PE/K) . ICEBs1 tRICEBs1  and S2 (tRICEBs1 . The intpressor) C (Auchtupressor) . We inveRICEBs1) B and intain (Δ3) B. SPβICE3 strain A. Next, lysogens A and 2B.r SPβkan . Even thBs1. We engineered a 22-kb chimeric ICEBs1SPβ carrying a chloramphenicol resistance cassette and the SPβ-derived SSR unit instead of the ICEBs1-derived SSR unit  sequence of xisBs1ICE. To reduce sprB expression, we introduced a GGAGG to GcAGG mutation into the SD sequence, 11 bp upstream of the start codon (TTG) of xisBs1ICE. This second chimeric ICE construct (designated as ICEBs1SPβ) was stably integrated into attBSPβ under non-MMC treatment conditions and was excised following addition of MMC (0.5 μg/mL). We performed PCR and qPCR-based detection assays and showed that the excision rates of both ICEBs1SPβ and the parent ICEBs1cat were increased in response to MMC-mediated induction . The integrase gene spoIVCA encodes an LSR that catalyzes the joining of the truncated 5′-sigK and sigK-3′ portions of the sigK gene . Because the promoter of spoIVCA (PσE) is located over 200 bp upstream of the translational start site, we hypothesized that an open reading frame (ORF) was present immediately upstream of the spoIVCA coding sequence. To confirm the presence of the ORF and its role as the rdf of skin, we introduced the IPTG-inducible Pspac promoter at positions +47, +18, and −17 nucleotides (nt) from the first nucleotide of the putative ORF (skin was only induced following expression from the −17 nt position (skr. It encodes a 64-amino-acid (aa) protein and is required for the reconstitution of a full sigK gene from the 5′-sigK and sigK-3′ portions. Unexpectedly, the 3′-end of skr overlaps with the 5′-end of spoIVCA by 101 nt, owing to a +1 frameshift  was eliminated. The resulting lysogen contained the chimeric SPβskin prophage. Upon treatment with MMC, the phage lysate was used to infect the Δ3 host strain and the SPβskin lysogen was selected for spectinomycin resistance. DNA sequences of the flanking regions of attLskin and attRskin were then determined to confirm that SPβskin was integrated at attBskin in the Δ3 strain genome . Rearrangement of sigK was as accurate as skin , we found that 16 B. subtilis strains had SPβ phage-like sequences inserted into the spsM gene. Each sequence was of similar size (131–134 kb) and gene organization was highly conserved , indicating that they are unlikely to be active prophages  is in the vicinity of spsM (formerly yodU), with just a few genes separating the two phage insertion sites (φ12-5) of strain D12-5 as query revealed that seven phage-like (128–136 kb) and one non-phage-like (11 kb) sequences are inserted into kamA in various Bacillus species (B. subtilis reportedly (φ3T (displaying 18.9% identity with SprA of SPβ) and its cognate RDF, Rdfφ3T (no similarity with SprB of SPβ), were encoded by genes located in the flanking regions of the φ3T prophage  were only 63% similar (comparison percentage generated using tBLASTx), whereas those of SPβ (spsM) and φ3T (kamA) were 69% similar. Thus, the degree of genomic similarity between prophages containing heterogeneous SSR units (SPβ versus φ3T) is higher than that between homogeneous SSR units (φ3T versus φ12-5) (In t of SPβ A. φ12-5 ol of σE . In B. son sites B. Althou species A. Among portedly  functionprophage A and 4B.s φ12-5) C. The prB. subtilis BEST7003 genome . Because the attPφ3T sequence is conserved between attLφ3T and attRφ3T, its presence was determined by comparison of DNA sequences before (attLφ3T and attRφ3T) and after (attBφ3T) excision of φ3T. These analyses showed that φ3T excision combines the 5′-kamA and kamA-3′ in frame during sporulation (int gene and its cognate rdf gene as components of the SSR unit of φ3T (attLφ3T-rdfφ3T-intφ3T-attRφ3T) by replacing the native promoter of each gene with the IPTG-inducible promoter Pspac. In both intφ3T- (ESI-φ3T) and rdfφ3T- (ESR-φ3T) inducible strains, no excision was detected in the absence of IPTG, neither following MMC treatment nor during sporulation , rdfφ3T-lacZ (in ESR-φ3T), and kamA-lacZ (in INDkamA) were constructed and analyzed. During vegetative growth and sporulation, intφ3T-lacZ was constitutively expressed. Yet, rdfφ3T-lacZ and kamA-lacZ were expressed concomitantly 2 h after the initiation of sporulation, indicating that rdfφ3T controls the timing of φ3T excision . Therefore, acquisition of individual SSR units is a key factor driving MGE evolution. Many different types of SSR units have been found in bacterial genomes. Each SSR unit carries a gene encoding an individual member of the integrase family (either a Tyr- or a Ser-type enzyme) that recognizes cognate attP and attB sites with high selectivity. Some site-specific recombination systems, including P1 (Cre), Bxb1, TP901-1, R4, and φC31 integrases, have been widely used as tools to introduce foreign genes, carried, for instance, on site-specific integration plasmids, in a range of organisms, including other microbes, plants, and mammalian cells  can be viewed as highly adaptable phage components. By extension, adaptability of SSR units may constitute an important factor modulating plasticity among MGEs in their interaction with host genomes.In bacteria, mobilization of lysogenic phages and ICEs consists of a series of successive steps, starting with excision, followed by intercellular transfer and finally integration of the genetic material into new host cells. Excision and integration are mediated by SSR units , expression of the RDF (xisBs1ICE) is repressed by ImmR, whose degradation by ImmA is dependent on SOS and cell density signals (sprB (the gene encoding the RDF of SPβ) immediately downstream of Pxis to ensure control by the immR-immA system. As a result, excision of the chimeric SPβBs1ICE prophage in the lysogen was detected following addition of MMC and induction of the SOS response, thus showing that regulation by the immR-immA module can be co-opted for prophage excision. This result agrees with a previous report that the B. subtilis lysogenic phage φ105 relied on a similar immR-immA system . After excision, however, int and rdf are only separated by the attP locus. In this circular genome state, RecA-mediated homologous recombination could promote exchange of int-attP-rdf cassettes between MGEs, especially between circular phage genomes. The presence of several lysogenic phages (SPβ and φ3T) in a single strain further supports our hypothesis that SSR units are transferable between MGEs . Thus, although the degradation of a prophage causes defectiveness, defective prophages can turn back into lysogenic phages by addition of SSR units (and possibly adjacent genes) under natural conditions   . We haverulation .Although we identified closely related prophages with distinct SSR unit, such ICEs were not found in genomic databases. These data were results based on the basic local alignment search tool (BLAST) analysis utilizing the database from the national center for biotechnology information (NCBI). To clarify the detailed SSR unit distribution, more advanced bioinformatics approaches would be required.All methods can be found in the accompanying