SMARCB1-deficient sinonasal carcinoma (SDSC) is an aggressive subtype of head and neck cancers that has a poor prognosis despite multimodal therapy. We present a unique case with next generation sequencing data of a patient who had SDSC with perineural invasion to the trigeminal nerve that progressed to a brain metastasis and eventually leptomeningeal spread.A 42 year old female presented with facial pain and had resection of a tumor along the V2 division of the trigeminal nerve on the right. She underwent adjuvant stereotactic radiation. She developed further neurological symptoms and imaging demonstrated the tumor had infiltrated into the cavernous sinus as well as intradurally. She had surgical resection for removal of her brain metastasis and decompression of the cavernous sinus. Following her second surgery, she had adjuvant radiation and chemotherapy. Several months later she had quadriparesis and imaging was consistent with leptomeningeal spread. She underwent palliative radiation and ultimately transitioned quickly to comfort care and expired. Overall survival from time of diagnosis was 13 months. Next generation sequencing was carried out on her primary tumor and brain metastasis. The brain metastatic tissue had an increased tumor mutational burden in comparison to the primary.This is the first report of SDSC with perineural invasion progressing to leptomeningeal carcinomatosis. Continued next generation sequencing of the primary and metastatic tissue by clinicians is encouraged toprovide further insights into metastatic progression of rare solid tumors.The online version of this article (10.1186/s12885-019-6043-0) contains supplementary material, which is available to authorized users. Sinonasal carcinomas have recently gained increased interest for their histogenetically diverse characteristics brought to light by advances in molecular genetics . These aSDSC is a rare, often fatal tumor characterized by distinct inactivating alternations of the tumor suppressor gene SMARCB1 located on chromosome 22q11.2, basaloid/rhabdoid differentiation, and histologic loss of INI1 expression . With feA 42 year old female presented with a right sided headache with associated right sided facial pain. A Computed Tomography (CT) scan showed a mass on the V2 division of the trigeminal nerve Fig. a. A maxiA few months after the GKSRS, the patient developed a right cranial nerve 6 palsy and severe facial pain refractory to medications. Interval imaging revealed an increase in the mass with extension along the trigeminal nerve into the cavernous sinus Fig. b and an DNA and RNA sequencing were performed on two patient’s tumor specimens, one from right temporal lobe of the brain, and the other from the primary sinonasal tumor, using the xT Laboratory Developed Test at Tempus’ Clinical Laboratory Improvement Amendments/College of American Pathologists-accredited laboratory in Chicago, IL. Tumor DNA was extracted from tumor tissue sections with tumor cellularity higher than 20% and proteinase K digested. Total nucleic acid extraction is performed with a Chemagic360 instrument using a source-specific magnetic bead protocol. Total nucleic acid is utilized for DNA library construction, while RNA is further purified by DNaseI digestion and magnetic bead purification. The nucleic acid is quantified by a Quant-iT picogreen dsDNA reagent Kit or Quant-iT Ribogreen RNA Kit (Life Technologies), and quality is confirmed using a LabChip GX Touch HT Genomic DNA Reagent Kit or LabChip RNA High HT Pico Sensitivity Reagent Kit (PerkinElmer).For the DNA library construction, one hundred nanograms of DNA for each tumor and normal sample was mechanically sheared to an average size of 200 base pairs using a Covaris ultrasonicator. The libraries were prepared using the KAPA Hyper Prep Kit. Briefly, DNA underwent enzymatic end-repair and A-tailing, followed by adapter ligation, bead-based size selection, and PCR. After library preparation, each sample was hybridized to a custom designed probe set. Recovery and washing of captured targets was performed using the SeqCap hybridization and wash kit. The captured DNA targets were amplified using the KAPA HiFi HotStart ReadyMix. The amplified target-captured libraries were sequenced on an Illumina HiSeq 4000 System utilizing patterned flow cell technology.Generated reads were aligned to the human reference genome (hg19) using BWA aligner , and subRNA Library construction. One hundred nanograms of RNA per tumor sample was fragmented with heat in the presence of magnesium to an average size of 200 base pairs. The RNA then underwent first strand cDNA synthesis using random primers, followed by combined second strand synthesis and A-tailing, adapter ligation, bead-based cleanup, and library amplification. After library preparation, samples were hybridized with the IDT xGEN Exome Research Panel. Target recovery was performed using Streptavidin-coated beads, followed by amplification using the KAPA HiFi Library Amplification Kit. The RNA libraries were sequenced to obtain approximately 65 million reads on an Illumina HiSeq 4000 System utilizing patterned flow cell technology.From RNA sequencing variant calling, the best practices published by the Broad Institute were followed. Namely, after a double pass alignment step with Star aligner  to the hThe patient’s primary tumor and brain metastasis underwent next generation sequencing. We found over 500 variant calls on both samples that were screened and selected based on their mapping quality and coverage (>20x). Due to the rarity of this malignancy, we overlapped those selected variants to the mutations reported on The Cancer Genome Atlas (TCGA) using ActiveDriverDB database. We reported calls that were up to three amino acids away from the variant location and were implicated in changes in protein signaling or posttranslational modification in any malignancy Fig. .Fig. 6SThree mutations matched TCGA cases of head and neck squamous cell carcinoma (HNSC). Namely, glutamate metabotropic receptor 3 (GRM3 D279E), ret. proto-oncogene (RET T946A), and BCR (BCR K724 N), but those mutations only appeared on the sinonasal sample. We also identified a mutation on the marker of proliferation Ki 67 MIK67 A1218T) found on brain lower grade glioma only on the sinonasal sample. Additional file 218T founCommon variants overlapping TCGA mutations on the sinonasal and brain samples were found primarily on the filaggrin (FLG) gene, notably a missense mutation on FLG R1469H for lung squamous cell carcinoma, and another on FLG R1469L for colon adenocarcinoma. Moreover, other cancer types shown mutations close by the SNP rs768563961: skin cutaneous melanoma, lung adenocarcinoma, rectum adenocarcinoma and glioblastoma multiform. Table Brain variants overlapping TCGA mutations were considerably less than the sinonasal counterpart. There was one variant on major histocompatibility complex I gene HLA-C with one case of lung adenocarcinoma (HLA-C G276 V) and another mutation found on colon adenocarcinoma (HLA-C G276R). Another variant was found on proto-oncogene MAF  close to a reported mutation on stomach adenocarcinoma (MAF V286A). Finally, on the Aurora kinase gene AURKB (rs101079278) a close mutation was reported on bladder urothelial carcinoma (AURKB S37Y).A variant found on the apoptotic activator BCL2L11 (rs762818079) in a case of uterine corpus endometrial carcinoma shows post transcriptional modifications of the binding sequence motif for the kinases AKT1, MAPK10, MAPK8, and MAPK9.Gene expression data demonstrated that ten genes  had at least a one log fold change in decreased expression when comparing the brain sample to the primary sample. All other genes were all less than one log fold change in expression  on the SNP rs5751602 at the RNA level. Another place that shows LOH is at rs140855897 (deletion in the brain specimen) in the major histocompatibility complex I gene HLA-A. Also, the immune system genes HLA-A and HLA-C were both highly upregulated on the sinonasal sample. Interestingly, in both samples immunohistochemistry demonstrated less than 1% PD-L1 expression in both tumor cells and tumor associated immune cells.We report a rare case of a patient with SMARC-B1 deficient tumor with perineural spread that progressed to develop into an intradural metastasis. Following treatment failure, the patient eventually developed leptomeningeal spread. Previous studies have reported the presence of brain metastasis from this tumor in 3 patients but only one patient has been previously reported as having concordant perineural spread and intradural disease, however no sequencing data was reported , 17. TheTo our knowledge, this is the first report that sequencing data has been reported for the primary SMARCB1 tumor with perineural spread and a matched brain metastasis. Previous whole exome sequencing of primary tumors and their accompanying brain metastasis demonstrated the potential to identify actionable alterations . With thIn other rhabdoid tumors, such as atypical teratoid/rhabdoid tumors (AT/RT) of the CNS, loss of INI1 is associated with overexpression of cell-cycle regulatory protein cyclin D1, leading to cell cycle progression and untethered cellular proliferation , 20. IntHistopathological diagnosis of this tumor can be complex as a number of malignant neoplasms can show similar histologic features and rhabdoid morphology in this location and should be considered in the differential diagnosis. ATRT has been reported in the sellar region of adults but these tumors were confined to the intracranial compartment and did not appear to predominantly involve the skull base and sinonasal regions . Given tThe progression of this patient’s tumor to leptomeningeal spread is an extremely rare occurrence. For cutaneous carcinomas, perineural invasion progressing to leptomeningeal spread has been previously reported . HoweverAdditional file 1:List of DNA mutations identified in each sample. (XLSX 22 kb)Additional file 2:Quantitative Gene Expression differences between samples. (XLSX 10 kb)Additional file 3:List of RNA mutational variants identified in each sample. (XLSX 13 kb)


RNA viruses are highly successful pathogens and are the causative agents for many important diseases. To fully understand the replication of these viruses it is necessary to address the roles of both positive-strand RNA ((+)RNA) and negative-strand RNA ((−)RNA), and their interplay with viral and host proteins. Here we used branched DNA (bDNA) fluorescence in situ hybridization (FISH) to stain both the abundant (+)RNA and the far less abundant (−)RNA in both hepatitis C virus (HCV)- and Zika virus-infected cells, and combined these analyses with visualization of viral proteins through confocal imaging. We were able to phenotypically examine HCV-infected cells in the presence of uninfected cells and revealed the effect of direct-acting antivirals on HCV (+)RNA, (−)RNA, and protein, within hours of commencing treatment. Herein, we demonstrate that bDNA FISH is a powerful tool for the study of RNA viruses that can provide insights into drug efficacy and mechanism of action. Positive-strand RNA, (+)RNA, viruses include many human pathogens, such as the families picornaviridae , togaviFollowing infection of a target cell, the viral RNA first serves as mRNA, exploiting the host-cell translational machinery to direct synthesis of the viral proteins; these proteins include enzymes that are responsible for synthesis of first the negative, then the positive strand of the genome, and proteins that modify the environment of the cell to support viral replication. Positive-strand RNA viruses replicate their genomes in the cytoplasm of infected cells, in association with virus-induced membrane structures, often termed the “membranous web” . These mWhile there are thousands of antibodies available to specifically identify viral and cellular proteins, and sufficient fluorescent tags to allow co-visualization of multiple proteins in a single sample, these imaging approaches are often incompatible with conventional methods for visualization of nucleic acids . Branched DNA (bDNA) in situ hybridization is a technique that exploits sequence specific probes, and branching preamplifier and amplifier DNAs, to produce an intense localized signal . Unlike Similar to the integrated provirus of retroviruses, the (−)RNA of (+)RNA viruses is present in low amounts relative to the (+)RNA ,28,29,30Huh-7.5.1 cells are derived from the human hepatoma 7 cell line, and have been previously described . Cells wDaclatasvir  and Danoprevir  were purchased from Selleckchem. Ledipasvir  was purchased from MedChem Express. Sofosbuvir  was purchased from Acme Bioscience. Mouse monoclonal primary antibody 9E10 specific for NS5A was used as previously described . Mouse mTM reverse transcriptase (Invitrogen) was added to the denatured RNA template and incubated at 60 °C for 1 h, followed by RNase H treatment for 20 min at 37 °C. Reverse transcribed cDNA was mixed with RC1 (5′-GCCTAGCCATGGCGTTAGTA-3′) and RC21 primers for positive strand amplification and tag (5′-ggccgtcatggtggcgaataa-3′) and RC21 primers for negative strand amplification. Amplification was conducted by denaturation at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 15 s and annealing/extension at 60 °C for 1 min using PerfeCTa SYBR Green FastMix . Amplification was carried out in an Applied Biosystems®® 7500 Fast Real-Time PCR Instrument (ABI). In vitro transcribed RNA from the HCV infectious clone was used to generate a standard curve.The initial reverse transcription step of the HCV 5′ UTR was carried out as previously described . BrieflyZIKV RT-qPCR was performed using the same general approach, with the following differences. RC21 and RC1 were replaced by Tag-ZK21 primer (5′-ggccgtcatggtggcgaataaCCTGACAACACTAAaATTGGTGC-3′) and Tag-ZK1 primer (5′-ggccgtcatggtggcgaataaAGGATCATAGGTGATGAAGAAAAGT-3′). cDNA synthesis was performed using SuperScript III First-Strand Synthesis System (Invitrogen), following the manufacturers’ instructions. qPCR amplification was conducted using PowerUp SYBR Green Master Mix , in a PikoReal 96 Real-Time PCR system (ThermoFisher Scientific). The cycle conditions were uracil-DNA glycosylase (UDG) activation at 50 °C for 2 min, dual-lock DNA polymerase at 95 °C for 2 min, followed by 40 cycles of denaturation at 95 °C for 15 sec, annealing at 55 °C for 15 sec, and extension at 72 °C for 1 min. An MR766 infectious clone  was usedbDNA FISH for cultured adherent cells was used for HCV RNA detection using the RNAscope method, with some modifications . Cells wZIKV-infected cells were stained by the same protocol as HCV, with the following changes: the protease pre-treatment used a 1:2 dilution, rather than 1:15; and the ZIKV-specific probes were V-ZIKA-pp-O2 for the (+)RNA  and V-ZIKA-pp-O2-sense-C2 for the (−)RNA .In order to quantify the differential drug effects on (+) and (−) strands of HCV RNA, we manually acquired 30 images of each biological replicate drug treatment experiment and performed cellular analysis and image analysis using BioTek Gen5 software. Abundance of (+) and (−)RNA was quantified and plotted in two ways: number of fluorescent foci per infected cell, and fluorescence intensity per infected cell. ZIKV staining was performed similarly, with an additional calculation of the area of (−)RNA foci.Graphs were plotted using Microsoft Excel. For significance testing, GraphPad Prism 6 was used to perform one-way ANOVA with Dunnett’s post-test for multiple comparisons, comparing each test condition to a control sample, or Tukey’s post-test for multiple comparisons comparing all conditions to one another.As proof of concept for the labelling of (+)RNA and (−)RNA, we chose to use HCV-infected cells, as HCV is an important human pathogen and one of the most extensively studied (+)RNA viruses. Furthermore, in their initial description of the RNAscope method, Wang and colleagues showed the labeling of the HCV (+)RNA, co-labeled with a probe against 18S rRNA [In the course of developing the sequential RNA staining protocol we performed similar staining with ZIKV-infected cells . We wereFollowing establishment of protocols for sequential staining of (−)RNA and (+)RNA, in virus harboring cells, we additionally stained protein for simultaneous imaging. HCV-infected Huh-7.5.1 cells were stained for RNA and NS5A or core proteins . During 50). Cells were fixed and examined 8 h after the addition of inhibitors . Two types of analyses were performed, quantifying either the number of fluorescent foci per cell, or the fluorescence intensity per cell, for both RNA strands C,D. ThesStudying the (−)RNA of (+)RNA viruses is particularly challenging, owing to its relatively low abundance in infected cells ,28,29, aIn the current study, we specifically labeled the (−)RNA of HCV, both in singly and dual labeled infected cells. In addition, we performed cell-level quantitation of the two RNA species, and compare this imaging-based quantitation to RT-qPCR; we found an approximately five-fold excess of the HCV (+)RNA by both techniques, consistent with a previous report that used RT-qPCR .In contrast with HCV, ZIKV (+)RNA was so abundant in infected cells that it was not possible to count foci, making direct comparisons to the abundance of (−)RNA unreliable. We also found that the ZIKV (−)RNA required more rigorous pre-treatment with protease than HCV (−)RNA to permit FISH staining. We assume this reflects differences in the nature of the RC, the amount of protein bound, and accessibility to protease treatment; these properties very likely vary extensively between viruses. Nevertheless, both bDNA FISH and strand-specific RT-qPCR approaches revealed a large excess of (+)RNA over (−)RNA in Zika virus-infected cells, comparable to a previous estimate using another flavivirus, the West Nile virus . CollectIt was striking that our ability to detect colocalized RNA strands was extremely limited. In the case of ZIKV we saw very few (−)RNA strands by bDNA FISH, while in HCV harboring cells, (−)RNA was reasonably abundant, but rarely colocalized with the (+)RNA signal. As the replication of the viruses requires transcription of one strand from the other, we would anticipate a certain proportion of the (+)RNA and (−)RNA signals should colocalize. While we have no single definitive explanation for the lack of colocalized RNA molecules, there are potential explanations. In the Zika imaging, we found that it was necessary to increase the concentration of protease. This may indicate that the (−)RNA, in particular, is sequestered and unavailable for labeling, potentially due to extensive interactions with the RC. A related potential contributing factor is the extremely high melting temperature reported for long double-stranded RNA ,53,54. TAn area of particular importance in antiviral research is the mechanism of action of DAAs, understanding their impact on virus replication and their role in therapeutic regimens. A strength of this imaging-based technique is to permit analysis of viral RNA and protein in the same cell, and to select those cells desired for analysis. All DAAs will prevent the spread of infection over time, irrespective of their specific mechanism, so long as they inhibit some part of the replication cycle. In contrast to other methods, such as RT-qPCR or Western/Northern blot analyses, which can phenotypically characterize only mixtures of cell populations, the use of an imaging-based approach allows us to specifically examine cells that are already infected, and determine how different DAAs influence on-going viral replication. Recently, we used another imaging-based strategy to assess the effects of DAAs against HCV by following NS5A staining . SimilarAs a solution to the difficulties in counting discrete RNA foci, we used an alternative form of quantification, measuring the total fluorescence in the field of view, expressed per cell. This greatly improved the consistency of the data, and confirmed that the NS5A inhibitors were particularly effective at rapidly inhibiting HCV, as we have previously reported . The lacIt has been previously reported that NS5A inhibitors suppress levels of (−)RNA to a greater degree than the levels of (+)RNA . In our Collectively our data demonstrate the utility of branched DNA in situ hybridization as a tool to help address questions relating to the biology of RNA viruses. The high sensitivity of bDNA FISH allows sensitive visualization of incoming viral RNA, before any amplification has taken place, and early stages of viral genome replication. The ability to select specific cell populations for analysis enables in-depth investigation of viral RNA strand-specific effects, localization, and interactions with proteins, owing to the compatibility with immunofluorescence. Thus, this approach may be particularly suitable, not only to the study of drug mechanisms of action, but also to studies involving over-expression or silencing of host cell-factors. The high specificity and sensitivity of staining offered by these methods should help illuminate the critical role of (−)RNA, until now a concealed player in the biology of (+)RNA viruses.


The purity of the RNA was higher in soft vial tubes than in rigid vials, as measured by A260/280 and A260/230 ratios. The RNA concentration, purity, and GAPDH density of groups 1, 2 and 3 were significantly higher than those of groups 4 and 5. Moreover, E6 density of group 1 and 2 was significantly higher than that of group 3, 4 and 5. The use of soft tubes enhanced the mRNA quantity and quality in cervico-vaginal cytology. The products of mRNA extraction using RNAlater RNA Stabilization Reagent and RNAprotect Cell Reagent at room temperature were comparable to those obtained by conventional frozen storage. Our protocol improved the yield and quality of RNA and might produce better results for molecular analysis in cervico-vaginal cytology.The separation of exfoliated cells from the brushes used during cervico-vaginal smears is difficult, a problem which may affect the quality of ribonucleic acid (RNA) extracted. We compared the results of RNA extraction from cervico-vaginal cytology samples according to the type of tubes, preservative solutions, and storage temperature. The samples included exfoliated cervico-vaginal cytological specimens from patients with human papilloma virus 16, positive for cervical intraepithelial neoplasia or cervical cancer. Exfoliated cells were obtained by shaking a brush in a conventional rigid vial tube or squeezing the brush in a soft vial tube. RNA quantity and quality were compared between the two tubes. The concentration and purity of RNA (A260/A280 and A260/A230 ratios) was compared amongst five groups: Group 1, standard frozen storage; Group 2–4, RNA stabilization reagents with room temperature ; and Group 5, Surepath Preservative fluid. To demonstrate the utility of the extracted RNA for PCR-based cDNA synthesis, GAPDH and E6 were targeted and gel band densities of GAPDH and E6 were measured. The median RNA concentration was significantly higher in the soft tubes compared with the rigid tubes (100.2 vs. 7.1 ng/μL,  Exfoliated cytology has been proposed as a method which can provide information about epithelial cells such as screening for precancerous lesions or RNA expression . CytologPrevious studies have demonstrated the important role of long non-coding RNAs, circular RNAs, microRNAs and mRNAs in cervical cancer development and progression –4. High-The separation of exfoliated cells from the brush is important for RNA extraction. Because most RNA stabilization reagents are sticky, and the tube is rigid, separation of the exfoliated cells from the brush is difficult. Traditionally, RNA is stored at − 20 °C, − 80 °C or in liquid nitrogen to provide protection from degradative reactions. However, shipping and storage of frozen RNA is expensive, requires special handling and Ultra-Low-Temperature freezers, and is time sensitive. The extraction of DNA and RNA from liquid-based cytology specimens has previously been studied , 7.To the best of our knowledge, comparison of mRNA extraction methods for molecular analysis of cervico-vaginal cytology samples has not been reported. Therefore, the aim of this study was 1) to compare methods of RNA extraction of cervico-vaginal cytology as they relate to the type of tubes, preservative solutions, and storage temperature and 2) to establish the best technique for molecular tests such as real-time reverse transcriptase reaction (qRT PCR) or RNA-seq at room temperature.After obtaining approval from the Institutional Review Board of our institution, and informed written consent from the subjects, cervico-vaginal smears were collected using pap brush-lines  in patients with HPV 16-positive cervical intraepithelial neoplasia or cervical cancer. The brush was placed into the cervix and rotated 360 degrees in a clockwise direction. The samples were transported in DNase, RNase and pyrogenic free tubes. To obtain the cells from the cytobrush, the cytobrush was shaken in rigid tube, or the tube and brush were squeezed in a soft tube . Samples from Group 3 were kept in RNAprotect cell Reagent  and those from Group 4 were stored in the AllProtect Tissue Reagent . Samples from Group 5 were kept in BD SurePath™ Preservative Fluid . Samples from Groups 1 and 5 were stored at − 20 °C for 7 days, and those from Groups 2, 3, and 4 were stored at room temperature for 7 days. We compared the RNA concentration and quality of each of the five types of samples for each patient using previously described methods. cDNA was synthesized from mRNA using TOPscript™ cDNA Synthesis Kits  according to the manufacturer’s protocol.Reverse Transcription Polymerase Chain Reaction (RT PCR) was performed to compare the expression of human housekeeping genes and human Papillomavirus E6 gene in each sample. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primers were used to detect human housekeeping genes, and E6 primers were used to detect HPV E6 genes. Primer sequences are described in Additional file PCR temperature cycling for GAPDH was as follows: pre-heating for 5 min at 95 °C, followed by 33 cycles of 30 s at 95 °C, 30 s at 58 °C, 25 s at 72 °C, and a final extension of 5 min at 72 °C. PCR conditions for the E6 region of the HPV primer set were as follows: pre-heating for 5 min at 95 °C, followed by 33 cycles of 30 s at 95 °C, 30 s at 57 °C, 30 s at 72 °C, and a final extension of 5 min at 72 °C. After confirming the GAPDH PCR results, E6 PCR was performed by selecting 6 samples with high GAPDH expression levels. All PCR amplifications were performed with appropriate positive and negative controls. The PCR products of the GAPDH and E6 were separated by electrophoresis on 1.8% agarose gels. The electrophoresis images of PCR products were analyzed and quantified using ImageJ, an open source image processing program designed for the analysis of scientific images.post-hoc tests were performed using Student-Newman-Keuls. Spearman’s correlation analysis was used to clarify the relationships between gene expression and RNA quantity and quality. A p value of < 0.05 was considered statistically significant.Statistical analysis was performed using SPSS version 21.0 software . Differences between subsets were evaluated using Student’s t-tests and Mann-Whitney tests, and differences between proportions were compared using chi-squared tests. ANOVA was used to compare the mean of each value among the groups and p = 0.0209). 260/280 ratios of between 1.8 and 2.0 were observed for all four samples in soft tubes, but for only one sample in rigid tubes. 260/230 ratios of between 1.8 and 2.2 were observed for three samples in soft tubes, but for none in rigid tubes  than in rigid tubes . The mean 260/280 ratio values were similar among the five groups ; however, 81.8% of Groups 1, 2 and 3, 9.1% of Group 4 and 50.0% of Group 5 had a260/280 ratio between 1.8 and 2.0 (p = 0.003). The mean 260/230 ratio values of Group 1 (1.24 ± 0.93 nm), Group 2 (1.35 ± 0.60 nm) and Group 3 (1.26 ± 0.70 nm) were significantly higher than those of Group 4 (0.07 ± 0.10 nm) and Group 5 . Moreover, 45.5% of Groups 1 and 2, 27.3% of Group 3, and 0% of Groups 4 and 5 had a 260/230 ratio between 1.8 and 2.2 , Group 2 (52.58 ± 71.42 ng/μL) and Group 3 (63.76 ± 57.35 ng/μL) were significantly higher than those of Groups 4 (2.11 ± 3.38 ng/μL) and 5 . The mean E6 densities of Group 1  and Group 2  were significantly higher than those of Group 3 (9593.98 ± 8535.84), Group 4 (2273.92 ± 1423.42) and Group 5 , Group 2 , and Group 3  were significantly higher than those of Group 4  and Group 5 (8633.20 ± 20,804.91; F = 17.682; This study provides a comparative analysis of the mRNA quantity and quality of cervico-vaginal cytology samples as affected by tube type, storage temperature, and preservative solution. Soft tubes enhanced the mRNA quantity and quality compared to the conventional rigid tubes usually used in cervico-vaginal cytology. We developed a novel method to separate exfoliated cells from cervico-vaginal smears from the brush using soft, elastic tubes. We could separate the exfoliated cells from the brush by squeezing the soft tube. The quantity and quality of the mRNA extracted using the soft tube was excellent compared to that obtained from the conventional rigid tubes in this study. The productivities of GAPDH mRNA extraction using RNAlater RNA Stabilization Reagent and RNAprotect Cell Reagent, and that of HPV 16 E6 using RNAlater RNA Stabilization Reagent at room temperature were comparable to those of conventional frozen samples.RNA samples obtained by cytology are partially degraded, leading to low levels of transcript detection . RNases Previous studies have demonstrated that RNA can be extracted from cervical cell lines and cytology specimens can be stored in BD Surepath™ preservative fluid, and the RNA is suitable for RT PCR . In our To the best of our knowledge, this is the first study into enhancing RNA extraction methods for cervico-vaginal cytology samples according to tube types, storage temperatures, and preservative solutions. By squeezing the soft tube and brush, we could acquire more cells from the brushes, and hence could enhance the quality and quantity of RNA extracted, compared to conventional rigid tubes. Moreover, room temperature storage in RNAlater RNA Stabilization Reagent and RNAprotect cell Reagent was comparable to conventional frozen storage in terms of RNA quantity and quality. Our improved RNA extraction method may enhance the quantity and quality of RNA cervico-vaginal cytology samples and contribute to successful molecular studies using cervico-vaginal cells and other exfoliated cell.Additional file 1: Table S1. Primer sets used for Polymerase Chain Reaction of GAPDH and E6 genes.


New analytics of post-transcriptional RNA modifications have paved the way for a tremendous upswing of the biological and biomedical research in this field. This especially applies to methods that included RNA-Seq techniques, and which typically result in what is termed global scale modification mapping. In this process, positions inside a cell’s transcriptome are receiving a status of potential modification sites , typically based on a score of some kind that issues from the particular method applied. The resulting data are thought to represent information that goes beyond what is contained in typical transcriptome data, and hence the field has taken to use the term “epitranscriptome”. Due to the high rate of newly published mapping techniques, a significant number of chemically distinct RNA modifications have become amenable to mapping, albeit with variegated accuracy and precision, depending on the nature of the technique. This review gives a brief overview of known techniques, and how they were applied to modification calling. Precise and reliable detection of the numerous modified nucleotides in RNA is still a highly challenging task in the epitranscriptomics field. In addition to traditional detection and quantification strategies, including various types of chromatography such as TLC, HPLC, and/or LC-MS(MS), approaches relying on deep sequencing have emerged in force, and numerous applications to large transcriptomes were reported, starting in 2012 ,4,5, wit6A-Ab-based enrichment dates back to the 1980s [6A-methylomes by MeRIP-Seq immunocapturing and sequencing [6A antibodies, mapping of this modification, especially in mRNA, is arguably the most frequent and visible application in the field. The initial embodiments of MeRIP-Seq had only low resolution and provided broad peaks of ~100–200 nt in which the actual m6A modification site was supposed to be contained. Further improvements of this technique came from the combination of MeRIP with Ab cross-linking, allowing single nucleotide resolution. The latter technique is a specialized derivative of CLIP, termed miCLIP , plausiLAIC-Seq , where b6A position or at the +1 site [6Am, which is frequently present at m7G-mRNA cap, was detected in the form of a mutational signature [6A-Seq) used s4U incorporation for PhotoActivatable-Ribonucleoside-Enhanced Cross-Linking and Immunoprecipitation -stops and/or misincorporations, which occur as a consequence of the cross-link events either at the m +1 site ,23. SimiAR-CLIP, ). 3C, m1A, m1I, m22G, and m1G sites in human tRNAs, of which a selection of predicted m3C sites were experimentally validated. Modifications in an RNA template are known to potentially cause reverse transcriptases to either arrest, to incorporate a dNTP cognate to the unmodified template, or to misincorporate non-Watson-Crick compatible dNTPs into the nascent cDNA. The combination of these events constitutes the so-called RT-signature, which varies with the type of modification, and likely with reaction conditions including the type of enzyme used . One of 1A detection in rRNA and tRNAs [The idea of a specific RT-signature was developed in application to mnd tRNAs ,28. It w1A-generated RT-signatures was extended to analysis of the human transcriptome [1A-induced RT-signature, but this level can be increased by prior MeRIP type enrichment with anti-m1A-Ab, indicating that modification levels at the relevant sites were most probably sub-stoichiometric.More recently, the analysis of mcriptome . Non-enrThe detection of modifications via their RT-signature can only be applied to a limited subset of known RNA modifications, since many do not affect Watson-Crick base pairing during cDNA synthesis and thus are RT-silent. Interestingly, for some presumed RT-silent modifications, particular conditions were identified in which they become visible ,30. For 6A  was shown to efficiently and selectively de-methylate metection .5C is thought to increase the electron density of the nucleobase, thus leading to repulsion of the nucleophile and thus inertness of m5C to deamination under these specific conditions. Consequently, the readout consists in detection of cytidines in deep seq data sets, and is therefore, in principle, well suited for quantification and single nucleotide resolution mapping. The method is generally considered robust for abundant RNAs such as tRNA and rRNA [The property of naturally occurring RNA modifications to affect the performance of RT enzymes during cDNA synthesis in terms of misincorporation or RT-arrest can be modulated by chemical treatment prior to the RT reaction. Various reagents are known to either selectively react with modified nucleosides, or to selectively affect non-modified nucleotides. An example for the latter is bisulfite sequencing, which was adapted from 5mC detection in DNA to application in RNA by Schäfer and Lyko . The bisand rRNA ,41,42,43and rRNA ,44,45, aand rRNA .5C were developed, which were, again, first applied on the respective DNA modifications. While hm5C yields signals that are indistinguishable from m5C in bisulfite sequencing, f5C remains invisible. However, the two species can be converted into each other by mild redox agents: f5C can be reduced to hm5C by NaBH4 , or be xylamine , applicaxylamine . Permutasequence , and the4U displays a certain percentage of misincorporation resulting in an U- > C transition in the cDNA. SLAM-seq [4U after alkylation with iodoacetamide (ICH2-CONH2), which causes s4U to be retrotrancribed as a C, increasing the apparent transition rate to near-quantitative carbodiimide metho-p-toluenesulfonate, usually abbreviated as CMCT) [In contrast, detection of pseudouridine must, so far, entirely rely on chemical treatment for the generation of RT-stops, as the nucleoside itself leaves no exploitable RT-signature. To this end, several labs have performed mapping experiments using soluble carbodiimide (as CMCT) ,56,57,58as CMCT)  for mappas CMCT) ,61. Openas CMCT) , potentias CMCT) .1A, which was already mentioned [1A, whose product m6A does not display any significant signature. This reaction was used as a means of validation, whereby the RT-signature of m1A was altered by incubation at high temperature under alkaline conditions, leading to the m1A- > m6A conversion [1A signals would be expected to coincide with the appearance of an m6A signal in, e.g., MeRIP-Seq data. Since the alkaline treatment causes significant RNA degradation, and because m6A detection is still inaccurate, applications of the Dimroth rearrangement so far have been limited to validation of predicted m1A sites, rather than for their global profiling.The reverse transcription signature of an RNA modification is, in principle, understood to also contain abortive RT events resulting in truncated cDNA, for which library preparation protocols must be specifically tailored. The composite nature of the RT-signature of mentioned , can be nversion ,17. The 7G with NaBH4 renders the corresponding site in the RNA chain susceptible to aniline induced cleavage, which was then analyzed as RT-stop in RNA-Seq data in a combination termed TRAC-seq [One of the oldest combination of chemical treatments known to specifically react with a modified nucleotide ,64 has oTRAC-seq .4C with NaBH4, leading to saturation of the 5,6 double-bond in acetylated cytidines. This, in turn, produced ~20–30% of misincorporation signals in cDNA, as determined by Sanger sequencing [A chemically related treatment is the reduction of acquencing . This mequencing .Variations of the so-called RiboMethSeq approach, first published by the Nielsen lab and further independently developed by two other groups ,68,69,704 oxidation, under conditions where the unmodified cis-diol of a normal ribose is rapidly oxidized and cleaved, thus making it inaccessible to 3′-adapter ligation. In this protocol, RNA was randomly cleaved by nuclease to release 5′-phosphates and 3′-riboses, and fragments were subjected to multiple rounds of a sequence consisting of NaIO4 oxidation treatments, subsequent removal of the 3′-end nucleotide, and de-phosphorylation. After several repetitions, an accumulation of Nm at the 3′-terminal position was to be expected. Positive enrichment of 3′-Nm-containing fragments, and thus improvement of Signal/noise ratio, is a very valuable feature for transcriptome-wide RNA analysis. However, the Nm-Seq protocol with repetitive oxidation/cleavage/de-phosphorylation cycles requires considerable amounts of input RNA, due to losses during such treatment.A novel trend in the field is the development of library preparation protocols which selectively produce RNA fragments with modified nucleotides at the 5′- or 3′-end extremity, properties which can then be exploited for enrichment of these fragments in RNA Seq libraries. Two recently developed methods, Nm-Seq  and RibO7G and m3C, ultimately led to a positive selection of fragments resulting from modifications, and thus to exquisite sensitivity, because large populations of irrelevant RNA sequences were excluded from the library. This procedure, which was also shown to be partially sensitive to D and ho5C residues, thus required very limited amounts of input RNA and showed exceptional selectivity and specificity because of extremely low background.Another recently established technique, named AlkAnilineSeq , relies The intrinsic reactivity of RNA (or DNA) abasic sites can be explored for detection of RNA modifications. For DNA, formation of abasic site is generally catalyzed by specific DNA-glycosylase, which removes damaged DNA bases, and subsequently the aldehyde group of abasic site can be chemically ligated to, e.g., a reactive amine, which in turn is coupled to biotin ,76. A si6A in mRNA, that field is in full swing, with high impact papers appearing on a weekly basis, despite the fact that comprehensive mapping of m6A at single nucleotide resolution in a quantifiable manner is still a largely unresolved problem.At first glance, this overview might convey the impression that RNA-Seq based detection methods can be successfully applied for mapping of almost any desired RNA modification throughout the transcriptome. However, while ever-new concepts for modification mapping get published, the community looks to the biology emerging, based on the methods published since 2012. Now, about six years after the furious resurrection of research on mhttps://doi.org/10.1101/271916).Detection of 2′-O-methylations in abundant RNAs like rRNA and tRNA is quite robust and is now applied even to quantitative analysis of RNA modification modulation under different conditions ,80,81, b5C and pseudouridine in mRNA are much better, papers on this topic issue from only a handful of research groups, and especially the numbers of reported modification sites have been met with skepticism [1A in mRNA, as such, is agreed upon, but the opinions on numbers differ by two orders of magnitude [In contrast, although the performance, at least on paper, of published methods for the mapping of mepticism ,62,63. Sagnitude ,19.Clearly, such controversies present a formidable obstacle for new groups to engage in targeted research on the effect of mRNA modifications. Research on the impact of defined modification sites is likely to focus on those sites identified by several labs in agreement. This strongly suggests that conservative “candidate-calling” might benefit the field. This means avoidance of false positives at the risk of overlooking some residues, i.e., producing false negatives out of caution.


Out of 756 mRNAs and 206 lncRNAs shared by enrolled patients, 135 RNAs were screened to be significantly associated with the IRI. Furthermore, combined with lncRNA‐mRNA, lncRNA‐miRNA and miRNA‐mRNA network, 51 RNAs and 131 relationship pairs were ascertained in the competing endogenous RNAs (ceRNA) network. Among these nodes, SH2D3C and GTF2H4 were significantly enriched in cellular response to stress and their interaction module were isolated from functional ceRNA network. Subsequently, their critical role was confirmed via down‐regulation of SH2D3C and GTF2H4 expression in vitro model. These results identified that lncRNA‐mRNA ceRNA network, associated significantly with IRI, functioned as critical regulative pivotal roles after PCI‐AMI, and SH2D3C and GTF2H4 may be the most responsive transcriptional regulator in the early‐phase of IRI.The study aimed to investigate time‐course transcriptomes in myocardial ischaemia reperfusion injury (IRI) via RNA‐Seq. Transcriptomes of 10 samples derived from patients with acute ST‐segment elevation myocardial infarction (ASTEMI) who were assigned to percutaneous coronary intervention (PCI), were sequenced at the time of 0 (before PCI), 2, 12, 24 and 72 hours after PCI, respectively. Using the genefilter package in  Subsequently, different expression lncRNA (DEL) and mRNA (DEM), significantly (P < 0.05) changed along with the tendency of times, were screened with the threshold of CV > 0.6. Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) functional analyses were performed for DEMs using the DAVID online tool (http://david.ncifcrf.gov/)P < 0.05. wgcna package http://www.cs.cmu.edu/~jernst/stem/) was utilized to cluster the DELs and DEMs with significant similar expression models (P < 0.05) according to RNAs expression at different time‐points. The lncRNAs‐targeted mRNAs and mRNAs were combined and the lncRNA‐mRNA regulatory network was visualized using Cytoscape 3.3  were used to store the peripheral blood samples of patients with acute ST‐segment elevation myocardial infarction (STEMI) at the time of 0 (before PCI), 2, 12, 24 and 72 hours after PCI respectively. Then the purified RNA was eluted and collected, according to the instructions of PAXgene Blood RNA kit (BD). This study protocol (Registration number: ChiCTR‐IPR‐17010501) was approved by the Ethics Committee of Guangdong Provincial Hospital of Chinese Medicine. All participants have been provided informed consents and all patients satisfied the inclusion and exclusion criteria. RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system . About 3 μg RNA per sample was used as input material for the RNA sample preparations. The products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. Then, the libraries were sequenced on an Illumina Hiseq 2500 platform and 125 bp paired‐end reads were generated. The mapped reads of each sample were assembled by both Scripture (β2)  and Cufflinks (v2.1.1)  in a reference‐based approach. Co‐efficient of variations (CVs) for samples in enrolled patients at different time‐points were calculated using genefilter    enrich in GO_BP: GO:0033554 ~ cellular response to stress (Figure About 18 997 lncRNA‐mRNA pairs and 105 188 RNA expression associated pairs were combined to select the overlapped lncRNA‐mRNA pairs; 42 pairs were selected to construct the lncRNA‐mRNA regulatory network (Figure 4Our results identified that lncRNA‐mRNA ceRNA network, associated significantly with IRI, functioned as critical regulative pivotal roles after PCI‐AMI and SH2D3C and GTF2H4 could be the most responsive transcriptional regulators in the early‐phase of IRI.The authors declare that there is no conflict of interests regarding the publication of this paper. Click here for additional data file.


Altogether, our results indicate that the use of ZFs in RBP design remains attractive even though engineering of specificity changes is challenging.Transcriptomes consist of several classes of RNA that have wide-ranging but often poorly described functions and the deregulation of which leads to numerous diseases. Engineering of functionalized RNA-binding proteins (RBPs) could therefore have many applications. Our previous studies suggested that the RanBP2-type Zinc Finger (ZF) domain is a suitable scaffold to investigate the design of single-stranded RBPs. In the present work, we have analyzed the natural sequence specificity of various members of the RanBP2-type ZF family and characterized the interaction with their target RNA. Surprisingly, our data showed that natural RanBP2-type ZFs with different RNA-binding residues exhibit a similar sequence specificity and therefore no simple recognition code can be established. Despite this finding, different discriminative abilities were observed within the family. In addition, in order to target a long RNA sequence and therefore gain in specificity, we generated a 6-ZF array by combining ZFs from the RanBP2-type family but also from different families, in an effort to achieve a wider target sequence repertoire. We showed that this chimeric protein recognizes its target sequence (20 nucleotides), both  In addition, non translated or, so-called, non-coding RNAs (ncRNAs) have emerged as important player of gene regulation and diseases with wide-ranging effects, including in tumors8 and neurological disorders11. Therefore, functionalized RNA Binding Proteins (RBPs) that are able to target a chosen RNA sequence would be valuable tools for RNA manipulation. Various protein scaffolds have been trialed for RBP engineering (reviewed in ref.12). The most promising data generated to date were obtained with the Pumilio (PUF) proteins. These proteins include eight Pumilio repeats and bind a 8-nt long single-stranded RNA (ssRNA) in a sequence-specific manner through their concave surface. Structural data has permitted the establishment of a complete recognition code for these proteins. In this code, each of the eight repetitions recognizes one base using two amino acid side chains located at specific positions in the repetitions. Specificity changes of PUF proteins can therefore be easily achieved by changing the identities of these residues. Nowadays, RBPs based on PUF domains have been successfully used to manipulate specific RNA targets in living cells15. However, despite these promising studies, PUF domains exhibit a high repetitive nature, allowing different RNA-binding modes that can lead to off-target binding16. Noticeably, the pentatricopeptide repeat (PPR) protein family is also considered as a suitable scaffold for RBP design. Recently, the endonuclease activity of the PPR protein SOT1 has been engineered to target predicted RNA substrates17. However, like PUF domains, their repetitive nature and the fact that RNA recognition is limited to one nucleotide per PPR repeat19 can also promote non-canonical RNA-binding modes that can potentially affect binding specificity20.Recent advances in genome biology have revolutionized our notion of the transcriptome. Over the last past years, RNA has proven to be a main player in eukaryotic biology in many different guises. Alterations in mRNA processing are implicated in different diseases such as Myotonic dystrophy (DM)21 and they have been successfully used to design DNA-binding proteins. Engineered DNA-binding ZFs were the first proteins used for genome editing and gene therapy22 and have been successfully transposed to human therapy23. Although, the molecular basis for ssRNA versus double-stranded DNA (dsDNA) recognition are very different, this DNA-binding ZF technology has set good precedent for the use of ZFs to manipulate nucleic acids in the context of living cells as well as full-organisms.Zinc-finger (ZF) proteins could potentially represent an attractive alternative candidate for RBP engineering since they are modular, robust to mutations25 and was initially discovered in the human splicing factor ZRANB2. This protein contains two RanBP2-type ZFs (ZF1 and ZF2) and we previously reported that each of these ZFs bind to ssRNA with a μM binding affinity and each of them was shown to target the trinucleotide sequence GGU24. The structure of the ZRANB2-ZF2:RNA complex26 , and in addition the observed binding affinity of the engineered ZF variant was much lower than that of the WT ZF for its WT GGU target28.In our case, since we wanted to target ssRNA, we have chosen a particular class of ZFs that naturally recognizes ssRNA with high affinity and specificity. This family, named the RanBP2-type ZF family, was shown to specifically bind to ssRNAx26 Fig.  has revex6 protein that combines RanBP2-type ZFs but also ZFs that belong to other families. We validated this chimeric protein using in vitro binding assays as well as a new cell-based assay that we developed in living bacteria.In the present work, we have adopted an alternative strategy in order to obtain a larger repertoire of recognized RNA sequences. Instead of engineering specificity changes, we evaluated the natural substrate specificity of various members of the RanBP2-type ZF family that, based on the ZRANB2-ZF2:RNA structure, exhibit different RNA-binding residues and, supposedly, bind to different sequences. In addition, the second main goal of this study was to show that we could engineer a functional tandem array of ZF domains in order to target a longer and therefore more specific RNA in the context of a living cell. This is why we generated a (ZF)Homo sapiens EWS ZF, (ii) the Arabidopsis thaliana ABI3-5Sup ZF, (iii) the Danio rerio ZRANB1B ZF, (iv) the Caenorhabditis elegans T0B2.5 ZF and (v) the Mus musculus RBM10 ZF x2 per liter of culture. For each protein, we examined their RNA-binding specificity using Systematic Evolution of Ligands by EXponential enrichment (SELEX)29 experiments, in which high-affinity RNA sequences were selected from a random 25 nt-long ssRNA library  the  ZF Fig. , describ30 . For GST-(EWS-ZF)x2, the identified consensus  indicated specificity of the EWS-ZF for either GGU or GGG motifs. However, we observed a higher occurrence of the GGU over the GGG trinucleotide: 69% of the selected sequences exhibited at least two GGU motifs against the 6% presenting two GGG motifs. For GST-(ABI3-5Sup-ZF)x2, 81% of the enriched sequences contained either GGA or GGAGGA motifs. The enriched consensus  revealed the specificity of the full (ABI3-5Sup-ZF)x2 array for (GGA)x2 sequences. No clear enrichment was observed after 4 selection rounds for GST-(ZRANB1-B)x2, GST-(T0B2.5)x2, GST-(mRBM10)x2 or for the GST control . Altogether, these data suggest that the characterized ZFs fall into two categories: those that, supposedly do not bind to ssRNA and those that do. In the latter case, they strongly favour GGU or closely related motifs.We used the ZRANB2- as a positive control since its specificity is known, whereas GST alone provided a negative control. After 4 selection rounds we sequenced 16 cherry picked clones and analyzed them using the motif-based sequence analysis tool MEME (Multiple Em for Motif Elicitation)30 Fig. . MEME anTo corroborate our SELEX data, we measured the binding affinities of the different ZFs for their selected RNA sequences x2 arrays by size exclusion chromatography, with an average yield of 8 mg of purified and isolated (ZF)x2 per liter of culture x2 and (FUS-ZF)x2 bound to (GGU)x2 with equilibrium dissociation constants (KD) similar to that observed for ZRANB2- (Table x2 showed a similar affinity for a (GGU)x2 motif compared to the SELEX-selected (GGA)x2 motif. As expected, no binding to RNA (using GGU or polyA control sequences) could be detected for either (ZRANB1-B-ZF)x2 and (T0B2.5-ZF)x2 constructs, whereas a lower affinity binding (KD = ~10 µM) with (GGU)x2 was observed for (mRBM10-ZF)x2 ZRANB2-ZF,2 (Table2) Table . Surpris31. As expected, the values of binding reaction stoichiometry are close to 1:1 or a bit lower, likely due to difficulties in accurate estimation of RNA or protein concentration, as described in our previous study32. Overall the BLI and ITC measurements are in good agreement (Table off and kon) were also closely similar for all the studied ZFs.It is interesting to note that all the binding reactions measured by ITC were always characterized by large favorable enthalpy changes and unfavorable entropy changes, as observed for other ssRNA-binding domainsnt Table  and S4, 25 with low μM binding affinity. In order to analyze the importance of these two residues in the RNA-binding affinity, we generated single mutations  in the mRBM10-ZF variant. The corresponding mutated proteins and the RBM10 ZFs were expressed and purified as isolated (ZF)x2 arrays  compared to its human homolog (hRBM10-ZF)x2. Interestingly, the single N4D mutation was sufficient to restore the RNA binding affinity measured for (hRBM10-ZF)x2, whereas M26V had no observable effect on the RNA-binding affinity, suggesting that D4 was solely responsible for the difference in binding affinity between the RBM10 ZF homologs. Furthermore, this residue seems to be highly conserved among the putative RNA-binding RanBP2-type ZFs  that we studied herein exhibited no enrichment in SELEX and presented a lower binding affinity for GGU motifs compared to the other studied ZFs that were shown to bind RNA. This observation is intriguing given that this protein only differs by 2 amino acids (at position 4 and 26) from the human RBM10 ZF x2, mutation of any guanine in the first and second positions of the GGX motif completely abolished the RNA-binding activity x2, RNA binding was only observed in the case of the U3 → G substitution. However, the binding affinity for a GGG motif was 2.3-fold reduced for ZRANB2- and 1.3-fold reduced for both (EWS-ZF)x2 and (FUS-ZF)x2 compared to the GGU sequence. Interestingly, (ABI3-5Sup-ZF)x2 and (hRBM10-ZF)x2 showed higher tolerance to modifications of the third base. Despite a 3-fold preference for U in the third position, (ABI3-5Sup-ZF)x2 bound to GGG as well as to GGA motifs. In contrast, protein binding to GGC RNA was too low to allow accurate fitting, therefore we could not report affinity for this sequence. Finally, although (hRBM10-ZF)x2 bound to all tested GGN sequences, it showed a preference for GGG over GGU (1.6-fold), GGA (4.6-fold) and GGC (8.6-fold) motifs.BLI experiments were also conducted to analyze the discrimination ability of the different studied (ZF)ed Table  in the fty Table . For ZRA33, RanBP2-type ZRANB2 ZF2, a phage display mutant of ZRANB2 ZF228, the RBM5 RanBP2-type ZF27 and the Ts11d CCCH-type ZF133  that were shown to bind ssRNA with different sequence specificity. The designed protein sequence included from N- to C-terminal: the EWS RanBP2-type ZF, the Ts11d CCCH-type ZF2133 Fig. .Figure 428. Both Ts11d ZFs bound to a UAUU motif34. The (ZF)x6 array was expressed in fusion with the Maltose Binding Protein (MBP) and purified by affinity chromatography followed by size exclusion chromatography. On average, we recovered ~20 mg of pure MBP-(ZF)x6 per liter of culture  with an affinity of 24 ± 0.6 nM x2 constructs. Since we previously reported that the addition of a third RanBP2-type ZFs to a 2-ZF construct led to a 4-fold increase of binding affinity for the target RNA27, this observation suggests that the ZFs included in this (ZF)x6 chimeric protein are at least partially functional.In contrast to the GGU-specific EWS and ZRANB2 ZFs, the ZRANB2 ZF2 mutant and RBM5 ZF recognized GCC and GGG motifs, respectivelynM Table  suggestix6 protein, we measured the binding of the protein binding to a library of random 20 nt-long RNA sequences (polyN) as well as to an RNA in which the 3/4-nt subsites were permuted to give the sequence: UAUU GGU GGG UAUU GCC GGU (PT RNA). Our data indicated that control RNAs bound with only a 10-fold reduced binding affinity compared to the designed target  values that are identical within the error limit, whereas the dissociation was 17-fold faster for random RNAs compared to the target. Noticeably, the binding kinetic profiles measured for the permuted target RNA (PT RNA) were more complex with increased non-specific contributions et Table . In partFinally, BLI experiments conducted on polyU, polyA and polyC RNA controls resulted in very low signal intensities that we attributed to nonspecific binding Fig. . Other bin vitro binding assays performed with the (ZF)x6 chimeric protein, we asked whether the 17-fold slower dissociation rate observed for the target RNA compared to random RNA sequences would be sufficient to see the specific binding of the (ZF)x6 protein to its target RNA in a living cell. For this purpose, we designed a new bacteria-based assay. In these experiments, we used genetic constructs derived from the pACYDuet vector for dual expression of two gene cassettes  the (ZF)x6 array alone or iii) the MBP alone. In all these constructs, the second gene cassette coded for a variant of the green fluorescent protein (GFP LVA) that exhibited a reduced half-life when expressed in Escherichia coli compared to normal GFP35. Importantly, upstream of the ribosome-binding site (RBS) in the second cassette, we replaced the vector sequence corresponding to positions +33 to +53 with the sequence coding for the 20 nt-long target RNA. These three vectors were named: (ZF)x6-MBP-target-GFP, (ZF)x6-target-GFP and MBP-target-GFP vector x6-GFP, MBP-GFP. The idea behind this new bacteria-based assay is that specific interaction of the (ZF)x6 chimeric protein with its target sequence would interfere with proper assembly of the ribosome machinery and consequently reduce GFP translation levels. In other words, this experiment can be seen as a cell-based assay to study the binding specificity of the chimeric protein where the (ZF)x6 has to locate its target RNA sequence among an ocean of RNAs that are present in the bacteria and compete with specific binding.Based on the tes Fig. . We genetor Fig. . In addi36 using our ZF technology.It is also important to mention that we chose bacteria and not eukaryotic cell-based assays for several reasons: i) bacteria do not have any cell compartment (no nucleus), therefore all the RNAs present in the cell are available as competing molecules, and ii) bacteria antibiotic resistance has always been the main interest of our lab and our long-term goal is to be able to target regulatory RNAs involved in antibiotic resistanceE. coli krx cells over time, after IPTG and L-rhamnose induction  exhibited normal growth behavior and phenotype which suggested that non-specific binding, if present, was negligible and compatible with the bacteria normal metabolism and growth.It is also important to note that all the bacteria transformed with the plasmid encoding the (ZF)41, and because the GGU motif resembles the 5′ splice site consensus motif, the redundant specificity of ZFs may reflect the high degree of conservation at splice site junctions and regulatory sequences. ZRANB2 has been proposed to recognize 5′ splice sites characterized by the consensus AG│GUR (R = purine) in mammals24. It is therefore plausible that EWS, FUS, ABI3 and RBM10 carry out a similar function. In addition, splicing intronic/exonic enhancers like UGGG repeats42, G-triplets44, GGAGA46, UAGG and GGGG47 sequences are close to the SELEX-selected motifs and therefore could be the targets of these proteins as well.In this study, we have attempted to engineer functional RanBP2-type ZFs tandem arrays in an effort to generate tools for RNA study and manipulation. First, we have focused our work on extending the sequence specificity repertoire of the RanBP2-type ZF family by analyzing the substrate specificities of various ZF members of this family that presented different amino acid composition on the predicted RNA-binding surface. Our SELEX experiments and binding assays have revealed that these ZF domains exhibit an unexpected highly conserved sequence specificity for GGU/GGN motifs. Since their associated full-length proteins are mostly predicted to be splicing factors24. Both residues are conserved in RBM10 ZF whereas EWS and FUS ZFs showed only conservation of R20. Notably, this substitution of R19 for tryptophan in EWS and FUS ZFs did not appear to affect the RNA specificity. We hypothesized that this side chain can potentially stack with the guanine base, perhaps compensating for the loss of the R19-mediated hydrogen bonds. In contrast, in the ZF from the zebrafish protein ZRANB1-B, which did not bind RNA, a lysine and a serine replace R19 and R20, respectively. These amino acid substitutions reduce the number of possible hydrogen bonds and preclude any compensatory stacking interaction. It is important to note that this ZF shares high sequence conservation with its human counterpart that is reported not to bind RNA but ubiquitin50. In contrast, we did not identify any specific feature that could explain the inability of the ZF from the worm protein T0B2.5 to bind to RNA. The R19K exchange found in T0B2.5 ZF is also found in its human homolog RBM5 that is able to bind to GGG RNA27. Furthermore, T0B2.5 and RBM5 ZFs share all putative RNA-binding residues, with the only exception of position 14, a residue that contacts the third nucleotide in the ZRANB2 ZF2:RNA structure. However, these two homologs exhibit poor conservation of non RNA-binding residues; therefore conformational changes may explain their opposite RNA-binding behaviors.Furthermore, a critical interaction for RNA recognition in the ZRANB2-ZF2:RNA structure is the hydrogen bond network formed between both guanines and two arginine sidechains  on this third base can explain this behavior. Indeed, in the ZRANB2 F2:GGU structure  that are conserved in EWS and FUS ZFs. In contrast, ABI3-5Sup-ZF and hRBM10-ZF, who tolerate any base on third position, exhibit substitution of both residues N14 and N24 which reduces the number of possible hydrogen bonds with the third nucleotide. For ABI3-5Sup ZF, the N14 is replaced by C14 that could only form a single hydrogen bond via its thiol group, whereas F24 could potentially stack any base. In hRBM10 ZF, hydrophobic residues replaced both asparagines , which explains the reduced specificity on the third base. Interestingly, both of these ZFs are surrounded by RRM domains in the full-length protein x6 can be seen as a proof of concept experiment that shows that we can combine ZFs from different families in an effort to extend the sequence-specificity repertoire of the generated ZF arrays. Our in vitro binding assays indicated that the resulting 20-kDa protein exhibited a ≈ 25 nM affinity for its 20-nt long target RNA. These data are very encouraging since higher affinity (low nM) RNA-binding domains such as PUF and PPR repeats include eight repeats that are 35/36-residues long and each of these repeats specifies and interact with a single nucleotide, which means that a 70-kDa protein would be required to target a 20-nt long sequence.In this study, we also generated for the first time a (ZF)in vitro binding specificity of our chimeric (ZF)x6 protein revealed a relatively poor discrimination ability between cognate and non-cognate RNAs  using a dual expression plasmid encoding our (ZF)x6 protein  and the GFP, with the region upstream its RBS replaced by the sequence encoding the target RNA. Our data suggest that in a living cell, our (ZF)x6 protein is able to bind its cognate RNA sequence and thereby decrease the yield of expressed GFP, most likely by diminishing the accessibility of the RBS to the translational machinery.This is why, we went on to determine whether the observed 20-fold affinity difference was sufficient to provide specific binding in a living cell. For this purpose, we designed a new cell-based assay in bacteria (in vivo experiments should be done to characterize the RNA-binding specificity of the (ZF)x6 array as well as the contribution of each zinc finger to the modular recognition of protein target, this new bacteria-based assay offers a quick and simple approach to assess any protein-RNA interaction in vivo.Although additional GGU: ZRANB2-ZF1, ZRANB2-ZF2, EWS-ZF, FUS-ZF, ABI3-5-Sup-ZF/GCC: ZRANB2-ZF2-HN/GGG: RBM5-ZF, RBM10-ZF/UAUU: Ts11d-ZF1, Ts11d-ZF2) is estimated to be close to ≈46 = 4096, namely 4096 possible ssRNA targets. This rough estimation makes the assumption that we can combine 4 different target subsites (3 or 4 nt- long) to create these 20 nt-long target sequences.It is also interesting to mention that, although, the sequence specificity repertoire of the individual ZF domains that we studied herein is limited, by combining these ZFs in different ways, it is possible to target numerous RNA sequences. Indeed, the different 20 nt-long RNA sequences that we could target by combining all the ssRNA-Binding ZFs known up to date  that can be fused to many different effector domains  and therefore constitutes a versatile tool for RNA study and manipulation. In addition, ZF-based proteins can be easily delivered, expressed under the regulation of various tissue-specific promoters or addressed to specific cellular compartments via specific signal peptides. However, the main limitation of this technology lies on the restrained sequence repertoire targeted by RanBP2-type ZFs. The RanBP2-type ZFs only recognize GGU or GGN motifs despites our previous engineering attempt that led to the generation of a single ZF variant displaying specificity (but reduced affinity) for a GCC sequenceH. sapiens), NP_976225, residues 8–38 (ZF1) and 65–95 (ZF2); EWS (H. sapiens), NP_053733, residues 523–555; RBM10 (M. musculus), NP_001155288, residues 135–166; Fus (X. laevis), NP_001080383, residues 435–467; ZRANB1-B (D. rerio), NP_001071236, residues 152–182; T08B2.5 (C. elegans), NP_871822, residues 86–116; ABI3–5 Suppressor , NP_001190084, residues 379–406. Zinc finger domains were duplicated and covalently linked by a 5 amino acids linker (GSGSG). In contrast, the ZRANB2 fingers  were connected by a shortened version of their natural linker, previously optimized (TTEAKM)27. Genes encoding these constructs were commercially purchased  and cloned into the expression vector pGEX6P-1. The integrity of the genetic constructs were confirmed by DNA sequencing. Protein expressions were carried out in E. coli (DE3) Rosetta cells at 18 °C (OVN) upon induction with 0.1 mM IPTG and supplementation with 0.1 mM ZnCl2. Cell lysis was performed using a cell destroyer equipment  in 50 mM Tris-HCl pH 8, 1 M NaCl, 1 mM dithiothreitol (DTT), 0.1 mM ZnCl2 and one Complete EDTA-free protease inhibitor mixture tablet (Roche Applied Science). GST fusion proteins from the soluble fraction were purified on Glutathione-Sepharose® 4B beads  and eluted using a 100 mM reduced gluthation solution. The GST tags were cleaved off the (ZF)x2 constructs using HRV-3C  in 50 mM Tris pH 8, 150 mM NaCl, 1 mM DTT and 0.1 mM ZnCl2 and all (ZF)x2 constructs were further purified by gel filtration . Protein folding was analyzed by both intrinsic fluorescence and one-dimensional 1H NMR spectroscopy.GenBank accession codes and residues from the RanBP2-type ZF variants chosen for analysis are as follows: ZRANB2 , Ts11d F1 . All these domains were connected by a 5 amino acids linker (GSGSG), excepted for those followed by classical fingers. In this latter case, four additional residues (GSGSGSGSG) were introduced in order to reduce potential steric hindrances. The choice of the selected ZFs required consistency in domain polarities since our ZFs bind to ssRNA in a directional manner (in our case: N → Cter to 5 → 3′). The gene encoding this chimeric protein was purchased  and cloned into the pMALC2X vector. Protein expressions were carried out in E. coli (DE3) Rosetta cells at 18 °C for 4–5 hours upon induction with 0.1 mM IPTG and supplementation with 0.1 mM ZnCl2. Cells were lysed by sonication in 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT, 0.1 mM ZnCl2 and one Complete EDTA-free protease inhibitor mixture tablet (Roche Applied Science). The MBP fused protein from the soluble fraction was purified on Amylose resin (NewEngland Biolabs) and eluted using a 10 mM maltose solution. An additional step of purification by gel filtration  was carried to ensure size-homogeneity in the final sample.The designed 6-ZFs chimeric protein Fig.  includesSDS-PAGE data were acquired by digital camera or by Gel Doc™ EZ Imaging System with Image Lab™ Software 5.0 (Biorad). All images were edited and annotating by using Adobe Illustrator CS6.in vitro transcription reaction . After removal of unincorporated nucleotides , RNA was extracted with phenol/chloroform, ethanol-precipitated and finally resuspended in RNAse-free water (36 µL). Final RNA concentration was calculated by absorbance at 260 nm. Binding reactions were carried out in 200 µL of SELEX buffer . Samples containing 40 pmol of GST-fused protein immobilized on GSH beads , 1–4 µg of heparin sulfate, and 0.4–3.2 nmol of RNA, were gently mixed at 4 °C for 60 min. Unbound RNA was removed by washing the beads (five times in 500 µL of SELEX buffer). RNA-protein complexes were dissociated by acidic elution in HCl-glycine 25 mM pH 2.2 . RNA was then purified , ethanol-precipitated, and reverse-transcribed  using a complementary primer. Resulting cDNA was then amplified by 10 or 15 rounds of PCR and purified . A new RNA library was prepared using the amplified cDNA as a template for the following SELEX round. During the different cycles, selection stringency was intensified by increasing the RNA/protein ratios  together with the heparin amount (+1 µg/round). After 4 selection rounds, the final PCR products were subcloned into pJET vector  and individual sequences were analyzed.The template DNA library was prepared by Klenow reaction using a oligonucleotide harboring a 25-nt random sequence surrounded by 2 primer binding sites and a forward primer (carrying the T7 promoter region) x2 constructions, were carried out using 5′ biotinilated RNAs. For the MBP-(ZF)x6 chimeric protein, 3′biotinilated RNAs including a triethylenglycol (TEG) spacer were used (3′biotin-TEG), to reduce steric hindrance eventually induced by the MBP tag.ssRNA oligonucleotides (Supplementary Table 2). Samples were kept RNase free by adding all of the time RNase inhibitor (0.4 u/µL) . All experiments were carried out by titrating proteins (100 μM) into RNA (10 μM), on a MicroCal i200 ITC microcalorimeter  at 25 °C. For each titration, an initial injection of 0.5 μL (discarded data) and 20 injections of 2 μL of titrant were made at 120 s intervals. Experimental data were corrected by subtraction of dilution heats from control experiments (protein titration into buffer) and finally analyzed . The data were fitted to a 1:1 (one-site) model.Concentrated proteins and ssRNA oligonucleotides stocks were diluted into the ITC buffer  were immobilized on streptavidin-coated biosensors  for 300 s. The assays conducted on the (ZF)x6 chimeric protein, were performed with a loading RNA concentration of 0.125 µg/mL and a duration of 500 s in order to increase the spacing between immobilized RNAs and prevent possible dimerization. Biosensor tips were then saturated in biocytin (10 µg/mL) for 500 s to block any remaining free streptavidin, and equilibrated in the experimental buffer for 180 s prior to binding assessment. We tested different protein concentrations ranging from 1.5 to 0.31 µM (1.3-fold dilution series) and from 5 to 0.44 µM (1.5-fold dilution series) for the (ZF)x2 and (ZFs)x6 chimeric proteins, respectively. In all cases, binding was performed for 10 s and dissociation in the experimental buffer for 20 s. Recorded data were corrected by subtraction from the measurements of a reference sensor immobilized with biotinylated RNA (baseline drift). Data were analyzed using Octet software, version 8.0  and fitted using a 1:1 interaction model. A single set of kinetic parameters was obtained each time for all tested concentrations by nonlinear least-squares fitting.BLI experiments were performed at 30 °C in 96-well microplates , with agitation set to 1000 rpm, on a Octet HTX instrument . All assays were carried out in Tris 50 mM pH 7.5, NaCl 150 mM, DTT 1 mM, supplemented with BSA 0.1% and Tween 0.02% to minimize non-specific interactions. Proteins and RNAs were directly diluted in this buffer and RNase inhibitor (0.4 u/µL)  was added to ensure RNase free conditions. For all experiments on (ZF)® high-throughput platform for protein production and analysis (www.robotein.ulg.ac.be).The Microlab STAR liquid handling workstation (Hamilton) and the Octet HTX instrument  are available at the Robotein51, providing a graphical representation of the developed sequence alignment.In order to select the putative RNA-binding RanBP2-type ZFs, we extracted from the RanBP2-type family (accession code pfam00641) all protein sequences showing an aromatic residue at position 17. This initial database was reduced to 1704 entries by eliminating redundancy. An additional correction was carried out by removing sequences not displaying to the Trp-X-Cys-X2-4-Cys-X3-Asn-X6-Cys- consensus. The resulting 1526 sequences were analyzed by WebLogo softwarex6 array and a C-terminal MBP-tag, fused by a 5-amino acids GS-linker. Both PCR-amplified (ZF)x6 and MBP DNA sequences harbored at their ends the BamHI (5′) and EcoRI (3′) restriction sites. The full sequence coding for (ZF)x6MBP was assembled between the restriction sites NcoI and HindIII (first MCS) of the pACYDuet-1 vector. Furthermore, we redesigned the second expression cassette of the vector to accommodate the target sequence of the (ZF)x6 array upstream the RBS (+33 to +53). Downstream the RBS and between the EcoRV and XhoI restriction sites, we assembled the sequence coding for the green fluorescent protein unstable variant GFP LVA35. Both target and GFP LVA sequences were commercially purchased as gBlocks® Gene Fragments . The pACYDuet-(ZF)x6MBP-target-GFP vector was obtained by assembling multiple DNA fragments in a single step using the NEBuilder® High-Fidelity Master Mix (New England Biolabs), following the manufacturer’s instructions. In addition, we assembled a control plasmid pACYDuet-(ZF)x6MBP-GFP that carried no target sequence. From these vectors, we excised either the (ZF)x6 or the MBP coding sequence by single digestion. Then, the digested plasmids were gel-extracted, purified and self-ligated. Using this approach, we obtained both negative control (pACYDuet-MBP-target-GFP) and positive control (pACYDuet-(ZF)x6-target-GFP) from digestion of the pACYDuet-(ZF)x6MBP-target-GFP x6-GFP), therefore providing a full set of control vectors with no target sequence  by heat-shock transformation. This strain ensured a dramatic control of the GFP LVA expression, thanks to the T7 RNA polymerase driven by a rhamnose promoter. For each transformed construct, various dilutions of a single colony were cultured in 160 µL of LB with chloramphenicol (30 μg/mL) in a 96 well plate at 37 °C with shacking. When an OD of 0.3–0.6 was achieved, bacterial dilutions exhibiting similar density were selected for culture inoculation. We prepared the assay plate  by diluting 2 µL of each cell suspension in 160 µL of LB supplemented with chloramphenicol (30 μg/mL). Cells were cultured at 37 °C with shacking in a plate reader TECAN infinite® 200 PRO . When cultures reached an OD of 0.3–0.35, protein expression was induced by adding 0.1% L-rhamnose and 0.5 mM IPTG, at 37 °C. As for all ZF productions, 0.1 mM ZnCl2 was added as well. The OD measurements were performed at 600 nm with a bandwidth of 9 nm. Fluorescence measurements were carried out using a excitation wavelength of 485 nm with a bandwidth of 9 nm, and the emission wavelength of 525 nm with a bandwidth of 20 nm. For each measurement,10 reads were performed every 15 min, with gain set to 50; lag, integration and settle time were set to 0 μs, 20 μs and 0 ms, respectively.In these experiments, we employed the pACYDuet-1 vector for the co-expression of two genes, each under the control of IPTG inducible T7 promoter. We designed a sequence coding for the chimeric (ZF)tables and figures


Mann-Whitney U test revealed that Ktrans and Kep of oesophageal SCC were higher while TTP was shorter when compared to normal oesophagus ; and areas under receiver operating characteristic [ROC] curves displayed that Kep was superior to TTP or Ktrans for identifying oesophageal SCC (0.903 vs. 0.832 or 0.713). Mann-Whitney U test also demonstrated that Kep was higher and TTP was shorter in patients with lymphatic metastasis when compared to non-metastatic cancer patients , and area under ROC curve also showed that TTP was superior to Kep for predicting lymphatic metastasis (0.696 vs. 0.659). In conclusion, the combination of quantitative and semi-quantitative parameters derived from DCE-MRI can aid in the identification of oesophageal SCC and lymphatic metastasis.This study aimed to determine whether dynamic contrast-enhanced MRI (DCE-MRI) derived parameters can identify oesophageal squamous cell carcinoma (SCC) and lymphatic metastasis. Thirty-nine oesophageal SCC patients underwent DCE-MRI. Quantitative parameters including endothelial transfer constant (K Among the various forms, the most prevalent histological type is oesophageal squamous cell carcinoma (SCC)2. Early detection of oesophageal SCC and prediction of lymphatic metastasis is essential for timely and effectively treatment, which can potentially be life-saving. Neovascularization provides nourishment for the growth and lymphatic spread of oesophageal SCC. Therefore, a better understanding of the angiogenic behavior of oesophageal SCC may be useful in identifying oesophageal SCC and predicting lymphatic metastasis.Oesophageal cancer is the eighth most common malignant tumour and is the sixth leading cause of cancer-related death worldwide4. However, perfusion CT is limited in clinical use due to concerns of radiation exposure. Magnetic resonance imaging (MRI) is increasingly used for diagnosing and N staging for oesophageal cancer. This is explained largely by the technical improvements (e.g. breath-hold sequences) and the addition of functional MRI techniques such as diffusion-weighted imaging (DWI) and dynamic contrast-enhanced MRI (DCE-MRI)8. Due to its noninvasive and non-ionising technique, DCE-MRI is often preferred and is being widely used in studies of malignant tumour including breast cancer, prostate cancer and rectal cancer11. DCE-MRI not only can visually judge the enhancement of a region of interest and semi-quantitatively characterize tumours by analyzing the signal variation with respect to time, but also can quantitatively evaluate tumours with parameters derived from pharmacokinetic models, which demonstrates the dynamic distribution of gadolinium-related contrast agent in the different compartments of the tumour15. The two-compartment model of DCE-MRI presumes that gadolinium-related contrast agent exchanges between the extravascular-extracellular space (EES) and the plasma space, and the transfer rate of forward and backward can reflect the permeability of the microvascular16. Despite the publication of relevant paper regarding DCE-MRI in oesophageal cancer7, the published study aimed to reveal the therapeutic effects with DCE-MRI. To our knowledge, there were no publications regarding the combination of quantitative and semi-quantitative parameters derived from DCE-MRI to identify resectable oesophageal SCC and predicting status of lymphatic metastasis. Therefore, the present study was undertaken to evaluate the feasibility of DCE-MRI for the discrimination of microcirculation differences between oesophageal SCC and the normal oesophagus and between oesophageal SCC with and without lymph node metastasis.The development of perfusion computed tomography (CT) has made it possible to capture the parameters reflecting the vasculature of oesophageal SCC, facilitating the identification of oesophageal SCC. It has also made lymphatic metastasis more predictableThe institutional review board of North Sichuan Medical College approved this study, and written informed consent was obtained from each participant before the prospective study. All methods were performed in accordance with the relevant guidelines and regulations.17; and (d) the quality of the DCE-MRI images was good, suggesting that the motion artifacts resulting from random autonomous movement, breathing, heart pulse and vascular pulse was slight enough for us to perform the data analysis. The exclusion criteria were: (a) the patient had received neoadjuvant radiotherapy and/or chemotherapy before surgery (n = 2); (b) the patient had contraindications for DCE-MRI  or surgery  (n = 4); or (c) the quality of the DCE-MRI images was poor (n = 2). The initial population consisted of 47 consecutive patients with biopsy-confirmed oesophageal SCC, 8 of which were excluded. Consequently, this study involved 39 patients .From February 2016 to October 2017, patients with biopsy-confirmed oesophageal SCC were enrolled into this study according to the following inclusion criteria: (a) the patient did not receive any preoperative tumour-related treatment (e.g. radiotherapy or chemotherapy); (b) the patient had no contraindications for DCE-MRI or surgery; (c) the tumour was considered resectable by endoscopic biopsy and CT18. They were categorized as N0, N1, N2 and N3 in 21, 9, 7 and 2 patients, respectively.With regards to the site of the tumours in the enrolled participants, 5.1% (2 of 39) were located at the upper thoracic portion of the oesophagus, 66.7% (26 of 39) were located at the middle thoracic portion, and 28.2% (11 of 39) were located at the lower thoracic portion without oesophagogastric junction invovement. All participants underwent double-contrast barium examinations, endoscopic biopsy, CT and thoracic DCE-MRI examinations before surgery. Subsequently, they were scheduled for radical oesophagectomy with three-field lymphadenectomy. All 39 oesophageal cancers were pathologically diagnosed as SCC, and it was confirmed that the cutting edges of the resected oesophageal segment demonstrated no signs of neoplasia. The time interval between DCE-MRI and surgery was less than 2 weeks , and none of patients received any preoperative tumour-related treatment. The N stage of tumour was clinically determined according to the postoperative histopathologic examination and American Joint Committee on Cancer criteria19, the initial pre-injection dynamic acquisitions of the axial DCE scans would provide the baseline images for generating the time courses when the DCE data analysis was performed.DCE-MRI was performed on a 3.0 T superconductive magnet  for all enrolled patients using 32-channel phased array body coil in the chest region with respiratory and electrocardiogram gating. The patients underwent breath training before the examination and were examined in the supine position. Axial and sagittal T2-weighted sequences with fat saturation were obtained for tumour localization using the following scanning parameters: repetition time (TR)/echo time (TE) of 3000–4000/85–95 ms, field of view (FOV) of 360 mm × 360 mm, matrix of 352 × 352, and slice thickness of 4 mm. Prior to the DCE acquisitions, five consecutive axial three-dimensional spoiled-gradient recalled-echo sequences for liver acquisition with volume acceleration were performed by using TR/TE of 3.3/1.5 ms, FOV of 360 mm × 360 mm, matrix of 256 × 192, and slice thickness of 6 mm with different flip angles of 3°, 6°, 9°, 12°and 15° for determination of pre-contrast T1 values. Subsequently, an axial DCE sequence was performed before and after elbow intravenous injection of 15 ml Gadodiamide  of 0.5 mmol/ml; the parameters for this test were arranged as follows: TR/TE of 3.3/1.5 ms, flip angle of 15°, FOV of 360 mm × 360 mm, matrix of 256 × 192, slice thickness of 6 mm, 40 dynamics, temporal resolution of 7 s, and duration of 5 min 4 s. The contrast agent was injected intravenously at the fourth dynamic acquisition using a high pressure injector system , immediately followed by a 20 ml saline flush at a rate of 2.5 ml per second. Based on the published literaturetrans, in ml/min), reflux rate , fractional extravascular extracellular space volume  and fractional plasma volume  were automatically generated on the basis of a two-compartment modified Tofts model20. The semi-quantitative parameters including time to peak , max concentration , Max Slope (in mmol/min) and area under the concentration-time curve  were also automatically generated. The previous software automatically derived the parametric maps of Ktrans  which provides pharmacokinetic measurement and calculation on a pixel-by-pixel basis. Two radiologists with experience in digestive radiology  who were blinded to the pathological results independently performed the data analysis. After the dynamic images were downloaded into this software, motion correction was automatically performed. A T1 mapping was computed from T1-weighted acquisitions with different flip angles . An arterial input function was extracted by randomly manually drawing a circle region of interest (ROI) with the diameter of 1 cm on the descending aorta. Next, the tumoural ROI was freehand outlined Fig.  randomlyans Fig. , Kep . Data are expressed as mean ± standard deviation.ICC). The agreement was defined as excellent (ICC > 0.90), good (ICC = 0.75–0.90), moderate (ICC = 0.5–0.75), or poor (ICC < 0.5)22. If the inter- and intra-observer agreements of the measurements of oesophageal SCC DCE-MRI parameters were good (ICC > 0.75), values of the first measurement by Y.L.C. were regarded as the final pharmacokinetic parameters for the oesophageal SCC. If not, the mean value of the two measurements by Y.L.C. and the measurement by T.W.C. was used as the final values.Inter-observer and intra-observer agreements for the measurements of oesophageal SCC DCE-MRI parameters were evaluated by using the inter-class correlation coefficient  analysis was performed to determine if the cutoff of any DCE-MRI derived parameter could help identify oesophageal SCC and lymph node metastasis.ICC 95%CI range, both 0.984~0.999; Tables Based on the DCE-MRI derived parameters of oesophageal SCC obtained independently by the two radiologists and repeatedly by Y.L.C., the inter- and intra-observer agreements were both good  as shown in Table ep was better than Ktrans or TTP for oesophageal SCC diagnosis according to the ROC analysis .The Mann-Whitney U tests showed that the quantitative parameters including Kis Table . The Manep and semi-quantitative parameter TTP between tumours with and without lymph node metastases. The Kep was higher whereas the TTP was shorter in tumours of the former as shown in Table P-values < 0.05). In comparison with the Kep, the ROC analysis showed that the TTP was better for predicting lymphatic metastasis .According to the Mann-Whitney U tests, there were significant differences in the quantitative parameter Kis Table . The Mantrans, Kep and TTP displayed significant differences between oesophageal SCC and normal oesophageal wall. In detail, Ktrans and Kep were higher while TTP was shorter in oesophageal SCC in comparison with normal oesophagus. This finding may be explained by the angiogenesis characteristics of malignancy. Zhao et al.23 reported that RhoC mRNA expression was mainly located in the cytoplasm of the tumour cells and were higher in oesophageal SCC than in normal oesophagus, and RhoC mRNA expression showed a positive correlation with vascular endothelial growth factor (VEGF) protein levels. Dvorak24 demonstrated that VEGF induced endothelial cell division and migration, enhanced microvascular permeability, promoted stromal proteolysis, and reduced endothelial cell apoptosis. Knopp et al.25 also indicated that the endothelium within tumour microvessels may exhibit increased permeability. These characteristics will allow a more rapid transfer and an accelerated clearance of the contrast agent from the interstitium, ultimately resulting in the higher Ktrans and Kep and shorter TTP in oesophageal SCC than in normal oesophageal wall.In our study, we found that several DCE-MRI derived parameters including Kep was higher but the TTP was shorter in patients with lymph node metastasis than without that metastasis. Zhao et al.23 reported that the expression of RhoC mRNA in oesophageal SCC with lymphatic metastasis was significantly higher than without lymph node metastasis, and the expression of VEGF protein in the tumour with lymphatic metastasis was significantly higher than in the tumour without lymphatic metastasis. Zhang et al.26 also indicated that there was a significant correlation between a high level of VEGF-C expression in oesophageal SCC and lymphatic metastasis. Some published reports indicated that microvessel density of oesophageal SCC with lymph node metastasis was significantly higher than without that metastasis31. According to the above mentioned reports, the microvascular permeability of increased microvessels was higher in oesophageal SCC with lymph node metastases than without this metastasis, which could lead to the higher Kep and shorter TTP in the cancer with nodal disease.As shown in our study, the Ktrans, Kep and TTP were significantly different between oesophageal SCC and normal oesophageal wall, ROC analysis was performed in this study to determine whether these parameters could be used for differentiating the microcirculation of oesophageal SCC from that of the normal oesophageal wall and applied to the diagnosis of oesophageal cancer. With ROC analysis, the areas under the ROC curves of Ktrans, Kep and TTP were 0.713, 0.903 and 0.832, respectively, which suggested that the Kep was the best parameter for aiding the diagnosis of oesophageal SCC. In addition, the current study showed that the Kep and TTP were significantly different between oesophageal SCC with and without lymph node metastasis, and the areas under the ROC curves of Kep and TTP were 0.659 and 0.696 for determining the microcirculation of oesophageal SCC with lymph node metastasis, respectively. This finding implies that DCE-MRI derived parameters could help identify oesophageal SCC with lymph node metastasis, and TTP could be the better parameter for this purpose.Because the DCE-MRI derived parameters including K2, and we performed this study focusing on assessing the microcirculation of oesophageal SCC rather than that of oesophageal adenocarcinoma with DCE-MRI. We confirmed that microcirculation assessment of oesophageal SCC could help identify this cancer and regional lymphatic metastasis, and we would lead to a new research approach for tumour diagnosis and lymphatic metastasis prediction of oesophageal adenocarcinoma by microcirculation assessment with DCE-MRI. Secondly, the mean values in the ROIs on two-dimensional (2D) image may not reflect spatially rich information within the tumour resulting from the tumour heterogeneity32. Thus, prospective studies with heterogeneity analysis on whole-tumour may eliminate the confounding effect in ROI average studies in the future. Thirdly, one of the criteria for the study was that the quality of the DCE-MRI images was good, however this criteria is a little vague. Fourthly, we did not explore the effects of the location of the tumours, and the gender and age of the samples on the accuracy of the findings in this study. We will perform the relevant study in the future.There were several limitations in our study. Firstly, as squamous cell carcinoma is the most common oesophageal carcinoma worldwideep could be the optimal parameter for identifying oesophageal SCC. The semi-quantitative parameter TTP could be more suitable for predicting lymph node metastasis. We hope that the findings in our study will be helpful for the identification and predication of lymphatic metastasis for treatment decision making.The quantitative and semi-quantitative parameters derived from DCE-MRI may potentially be helpful for determining the microcirculation within oesophageal SCC and predicting the status of lymphatic metastasis. The quantitative parameter K