The increased integration of molecular alterations to define tumor type or grade in central nervous system (CNS) tumor classification brings new challenges for the pathologist to make the best use of a precious limited tissue specimen for molecular studies. Within the different methods available to identify gene alterations, the droplet digital PCR (dPCR) constitutes a rapid, cost-effective, and very sensitive tool. In this study, we describe the development and validation of five multiplexed dPCR assays to detect major CNS biomarkers by using only small amounts of DNA extracted from formalin-fixed paraffin-embedded specimens. When compared to HRM-sequencing, NGS-sequencing, RNA-sequencing, or simplex digital PCR assays used as “gold standard” methods, these multiplexed dPCR assays displayed 100% specificity and sensitivity for the simultaneous detection of: 1/BRAF V600E mutation and KIAA1549:BRAF fusion; 2/FGFR1 N546K and K656E mutations and FGFR1 duplication; 3/H3F3A K27M and G34R/V mutations; 4/IDH1 R132X and IDH2 R172X mutations; and 5/TERT promoter mutations C228T and C250T. In light of the increased integration of molecular alteration, we believe that such strategies might help laboratories to optimize their screening strategies for routine diagnosis of pediatric and adult CNS tumors. For other entities, although the diagnosis does not yet require the characterization of the underlying molecular abnormalities, it is nevertheless specified in the 2016 WHO classification that certain molecular alterations constitute a major additional putative diagnostic marker   tumors in 2016  and inevitably leading to the exhaustion of a valuable diagnostic material. Finally, frozen material may not always be available and DNA or RNA extracted from formalin-fixed paraffin-embedded samples (FFPEs) may be of limited use for molecular analyses due to chemical modifications of nucleic acids and degradation over time , 3.BRAF duplication which is sufficient to predict a KIAA1549:BRAF fusion allowing a single analytical procedure limited to extracted DNA  constitutes an interesting strategy as it is a rapid, cost-effective with low turnaround time, and a very sensitive tool. It is particularly useful on FFPE specimen since dPCR is a very robust approach on altered or fragmented DNA . Indeed,cted DNA . HoweverBRAF V600E mutation and BRAF exon 14 duplication associated with the KIAA1549:BRAF fusion; 2/FGFR1 N546K and K656E mutations and FGFR1 internal tandem duplication of the tyrosine-kinase domain; 3/H3F3A K27M and G34R/V mutations; 4/IDH1 R132X and IDH2 R172X mutations; and 5/TERT promoter (pTERT) C228T and C250T mutations.In this study, we developed and evaluated the efficacy of five multiplexed dPCR assays allowing the simultaneous detection of: 1/BRAF V600E mutation, five cases with KIAA1549:BRAF fusion (including three with 16:9 fusion and two with 16:11 fusion), five cases with FGFR1 N546K or K656E mutations (including one with N546K and four with K656E mutations), and five cases with FGFR1 exon 16 duplication. These cases were retrieved from previously published cohorts , five cases with IDH2 R172X mutation , five cases with pTERT C228T mutation, and five cases with pTERT C250T mutation, and 10 cases without IDH1/2, H3F3A nor pTERT alteration. These alterations were evaluated by targeted HRM-sequencing (eight cases) or next generation sequencing  performed on formalin-fixed paraffin-embedded (FFPE) tissue sections. H3F3A mutation status was known for 39/40 cases, IDH1/2 mutation status for 37/40 cases, and pTERT mutation status for 35/40 cases. It should be noted that four cases presented both IDH2 and pTERT mutations. Selected cases with known gene alteration were used to evaluate the H3F3A, IDH1/2, and pTERT multiplex digital PCR assay sensitivities while negative cases for which the gene status was known were used to evaluate assays specificities.The second cohort comprised samples from 40 French patients with a diagnosis of diffuse glioma for which For the low grade CNS neoplasm cohort we retrieved the extracted DNA used for previously published analysis , 7. RegaEach multiplexed digital PCR (mdPCR) assays were developed in collaboration between APHM and ID-Solutions . After analytical validation of each assay, they were produced and packaged as a commercial kit before being clinically validated. The aim was to validate each assay in its final version before its commercialization. To allow clinical validation, target positive controls (TPCs) were generated for each assay. TPCs allow the validation of the PCR workflow and the PCR mix, as they are composed of synthetical DNA harboring every alteration to be detected. These TPCs are also calibrated at low concentration with a known allele frequency or CNV (copy number variation). DPCR analyses were performed as previously described . ExtractThe dPCR partition samples into thousands of PCR reaction chambers with individual endpoint detection. Thus, different fluorescence profiles can be read and recorded individually and different strategies for multiplexing in dPCR have been developed in recent years . Our mdPBRAF V600E mutation and BRAF exon 14 duplication, associated with the KIAA1549:BRAF fusion as previously published , equal to the ratio of mutant-DNA to wild-type DNA, was calculated as follows:Following the analysis and quantification of positive droplets on each channel, The CNV represents for a haploid genome the ratio of target-DNA to reference-DNA multiplied by two. As previously published , a samplFGFR1 mdPCR assay to screen simultaneously the presence of FGFR1 N546K and K656E mutations as well as FGFR1 exon 16 duplication. In this assay, FGFR1 N546K and K656E mutations were targeted using 100% FAM labeled probe; FGFR1 exon 16 was targeted using 100% HEX labeled probes; and FGFR1 exon 8, used as reference, was targeted using a mixture of both FAM and HEX labelled probes. This design resulted in a 2D plot with four clusters . Thus, one FAM-labeled probe bound a “reference” sequence distant from the IDH1 R132 locus and a second Drop-off HEX-labeled probe bound the R132 wild-type locus, both within the same amplicon. In addition, one HEX labeled probe bound a “reference” sequence distant from the IDH2 R172 locus and a second Drop-off FAM-labeled probe bound the R172 wild-type locus both within the same amplicon. Finally, the separation of IDH1 and IDH2 wild-type clusters was achieved by varying ratio of FAM and HEX labeled probes. This design resulted in a 2D plot with five clusters  or FGFR1 N546K or K656E mutation (5/5) evaluated by HRM-sequencing were also detected with the mdPCR assays. Mutant allele frequencies ranged from 3.8 to 26.1% (FGFR1 G539R mutation was not detected by the FGFR1 mdPCR assay. All cases without BRAF V600E mutation (7/7) or FGFR1 N546K or K656E mutation (12/12) were also negative with the mdPCR assays. These included two cases with BRAF V600E mutant allele frequency of 0.3 and 0.1% and thus considered as negative.Among the low grade glial or glioneuronal tumor cohort, all selected cases with KIAA1549:BRAF fusion evaluated by RNA-sequencing were also positive with the BRAF mdPCR assay (5/5). Similarly, all cases with known FGFR1 duplication evaluated by dPCR were also detected with the FGFR1 mdPCR assay (5/5). Cases without KIAA1549:BRAF fusion (10/10) or FGFR1 duplication (12/12) were also negative with the mdPCR assays.In addition, all selected cases with already known BRAF and FGFR1 mdPCR assays had a sensitivity and specificity of 100% , IDH1, or IDH2 mutation (11/11) or pTERT mutation (13/13) evaluated by HRM-sequencing or NGS were also positive with the mdPCR assays. The detected alterations included: five H3F3A K27M mutations, five H3F3A G34R, two IDH1 R132H, one IDH1 R132C, one IDH1 R132S, one IDH1 R132L, three IDH2 R172K, one IDH2 R172L, one IDH2 R172M, one IDH2 R172W, eight pTERT C228T, and five pTERT C250T mutations. All cases without H3F3A K27M or G34R mutation (29/29), IDH1 R132X or IDH2 R172X mutation (24/24) or pTERT C228T or C250T mutation (21/21) were also negative with the mdPCR assays. These included one case with H3F3A K27M mutant allele frequency of 0.04% and one case with IDH1 R132X mutant allele frequency of 0.6% and thus both considered as negative. Unfortunately, the IDH1/2 and pTERT mdPCR assays could not be performed for respectively two and one cases because all the sample and extracted DNA had been used.Among the diffuse glioma cohort, all selected cases with known H3F3A, IDH1/2, and pTERT mdPCR assays had a sensitivity and specificity of 100%  which analysis is limited to two fluorescence reading channels (FAM and HEX). Different approaches can be applied for multiplexing dPCR . The ratBecause of the multiplexing limitations of digital PCR, other approaches for molecular screening may have specific advantages depending on the context Table 2.The present study showed the usefulness of five multiplexed digital PCR assays to quickly, easily and simultaneously assess the status of several major CNS biomarkers by using small amounts of DNA extracted from FFPE tissue. We hope that such strategies might help laboratories to optimize their screening strategies for routine diagnosis of pediatric and adult CNS tumors.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the APHM ethics committee. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.RA, DF-B, and CC supervised the study and wrote the manuscript. DS contributed to build the tissue cohorts. DF-B and RA made the selection of the cohorts  and performed pathological review. FF, DH, JV, LG, and PP designed the multiplexed droplet digital PCR assays. IN-M performed genomic DNA extraction. FF, DB, and CG performed the multiplexed droplet digital PCR assays and contributed to data analysis. All authors contributed to the article and approved the submitted version.Société Française de Lutte contre les Cancers et Leucémies de l’Enfant et de l’Adolescent) the Cancéropôle PACA, and the GIRCI Méditerranée (GlioMark protocol) for their financial support.We thank the ARTC-Sud patients’ association , the Association Cassandra, the Imagine For Margo Association, the SFCE (FF, DH, JV, LG, and PP are all members of Id-Solutions, Grabels, France.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Litomosoides sigmodontis. S100A9 forms with S100A8 the heterodimer calprotectin, which is expressed by circulating neutrophils and monocytes and mitigates or amplifies tissue damage as well as inflammation depending on the immune environment. Mice deficient for S100A8/A9 had a significantly reduced worm burden in comparison to wildtype (WT) animals 12 days after infection (dpi) with infective L3 larvae, either by the vector or subcutaneous inoculation, the latter suggesting that circumventing natural immune responses within the epidermis and dermis do not alter the phenotype. Nevertheless, upon intradermal injection of L3 larvae, increased total numbers of neutrophils, eosinophils and macrophages were observed within the skin of S100A8/A9-/- mice. Furthermore, upon infection the bronchoalveolar and thoracic cavity lavage of S100A8/A9-/- mice showed increased concentrations of CXCL-1, CXCL-2, CXCL-5, as well as elastase in comparison to the WT controls. Neutrophils from S100A8/A9-/- mice exhibited an increased in vitro activation and reduced L3 larval motility more effectively in vitro compared to WT neutrophils. The depletion of neutrophils from S100A8/A9-/- mice prior to L. sigmodontis infection until 5dpi abrogated the protective effect and led to an increased worm burden, indicating that neutrophils mediate enhanced protective immune responses against invading L3 larvae in S100A8/A9-/- mice. Interestingly, complete circumvention of protective immune responses in the skin and the lymphatics by intravenous injection of L3 larvae reversed the phenotype and resulted in an increased worm burden in S100A8/A9-/- mice. In summary, our results reveal that lack of S100A8/S100A9 triggers L3-induced inflammatory responses, increasing chemokine levels, granulocyte recruitment as well as neutrophil activation and therefore impairs larval migration and susceptibility for filarial infection.Neutrophils are essentially involved in protective immune responses against invading infective larvae of filarial nematodes. The present study investigated the impact of S100A8/S100A9 on protective immune responses against the rodent filarial nematode  Litomosoides sigmodontis. Investigations in L. sigmodontis-infected mice deficient in S100A8/S100A9 and wild-type controls demonstrate that the lack of S100A8/S100A9 promotes protective responses that trigger L3-induced inflammation by increasing chemokine production, granulocyte recruitment and neutrophil activation, triggering transient inflammatory responses and therefore impairing larval migration and worm recovery.Human pathogenic filariae are responsible for several devastating diseases such as lymphatic filariasis and onchocerciasis. Neutrophils are essentially involved in protective immune responses against invading infective L3 larvae of filarial nematodes. The S100A8/S100A9 heterodimer, also known as calprotectin, is abundantly expressed by neutrophils and markedly increased during infection, tissue damage, neutrophil extracellular trap formation (NETosis) and cellular necrosis. However, calprotectin is also known to have an anti-inflammatory or protective role during infection and inflammation and there is an ongoing controversy on whether S100A8/A9 is pathogenic or rather protective. The present study investigated the impact of calprotectin on protective immune responses against the rodent filarial nematode  Inflammation represents an essential process in the initiation, development and pathogenesis of several human diseases. During stress-mediated responses caused by infections or tissue injuries, damage-associated proteins from the S100 protein family are released locally . S100 prIn the past neutrophils have been shown to mediate protection against parasitic filarial nematodes. These filariae represent a huge global health problem in numerous tropical and subtropical areas and are responsible for several devastating diseases such as lymphatic filariasis (elephantiasis) or onchocerciasis (river blindness) ,17. ParaLitomosoides sigmodontis has essentially helped to improve our understanding of the protective immune responses during filarial infection, the modulation of the host’s immune system [The rodent filarial nematode e system –29 and te system ,30–37.Ornithonyssus bacoti), infective L. sigmodontis L3 larvae are transmitted into the murine host’s skin and migrate via the lymphatic system, reaching the pulmonary blood circulation, transiting the lung and entering the thoracic cavity, where the filariae finally reside (2–6 days after infection (dpi) in BALB/c mice) [Through the bite of the tropical rat mite vector (/c mice) . Within L. sigmodontis. As for type 2 immune responses the absence of IL-4 [L. sigmodontis infection and increased the adult worm burden and/or microfilaremia [et al. demonstrated that the deficiency of IFN-γ led to an increased worm burden due to reduced neutrophil recruitment and encapsulation of adult worms compared to wildtype (WT) mice [L. sigmodontis L3 larvae [et al. showed that migrating Brugia pahangi third-stage larvae induce neutrophil accumulation around the invading larvae in the skin with increased associated inflammatory cytokines such as IL-6 and TNF [L. sigmodontis L3 infection in the skin of NOD2-/- and IL-6-/- mice as well as neutrophil depletion in WT animals increased the worm recovery, indicating that neutrophils are essential for protective immune responses against invading L3 larvae [+/1013 mice, which have an increased number of neutrophils in the skin, reduced the L. sigmodontis worm recovery in a neutrophil-dependent manner [During these investigations type 1 –42 and t of IL-4 ,44, IL-5 of IL-4 –47, IL-6 of IL-4 , eosinop of IL-4  and eosi of IL-4  led to ailaremia . As for 3 larvae ,41 as we3 larvae . Porthou and TNF . Accordi3 larvae ,48. AccoL. sigmodontis L3 larvae trigger the expression of S100A9 in neutrophils during their migration through the lung into the thoracic cavity [L. sigmodontis infection in C57BL/6 mice lacking S100A9 and compared the worm burden and immune responses to C57BL/6 WT controls. Of note, S100A9-/- mice are also deficient of S100A8 on the protein level representing a functional double knockout mouse [Recently, it was demonstrated that c cavity . In the ut mouse ,54. TherOur data indicates an anti-inflammatory role of S100A8/S100A9 on neutrophil activation, which reduced inflammatory responses and facilitated the migration of infective L3 larvae into the thoracic cavity.er fevrier 2013, Ministère de l'Agriculture, de l'Agroalimentaire et de la Foret). Animal welfare was scored on a scale from A-C for symptoms considering appearance, injuries, weight loss, and behaviour. A score of A required daily observations of the symptoms, a score of B, consultation of the project leader or a veterinary, and a score of C the immediate euthanasia of the affected animal. For euthanasia, animals were exposed to an overdose of isoflurane.Animal housing conditions and animal experiments were approved by the local authorities  as well as the ethical committee of the MNHN  and by the DDCSPP (C75-05-15). Experiments were performed according to the Directive 2010/63/EU guidelines and the relevant national legislation  and corresponding C57BL/6J WT controls (Janvier) were bred and housed in individually ventilated cages on a 12 hour light/dark cycle at the animal facility of the Institute for Medical Microbiology, Immunology and Parasitology, University Hospital Bonn, Germany. Mice had access to food and water ad libitum. Only female mice were included in the experiments and mice were infected at 8–10 weeks of age with L. sigmodontis via natural infection with the intermediate host Ornithonyssus bacoti. Mice were exposed simultaneously to the same batch of O. bacoti mites to ensure a comparable infection of both groups [Meriones unguiculatus) [L. sigmodontis infection using an overdose of Isoflurane (Forene) and worms were isolated from the thoracic cavity by lavage and quantified using a binocular microscope.C57BL/6JS100A8/A9h groups . For subh groups  or 5 dayculatus)  and 40 LMice were euthanized per inhalation of an overdose of Isoflurane. In order to isolate thoracic cavity cells the thoracic cavity was initially flushed with 1ml PBS (Gibco) and the lavage centrifuged at 400g for 5min at 4°C. The supernatant was frozen at -20°C for further cytokine and chemokine measurements. The separated cells were combined with the cells gathered during a second lavage with 9ml PBS.To assess bronchoalveolar cells, the trachea of euthanized mice was exposed and a venous catheter  was inserted at the cervico-thoracic junction. The bronchoalveolar space was washed with 10ml PBS and the cell isolation was performed as described above.Spleens of dissected mice were removed after necropsy, homogenized and pressed through a 70μm cell strainer. Cells were centrifuged at 400g for 10min at 4°C, the supernatant was discarded and red blood lysis was performed with 1ml of RBC lysis buffer (ThermoFisher) for 5min at room temperature. Lysis was stopped by adding 9ml of RPMI 1640 medium (Gibco). After centrifugation the supernatant was removed and the pellet was resuspended in 10ml of RPMI 1640 media. Thoracic cavity, bronchoalveolar lavage (BAL) and spleen cell numbers were determined by Casy Cell Counter (Roche) and prepared for flow cytometry or cell culture stimulation experiments.2) were taken and incubated with liberase (containing DNase (Thermofisher)) for 50min at 37°C. Digested tissue was filtered through a 70μm strainer (Milteny). RBC lysis was performed with 1ml of RBC lysis buffer as described above and subsequently the cell suspension was washed with PBS. Cell numbers were determined by Casy Cell Counter and cells were subsequently prepared for flow cytometric analysis.For skin analyses, mice were shaved 48h prior injection on the upper hind leg region. 10 infective L3 larvae isolated from infected gerbils by pleural lavage  were susAdult worms, freshly isolated from the thoracic cavities of infected gerbils were scavenged in endotoxin-free PBS, before being homogenized using a mortar under sterile conditions. After centrifugation at 300g for 10min at 4°C, the supernatant was collected and protein concentration of the crude extract (LsAg) was determined using the Advanced Protein Assay (Cytoskeleton) ,57.6 cells/ml of isolated splenocytes were plated in enriched media  . Isolated cells remained either unstimulated or were stimulated with 2.5μg/ml Concanavalin A (Sigma) or with 25μg/ml L. sigmodontis adult worm extract (LsAg). Cells were cultured at 37°C, 5% CO2 and 90% humidity for 72h and then centrifuged at 400g for 5min. Cell culture supernatants were taken for cytokine measurements and stored at -20°C.2x10Measurements were performed from the first ml of thoracic cavity lavage, BAL and splenocyte culture supernatants by ELISA. IFN-γ and TNF (ThermoFisher) as well as CXCL-1, CXCL-2, CXCL-5, elastase, myeloperoxidase and S100A8/A9  were measured according to manufacturers’ guidelines.+ T cells and CD8+ T cells were identified as CD4high or CD8high cells, respectively; neutrophils as Ly6Ghigh, CD4low; eosinophils as SiglecFhigh, F4/80low; macrophage populations were identified as F4/80high, SiglecFlow. BAL macrophages were identified as F4/80high, SiglecFhigh and neutrophils as Ly6Ghigh/CD11bhigh. Cells were further analyzed with MHCII-PE (BioLegend) in a different panel. Cells were stained and after another wash and centrifugation step cells were analysed by flow cytometry. Analysis was performed using a BD FACS Canto system and data was subsequently analyzed using the FACS Diva 5.1 software (BD Biosciences). During analysis, cut-offs were set using the fluorescence minus one (FMO) approach. The gating strategy is shown in Thoracic cavity, spleen, BAL and skin cells were analyzed by flow cytometry. Cells were isolated as described above and subsequently blocked with PBS/1% BSA including 0.1% rat IgG (Sigma) and stained. Flow cytometric analysis was performed using a combination of the following surface markers: CD4-FITC, CD8-APC, SiglecF-PE, F4/80-PerCP-Cy5.5, CD11b-APC-Cy7 , and Ly6G-PE-Cy7 (ThermoFisher). Thoracic cavity and spleen CD4-/- and WT controls were removed surgically and remaining tissue was detached. The condyles of the bones were cut and subsequently the bone marrow was flushed with 5ml of cold sterile PBS using a syringe. The flow through was filtered (70μm strainer) and after a centrifugation step at 400g for 5min at 4°C the supernatant was discarded and red blood lysis was performed by adding 1ml RBC lysis buffer for 5min. Lysis was stopped by adding 9ml of cold, sterile PBS. Cells were centrifuged again at 400g for 5min at 4°C and the pellet was resuspended in 3ml of PBS. Murine bone marrow-derived neutrophils were purified by a 3-layer Percoll gradient method using 72%, 64% and 52% Percoll solutions in RPMI 1640 with the cells layered on top. After centrifugation at 1500g for 30min without brake, neutrophils were recovered from the interface between the 72% and 64% layers. Cells were washed with PBS and centrifuged at 400g for 5min. Next, the supernatant was removed and cells were resuspended in 1ml of PBS. Cell numbers were determined by Casy Cell Counter and used for flow cytometric analysis or cell culture stimulation experiments.Experimental animals were euthanized per inhalation of an overdose of Isoflurane. Femurs of S100A8/A95 WT and S100A8/A9-/- bone marrow-derived neutrophils were cultured in a total volume of 200μl of RPMI 1640 media containing 10% FCS, 1% L-Glutamine, 100U/mL Penicillin, 100μg/ml Streptomycin for 7h at 37°C  in triplicates. Quantification of apoptosis (FITC Annexin V) and necrosis (Propidium Iodide) was performed according to manufacturer’s protocols (BD). The supernatant was collected and frozen at -20°C for subsequent cytokine and chemokine analysis and cells were used for further flow cytometric analysis.2x10L. sigmodontis infection in S100A8/A9-/- and corresponding WT mice by intranasal treatment with 250μg anti-Ly6G antibody  in 50μl pre-warmed sterile PBS. Treatment was repeated every second day until 5 days after L. sigmodontis infection. Corresponding control mice were treated with an IgG2a isotype antibody . Neutrophil depletion within the lung was confirmed in a pilot experiment, where IgG2a isotype control and anti-Ly6G depletion antibody were given every second day with a total of three injections and confirmation of the depletion 24h after the last injection. The systemic effect of intranasal neutrophil depletion was tested within the blood, pleura, spleen, lung as well as skin in naïve mice, which were treated trice (every second day) either with the isotype control or with the neutrophil depleting anti-Ly6G antibody (250μg). Results are shown in Neutrophil depletion was performed 18h prior to natural 5 WT and S100A8/A9-/- neutrophils were cultured in triplicates in 200μl enriched media  for 48h at 37°C  with 10 L3 larvae. Motility inhibition was assessed daily by a five point scoring system; Score 4: vigorous, fidgeting movements. Score 3: slightly impaired vigorous movements. Score 2: impaired motility/slow movements. Score 1: mostly immobile/single movements. Score 0: fully immobile within 20s observation period.For motility experiments, 1x10http://www.ihcworld.com/_protocols/special_stains/congo_red_highman.htm; 3) Alcian Blue/Periodic Acid Schiff (AB-PAS) staining to visualize mucus producing cells using the protocol: http://www.ihcworld.com/_protocols/special_stains/alcian_blue_pas_ellis.htm; 4) a Ly6G/Ly6C immunostaining to visualize neutrophils. Briefly, sections were washed, deparaffinized, rehydrated, and rinsed in PBS. A proteinase K solution (0.004%), diluted in a 1:1 glycerol-modified Tris Buffer , was used for antigen retrieval for 10min at 37°C, then tissue endogenous peroxidases were blocked by utilizing a dual endogenous enzyme block (Dako) and biotin/avidin by a biotin blocking kit (Vector). Neutrophils were stained in a 1/200 dilution with a primary antibody against Ly6G/Ly6C  dissolved in blocking serum . Detection was conducted by the Vectastain Elite ABC kit (Vector) and revelation was performed with a high sensitivity AEC substrate (Dako), which was subsequently counterstained with a Mayer’s hematoxylin solution (Merck).After necropsy, the lung was filled with 4% formalin-PBS (Sigma Aldrich) and fixed overnight. Afterwards, the fixative was renewed for another 24h, before it was replaced by 70% ethanol for 2–7 days prior paraffin embedding. Five-micron-thick serial sections were prepared. Lungs were stained with 1) hematoxylin-eosin to analyse the general structure of the lung; 2) Congo red to visualise eosinophils following the online protocol in vivo vascular permeability was determined via the intracutaneous injection of 10μg LsAg in one ear and PBS as corresponding control in the other ear. After 3min, mice were injected i.v. with NaCl 0.9% containing 0.9mg of Evans Blue (Sigma-Aldrich). After 10min, mice were euthanized by cervical dislocation. Ears were removed and transferred into 500μl of >99% formamide (Sigma-Aldrich) and incubated at 56°C for 24h. Evans Blue leakage was then determined by optical density at 620nm .Mice were anaesthetized with Rompun  and Ketamine  and Statistical analyses were performed with GraphPad Prism software Version 5.03 (GraphPad Software). Normal distribution of the data was tested with D’agostino test. Non-parametrically distributed data and data of non-sufficient animal numbers for parametric testing were analyzed by Kruskal-Wallis test followed by Dunn’s posthoc test. Differences between two unpaired groups were tested for statistical significance with the Mann-Whitney-U-test. P-values <0.05 were considered statistically significant. Power analysis with G*Power (3.1.9.4) indicated that a sample size of 8 mice per group provides a power >95% and an α-error of 0.05. Data from pooled experiments were tested by two-way ANOVA and Spearman’s test for heteroscedasticity using GraphPad Prism software Version 8. Only experiments that did not pass the heteroscedasticity test were pooled.-/- mice were subcutaneously infected with 40 L. sigmodontis L3 larvae. Concentration of calprotectin within the thoracic cavity lavage was analysed between naïve and infected WT mice and necropsy of WT and S100A8/A9-/-L. sigmodontis-infected animals was performed at 12dpi, a time-point where all L3 larvae reached the thoracic cavity and molted into L4 larvae. Following necropsy, analysis of the worm burden, cellular composition, neutrophil-associated chemokines, and neutrophil elastase were performed.In order to investigate the impact of S100A8/A9 during filarial infection, WT and S100A8/A9L. sigmodontis infection led to a significant increase of S100A8/A9 levels within the thoracic cavity lavage of infected WT animals compared to naïve WT mice . In comparison to the subcutaneous infection, mite-transmitted L3 larvae have to additionally bypass the epidermis containing Langerhans cells and dermis-containing dendritic cells, macrophages and T cells. Nevertheless, natural L. sigmodontis infection led to similar results as observed during subcutaneous L sigmodontis infection, showing a significantly reduced worm burden  and in the PVS (4dpi) upon migration of L3 larvae and were associated with an increased expression of S100A9 [Furthermore, no influx of neutrophils were observed in the PVS of infected animals at 12dpi surrounding congested  and intaf S100A9 .L. sigmodontis L3 larvae within the lung, concentration of S100A8/A9 in naïve and infected WT mice as well as absolute bronchoalveolar lavage (BAL) cell numbers and the cellular composition of the BAL in L. sigmodontis-infected WT and S100A8/A9-/- mice were determined. L. sigmodontis infection led to a significant increase of S100A8/A9 within the BAL compared to naïve WT controls  as well as elastase, enzymes which are abundantly expressed in neutrophils, were increasingly released  from bone marrow-derived neutrophils of S100A8/A9-/- mice compared to WT neutrophils at baseline conditions in vitro  were analysed for their expression of CD4, CD8, Ly6G, CD11b, SiglecF and F4/80 to differentiate CD4+ (CD4+ CD8-), CD8+ (CD8+ CD4-) T cells, neutrophils , eosinophils (SiglecF+ F4/80 low) and macrophages (SiglecF low/int F4/80+). SiglecF- F4/80- cells (P3 gate) were analyzed for their expression of Ly6G to identify neutrophils. Bronchoalveolar macrophages were identified by their expression of SiglecF+/F4/80+. Gating was performed using the fluorescence minus one (FMO) approach.Shown is a representative gating strategy from thoracic cavity and bronchoalveolar cells of a wildtype mouse infected for 12 days with (TIFF)Click here for additional data file.S2 FigA-E). Statistical significance was analyzed by two-tailed non-parametric Mann-Whitney-U-test (A-D) and by Kruskal-Wallis followed by Dunn’s multiple comparison test (E). One experiment with 3 mice per group.Frequency of neutrophils within the bronchoalveolar lavage (BAL), thoracic cavity lavage, blood, spleen and skin in naïve WT mice treated either with an isotype control or with an anti-Ly6G depleting antibody. Results are shown as median ((TIFF)Click here for additional data file.S3 Fig-/- mice 12 days after subcutaneous L. sigmodontis infection. Total number of splenocytes (A), CD4+ T cells (CD4+ T), CD8+ T cells (CD8+ T), neutrophils (NØ), macrophages (Macs), and eosinophils . Concentrations of IFN-γ (C) and TNF (D) after in vitro spleen cell culture without stimulation (US) or after stimulation with ConA or L. sigmodontis extract (LsAg) of WT and S100A8/A9-/- mice 12 days after subcutaneous L. sigmodontis infection. Results are shown as median. Statistical significance was analyzed by two-tailed non-parametric Mann-Whitney-U-test. *p<0.05, **p<0.01, ***p<0.001. Data are pooled from two independent experiments (A-B) with 8 mice per group. Data shown in C and D are pooled from two independent experiments with 4 mice per group.Total number of spleen cells in WT and S100A8/A9(TIFF)Click here for additional data file.S4 FigL. sigmodontis infection in WT and S100A8/A9-/- mice at 12 days after infection (A). Number of thoracic cavity cells (B) and total cell count of the thoracic cavity lavage 12 days after L. sigmodontis infection in WT and S100A8/A9-/- mice showing CD4+ T cells (CD4+ T), CD8+ T cells (CD8+ T), neutrophils (NØ), macrophages (Macs) and eosinophils (Eos) (C). Concentrations of CXCL-1 (D), CXCL-2 (E), CXCL-5 (F), and elastase (G) in the thoracic cavity lavage 12 days after L. sigmodontis infection in WT and S100A8/A9-/- mice. Results are shown as median (A-G). Statistical significance was analyzed by two-tailed non-parametric Mann-Whitney-U-test. *p<0.05, **p<0.01. One experiment with 5 mice per group.Worm burden following natural (TIFF)Click here for additional data file.S5 Fig-/- mice (A) and total number of thoracic cavity cells (B) and total cell counts of neutrophils (NØ), macrophages (Macs) and eosinophils (Eos) in the thoracic cavity lavage (C) 29 days after natural L. sigmodontis infection. Results are shown as median (A-C). Statistical significance was analyzed by two-tailed non-parametric Mann-Whitney-U-test (A-C). *p<0.05, **p<0.01. Data shown is of one experiment with 9–10 mice per group.Worm burden in WT and S100A8/A9(TIFF)Click here for additional data file.S6 FigA), eosinophils (B), neutrophils (C), and macrophages (D) and their respective MHCII expression  in WT and S100A8/A9-/- mice 3h after intradermal L. sigmodontis L3 or PBS injection. Results are shown as median. Statistical significance of not normally distributed data (A-G) was analyzed by Kruskal-Wallis followed by Dunn’s multiple comparison test. p<0.05, **p<0.01. Representative data from one out of two independent experiments with 5 mice per group (A-G).Total number of skin cells ((TIFF)Click here for additional data file.S7 Fig-/- mice were naturally infected with L. sigmodontis. Representative pictures of a WT mouse lung 12 days after natural L. sigmodontis infection (A) and an uninfected WT mouse (B). Pathology of lung blood vessels (bv) and mucus production in 12-day L. sigmodontis-infected mice (C) (left panel) and naïve mice (D) . Number of congested blood vessels (E) and bronchi with goblet cells (F) from naïve and 12-day L. sigmodontis-infected WT and S100A8/A9-/- mice. Spearman correlation of congested blood vessels and worm counts (G) as well as bronchi with goblet cells and worm burden (H). Total number of cells in PVS in intact and congested blood vessels of WT and S100A8/A9-/- mice 12 days after L. sigmodontis infection, as well as naïve controls (I). Influx of neutrophils  and eosinophils  in the perivascular space (PVS) of congested  and intact blood vessels  of 12-day L. sigmodontis-infected WT mice. Results are shown as median . Data are pooled from 2 independent experiments  with 3–8 mice per group. Remaining figures are representatives of two experiments with at least 2–5 mice per group.C57BL/6 WT and S100A8/A9(TIFF)Click here for additional data file.


Increasing studies have shown that long noncoding RNAs (lncRNAs) are pivotal regulators participating in carcinogenic progression and tumor metastasis in colorectal cancer (CRC). Although lncRNA long intergenic noncoding RNA 460 (LINC00460) has been reported in CRC, the role and molecular mechanism of LINC00460 in CRC progression still requires exploration.The expression levels of LINC00460 were analyzed by using a tissue microarray containing 498 CRC tissues and their corresponding non-tumor adjacent tissues. The correlations between the LINC00460 expression level and clinicopathological features were evaluated. The functional characterization of the role and molecular mechanism of LINC00460 in CRC was investigated through a series of in vitro and in vivo experiments.LINC00460 expression was increased in human CRC, and high LINC00460 expression was correlated with poor five-year overall survival and disease-free survival. LINC00460 overexpression sufficiently induced the epithelial–mesenchymal transition and promoted tumor cell proliferation, migration, and invasion in vitro and tumor growth and metastasis in vivo. In addition, LINC00460 enhanced the protein expression of high-mobility group AT-hook 1 (HMGA1) by directly interacting with IGF2BP2 and DHX9 to bind the 3′ untranslated region (UTR) of HMGA1 mRNA and increased the stability of HMGA1 mRNA. In addition, the N6-methyladenosine (m6A) modification of HMGA1 mRNA by METTL3 enhanced HMGA1 expression in CRC. Finally, it suggested that HMGA1 was essential for LINC00460-induced cell proliferation, migration, and invasion.LINC00460 may be a novel oncogene of CRC through interacting with IGF2BP2 and DHX9 and bind to the m6A modified HMGA1 mRNA to enhance the HMGA1 mRNA stability. LINC00460 can serve as a promising predictive biomarker for the diagnosis and prognosis among patients with CRC.The online version contains supplementary material available at 10.1186/s13046-021-01857-2. Long noncoding RNAs (lncRNAs) are a class of large transcripts with more than 200 nucleotides having no or limited protein-coding capacity, lncRNAs are pervasively transcribed from the human genome , 2. WithCRC is one of the most lethal malignancies worldwide and is also the leading cause of cancer incidence rate and mortality in China . Most paLncRNAs are increasingly being discovered, and they may affect the complicated metastasis progress. In the present study, we assayed clinical specimens and adjacent normal tissues from patients with CRC to screen differentially expressed lncRNAs. We identified a novel lncRNA, called long intergenic noncoding RNA 460 (LINC00460), which is positively correlated with CRC proliferation and metastasis. LINC00460 is located on chromosome 13q33.2 and is transcribed into a 935 bp transcript; this lncRNA has recently been reported in several cancers. Most recently, several studies have shown LINC00460 was overexpressed in multiple cancers, such as gastric cancer , lung ca6A) reader enhancing mRNA stability through recognizing m6A modification sites [6A modification of mRNAs [6A modification in mRNAs have been shown regulating mRNA stability [6A modification in regulating HMGA1 has not been reported.Most recently, Marco Mineo et al. have demonstrated that lncRNA HIF1A-AS2 binding proteins insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) and ATP-dependent RNA helicase A (DHX9) are engaged in downstream mRNA stabilization . The Higon sites . Methyltof mRNAs . The m6Atability . However6A) modification of HMGA1 mRNA by METTL3 enhanced its expression in CRC and LINC00460 regulate HMGA1 expression depending on METTL3. It suggested LINC00460/DHX9/IGF2BP2 complex may regulate HMGA1 expression through recognizing the m6A modification sites of HMGA1 to enhance its mRNA stability.In the present study, we showed that LINC00460 is highly expressed in CRC and indicates poor prognosis. We also revealed that LINC00460 regulated the CRC growth and metastasis in vitro and in vivo. LINC00460 interacted with IGF2BP2 and (DHX9) to promote the mRNA stability of HMGA1 and further leading to biological response to CRC malignant proliferation and extensive metastasis. In addition, the  slides included examination of 498 pairs of tissue specimens including CRC tissues and the matched corresponding adjacent normal colorectal tissues from CRC patient cohorts that were enrolled at Affiliated Hospital of Xuzhou Medical University from 2010 to 2015 in China. Clinical and pathological information was obtained from the medical records of the Affiliated Hospital of Xuzhou Medical University. Survival time was calculated based on the date of surgery to the date of death or to the last follow-up. The patient studies were conducted in accordance with Declaration of Helsinki. The use of these specimens and data for research purposes were granted approval by the Ethics Committee of the Affiliated Hospital of Xuzhou Medical University.2. 1% O2 was generated by flushing a 94% N2/5% CO2 mixture into the incubator.The CRC cell lines and HEK293T cell were obtained from the cell bank of Chinese academy of sciences. SW480, SW620, HCT116, DLD1, LOVO, HT29, FHC and HEK293T cells were cultured in 1640 and DMEM Medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and incubated in a 37 °C humidified incubator with 5% COThe small interfering RNAs  against human IGF2BP2 and DHX9 were transfected into the CRC cells SilenFect reagent , while non-specific siRNA was used as negative controls. All siRNAs were purchased from Genepharma Technology . The sequences of the siRNAs were listed in below:siIGF2BP2:CAGUUUGAGAACUACUCCUTTAGGAGUAGUUCUCAAACUGTT.siDHX9:GCCUCCAAGAAAGUCCAAUTTAUUGGACUUUCUUGGAGGCTT.The cDNA of LINC00460 was inserted into pCDH-CMV-MCS-EF1-GreenPuro lentivirus vector at ECOR1/BamH1 sites. LINC00460 shRNA sequences were cloned to the vector pLko.1 at Age1/ECOR1 sites. The METTL3 shRNA vectors were purchased from GENE company . The overexpression and knockdown lentiviruses were generated by co-transfecting 293 T cells with the other two packing vectors pMD2G and psPAX. The supernatants of 293 T cultured medium were collected 48 h later, filtered through 0.45-mm filters  and concentrated using Amico Ultra centrifugal filters (Millipore 100KD MWCO). The concentrated virus was used to infect HCT116 and SW480 cells and stable LINC00460 overexpression or knockdown cells were generated by lentivirus infection. The relative LNC00460 and METTL3 shRNA sequences were described as below:ShLINC00460#1-For: CCGGGGTACCCAGACATTGTTATGACTCGAGTCATAACAATGTCTGGGTACCTTTTTG;ShLINC00460#2-For: CCGGGGAGGCGTCTGTGTAGCAATTCTCGAGAATTGCTACACAGACGCCTCCTTTTTG;shMETTL3#1-For: CCGGGCAAGTATGTTCACTATGAAACTCGAGTTTCATAGTGAACATACTTGCTTTTTG.shMETTL3#2-For:CCGGGCCAAGGAACAATCCATTGTTCTCGAGAACAATGGATTGTTCCTTGGCTTTTTG.P-values were less than 0.05.Total RNA was extracted from five paired CRC tissues and NCTs using TRIzol Reagent . RNA quantity and quality were measured by NanoDrop ND-1000. RNA integrity was assessed by standard denaturing agarose gel electrophoresis. LncRNA microarray was performed by . Arraystar Human LncRNA Microarray V3.0 is used for detecting the global profiling of human LncRNAs. Agilent Feature Extraction software (version 11.0.1.1) was used to analyze acquired array images. LncRNAs were deemed differentially expressed if their fold change between the CRC and NCT  groups exceed 2.0 and their RNA was extracted using TRIzol (Invitrogen) and cDNA was synthesized using the HiScript 1st Strand cDNA Synthesis Kit . Real-time PCR was carried out on ABI-7500 using UltraSYBR One Step RT-qPCR Kit . The primers using for quantitative RT-PCR analysis were listed in Additional file Cells were harvested, total protein from cells was extracted by using RIPA lysis buffer and was qualified by using a BCA detecting kit . Proteins samples were subjected to 7.5% or 10% SDS-PAGE and transferred onto a PVDF membrane, and then incubated with specific antibody at 4 °C overnight, respectively. The next day, the membranes were incubated with secondary antibodies included HRP-goat anti-mouse, HRP-goat anti-rabbit  at room temperature for 1 h. Protein bands were detected on Tanon 5200 automatic chemiluminescence imaging analysis system using ECL reagent . Antibody against GAPDH was used as control . HMGA1 , IGF2BP2 , DHX9 , E-cadherin , N-cadherin ,β-catenin , were used for Western blot assays.The in situ hybridization of LINC00460 was performed on formalinfixed paraffin-embedded sections using specific oligonucleotide probe , following the manufacturer instructions. Positive control probe (Hs-PPIB) and Negative control probe (DapB) were included for each hybridization procedure and analyzed using an OLYMPUS microscope with OLYMPUS OlyVIA V2.9 software.IHC assays was implemented following a standard streptavidin-peroxidase (SP) method as previously reported  and heatCells were seeded on glass bottom cell culture dish  for about 24 h, then cells were fixed with 10% neutral buffer formalin, incubation with hydrogen peroxide  and protease III  for about 10 min separately. After cell preparation and pretreatment, following the manufacturer instructions and used specific oligonucleotide probe  to visualize the single RNA in each cell. The nuclei were stained with DAPI for 15 min. Finally, images were taken under a confocal microscope.CCK-8 assay was applied to measure the cell proliferation according to the Cell Counting Kit-8 manufacturer protocol . Colony formation assay was performed in 60 mm dishes one thousand cells seeded and two weeks cultured. Colonies were counted manually after staining with 0.1% crystal violet.The Real Time Cell Analyzer  were used for examined the effect of LINC00460 on cell proliferation. The xCELLigence system was used according to the instructions of the supplier (ACEA Biosciences). The system measured impedance differences to derive cell index values at time points and it may be set by the operator.The xCELLigence system consists of four main components: the RTCA analyzer, the RTCA DP station, the RTCA computer with integrated software and disposable E-plate 16. Firstly, the optimal seeding concentration for proliferation assay was determined. After seeding the number of 5000 cells in 200 ml medium to each well in E-plate 16, the attachment and proliferation of the cells were monitored every 15 min. All experiments were carried out for 96 h .Cells were starved in serum-free media for 24 h then added to the top chamber of 24-well transwell chambers plates . Specially, for the invasion assay, cells were seeded into the top chamber coated with Matrigel (BD Biosciences). Complete medium was added to the bottom chambers to stimulate migration or invasion. After incubation for 24–48 h, the cells those adhered to the lower surface of the membrane were stained with 0.1% Crystal Violet, and then the percentages of migrated or invasion cells were calculated. Five randomly selected fields per filter were counted.6 cell/well onto six-well plates and cultured to about 80% confluence. Then, a sterile 10-μl pipette tip was used to form artificial scratches for each well. The suspended cells were washed away with PBS, and then the cells were cultured in medium with 1% FBS. Cell migration distance was photographed at 0 h and 24 h under an inverted light microscope.In wound healing assay, cells were seeded at a density of 1 × 10RNA pull-down assay was carried out as described briefly: in vitro biotin-labeled RNAs (LINC00460 and the antisense RNA) were transcribed with Biotin RNA Labeling Mix  and T7 RNA polymerase  treated with RNase inhibitor, and purified with Clean-up kit . The biotinylated LINC00460 probes were dissolved in binding and washing buffer and incubated with Streptavidin Agarose Resin . Then cell lysates of HCT116 and SW480 were incubated with probes-coated streptavidin beads, and pull-down proteins were run on SDS-PAGE gels, and then gels were stained by Coomassie Blue staining, and differential bands were cut off for Mass spectrometry .The RIP experiment was carried out with the EZ-Magna RIP Kit (Millipore) according to the manufacturer’s protocol using 5 mg of antibody. HCT116 and SW480 cells were lysed in complete RIP lysis buffer, and the cell extract was incubated with protein A/G agarose beads conjugated with specific antibodies or control IgG for 2 h at 4 °C. Beads were washed and incubated with Proteinase K to remove proteins. Finally, purified RNA was subjected to quantitative RT-PCR analysis. Antibody IGF2BP2 , DHX9 .MeRIP, we used Anti-N6-methyladenosine (m6A) antibody (ab151230) to pull down m6A modified HMGA1. Total RNA was extracted from cells using Trizol (Thermo-Fisher). 100 μg RNA was added to 500 μl MeRIP buffer  and incubated briefly with 1 μl rabbit IgG and then the IgG was removed by protein A/G beads. The pre-cleaned lysates were transferred to new tubes and incubated with rabbit IgG or m6A antibody for 2 h at 4 °C with rotation. Finally, m6A bound RNA was extracted with Trizol and the RNA level of HMGA1 was measured by qRT-PCR.6) were injected subcutaneously into the flanks of mice correspondingly. Tumors volume (V) was monitored every 2 days by measuring the long axis (L) and the short axis (W) of xenograft tumor and calculated with the following formula: V = (L × W2)/2.The female BALB/c nude mice (6–8 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. . All animal experiments were approved by the Animal Care and Use Committee at Xuzhou Medical University. Groups of HCT116-Luc-shCtrl, HCT116-Luc-shLINC00460, and HCT116-Luc-shLINC00460 + HMGA1 cells . GSE40967 contains 566 CRC tissues.p < 0.05 was considered statistically significant.Statistical analyses were carried out using SPSS 20.0 software  and GraphPad Prism 8 The association between LINC00460 and the clinicopathologic parameters of the CRC patients were evaluated by a Chi-square test. The Kaplan–Meier method and log-rank test were used to evaluate the correlation between LINC0046 expression and CRC patient survival. The unpaired t test was used to determine the statistical significance of differences between groups. Data were presented as mean ± SD. p < 0.05, Additional file p = 0.0048, Fig. p < 0.001, Fig. p < 0.0001, Fig. p = 0.0005, Fig. p < 0.001) and DFS    to screen differentially expressed lncRNAs by using lncRNA microaaray. Among the lncRNAs tested in our microarray, 462 were significantly upregulated, and 817 were significantly downregulated in CRC relative to NCT  is a well-known driving force for cell migration and invasion, we investigated the effects of LINC00460 on EMT. We observed that LINC00460 overexpression reduced the mRNA expression of E-cadherin and increased the mRNA expression of N-cadherin, whereas LINC00460 knockdown increased the mRNA level of E-cadherin and decreased N-cadherin mRNA expression Fig. e-h. ConsTo investigate the regulatory mechanism of LINC00460 in CRC, the cellular localization of LINC00460 in CRC cells was studied using RNA fluorescence in situ hybridization (FISH) and subcellular fractionation assays. The results showed that LINC00460 was mainly localized in the cytoplasm of colon cancer cells  [6A modification regulates the mRNA stability of HMGA1, silencing the expression of METTL3, an important m6A methyltransferase, decreased HMGA1 expression  , Resultsion Fig. a-b. Furtion Fig. c-d. FinaRNA Fig. e-f. ThesAccumulating evidence has indicated that lncRNAs play vital roles in human cancer. However, limited information is known about their pattern of action and potential role in the regulation of cancer-related processes. To our knowledge, LINC00460 has been reported to participate in many kinds of malignancies and promotes cancer progressions –20. AlthIn previous studies, LINC00460 have been proved overexpressed in CRC by using TCGA data and conforming by a small CRC cohort detected by qRT-PCR. In this study, RNAscope probes were used to detect the expression in a CRC cohort containing 498 CRC tissues and corresponding non-tumor adjacent tissues. Our findings showed that LINC00460 expression increased in human CRC, and high LINC00460 expression was correlated with clinicopathological parameters, such as TNM stage, lymph node metastasis, metastasis, and tumor size. Furthermore, a high LINC00460 expression indicated poor five-year OS and DFS via tissue microarrays. Univariate and Multivariate Cox regression analyses showed that high LINC00460 expression is an independent adverse prognostic factor for CRC patients. Our findings provided reliable evidence that LINC00460 could act as a potential clinical prognostic predictor for patients with CRC.To further validate the biological function of LINC00460 in CRC progression, we investigated the malignant features of LINC00460 in a series of in vitro and in vivo experiments. CRC cell lines HCT116 and SW480 were studied using LINC00460 knockdown or the overexpress lentivirus infection. Consistent with these previous data, our results showed that LINC00460 KD and OE respectively reduced or increased tumor cell growth, migration, and invasion abilities. LINC00460 was shown to support the proper functioning of the EMT transcription program, downregulate epithelial markers (such as E-cadherin), and upregulate mesenchymal markers (such as N-cadherin). Our data also showed that LINC00460 downregulation suppressed tumor growth and lung metastasis in a xenograft mouse model. These results consistently emphasized the importance of LINC00460 in CRC progression.6A reader and the 3’UTR of HMGA1 mRNA have a strong m6A modification signal. We then showed that silencing METTL3 could affect LINC00460 induced HMGA1 expression, suggesting LINC00460/DHX9/IGF2BP2 complex may stabilize the mRNA of HMGA1 through recognizing m6A modification.Several studies have suggested LINC00460 was mainly located in cytoplasm –19. In tHigh mobility group proteins are a family of non-histone chromatin-bound proteins without any transcriptional activity; however, they modulate transcription by altering the architecture of chromatin , 44 and In summary, we have exhibited a set of comprehensive data suggesting the clinical and functional significance of LINC00460 in CRC proliferation and metastasis. We uncovered a novel molecular mechanism of LINC00460 in regulating CRC progression. We conclude LINC00460/DHX9/IGF2BP2 complex may regulate HMGA1 expression through recognizing the m6A modification sites of HMGA1 to enhance its mRNA stability and promote CRC metastasis. LINC00460 may serve as potential biomarker and target in the prognosis and treatment of CRC.Additional file 1: Table S1. Primers used for qRT-PCR.Additional file 2: Dataset 1. Differentially expressed genes in the microarray.Additional file 3: Figure S1. PCR detection of LINC00460 in CRC tissues and corresponding adjacent normal colon tissues using gel electrophoresisAdditional file 4: Table S2. Univariate Cox regression analysis of LINC00460 expression and clinicopathologic variables predicting the survival of CRC patientsAdditional file 5: Table S3. Multvariate Cox regression analysis of LINC00460 expression and clinicopathologic variables predicting the survival of CRC patientsAdditional file 6: Figure S2. RTCA assays showed LINC00460 positively regulated CRC cells proliferation.  Effect of LINC00460 KD or OE on HCT116 and SW480 cells proliferation as assessed by Real-Time Cell Analyzer  assays .Additional file 7: Figure S3. Wound-healing assays showed LINC00460 positively regulated CRC cell mobility.  The ability of motility in HCT116 and SW480 cells ± LINC00460 KD/OE tested by wound-healing assay. Statistical analysis was performed using unpaired t-tests. All statistical tests were two-sided. *p < 0.05, **p < 0.01, ***p < 0.001.Additional file 8: Figure S4. LINC00460 intracellular localization in CRC tissues.Additional file 9: Figure S5. Relative mRNA expression levels of HMGA1 expression in tumor xenografts.Additional file 10: Figure S6. Prediction of HMGA1 mRNA m6A modification status. (A) HMGA1 mRNA m6A modification status in the MethylTranscriptome DataBase v2.0. (B) Potential METTL3 modified regions in the 3’UTR of HMGA1 mRNA.


Cucumis sativus L.) is a widely planted vegetable crop that suffers from various pathogen infections. Powdery mildew (PM) is typical disease caused by Sphaerotheca fuliginea infection and destroys the production of cucumber. However, the metabolic responses to S. fuliginea infection are largely unknown.Cucumber  and differentially expressed genes (DEGs) under S. fuliginea infection, providing some potential regulators for the improvement of PM resistance. PM resistance of cucumber was controlled by a complex network consisting of various hormonal and metabolic pathways.Many differentially expressed transcription factor genes were identified under  Cucumis sativus L.) is an economically significant vegetable crop widely planted in many countries (http://faostats.fao.org). Although cucumber is important, its production is largely limited by various infective agents, including bacterial, viral, fungal and oomycete  and B [C2H3N: CH3OH (9: 1)]. Rutin were purchased from Sigma-Aldrich  [The total flavonoid contents were measured using HPLC assay. In briefly, cucumber samples were extracted with 75% ethanol at 37 °C for 30 min. Cell debris was discarded by centrifugation, and the supernatant was used for HPLC analysis. For HPLC analysis, the supernatant was uploaded to a Waters HPLC e2695 series . HPLC was performed on an XBridge C18 (4.6 mm × 250 mm) column. The elution was performed using solution A [H U.S.A.) .t-test was performed to evaluate the differences in the metabolite levels between two sample groups. An FDR (Benjamini–Hochberg) method was used to adjust the P value for multiple tests. The detailed method of statistical analysis was performed according to the previous publication [Student lication .Additional file 1: Table S1 The detailed information of RNA sequencing.Additional file 2: Table S2 The detailed information of the mapped reads from different samples.Additional file 3: Table S3 The detailed information of 24,317 cucumber genes with annotation.Additional file 4: Table S4 The detailed information of all identified metabolites.Additional file 5: Figure S1 Analysis of several quality parameters of the metabolomes.Additional file 6: Table S5 The detailed information of metabolites with KEGG annotation.Additional file 7: Table S6 The detailed information of flavonoid metabolism-related genes.Additional file 8: Table S7 The detailed information of hormone-related genes.Additional file 9: Table S8 The detailed information of fatty acid metabolism-related genes.Additional file 10: Table S9 The detailed information of diterpenoid metabolism-related genes.Additional file 11: Table S10 The number of TFs.Additional file 12: Figure S2 Expression validation of the key genes involved in responses to Sphaerotheca fuliginea infection.Additional file 13: Figure S3 Differential accumulation of total flavonoids.Additional file 14: Table S11 The primer information.


Cupriavidus metallidurans BS1 was isolated from the Zijin gold–copper mine in China. This was of particular interest since the extensively studied, closely related strain, C. metallidurans CH34 was shown to not be only highly heavy metal resistant but also able to reduce metal complexes and biomineralizing them into metallic nanoparticles including gold nanoparticles. After isolation, C. metallidurans BS1 was characterized and complete genome sequenced using PacBio and compared to CH34. Many heavy metal resistance determinants were identified and shown to have wide-ranging similarities to those of CH34. However, both BS1 and CH34 displayed extensive genome plasticity, probably responsible for significant differences between those strains. BS1 was shown to contain three prophages, not present in CH34, that appear intact and might be responsible for shifting major heavy metal resistance determinants from plasmid to chromid (CHR2) in C. metallidurans BS1. Surprisingly, the single plasmid – pBS1 (364.4 kbp) of BS1 contains only a single heavy metal resistance determinant, the czc determinant representing RND-type efflux system conferring resistance to cobalt, zinc and cadmium, shown here to be highly similar to that determinant located on pMOL30 in C. metallidurans CH34. However, in BS1 another homologous czc determinant was identified on the chromid, most similar to the czc determinant from pMOL30 in CH34. Other heavy metal resistance determinants such as cnr and chr determinants, located on megaplasmid pMOL28 in CH34, were shown to be adjacent to the czc determinant on chromid (CHR2) in BS1. Additionally, other heavy metal resistance determinants such as pbr, cop, sil, and ars were located on the chromid (CHR2) and not on pBS1 in BS1. A diverse range of genomic rearrangements occurred in this strain, isolated from a habitat of constant exposure to high concentrations of copper, gold and other heavy metals. In contrast, the megaplasmid in BS1 contains mostly genes encoding unknown functions, thus might be more of an evolutionary playground where useful genes could be acquired by horizontal gene transfer and possibly reshuffled to help C. metallidurans BS1 withstand the intense pressure of extreme concentrations of heavy metals in its environment.The highly heavy metal resistant strain  Cupriavidus metallidurans BS1 was isolated from a gold copper mine in China and was found to have 99.9–100% 16S rRNA gene identity to available genomes in the National Center for Biotechnology Information (NCBI) database of C. metallidurans strains  using the Geneious alignment   and copper (Cu) handling in gold biomineralization. C. metallidurans CH34 was shown to accumulate toxic Au(I/III) complexes from solution which in turn induced Cu resistance gene clusters, to probably promote cellular defense against oxidative stress -species are subsequently imported into the bacterial cytoplasm (dentity) . The come stress . Au(III)ytoplasm  where thytoplasm .C. metallidurans CH34 was shown to be a facultative chemolitho-autotrophic β-proteobacterium belonging to the family Burkholderiaceae/order Burkholderiales and has been found to be highly resistant to Zn2+, Cd2+, Ni2+, AsO43– CrO42–, Hg2+, Ag+, Cu1+/2+, Pb2+, and Co2+  systems of the heavy metal efflux RND (HME) protein family are known  of the gold–copper mine located at the geography coordinates N25° 11.027′ and E116° 24.414′. The bacterium was isolated by inoculating the collected soil samples into Reasoner’s 2A (R2A) agar medium . There were three replicates of each sample and prior to inoculation of the soil samples on the R2A medium, 1 g of each sample, was mixed with 1000 μL double distilled water (ddw) and incubated for 30 min at 30°C with 150 revolutions per minute (rpm). After incubation, 100 μL of the supernatant was used for the inoculation in R2A medium. The broth culture was incubated at 30°C with shaking at 250 rpm while the agar plates were incubated in the dark at 30°C overnight. The bacterial strain was purified by continuously growing the strain on R2A agar solid plates, without any heavy metal addition, until single colonies were obtained. Finally, the isolates were stored at −80°C in 30% sterile glycerol for long term storage and in −20°C in 30% sterile glycerol for continuous laboratory experiments.The heavy metal resistant bacterium, 4, ZnSO4 7 × H2O, CdCl2 5 × 2H2O, NaAsO2, NiCl2 × 6H2O, HAuCl4 4 × H2O, CoCl2 × 6H2O and Pb(NO3)2 supplemented mineral salts media 284 (gluconate 2g/L as sole carbon source) cultures plates. Mineral salts medium 284 contained (1000 ml of DD water) Tris 6.06 g pH 7.0, NaCl 4.65 g, KCl 1.49 g, NH4Cl 1.07 g, Na2SO4 0.43 g, MgCl2 6∗H2O 0.20 g, CaCl2 2H2O 0.03 g, Na2HPO4 2H2O 0.23 g, Fe(III)NH4 citrate 0.005 g, sodium gluconate 2 g, 1 ml of the trace element solution SL 7 and Difco BactoAgar 15 g L–1  on CuSO15 g L–1 . Medium 15 g L–1 .®ZYM system test kit  was used for biochemical characterization of C. metallidurans strain BS1 according to manufacturer’s protocol.The bacterium strain was grown aerobically in 40 ml R2A broth culture incubated at 30°C with shaking at 250 rpm. After 24 h of growth, the genomic DNA of the bacterium strain was extracted using the TIANamp Bacteria DNA Isolation Kit following the standard protocol provided by the manufacturer . The quantity and purity of genomic DNA were assessed using an UV spectrophotometry . The OD260/280 value of the genomic DNA higher than 1.80 was picked to check the intact ones via agarose gel electrophoresis (0.8%). Samples containing greater than 25 μg of intact genomic DNA were sent out to perform complete genome sequencing. Molecular identification of the strain was carried out by partial sequencing of the 16S rRNA gene. The 27F/1492R primer pair was used to amplify 16S sequence and the PCR product was subsequently sequenced. Amplification of the 16S rRNA gene was performed using the polymerase chain reaction (PCR). BIO-RAD S1000TM Thermal Cycler  machine was used for the PCR and PCR cycle used for amplification was as follows: 5 min at 95°C, followed by 30 cycles of 30 s at 95°C, 30 s at 54°C, 1 min at 72°C and a final extension of 7 min at 72°C. The machine was finally set at 16°C for 2 min for final cooling before the storage of the amplified products. The PCR products were sequenced by Biosune Company  using the Sanger method. APIC. metallidurans strain BS1 genome was sequenced using a PacBio RS II platform and Illumina HiSeq 4000 platform at the Beijing Genomics Institute . Four SMRT cells Zero-Mode Waveguide arrays of sequencing, were used by the PacBio platform to generate the sub reads set. PacBio sub reads (length < 1 kb) were removed. The detailed methods of library construction and sequencing can be found at Illumina’s official website1. The program Pbdagcon was used for self-correction2. Draft genome contigs, which are uncontested groups of fragments, were assembled using the Celera Assembler against a high quality corrected circular consensus sequence subreads set. To improve the accuracy of the genome sequences, GATK3 and SOAP tool packages  were used to make single-base corrections. To trace the presence of any plasmid, the filtered Illumina reads were mapped using SOAP to the bacterial plasmid database4 .The 5. The 16S rRNA gene sequences were submitted for comparison and identification to the GenBank databases using the NCBI Blastn algorithm and to the EMBL databases using the Fasta algorithm. The phylogenetic tree of 16S rRNA gene sequences and the amino acid sequences of compared resistance determinants were inferred by Geneious prime 2020 0.4. (Geneious Tree Builder) Geneious prime 2020 0.4. (Geneious Tree Builder)6, global alignment, gap open penalty 12, gap extension penalty 3, Blosum62 cost matrix, Jukes-Cantor, Neighbor-Joining, Resampling Method – Bootstrap; with 100 replicates). Morphological and physiological experiments were done to further characterize the bacteria.Close relative and phylogenetic affiliation of the obtained 16S rRNA sequences were determined by using the BLAST search program at the NCBI websiteC. metallidurans BS1 was submitted to NCBI Prokaryotic Genome Annotation Pipeline (Annotation Software revision 4.6) and The RAST Server: Rapid Annotation using Subsystem Technology (version 2.0) for gene identification following the standard operating procedures. The genes were predicted using Glimmer 3.02  bacteria that are oxidase- and catalase-positive, produce indole from tryptophan, assimilate histidine, D-gluconate, adipate and L-malate but not D-glucose, L-arabinose, D-fructose, D-mannose, D-mannitol, N-Acetyl-D-glucosamine or maltose as C-source /(G + C) calculations based on Genomic sequences of C. metallidurans BS1, the results of gene distribution, ncRNA distribution and gene annotation are also shown on this C. metallidurans BS1 conferred resistance to Zn2+ displaying a MIC of 20 mM, Cd2+ (2.5 mM), Co2+ (20mM), Ni2+ (8 mM), As3+ (3.5 mM), Cu2+ (5 mM), Au3+ (1 μM) and Pb2+ (1.7 mM) on Mineral salts medium 284 plates. The MICs were compared to those obtained with C. metallidurans CH34: Zn2+ (25 mM), Cd2+ (3.5 mM), Co2+ (35mM), Ni2+ (7 mM), As3+ (2.5 mM), Cu2+ (3 mM), Au3+ (0.5 μM) and Pb2+ (1.6 mM) respectively  similarity to C. metallidurans CH34 but the cnr determinants were significantly different  with 6,91335 bp of size in total and identical 63.5% of G + C content. C. metallidurans BS1 contained one chromosome , one chromid  and one megaplasmid – pBS1  close in size to the combined two megaplasmids, pMOL28 and pMOL30  but missing almost 40,000 bp, present in type strain C. metallidurans CH34 , C. metallidurans NA1 (238 contigs), C. metallidurans NA4 (chromosome + chromid + pNA4_A-_D), C. metallidurans NE12 (87 contigs), C. metallidurans NBRC101272 (63 contigs), C. metallidurans NDB3NO24 (38 contigs) and C. metallidurans Ni-2 (chromosome + pNi-2_1-3) , compared with rRNA database and Rfam database   . The anai-2_1-3) . Rfam7 p 12 sRNA . RNA anadatabase . C. metaes (CDS)  while Mathetical . The cluthetical . CDS assthetical . COG anacarX (Rmet_5644) is 97%, crtB (Rmet_4149) is 99%, tolerance and utilization of acetone - acxR (Rmet_4104) 97% (acxABC (Rmet_4105-7) absent) similar to the chromid of CH34. A translation initiation factor IF-1 infA2 (Rmet_5176) in BS1 was 100% similar to the one present in CH34.The chromid in both CH34 and BS1 harbors genes specialized in the activities of sulfonate/taurine transport and utilization, sulfite oxidation, polyhydroxy alkanoic acid (PHA) synthesis and conversion, biofilm formation, and exopolysaccharide synthesis squalene/hopene synthesis, tannin degradation . The chrC. metallidurans strains CH34, BS1 and NA4is showing a high genome plasticity  , the gene locus encoding a zinc and cadmium PIB2-type ATPase (CadA) as well as one mercury resistance cluster  . All CMG located  , and ECF RpoQ  are not present on the genome of strain BS1 due to the lack of CMGI-D. Instead of the missing sigma factor, strain BS1 harbors one additional (SigX) with 74.5% similarity on amino acid level to RpoO  to chromid  and atpX (encoding an not existing P-type ATPase family protein in CH34) were brought into spatial proximity on the chromid  having three-component cation efflux systems such as a well-studied CzcCBA system , CnrCBA , CusCBA , NccCBA  and plenty of additional genes encoding cation diffusion facilitators  such as czcD, dmeF and fieF, a member of the DMT  family such as cnrT, major facilitator superfamily (MFS super family) such as nccT/nreB and also cation P-type ATPases for cytoplasmic detoxification  , czc  and ncc  in C. metallidurans BS1 could be identified with up to 100% AA sequence similarities to those found in C. metallidurans CH34. This high number of different resistance determinants enable multiple resistance mediation and ability to handle the following metals; Zn2+, Cd2+, Ni2+, Ag+, Cu1+/2+, Pb2+, and Co2+, from the micromolar to the millimolar concentration range.The encoded determinants of heavy metal resistance that were found on the genome of BS1 included chemiosmotic efflux of cations with proton antiporters of the HME-RND family (nd czcP)  detoxifynd czcP) . Memberseriplasm  and the eriplasm  as a twoIB2/4-type ATPases ZntA, PbrA and CzcP, which are primarily involved in Zinc, cadmium, cobalt and lead efflux, with a 97–100% conservation at the amino acid level  or calcium (Ca+-type) are present in both strains with a similarity of 98-99%  by duplication of the czc cluster on plasmid and chromid in BS1 and the plasmid encoded CDF protein showed a lower similarity with 91.5% in comparison to the chromid encoded version with 100% similarity on the amino acid level to CzcD of CH34  of E. coli as well as with an unknown and uncharacterized CDF (CdfY) member only encoded on chromid in BS1  and OMF (outer membrane factor) in synteny was detected and these could be sorted to the HME-RND efflux systems in BS1 which have been shown to be involved in heavy metal resistance/tolerance (2)], HME2 (CnrA and NccA), HME3a , HME3b (NimA and HmzA), HME3c (HmyA) and HME4 (CusA and SilA) group   group . The gen pMOL30)  (Supplem pMOL30) . In addi cluster .C. metallidurans BS1 genome revealed a high degree of similarity of metal resistant factors to the well-studied C. metallidurans strain CH34. Overall, the genome of this bacterium contained 42 genes coding for proteins indirectly or directly involved in copper homeostasis including Cu resistance proteins encoded by the 1R1A1C1B1D1copS gene cluster as well as another homolog but extended gene cluster copHMKNSRACBDIJGFLQHE   (IB1-type ATPases (CtpA) from the genome of strain CH34 encoded on CMGI-D is not present due to the absence of this genomic island in strain BS1   efflux pump. Many predicted protein homologies were found in both C. metallidurans BS1 and the well characterized strain C. metallidurans CH34. The cupR and cupA gene products from C. metallidurans CH34 have 99 and 98% amino acid (AA) sequences identity, respectively, to those found on the genome of C. metallidurans BS1  by reductive precipitation from Au(I/III) -complexes and provided evidence for the bacterial contribution to the authigenic formation of secondary bacterioform gold grains and nuggets -oxidase that detoxifies gold complexes by a reduction pathway of Au(III)-complexes via Au(I)-intermediates to Au(0) nanoparticles in the periplasm are highly conserved in both strains  , the RNDd CopAY)  and thrend RdxI) . The fourain BS1  (Table 4s operon . Furtherf Hg(II) . The e Momplexes   in BS1 which has a nonsense mutation in the middle of the reading-frame.The Czc determinant mediating resistance to Co, Zn, and Cd is encoded on pMOL30 in ans CH34  starting similar . CzcA, a similar . The duprans BS1 . Sequencczc cluster present on chromid of BS1 was more preserved and similar to the czc cluster on pMOL30 in CH34 than czc present on the pBS1 in BS1. This phenomenon of a czc determinant duplication was observed in C. metallidurans BS1 while previously a similar phenomenon was also reported in C. metallidurans NA4 where a large part of the resistance systems was found not to be plasmid-encoded. C. metallidurans NA4 was shown to have similar genome architecture in that the main large metal resistance clusters on pMOL30 were syntenic with gene clusters on NA4’s chromid instead of NA4’s plasmids (pNA4_A-D) as was also the case in BS1 (zntA_2B2A2_S2R2czcI core-determinant was present (DDF84_RS21985 - DDF84_RS22020) comprising structural and regulatory genes as well as encoding the PIB2-type ATPase, ZntA  with same pattern onward  as the determinant was present in strain BS1 with 98–99% similarity index  in same direction and close to the mer2 cluster on the chromid  operon in CH34 was similar to that found in BS1. Both the probable structural (cnrCBAT) and regulatory (cnrYXH) genes were present on chromid but with only 93, 89, 89, 93, 85, 83, and 61% AA sequence similarity to CnrA, CnrB, CnrC, CnrT, CnrH, CnrX, and CnrY from C. metallidurans CH34, respectively, indicating that this determinant might have undergone an accelerated mutation rate during evolutionary adaptation  between czc and cnr, while in type strain CH34, czc is present on pMOL30 and cnr and chr are present on pMOL28  and location upstream of the chr cluster as it is in strain CH34 , showed a 100% identical AA sequence in both strains, CH34 and BS1. A nonsense/frameshift mutation in NccB, indicated to be present in strain CH34 and NA1, NA4, H1130  on pMOL30 displayed 100% sequence identity to the protein found on the chromid of C. metallidurans BS1, which also encoded cnr putative structural genes, cnrCBA. The presence of the czc and cnr operons encoded on the same locus supported the idea of an ancestral cobalt or cobalt-zinc resistance operon that evolved divergently by duplication and acquired additional specificities to become cnr and czc  determinant was located upstream of the cnr operon in C. metallidurans BS1. Using the RAST functional based comparison, it was determined that both C. metallidurans BS1 and CH34 contained genes encoding all the proteins (ChrABCEF) responsible for resistance to chromium compounds. A Blast search of the ChrI sequence of C. metallidurans CH34 obtained from NCBI against the genome of C. metallidurans BS1 in RAST showed that ChrI in C. metallidurans BS1 had only 44% AA sequence similarity to ChrI from C. metallidurans CH34.The cobalt and nickel resistance  resistance operon are encoded on the genome of BS1 and four in CH34, including six genes coding for MerR < – > MerTPC/ADE (43–/AsO33–) resistance including ArsHR, 2 ACR3 , and 5 ArsC while 13 proteins were found associated with chromium (CrO42–) resistance including ChrABCEF  TraD, and TraN are predicted to be involved in plasmid transfer, were present on plasmid –pBS1 DNA displaying the same pattern as on pMOL30 in CH34. The pBS1 plasmid displayed more similarities with pMOL30 by having similar plasmid maintenance systems while, the pBS1 plasmid showed no similarities with pMOL28 with the sole exception of a 3.5 kbp region . Three copies of Polymyxin resistance protein amino-arabinose transferase ArnT were found on the chromid in BS1 and one copy on plasmid out of four ArnT locus in total while it is a component of pMOL30 in CH34. This last-resort antibiotic is affective against Gram negative bacteria by permeabilizing its outer membrane using electrostatic interactions between amino group of polymyxins and negatively charged glucosamine and 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) sugars of lipid A . Putative heavy metal resistance determinants of C. metallidurans BS1 were largely similar to those present on the genome of C. metallidurans CH34. A noticeable difference was observed in C. metallidurans BS1 as major determinants of metal detoxification systems were located on the chromid instead of the plasmids (pMOL30 and pMOL28) in C. metallidurans CH34. Known gold–copper detoxification systems  found in C. metallidurans CH34 were also present in C. metallidurans BS1. Two prophages detected on the genome are probably intact and one very likely intact. Metal resistance determinants from different origins in BS1 displayed critical differences between C. metallidurans BS1 and CH34. Future studies would be aimed to explore the mechanisms involved in the interplay between gold and copper in gold–copper detoxification systems. In addition, one future aim will be to better understand the evolutionary adaptation to the auriferous environment of isolation on the genetic level and further reconstruct genomic rearrangements.This study presents the complete genome sequence and annotation of https://www.ncbi.nlm.nih.gov/assembly/GCA_003260185.2 for more details.The data used to support the findings of this study are included within the article. Please check SM and SB performed the laboratory experiments, analyzed the annotated genome sequence, submitted genome to NCBI, wrote the draft manuscript, and participated in the design of the experiments. MH carried out the genomic characterization and constructed the phylogenetic trees. IF, YL, JS, and JX performed the description of the sampling environment, sampling, isolation and microbiological characterization of the bacteria. CZ, SM, and SB carried out the isolation and purification of the genomic DNA. CR, RF, SZ, and IF participated in the design of the study and revised the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


ITC showed the 1:1 DNA binding event to be endothermic  and entropy driven . Taken together, the experimental data suggest a dual DNA binding mode by TC. At low [DNA]/[TC] ratio, the dominant mode is intercalation. This switches to minor groove binding at higher [DNA]/[TC], only for sequences containing AnTn or (AT)n stretches. Turn-on fluorescence results only in the previously unreported minor groove bound state. Our results allow a better understanding of DNA-ligand interaction for the newly reported turn-on probe TC.Turn-on fluorescent probes show enhanced emission upon DNA binding, advocating their importance in imaging cellular DNA. We have probed the DNA binding mode of thiazole-coumarin (TC) conjugate, a recently reported hemicyanine-based turn-on red fluorescent probe, using a number of biophysical techniques and a series of short oligonucleotides. TC exhibited increased fluorescence anisotropy and decreased absorbance (~50%) at low [DNA]/[TC] ratio. Although the observed hypochromicity and the saturating value of [DNA base pair]:[TC] ratio is consistent with a previous study that suggested intercalation to be the DNA binding mode of TC, a distinctly different and previously unreported binding mode was observed at higher ratios of [DNA]:[TC]. With further addition of DNA, only oligonucleotides containing A DNA is a ubiquitous biological macromolecule whose specific detection under various conditions forms the basis of probing molecular mechanisms behind various cellular processes . A typicThe most versatile way of mapping molecular architecture of a cell is through fluorescence-detected imaging . TypicalAn ideal fluorescent probe should have certain attributes- excitation at longer wavelengths to prevent auto fluorescence of the cellular components, ability to show turn-on fluorescence in presence of dsDNA, high permeability to cellular membranes, low levels of toxicity to live cells, high specificity to dsDNA over ssDNA or RNA and responsive at low concentrations. The design of a recently reported hemicyanine-based fluorescent probe, thiazole-coumarin conjugate TC; , takes iAlthough preliminary experiments, based on FRET assays with Hoechst 33258, and DFT calculations suggested that TC binds DNA via intercalation, the DNA binding mode by TC was not probed directly. Here we have studied the DNA-binding mode of TC to a number of systematically designed short oligonucleotides containing AT or GC stretches  using a A2T2: 5’-(CG)2A2T2(CG)2−3’; (2) A3T3: 5’-(CG)2A3T3(CG)2−3’; (3) (AT)2: 5’-(CG)2(AT)2(CG)2−3’; (4) t-(AT)2: 5’-(AT)2(CG)2(AT)2−3’; (5) AT: 5’-(CG)2AT(CG)2−3’; (6) CG: 5’-(CG)6−3’. All oligonucleotides  leotides  were purleotides .ex = 530 nm) in buffer A. Fluorescence anisotropy was measured using a L-format configuration. Unless otherwise stated, all experiments were performed at 25°C with [TC] = 5 μM. Binding constants were estimated usingF, F0 and (F∞−F0) are the observed, initial and final (under saturating conditions) fluorescence, n is the number of binding sites, C0 and L are the receptor and ligand concentrations, respectively.The UV-visible spectra of TC, with and without DNA, were recorded on a Shimadzu UV-1700 spectrophotometer in buffer A. Spectra obtained from titrations were deconvoluted using the method of Convex Constrained Analysis (CCA) using the software CCA+  where thA3T3 (40 μM). The interval between two subsequent injections and the injection volume were 200 seconds and 2 μl, respectively. The observed heat rates were corrected by subtracting heat of dilution obtained by repeating the experiment without A3T3 (only buffer A). The thermograms were analysed using the in-built NanoAnalyze software.ITC experiments were performed on a TA Affinity ITC instrument. Samples were extensively degassed prior to the experiment, conducted in buffer A and at 25°C. Titrations were performed by injecting incremental amounts of TC into 2O  and trimethyl-silyl propionate . TC:DNA titrations were performed by recording 1D 1H spectra upon incremental addition of either TC or DNA to the other species (300 μM in buffer A). Resonance assignments and proximity of protons were determined from 2D TOCSY and NOESY (mixing time = 250 ms) experiments recorded under similar conditions /[TC] < 0.2) and then decreased before finally stabilizing at [DNA]/[TC] ~ 1.2. (AT)2 also showed similar trend. However, anisotropy increase for members of group B was smaller and the increase was monotonic. Changes in fluorescence and absorbance were correlated for A3T3  TC peaks did not reappear even at [TC]/[A3T3] = 4, although considerable shifts were observed for the DNA (A2T2 and A3T3) protons.The solution  that cousolution . By ~ /[TC] ~ 1 for AT-rich DNA, the associated peak shifts saturated at much less [DNA]:[TC] ratio (~ 0.2). Like the peak shift, anisotropy  also shoA3T3, A2T2 and (AT)2 (ii) t-(AT)2 and AT (iii) CG. While the UV-vis spectra of all intermediate species showed reduced absorbance, the extent of the associated red shift was sequence-dependent (no shift for CG). The absorbance signature for the other DNA interaction mode, that turns on beyond [DNA]/[TC] ~ 0.2, was the loss of the initial hypochromicity and further red shift. While this was true for A3T3, A2T2 and (AT)2, t-(AT)2 and AT showed a different signature: the hypochromicity remained but absorption spectra showed a small red shift. The absorption spectra of CG, on the other hand, showed no significant change beyond [DNA]/[TC] ~ 0.2.The presence of the dual DNA binding mode of TC, dependent on [DNA]/[TC] values, was also apparent from the UV-vis absorption data. The low [DNA]/[TC]-regime interaction manifested itself in the abrupt appearance of hypochromicity. CCA showed that three slightly different intermediary species were generated by three different DNA groups during this mode: (i) t-(AT)2 stand out from the other five DNA that contain GC base pairs at the termini, which was not observed. Further support for intercalation comes from the earlier report [20. Intercalation was also reported to be energetically favourable from DFT calculations.To understand the nature of the binding mode at low [DNA]/[TC] we focus on the remarkable hypochromicity (~ 50%) associated with this binding mode. Hypochromicity is known to be characteristic of π-stacked molecules . For ourr report  where FRA3T3, A2T2 and (AT)2. Since all sequential connectivities between the sugar H1’ and the aromatic H6/8 protons of the next nucleotide in TC-bound DNA (1:1) could be readily assigned for A2T2 and A3T3 . The docked model lacks any specific DNA-TC interaction like H-bonds which can enthalpically stabilize the bound state. This is compatible with the endothermic and entropy driven nature of TC: A3T3 interaction, as observed from ITC and van’t Hoff analysis. Interestingly, it is this 1:1 minor groove bound TC:DNA complex that exhibited the functionally important enhanced fluorescence for A3T3, A2T2 and (AT)2. For t-(AT)2 and AT, the 1:1 TC:DNA complex exhibited only medium fluorescence enhancement. For CG, the second binding mode seemed to be absent.We now look at the other binding event, dominating beyond [DNA]/[TC] ~ 0.2. As shown by ITC, NMR and Job Plot, this event yielded a 1:1 TC:DNA complex for and A3T3 , interca TC:A2T2 . The equ1H NMR spectrum. Just like the observed hypochromicity associated with this event, the line broadening was observed for AT-rich A3T3 (A2T2 (CG) sequences n stretches) the dominant DNA binding mode at [TC]/[DNA] ~1 is minor groove binding. At [TC]/[DNA] >> 1, in addition to minor groove binding, TC can also intercalate DNA. However, if the AT-stretch is at the termini (like in t-(AT)2) or if it is only two-nucleotide long (like in AT) a mixed binding mode  ensues at [TC]/[DNA] ~ 1 while intercalation is the dominant mode at [TC]/[DNA] >> 1.Taken together, the experimental data presented above suggest a model of DNA binding by TC  that depThe coumarin moiety, an integral part of TC, is known to intercalate DNA . So it iS1 Text(PDF)Click here for additional data file.S1 Fig(JPG)Click here for additional data file.S2 Fig(JPG)Click here for additional data file.S3 Fig(JPG)Click here for additional data file.S4 Fig(JPG)Click here for additional data file.S5 Fig(JPG)Click here for additional data file.S6 Fig(JPG)Click here for additional data file.S7 Fig(JPG)Click here for additional data file.