The characterizations of the resulting synthetic organoclay by scanning electron microscopy (SEM), X-ray diffraction, N2 sorption isotherms (BET method), Fourier transform infrared spectroscopy (FTIR), CHN elemental analysis and UV–Vis diffuse reflectance spectroscopy (UV–Vis-DRS) demonstrated the effectiveness of the process used for grafting of amine functionality in the interlamellar clay. The results indicate the presence of organic moieties covalently bonded to the inorganic lattice of talc-like magnesium phyllosilicate silicon sheet, with interlayer distances of 1568.4 pm. In an effort to use a talc-like material as an electrode material without the addition of a dispersing agent and/or molecular glue, the TalcNH2 material was successfully dispersed in distilled water in contrast to natural talc. Then, it was used to modify a glassy carbon electrode (GCE) by drop coating. The characterization of the resulting modified electrode by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) revealed its charge selectivity ability. In addition, EIS results showed low charge transfer resistance (0.32 Ω) during the electro-oxidation of [Fe(CN)6]3−. Kinetics studies were also performed by EIS, which revealed that the standard heterogeneous electron transfer rate constant was (0.019 ± 0.001) cm.s−1, indicating a fast direct electron transfer rate of [Fe(CN)6]3− to the electrode. Using anodic adsorptive stripping differential pulse voltammetry (DPV), fast and highly sensitive determination of Pb(II) ions was achieved. The peak current of Pb2+ ions on TalcNH2/GCE was about three-fold more important than that obtained on bare GCE. The calculated detection and quantification limits were respectively 7.45 × 10−8 M (S/N = 3) and 24.84 × 10−8 M (S/N 10), for the determination of Pb2+ under optimized conditions. The method was successfully used to tap water with satisfactory results. The results highlight the efficient chelation of Pb2+ ions by the grafted NH2 groups and the potential of talc-like amino-functionalized magnesium phyllosilicate for application in electrochemical sensors.A talc-like magnesium phyllosilicate functionalized with amine groups (TalcNH It consists of tetrahedral Si and octahedral Mg sheets forming the layered structure 2:1   and Ru(NH3)6Cl3 . NaNO3 , KCl , HCl , NaCl . Zn(NO3)2·6H2O (98%), Cd(NO3)2·4H2O (98%), Cu(NO3)2·xH2O (99.99%) and Hg(NO3)2·H2O (99.99%) were from Sigma-Aldrich .The natural talc clay mineral  used in this work is a commercial sample  was used. For the measurements, the samples were deposited on a conductive strip placed on a specimen holder and coated with gold by vacuum sputtering. Characterisation by Fourier Transform Infrared Spectroscopy (FTIR) was carried out using a Nicolet 8700 instrument equipped with a specular reflectance accessory (Smart Collector).® MYTHEN 1K detector . The crystallinity of the materials was assessed by X-ray diffraction analysis, using an STOE Stadi-p X-ray powder diffractometer  operated at 40 kV and 30 mA with Cu Kα1 radiation (λ = 1.54056 Å), in transmission geometry with an IP-PSD  and/or a DECTRIS2 adsorption-desorption at 77.13 K using a micrometrics model sorptometer . Before N2 adsorption, the samples were degassed at 307.13 K under vacuum. The linear part of the BET equation was used to evaluate the surface area.The Brunauer-Emmett-Teller (BET) specific surface areas of the samples were determined by means of NA CHNS Euro EA 3000 analyser  was used to determine the chemical composition of our materials.4 was used as the reflectance standard.The optical properties of our materials were determined using a Shimadzu UV-Vis 3101PC Diffuse Reflectance Absorption Spectrophotometer (DRS) in the wavelength range 200–800 nm. BaSO6]3− and [Ru(NH3)6]3+ ions were recorded from −0.15 V to 0.7 V and from −0.6 V to 0.2 V respectively, in 0.1 M KCl at a scan rate of 50 mV/s, unless otherwise stated and without stirring. Using anodic stripping differential pulse voltammetry (ASDPV), the electroanalysis of Pb2+ ions involved two successive steps: open circuit preconcentration of the analyte under gentle agitation followed by voltammetric detection in the potential range of −0.7 V to −0.3 V after 30 s electrolysis at −0.8 V. Electrochemical impedance spectroscopy (EIS) measurements were performed on a Palmsens3 potentiostat driven by PS Trace 4.2 software. It was performed over the frequency range of 0.01 Hz to 10 kHz with a potential amplitude of 10 mV in a 0.1 M KCl solution containing 1 mM [Fe(CN)6]3−/[Fe(CN)6]4−. Voltammetry measurements were performed on a µ-Autolab potentiostat running GPES software and using a standard three-electrode cell (bare or modified GCEs used as working electrodes (WE), the saturated silver chloride electrode (Ag/AgCl/KCl) as reference electrode (RE), and a stainless-steel bar as auxiliary electrode). Cyclic voltammograms of 3−) and positively charged ([Ru(NH3)6]3+) redox probes were probes were used to verify and confirm the ion exchange properties of TalcNH2. The analysis of [Fe(CN)6]3− was carried out within a potential window ranging from −0.15 V to 0.7 V in 0.1 M KCl and the results are depicted on 2/GCE (Ipa = 4.91 µA and Ipc = 5.15 µA) is lower compared to that observed on bare GCE (Ipa = 8.07 µA and Ipc = 8.93 µA). This is due to a gradual increase of ions on the electrode surface through the binding sites or pores of the electrode material. Not all [Fe(CN)6]3− ion binding sites on the electrode surface have the same accessibility. An increase in CV peak currents is then observed up to the 20th scan (Ipa = 24.5 µA and Ipc = 23.5 µA). The high peak currents (anodic and cathodic) and a strong accumulation up to the 20th scan could be due to the electrostatic attraction between the negatively charged redox system [Fe(CN)6]3− and the positively charged amino (-NH3+) groups present on the organosilane fragments used in the synthesis process. Several recently published works have highlighted the good affinity that protonated amine functions have in an acid medium to interact (through electrostatic interactions) with either negatively charged molecules or those having free pairs of electrons capable of reacting with other species in solution 3+ ions was carried out in the potential range from −0.6 V to 0.2 V in 0.1 M KCl, and the results are presented on 2/GCE . The lower peak current and non-accumulation obtained on TalcNH2/GCE could be explained by the fact that the synthesised and protonated TalcNH2 film in solution acted as an electrostatic barrier, preventing the absorption of the cationic species [Ru(NH3)6]3+. These electrochemical results support the results obtained during the physico-chemical characterisations which confirmed the effectiveness of the synthesis of a talc-like material and the incorporation of APTES into the structure of the synthesised product.Similarly, the analysis of [Ru6]3−/4− redox system and applying the Randles–Sevcik for a reversible Equation (1):Ip is the peak current, A the electrode electroactive area (cm2), n the number of electrons transferred, D the diffusion coefficient of [Fe(CN)6]3− in a 0.1 M KCl solution, C the concentration of [Fe(CN)6]3− (mol·cm−3) and v the potential scan rate (V·s−1) [1/2 plots for the [Fe(CN)6]3− oxidation process were 4.29 × 10−5 A·v−1/2·s1/2 for the GCE and 5.59 × 10−5 A·v−1/2·s1/2 for the TalcNH2/GCE. From the D value for [Fe(CN)6]3− equal to 7.6 × 10−6 cm2·s−1 3− probe is affected by modification on the GCE, with the TalcNH2 material allowed an increase in the electroactive area by 1.3 fold compared to the GCE.The electrochemically active surface areas of bare GCE and TalcNH (V·s−1) . Cyclic  cm2·s−1 , the corRct) [2/GCE in 0.1 M KCl containing 1 mM [Fe(CN)6]3−/4−. On the bare GCE, the value of Rct was 2725.7 Ω  3−/4− solution (10−6 mol·cm−3).Using electrochemical impedance spectroscopy (EIS), it is possible to elucidate the heterogeneous electron transfer properties at the electrode-solution interface. The EIS curves display semi-circular and linear areas corresponding respectively to a process limited by electron transfer and diffusion. The diameter of semicircle is equal to the charge transfer resistance (Rct) . Figure 2725.7 Ω a. On Tal 186.8 Ω b, which tion (2) :(2)ko = k° values were obtained for the bare GCE (0.0017 ± 0.0003 cm·s−1) and TalcNH2/GCE (0.019 ± 0.001 cm·s−1). The k° values give information on the kinetic ease of a reaction process. A system with a low k° value will reach equilibrium faster than a system with a high k° value. The k° value is greater on TalcNH2/GCE than on GCE, indicating a faster electron transfer on this electrode, a property that is very beneficial in electrochemistry both in terms of energy savings and analysis time.The 2 and the TalcNH2/GCE, the ability of the modified electrode to detect Pb2+ ions was investigated and preliminary experiments were carried out. The comparison between the anodic stripping differential pulse voltammetry responses on a bare GCE (curve a) and TalcNH2/GCE (curve b) is shown on 2/GCE. After the characterization of the TalcNH2+ ions on TalcNH2/GCE was about 3 times more important than that obtained on bare GCE. This shows the better affinity between Pb2+ ions and TalcNH2 synthesized clay which can be explained by the presence of amino functional groups -NH2 on its surface and in its structure that can easily chelate Pb2+ ions.The results demonstrated that the peak height of Pb2+ ions with the TalcNH2 modified GCE, parameters such as pH of the accumulation medium, the concentration of the detection medium, the deposition potential and the deposition time were optimised.To achieve a best detection of Pb2 clay surface depends on the pH of the solution. At pH < pKa (the pKa of amines being around 9–10), most of these functional groups are mainly in ionized form (protonated amine) and can exchange H+ with metal ions in solution. The effect of the pH of the accumulation medium (from 1 to 9) on the peak current was studied in the range of 1.0 to 9.0, in order to find the right pH value to define the optimal values of detection of lead ions. The results were shown in The pH of the accumulation or detection medium can influence the electrochemical response of the modified electrodes with respect to the detection of heavy metal ions . The ion2 material that can prevent the fixation of lead ions (predominant in accumulation medium) on its surface via electrostatic repulsions forces. The absorption of H+ (H+ coming from hydrochloric acid (HCl) used) hinders the adsorption of Pb2+, as it is a stronger acid than Pb2+. However, the peak currents increase when the pH increase from 1.0 to 7.0, and further decrease up to a pH value of 9.0. Yet, in the pH range 3.0–7.0, polymeric hydroxocomplexes of lead, mainly Pb(OH)- and Pb(OH)2 predominate due to hydrolysis reactions (M) + (2.66 × 10−6) (R2 = 0.997). The detection limit (DL) and quantitation limit (QL) were calculated from DL = 3 Sb/m [L = 10 Sb/m, where Sb is the standard deviation of the blank and m is the slope of the calibration curve. DL and QL were estimated to be 7.45 × 10−8 M and 24.84 × 10−8 M, respectively, and the sensitivity of the method of 3.6 µA.µM−1 was obtained. The performances of the sensor developed were compared with some other electrochemical sensors of Pb2+ ions . A relative standard deviation of 5.65% was obtained, showing the stability, repeatability and reproducibility of TalcNH2/GCE  during the detection step. It was found that some other ions, Na+, K+, Cl−, SO42− and NO3−, which were not listed in 2+.2/GCE as a sensing device was evaluated for the detection of traces of lead in tap water sample. Tap water was used to prepare accumulation medium at a final Pb2+ concentration of 5 µM. After analysis under optimal conditions, a recovery amount of 95.65% of added Pb2+ ions was obtained. A high recovery obtained indicated that the TalcNH2/GCE can be successfully applied to analyse water polluted by lead ions.The applicability of TalcNH2/GCE can be used for the analysis of Pb2+ ions by stripping voltammetry. The ability of the TalcNH2 to complex Pb2+ ions onto the electrode was precise and accurate. After optimization of the factors affecting the pre-concentration and stripping steps, a detection limit of 7.45 × 10−8 M was obtained. The amino-functionalized talc/glassy carbon electrode prepared in this study was applied in the determination of Pb2+ ions in tap water with good results. This study demonstrates the potential of TalcNH2 synthetic material, a hydrophilic talc-like magnesium phyllosilicate amino-functionalized to serve as electrode material for the preparation of sensing devices dedicated to the detection of heavy metals.Overall, this study has shown that it is possible to use a synthetic talc-type clay, easily functionalized with amine chelating groups as electrode material to improve the sensitivity and selectivity of the resulting modified electrodes. Indeed, a synthetic talc-like magnesium phyllosilicate amino-functionalized was obtained from the sol–gel process. Infrared spectroscopy, X-ray diffraction, CHN elemental analysis, SEM, BET method and UV–Vis diffuse reflectance spectroscopy study results confirmed the synthesis process. We have demonstrated that a TalcNH


Depletion of lncRNA-Smad7 dramatically impairs cardiomyocyte differentiation in mESCs. Moreover, lncRNA-Smad7 represses Bmp2 expression through binding with the Bmp2 promoter region via (CA)12-repeats that forms an R-loop. Importantly, Bmp2 knockdown rescues defects in cardiomyocyte differentiation induced by lncRNA-Smad7 knockdown. Hence, lncRNA-Smad7 antagonizes BMP signaling in mESCs, and similarly regulates cell fate determination between osteocyte and myocyte formation in C2C12 mouse myoblasts. Moreover, lncRNA-Smad7 associates with hnRNPK in mESCs and hnRNPK binds at the Bmp2 promoter, potentially contributing to Bmp2 expression repression. The antagonistic effects between Nodal/TGF-β and BMP signaling via lncRNA-Smad7 described in this work provides a framework for understanding cell fate determination in early development.Transforming growth factor β (TGF-β) superfamily proteins are potent regulators of cellular development and differentiation. Nodal/Activin/TGF-β and BMP ligands are both present in the intra- and extracellular milieu during early development, and cross-talk between these two branches of developmental signaling is currently the subject of intense research focus. Here, we show that the Nodal induced  CTGF, Gremlin) (Transforming growth factor β (TGF-β) family proteins are key components of the intra- and extracellular stem cell milieu of embryonic and somatic stem cells. Nodal/Activin/TGF-β  and BMP signaling each play diverse roles in controlling pluripotent and multipotent cell fates . These rGremlin) , and vicGremlin) ,10. AlthHAND1, HAND2, NKX2.5, MESP1)  , chromat, MESP1) , lncRNAs, MESP1) , and sig, MESP1) ,23. For , MESP1) ,24,25. W, MESP1) , Nodal r, MESP1) . Cardiom, MESP1) . It is tDIGIT in mESCs 12-repeats. Moreover, lncRNA-Smad7 antagonizes BMP signaling in mESCs, resulting in cell fate determination between osteocyte and myocyte formation in mouse myoblast C2C12 cells. Finally, we identified hnRNPK as an interaction partner of lncRNA-Smad7 in mESCs, and CUT&Tag assays showed that hnRNPK binds the Bmp2 promoter, indicating that lncRNA-Smad7 may repress Bmp2 expression through recruitment of hnRNPK. These findings thus provide new insight into the role of lncRNAs in regulating cross-talk between Nodal/TGF-β and BMP signaling during early development.In this work, we show that of Smad7 ,89, is aMice were obtained from Tsinghua University Laboratory Animals Resource Center. And mice were housed at 20–22°C . All mice we used had a C57BL/6 background including adult mice  and pregnant mice . Embryos were isolated at the developmental stages (E17.5). The dissected tissues were used for the qPCR to verified the gene expressions in different tissues. All animal experiments were carried out in accordance with institutional guidelines for animal welfare and approved by the Institutional Animal Care and Use Committee (IACUC) at Tsinghua University, China.2. Basic ES cells were cultured in DMEM , 15% fetal bovine serum , 1% penicillin/streptomycin , 1% non-essential amino acids (Hyclone), 1% l-glutamine , 1% sodium pyruvate , 100 μM β-mercaptoethanol (Sigma), 103 U/ml LIF. HEK293T (human embryonic kidney cells) and C2C12 cells are cultured in DMEM supplemented with 10% FBS .Mouse ESCs E14Tg2a.IV were maintained in feeder-free condition on 0.1% gelatin-coated dishes at 37°C with 5% CO5 cells/ml in EB media were seeded in low-attached 10-cm dish without LIF to initiate the differentiation. We passaged the cells at the ratio of 1:6 on day 2 and change the media every two days. Retinoic acid (RA) was added for ectodermal differentiation on day 2. Cells were harvested on day 0 and day 2 to day 6 for total RNA extraction during the differentiation.Mouse EBs formation and ectodermal differentiation were carried out as described by the supplier (ATCC) and previously reported . 1.5 × 1l-ascorbic Acid  for 32 h. EBs were then transferred to 0.1% gelatin-coated plates and incubated for 6–8 days. Cells were harvested for total RNA extraction at day 0, day 2, day 3.3, day 6, day 8 and day 10. The cells were collected for immunofluorescence staining at pH3 on day 3.3 (80 h) and cTnT on day 10.Cardiomyocyte differentiation was initiated via EB formation induced by hanging ∼20 μl suspensions of drops, with roughly equivalent embryonic stem cell counts (2500 cells) . The droMyogenesis differentiation of C2C12 cells was carried out in 2% fetal equine serum  at the confluence of 80–90%, we changed the media every day and collected the cells on day 0, day 2, day 4 to day 6 of for total RNA extraction. Osteoblast differentiation of C2C12 cells was induced by 50 ng/ml hBMP4 at the confluence of 40–50%, we collected the cells every 6 h from hour 0 to hour 48 for total RNA extraction. Alternatively, osteoblast differentiation of C2C12 cells was initiated with 500 cells in 6-well plate by 50 ng/ml hBMP4 for up to 10 days, we change the media with hBMP4 every day and set up alkaline phosphatase staining (AP staining) on day 10.lncRNA-Smad7 was cloned by overlap PCR according to the sequence information of 5’ and 3’ of RACE. Here we used the 3’ CDS primer to initiate the 3’ RACE: AGCAGTGGTATCAACGCAGAGTAC(T)30 V N . The sequence of other primers are listed in 5’ and 3’ RACE was performed using the SMART RACE cDNA Amplification Kit (Clontech) according to the manufacturer's recommendations. The full length of lncRNA-Smad7 was calculated by DNA/RNA Copy Number Calculator from website (http://endmemo.com/bio/dnacopynum.php). The linearized full length of lncRNA-Smad7 was cloned from a plasmid DNA (PUC19) containing lncRNA-Smad7 using primers listed in lncRNA-Smad7 cDNA was used to qRT-PCR to generate a standard curve for lncRNA-Smad7. To measure the lncRNA-Smad7 copy number in E14 or C2C12 cells, total RNA extracted from 1.5 × 106 and 1.2 × 106 cells of each line was reverse transcribed into cDNAs for qPCR analysis, and the copy number could be quantitated from the standard curve. The qPCR primers were listed in The copy number of the diluted full length of Smad2 or Smad4 reintroduction in the Smad2 KO or Smad4 KO mESCs: sequences harboring the Smad2 (Gene ID: 17126) or Smad4 (Gene ID: 17128) UTR, and CDS regions were introduced into Smad2 KO or Smad4 KO mESCs (PMID: 3762693) by PB-CAG system. The sequences of Smad2 and Smad4 were synthesized by Junbiao Dai lab.For lncRNA-Smad7 overexpression: The full-length transcript of lncRNA-Smad7 were retrieved and cloned from E14 cells according to the RACE data, inserted into the PiggyBac vector resistant to hygromycin, and driven by CAG promoter. The overexpression vectors were co-transfected with pBASE by lipofectamine 2000. After drug selection of hygromycin , the over-expressed cells were collected for RNA extraction and qPCR analysis.For lncRNA-Smad7 genomic DNA and two clones showing 4× polyA insertion were picked up for further analysis.The short guide RNAs (sgRNAs) were inserted into BbsI-linearized pSpCas9 (BB)-2A-GFP (PX458) vector (Addgene# 48138) respectively. The 4× polyA transcription stop cassette was cloned into the pMD19-T (TaKaRa) donor vector. We co-transfected the PX458 and donor vector containing homologous arms and 4× polyA sequence at a ratio of 1:4. Cells were transfected and GFP-positive cells were FACS sorted as single cells into 96-well plate at 48 h after transfection. The cells were cultured for 2 weeks followed by PCR-based genotyping. One clone showing deletion of the targeted region in lncRNA-Smad7, Bmp2 and Hand1 were listed in Short hairpins (shRNAs) were designed by Public TRC Portal  or as previously reported. Annealed oligonucleotides of shRNAs were cloned into AgeI and EcoRI restriction enzymes digested pLKO.1-puro lentiviral vector driven by U6 promoter. Scramble shRNA was used as the control shRNA. The shRNAs were transfected into HEK293T cells to package lentivirus by lipofectamine 2000  followed by infections of mESCs or C2C12 cells. After drug selection, cells were collected for RNA extraction and qPCR analysis. Sequence of shRNAs for Subcellular fractionation was performed as previously described with some subtle modifications . One 10-EBs of cardiomyocyte on day 3.3  were collected and fixed in 4% paraformaldehyde (PFA) at room temperature (RT). The EBs were collected to the 30% sucrose solution for 1–2 days to enable most EBs to sink to the bottom and embedded with O.C.T. compound. EB sections were permeabilized in 0.5% Triton X-100 in PBS (PBST) for 30 min at RT, incubated in primary antibody anti-pH3 overnight and AlexaFluor-488-conjugated secondary antibody for 1 h at RT. Signals were detected with A1/SIM/STORM-confocal . The antibodies used in this study are listed in Immunofluorescence of cTnT: EBs were seeded on 35 mm confocal dish on day 3.3 and cultured to day 10. CMs were fixed with 4% PFA for 10 min, permeabilized with 0.5% Triton X-100 in PBS for 10 min, stained with the primary antibody cTnT overnight at 4°C and AlexaFluor-594-conjugated secondary antibody for 1 h at RT. Signals were detected with A1R confocal microscopy . The antibody used in this study are shown in Immunofluorescence of MY-32 (Myosin Heavy Chain 1 Antibody): C2C12 cells were seeded on 35 mm confocal dish and induced to myogenesis differentiation with 2% fetal equine serum at the confluence of 80–90%. Myogenesis differentiated cells were fixed with 4% PFA for 10 min, permeabilized with 0.5% Triton X-100 in PBS for 10 min, stained with the primary antibody MY-32  overnight at 4°C. The following procedures were the same as we described above. The antibody used in this study are shown in For the ChIP (Chromatin Immunoprecipitation) assay, 200 drops on day 3.3 and 1 μl H3K27me3 antibody were enough for each ChIP assay. Cells were collected and crosslinked with 1% formaldehyde for 10 min at 37°C, then quenched by 0.125 M glycine. Cell pellets were lysed in SDS containing buffer  freshly supplied with protease inhibitors cocktail  and sheared to 200–500 bp fragments by sonication. Fragmented chromatin was centrifuged at 13 000 rpm for 20 min at 4°C and the supernatants were diluted in dilution buffer . The chromatin complex was incubated for 6 h with H3K27me3 antibody , then precipitated by 20 μl protein A/G beads  for 2 h rotating at 4°C. The precipitated chromatin was sequentially washed 3 times with RIPA buffer  and then twice with TE buffer . Precipitated chromatin complexes were eluted in elution buffer  for 1 h at 65°C, and the supernatant was incubated at 65°C for 6 h on a thermos shaker for de-crosslinking.RNA and proteins were digested using RNase A or proteinase K, respectively. And the DNA was purified by phenol chloroform extraction and ethanol precipitation. We eluted the samples with 30 μl water for ChIP-seq and qPCR analysis. The enrichment for ChIP-qPCR and ChIP-seq was normalized to the sample incubated without antibody. The primer sequences of qPCR and antibody are shown in Cleavage under targets & tag mentation (CUT & Tag)  were con7 mESCs were crosslinked in 10-cm dish by 3% formaldehyde for 10 min at 37°C, quenched by 0.375 M glycine and harvested at 1500 rpm for 10 min at 4°C. Cells were resuspended in 800ul lysis buffer  with addition of protease inhibitor cocktail, PMSF and RNase inhibitor. The lysate was sonicated to 200–500 bp, then centrifuged at 13 000 rpm for 20 min at 4°C. We used 100 pmol probe mix for 800 μl lysis, and incubated the chromatin complex for 6 h at 37°C rotating all the time. The chromatin complex was then immunoprecipitated by streptavidin beads  for 2 h at 37°C. The precipitated chromatin was sequentially washed 4 times by wash buffer . DNA was eluted sequentially by RNase elution buffer  at 37°C for 1 h. De-crosslinked chromatin was subjected to proteinase treatment and the DNA was prepared as described in ChIP-seq method. The enrichment of ChIRP-seq signals were normalized to the lncRNA-Smad7 knock-out cells. The sequences of probes used in this study are listed in ChIRP (Chromatin isolation by RNA purification) was performed as previously described with modifications , the 50–Mass spectrometry was performed followed ChIRP experiment. Equal amount of E14 cells were used for the ChIRP and control group. Proteins was eluted in 300 μl RIPA lysis buffer (Solarbio) with 10 μl RNaseA (20 mg/ml) and 10 μl RNaseH1 (10 U/μl) and incubated 2 h at 37°C. The beads were discarded by centrifugation, and the supernatant were recovered for the following experiment. 5× SDS loading buffer was added to the supernatant and the samples were prepared for 10 min at 100°C. Protein samples were separated in SDS-PAGE gel, and stained with Coomassie brilliant blue R-250. Each lane was cut into 2 pieces for the following mass spectrometry.The gel bands of interest were excised from the gel, reduced with 5 mM of DTT and alkylated with 11 mM iodoacetamide which was followed by in-gel digestion with sequencing grade modified trypsin in 50 mM ammonium bicarbonate at 37°C overnight. The peptides were extracted twice with 0.1% trifluoroacetic acid in 50% acetonitrile aqueous solution for 1 h and then dried in a speedVac. Peptides were redissolved in 25 μl 0.1% trifluoroacetic acid, and 6μl of the extracted peptides were analyzed by Orbitrap Fusion mass spectrometer.m/z, 70 000 resolution) followed by 20 data-dependent MS/MS scans at 27% normalized collision energy (HCD). The MS/MS spectra from each LC–MS/MS run were searched using an in-house Proteome Discoverer . Static peptide modification included carbamidomethylation (C), dynamic oxidation (M). Two trypsin missed cleavage was allowed. Precursor tolerance and ion fragment tolerance were set at 20 ppm and 0.02 Da, respectively. Confidence levels were set to 1% FDR (high confidence) and 5% FDR (middle confidence). The top ranked proteins of ChIRP-MS were listed in For LC–MS/MS analysis, the peptides were separated by a 120 min gradient elution at a flow rate 0.30 μl/min with a Thermo-Dionex Ultimate 3000 HPLC system, which was directly interfaced with an Orbitrap Fusion mass spectrometer . The analytical column was a home-made fused silica capillary column  packed with C-18 resin . Mobile phase A consisted of 0.1% formic acid, and mobile phase B consisted of 100% acetonitrile and 0.1% formic acid. The Q Exactive mass spectrometer was operated in the data-dependent acquisition mode using Xcalibur 2.1.2 software and there was a single full-scan mass spectrum in the orbitrap (300–1800 Bmp2 promoter region (chr2: 133551243–133551936) with or without GT-repeats and Pou5f1 promoter (chr17: 35503947–35504243) region were cloned into pGL3-promoter vector. mESCs E14 (1 × 105 in each well of 12-well plate) were co-transfected by 900 ng luciferase reporter vector and 100 ng renilla vector  with lipofectamine 2000. Cells were harvested 48 h after transfection. Luciferase activities were examined using the Dual-Luciferase Reporter Assay System (Promega Cat# E1910) and measured by enzyme-labeled instrument (PerkinElmer).The autocrine BMP2 protein levels were quantified using a commercially available ELISA kit . All samples were collected from the media culturing cells for 48 h. The media were centrifuged 1000 rpm for 5 min and measured immediately or stored at −80°C less than one month. All samples were assayed according to the manufacturer's instructions and tested in duplicates by personnel blinded for each group. The optical density of each well was determined using a microplate reader at an absorbance of 450 nm. No interference and no cross-reactivity were expected based on the manufacturer's instructions. The minimum detectable dose (MDD) of BMP2 ELISA ranged from 50 to 200 pg/ml. The dynamic range of BMP2 ELISA ranged from 15.625 to 1000 pg/ml.2; 40 mM HEPES–KOH, PH 7.0; 0.5% NP40), containing 1 mM DTT and EDTA-free protease inhibitor cocktail. Cells were lysed by sonication on ice for 30 s intermittently at 30% power. The resulting lysates were centrifuged at 4°C for 15 min at 13 000 rpm, then the supernatants were saved for purification experiments. The 50 μl anti-Flag resins were added to the supernatant of each sample and incubated for 6 h at 4°C. Then, the resins were washed with lysis buffer for four times at 4°C followed with incubation with the elution buffer  containing 300 μg/ml flag peptide for twice (125 μl each time) at 4°C. The hnRNPK protein was further purified with heparin column and gel-filtration column (HiLoad 16/60 Superdex200) by AKTA system . The protein purity was monitored by SDS-PAGE.The recombinant Flag-tagged hnRNPK of mouse was constructed in PCI backbone. The plasmid was transfected into the HEK293T cells when the optical density of the culture reached 0.7 (20 μg plasmid for each T75 flask cells). Each T75 flask cells were passaged to one 15 cm dish 12 h post-transfection and harvested 36 h post-transfection. The cell pellet of each dish was suspended in 1 ml cold lysis buffer 25 containing segments of lncRNA-Smad7 were transcribed in vitro. Briefly, the purified DNA templates of lncRNA-Smad7 fragment were transcribed in vitro by T7 polymerase (0.2 mg/ml) for 3 h at 37°C. DNase I was then added to the transcribed RNA for 30 min at 37°C. The mixture was centrifuged to remove any precipitate. The digested RNA was purified by phenol–chloroform (PH 5.2) at the ration 1:1 followed with centrifugation for 30 min at 13 000 rpm at 4°C. The supernatant was concentrated by ultrafiltration spin columns (Millipore) with DEPC water for 100 000 folds at least. Finally, the RNA was concentrated to ∼0.5–2.0 mg/μl.Full length, (CA)lncRNA-Smad7 fragments for 30 min at RT .For RNA–protein EMSA: Increasing amounts of purified hnRNPK was incubated with 1.5 pmol in vitro transcribed in vitro transcribed lncRNA-Smad7 fragments were incubated with 0.15 pmol Cy5 labeled single strand oligonucleotide [(TG)17], [(CA)17], and [TCTAGTGA-(TG)17-TCCATGTG] for 30 min at RT. Double strands 50nt oligonucleotides (CA:TG)17 were annealed by Cy5 labeled [TCTAGTGA-(TG)17-TCCATGTG] and [CACATGGA-(CA)17-TCACTAGA] oligonucleotide.For RNA-DNA EMSA: The Cyanine5 (Cy5) labeled single strand oligonucleotides were generated by incubating 5’NH2 labeled oligonucleotides (Sangon Biotech) and the reactive dye Cyanine5 NHS ester (LumiProbe) at 1:1 molar ratio and kept at RT for 4 h. Labeled nucleic acid were then separated from excess free fluorophores through ethanol precipitation. Increasing amounts of the 2, 2.5M Glycerol, 50 mM NaCl and 1 mM DTT] at RT for 30 min. Then, 2.5 U and 5.0 U RNase H1 (NEB), 0.01mg RNase A (Transgene) or vehicle was added to the DNA/RNA/S9.6 complex.For RNA-DNA and R-loop antibody S9.6 EMSA: the RNA/DNA hybrids containing 0.15 pmol 5′-cy5–labeled DNA and 1.8 pmol RNA (12 folds to the DNA), and increasing amount of S9.6 antibody were incubated in the buffer  and S9.6 antibody  in a shaker at 4°C overnight. After adding Protein A/G beads and incubating at 4°C for 3 h, the beads were washed four times with 1× DRIP binding buffer. Next, the S9.6 associated complexes were eluted by elution buffer  containing proteinase K at 55°C for 1 h. The complex was purified with phenol/chloroform extraction, and the supernatant was transferred into a new tube. 1/10 volume of 3 M NaAc, 1 μl Acryl carrier (Solarbio), and 2.5 volume ethanol was added to precipitated the complex at −20°C at least 2 h. The DRIPed DNA pellet was washed with 70% ethanol, air-dried, resuspended in DEPC water and used for qPCR. The sequence of the primers are listed in 2 fold change calculations were performed using Cuffdiff (version 2.2.1), with the implementation of two biological replicates. Heatmap was performed by Rstudio and Gene Ontology analysis was performed using DAVID (https://david.ncifcrf.gov/).Cells were collected by trypsin digestion and the RNA was isolated by RNA isolation kit . High-throughput sequencing was performed by Novogene on a Hiseq X. We mapped the RNA-seq data to the mouse reference genome (mm10) using HISAT2 (version 2.1.0). The levels of gene expression were calculated by Cufflinks (version 2.2.1) based on mm10 annotations. Differential testing and logn ≥ 3) unless otherwise stated, and compared statistically by unpaired Student's t test. Statistical significance was indicated as follows: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Statistical parameters for each experiment, including values of n and statistical significance, can be found in the figure legends. Graphs were generated using GraphPad Prism (6.01).The intensity of the western blotting bands was quantified by Image J and presented after normalization to the loading control. β-Actin (Actb) was used as the internal control for qPCR analysis. Quantitative data in this study are presented as mean ± S.D. from three independent experiments at least  and lncRNA-Smad7 and Smad7 expression were quantified by qPCR. The results indicated that both lncRNA-Smad7 and Smad7 are transcriptionally activated in the presence of Activin, suggesting that their expression did not require nascent synthesis of protein factors, and that lncRNA-Smad7 is a primary target of Nodal signaling , which revealed an alternative isoform that was 81 bp shorter at the 5’ end of exon1 compared with other previously reported transcript isoforms  cell lines (lncRNA-Smad7 KIpA) by inserting a 4× polyA transcription stop cassette in exon1  analysis showed that the differentially expressed genes (DEGs) were mainly involved in development  activity similar to WT mESCs  of lncRNA-Smad7 in these differentiation processes   formation for mesendoderm and ectoderm differentiation assays in WT and rocesses  53,54).,54.lncRNn day 3)  during tpression . No obviKD cells , but earKD cells  compareds Figure , suggestlncRNA-Smad7 in cardiomyocyte differentiation , whereas lncRNA-Smad7 expression was reduced on day 3.3 (80 h) of cardiac progenitor cell (CPs) differentiation  levels at this time point  .,55.lncRNn Figure . The carntiation . Changesntiation ,23. It it Figure . HoweverCM D3.3) .lncRNA-Smad7 KIpA cells compared to WT cells  was markedly reduced in lncRNA-Smad7 KD cells on day 8 of cardiomyocyte differentiation  were mainly associated with organismal development, including heart development  mESCs as the negative control. We found that lncRNA-Smad7 strongly associated with its own gene locus in WT mESCs but not in KO cells, validating the specificity of DNA affinity capture 17-repeats   for endo capture . Interess Figure . Moreoves Figure .Bmp2 promoter region harboring the (CA:TG)17-repeats into the pGL3-promoter vector to examine its transcriptional regulatory activity. The Pou5f1 promoter was used as a positive regulatory element control. Luciferase reporter assays in mESCs showed that the Bmp2 promoter region exerted repressive regulatory effects in a manner partially dependent on (CA:TG)17-repeats, since deletion of the (CA:TG)17-repeat region led to a significant reduction in transcriptional repression of luciferase signal  and binds to a promoter region containing (CA:TG)17-repeats.Collectively, these results indicated that lncRNA-Smad7 and the promoter region of Bmp2, we conducted gel-shift assays using variants of lncRNA-Smad7 transcribed in vitro 17-repeats, and that lncRNA-Smad7 exhibited a greater shift with ss DNA than ds DNA 17 repeats, but not with (CA)17-containing ss DNAs 25-repeats are present in lncRNA-Smad7 transcripts 12- and (UG)25-repeats  could bind with either (TG)17-containing ss DNAs or (CA:TG)17-containing ds DNAs in a similar manner to full length lncRNA-Smad7 12 sequence in the RNA 17 DNA 12-containing lncRNA-Smad7 segment 25 RNA segment with ss (CA)17 DNA 17 repeats) while inhibiting RNA binding with other sequences (i.e. (CA)17 repeats), which could be critical for its function suppressing Bmp2 expression. We further analyzed the CA-repeats containing lncRNAs responsive to nodal/TGF-β signaling in mESCs 17 DNA , althougin mESCs  and hESCin mESCs , indicat Sapiens .lncRNA-Smad7 preferentially associates with the ss (TG)17 repeats over interactions with ds (CA:TG)17 of the Bmp2 promoter in vitro, we hypothesized that lncRNA-Smad7 may form an R-loop with DNA fragments containing (TG) repeats at the Bmp2 promoter. Exposure to the R-loop antibody S9.6 in EMSAs resulted in a so-called supershift in DNA/RNA complex, while RNaseH1 treatment abolished the supershift band -seq analysis in iPSCs . This experiment revealed that the candidate lncRNA-Smad7 interacting proteins were RNA-binding proteins ..lncRNA-SXist and is necessary for PRC1 and PRC2 recruitment 12 or (UG)25 repeats .,70.Xist lncRNA-Smad7 may represent a general regulatory mechanism that is conserved among cells which recognize both TGF-β/Nodal and BMP ligands. C2C12 cells are progenitor mesenchymal cells with the potential to differentiate into osteoblasts or myoblasts. In these cells, BMP signaling is known to reverse cell fate determination from myocytes to osteocytes, while TGF-β signaling can negatively regulate osteoblast formation   Figure . Consists Figure . Moreoves Figure . In lined Figure , while es Figure . Additios Figure . These clncRNA-Smad7, regulated by Nodal/TGF-β, transcriptionally represses Bmp2 to control cell fate determination during early developmental stages. This involvement of lncRNA-Smad7 in the cross-talk between the Nodal/TGF-β and BMP signaling pathways thus defines a layer of regulation beyond that of the known coding genes in these pathways  and BMP ligands are present in niches of cells during early development ,71. Heres Figure .lncRNA-Smad7 17 repeats motif which is overlapped with lncRNA-Smad7 binding site and the lncRNA-Smad7 transcript contains both a (CA)12- and a (UG)25-repeat regions. The in vitro binding assays further confirmed that the (CA)12 containing RNA segment of lncRNA-Smad7 associates with ss (TG)17-repeats at the Bmp2 promoter. Studies showed that RNA could associate with DNA to form the R-loop  to the locus. In the future, elucidating the assembling of lncRNA-Smad7, the hnRNPK, and polycomb complex at Bmp2 promoter region is worth the investigation.Polycomb complex is required for maintaining pluripotency in embryonic stem cells ,84. Many factors ,67, it ilncRNA-Smad7 is not conserved among species based on the primary sequences. However, RNA molecules are folded into complex of three-dimensional structures which is more conserved than its primary sequences, and RNA structures often play essential roles in RNAs’ function 12-repeats in lncRNA-Smad7 is essential for the binding of lncRNA-Smad7 to the Bmp2 promoter region. There are some CA-repeats containing lncRNAs in human, and we speculate that there may be CA-repeats containing lncRNAs which function similarly to mouse lncRNA-Smad7 in Homo sapiens (lncRNA-Smad7, it is worth taking advantage of these techniques to reveal the RNA structure in the future.Mouse function ,40. It i sapiens . Recent  sapiens , especialncRNA-Smad7 and Smad7 share the same promoter (Smad7 embedded in the lncRNA-Smad7 locus, it is not feasible to generate lncRNA-Smad7 KO mice by truncating the whole locus of lncRNA-Smad7 since the truncation will affect Smad7 expression. Once the functional domain of lncRNA-Smad7 is identified, it would be a plausible way for us to generate KO mice by deleting the functional domain of lncRNA-Smad7, which will elucidate the function of lncRNA-Smad7 in early development.Given that promoter  and therRaw data have been deposited in the GEO database with the series accession GSE178136. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD036394.gkac780_Supplemental_FilesClick here for additional data file.


The communication between calcitonin gene-related peptide (CGRP) and cytokines plays a prominent role in maintaining trigeminal ganglion (TG) and trigeminovascular sensitization. However, the underlying regulatory mechanism is elusive. In this study, we explored the hypothesis that Src family kinases (SFKs) activity facilitates the crosstalk between CGRP and cytokines in sensitizing TG. Mouse TG tissue culture was performed to study CGRP release by enzyme-linked immunosorbent assay, cytokine release by multiplex assay, cytokine gene expression by quantitative polymerase chain reaction, and phosphorylated SFKs level by western blot. The results demonstrated that a SFKs activator, pYEEI (YGRKKRRQRRREPQY(PO3H2)EEIPIYL) alone, did not alter CGRP release or the inflammatory cytokine interleukin-1β (IL-1β) gene expression in the mouse TG. In contrast, a SFKs inhibitor, saracatinib, restored CGRP release, the inflammatory cytokines IL-1β, C-X-C motif ligand 1, C-C motif ligand 2 (CCL2) release, and IL-1β, CCL2 gene expression when the mouse TG was pre-sensitized with hydrogen peroxide and CGRP respectively. Consistently with this, the phosphorylated SFKs level was increased by both hydrogen peroxide and CGRP in the mouse TG, which was reduced by a CGRP receptor inhibitor BIBN4096 and a protein kinase A (PKA) inhibitor PKI (14–22) Amide. The present study demonstrates that SFKs activity plays a pivotal role in facilitating the crosstalk between CGRP and cytokines by transmitting CGRP receptor/PKA signaling to potentiate TG sensitization and ultimately trigeminovascular sensitization. Migraine is a recurrent primary headache disorder that afflicts approximately 15% of the population worldwide ,3,4. As 2+ sensitivity and contraction of the cerebral arteries, which contributes to vascular tone and brain perfusion abnormalities [Src family kinases (SFKs) activity has been previously found to mediate CGRP release in dorsal root ganglion neurons  and TG . SFKs acmalities . Similarmalities . Taken tIn the present study, we examined whether SFKs activity facilitates the crosstalk between CGRP release and cytokines release and gene expression to activate and sensitize the mouse TG. How SFKs activity mediates the communication between CGRP and cytokines in TG is also explored by investigating the involvement of CGRP receptor/protein kinase A (PKA) pathway.A total of 143 adult male C57BL/6J mice (21.4 ± 0.17 g) were used and purchased from Shanghai SLAC Laboratory Animal Corporation Ltd. . All studies in this paper were carried out in male rodents so that the effect of hormonal fluctuation in females is minimized. Mice were housed in the Experimental Animal Centre of Soochow University for at least one week to be acclimated to the housing room before use. Animal procedures were approved by the Ethical Review Panels of Xi’an Jiaotong–Liverpool University (XJTLU) under the agreement with Soochow University and performed in accordance with relevant China national and provincial guidelines. For each experiment, randomization of experimental groups was performed to reduce bias. All animals used were randomly allocated to different experimental groups.2·2H2O, 1.3 MgCl2·6H2O, 18 NaHCO3, 1.2 NaH2PO4, 10 glucose; pH 7.4) for 30 min at 37 °C and then washed with pre-oxygenated Kreb’s solution every 5 min for 30 min. Subsequently, the TG were incubated with each drug for 20 min or 1 h at 37 °C.Isolated TG culture is a commonly used model to study TG molecular and neurophysiological properties. Signaling molecules produced in TG cell bodies are delivered to the peripheral and central terminals via axonal transport to give rise to sensory transduction and neurotransmission ,29,30,31Series 1: whether activation of SFKs increases CGRP release and IL-1β gene expression was examined in cultured mouse TG. This model has been validated in our previous publication by using KCl, a known trigger for neuronal activation and CGRP release, which successfully induces CGRP release [+ Peptide . Two groups were designed: (i) 1 mM pYEEI; (ii) 1 mM YEEI (n = 8 for each). The level of CGRP released into the culture medium was detected by enzyme-linked immunosorbent assay (ELISA); the mRNA level of IL-1β in the TG was detected by quantitative polymerase chain reaction (qPCR). Series 2: whether deactivation of SFKs reduces CGRP release was examined in cultured mouse TG pre-sensitized by reactive oxygen species (ROS). Stress is the top trigger for migraineurs [2O2), a type of ROS, can induce CGRP release in dorsal root ganglion neurons [2O2 to induce CGRP release in the TG. To inhibit SFKs activity, a SFKs inhibitor saracatinib , which binds to the kinase (SH1) domains of SFKs [2O2 [2O2 for 20 min. For this series, five groups were designed: (i) Kreb’s (n = 8); (ii) 1 mM H2O2 (n = 8); (iii) 1 mM H2O2 + 1.5 µM saracatinib (n = 8); (iv) 1 mM H2O2 + 4 µM saracatinib (n = 7); (v) 1 mM H2O2 + 10 µM saracatinib (n = 7). Series 3: whether deactivation of SFKs reduces the release and gene expression of inflammatory cytokines induced by CGRP was examined in cultured mouse TG. CGRP , the potent neuroinflammatory mediator [n = 8 for each). To detect the release of multiple cytokines into the culture medium, a multiplex immunoassay was used to measure the levels of 12 pro-inflammatory cytokines, C-C motif ligand 2 (CCL2), C-C motif ligand 5 (CCL5), C-X-C motif ligand 1 (CXCL1), C-X-C motif ligand 10 (CXCL10), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon alpha (IFN-α), interferon beta (IFN-β), interferon gamma (IFN-γ), IL-1β, interleukin 6 (IL-6), interleukin 12 (IL-12), TNFα, and one anti-inflammatory cytokine, interleukin 10 (IL-10). Only the cytokines whose levels in the TG culture medium were significantly altered by both CGRP and saracatinib were selected to detect their mRNA levels by qPCR.In order to explore whether SFKs activity mediates the communication between CGRP and cytokines in TG, three series of experiments were designed.  release . A SFKs  release ,34, or i release ,36 for 2 release ,38. A ce release  was conjraineurs , and ROSraineurs . Since h neurons , the pre of SFKs , was use of SFKs ,45,46 an of SFKs ,48 in clFKs [2O2 , 1.5 µM,FKs [2O2 ,50 in thmediator , was usemediator , or 1.5 mediator ,50 in thSeries 4: to ensure that SFKs activity is increased by H2O2 in the mouse TG in Series 2, the TG treated with Kreb’s and 1 mM H2O2 were collected to measure SFKs activity represented by the level of phosphorylated SFKs at Y416 using western blot in order to minimize animal use. How SFKs activity is enhanced by H2O2 was then investigated by examining whether inhibition of CGRP receptor reduces H2O2-enhanced SFKs activity in cultured mouse TG. To inhibit CGRP receptor, a CGRP receptor inhibitor, BIBN4096 , was used. The cultured TG was treated with 10 µM BIBN4096 in the presence of 1 mM H2O2 for 20 min. One additional group was designed: 1 mM H2O2 + 10 µM BIBN4096 (n = 7). Series 5: to ensure that SFKs activity is increased by CGRP in the mouse TG in Series 3, the TG treated with Kreb’s and 3 µM CGRP for 20 min were collected to measure the level of phosphorylated SFKs at Y416 using Western blot. Next, how SFKs activity is enhanced by CGRP was investigated by examining whether SFKs activity transmits CGRP receptor/PKA pathway as PKA is known to transmit signaling downstream CGRP [n = 8 for each). Series 6: whether SFKs co-localize with CGRP or receptor activity modifying protein 1 (RAMP1), the unique and essential functional CGRP receptor subunit [In order to explore how SFKs activity mediates the communication between CGRP and cytokines in TG, the signaling pathway that SFKs transmit during these processes is investigated, for which three series of experiments were designed. eam CGRP ,53,54 aneam CGRP ,55,56. Seam CGRP  or 30 µMeam CGRP  in the p subunit ,60, was After TG tissue culture, the level of CGRP released into the culture medium was measured using a mouse CGRP ELISA kit . Briefly, 100 μL medium and each of 8 serially diluted standard solutions were added into an assay plate pre-coated with CGRP antibody, which was then incubated at 37 °C for 2 h. Next, after removing the remaining liquid in the wells, 100 μL 1 × biotin-conjugated antibody specific for CGRP was added to each well followed by incubating at 37 °C for 1 h. The wells were then aspirated and washed, after which each well was added with 100 μL 1 × avidin conjugated horseradish peroxidase (HRP) and incubated at 37 °C for 1 h. Following further wash to remove any unbound substances, each well was added with 90 μL TMB substrate and incubated at 37 °C for 30 min in the dark. The reaction was stopped by adding 50 μL stop solution to each well and the OD of the wells was read at 450 nm, 540 nm, and 570 nm using a colorimetric microplate reader . The mean reading at 540 nm and 570 nm were subtracted from that at 450 nm, which corrected for optical imperfections. A standard curve relating the OD values to the concentration of CGRP (pg/mL) in the standard solutions was plotted and an equation of the curve was obtained. The OD values of the media were used to calculate their CGRP concentration (pg/mL) using the equation.A multi-analyte flow assay kit  was used to detect the release of 12 pro-inflammatory cytokines, CCL2, CCL5, CXCL1, CXCL10, GM-CSF, IFN-α, IFN-β, IFN-γ, IL-1β, IL-6, IL-12, TNFα, and one anti-inflammatory cytokine—IL-10—into the TG culture medium. First, 25 μL medium and each of 8 serially diluted standard solutions were added into an assay plate followed by adding 25 μL assay buffer and 25 μL mixed beads, which was then shaken at 500 rpm at room temperature for 2 h in the dark. After aspirating and washing the plate, 25 μL detection antibodies was added into the plate followed by shaking at 500 rpm at room temperature for 1 h. Next, 25 μL streptavidin-phycoerythrin (SA-PE) was added, and the plate was shaken at 500 rpm at room temperature for 30 min. After aspirating and washing the plate again, the beads in the plate were resuspended, and the plate was read on CytoFLEX S Flow Cytometer . Each of the mixed beads was conjugated with a type of allophycocyanin (APC) fluorescence and an antibody specific to one of the 13 cytokines so that each cytokine in the medium was captured by its specific bead. The APC fluorescence conjugated to each bead had a differing level, which could be recognized by the flow cytometer at 660 nm to distinguish among different beads and identify the corresponding cytokine of each bead. The SA-PE bound to the detection antibody provided fluorescent signal in proportion to the amount of a certain cytokine bound to each bead, which was read as PE signal fluorescence intensity by the flow cytometer at 585 nm. Using LEGENDplexTM Data Analysis Software 8.0 , a standard curve relating the PE signal fluorescence intensities to the concentration of each of the 13 cytokines (pg/mL) in the standard solutions was plotted and an equation of the curve was obtained. The PE signal fluorescence intensities of the media were used to calculate the concentration (pg/mL) of each of the 13 cytokines using the equation.After 60 min of TG tissue culture, total RNA of mouse TG was extracted using TRIZOL reagent  and was reverse transcribed to cDNA by a GoScript Reverse Transcription System . The mRNA levels of specific genes were detected by qPCR using GoTaq qPCR Master Mix . The qPCR reaction was performed in QuantStudio 5 Real-Time PCR System  under the following thermal cycling conditions: 95 °C for 2 min, 95 °C for 15 s, 60 °C for 1 min, and 60–95 °C for 1 min. The mRNA level of each target gene was presented as relative fold change by normalizing the individual mRNA level of the gene to the geometric mean of the mRNA levels of two housekeeping genes, β-actin, and peptidylprolyl isomerase A (PPIA). Primers specific to the target genes were shown as follows: IL-1β forward 5′ACTACAGGCTCCGAGATGAACAAC3′, reverse 5′CCCAAGGCCACAGGTATTTT3′; CCL2 forward 5′CACTCACCTGCTGCTACTCA3′, reverse 5′ GCTTGGTGACAAAAACTACAGC3′; ACTB forward 5′CTGTCCACCTTCCAGCAGAT3′, reverse 5′CGCAGCTCAGTAACAGTCCG3′; PPIA forward, 5′TTGCTGCAGACATGGTCAAC3′, reverse 5′TGTCTGCAAACAGCTCGAAG3′.Total protein of mouse TG was extracted using sodium dodecyl sulfate , as described previously . The con2:N2O (1:2) and transcardially perfused with phosphate buffer saline  and 4% paraformaldehyde . The TG were collected and post-fixed in 4% paraformaldehyde at 4 °C overnight. The tissues were dehydrated in 10%, 20%, and 30% sucrose  solutions at 4 °C overnight. Before sectioning, the tissues were embedded in Tissue-Tek O.C.T. Compound . Coronal sections at 20 μm of the TG tissues were prepared using a cryostat  and fixed on glass slides. The TG slice was permeabilized in 0.25% Triton X-100  for 15 min and blocking in 2% donkey serum  with 2% bovine serum albumin  and 0.1% Tween20  for 1.5 h at room temperature. Subsequently, the TG slice was incubated with anti-SFKs antibody  with anti-CGRP antibody  or anti-RAMP1 antibody  respectively at 4 °C overnight. The next day, the TG slice was incubated with Alexa fluor 488 donkey anti-goat secondary antibody  with 568 goat anti-mouse secondary antibody  or 568 donkey anti-rabbit secondary antibody  at room temperature for 1 h in the dark, after which they were incubated in 4′,6-diamidino-2-phenylindole  for 5 min. After mounting in mounting solution , the expressions of SFKs, CGRP, and RAMP1 in the TG slice were imaged using a Confocal Laser Scanning Microscope . The co-localization of SFKs and CGRP or RAMP1 in the acquired images was analyzed qualitatively.As CGRP and CGRP receptor distribute differently in TG neurons and nerve fibers ,60, we tt-test; if not, the data were presented as median (interquartile range), and the significance of intergroup statistical difference was analyzed by two-tailed Mann–Whitney test. Four types of significant intergroup statistical difference were used: * p < 0.05, ** p < 0.01, *** p < 0.001, or **** p < 0.0001. Detailed data presentation and statistical analysis for each quantitative study was described in the respective figure legend.For quantitative studies, all raw data generated in experiments were statistically analyzed using GraphPad Prism 7.0  for testing if each dataset followed normal distribution and if significant difference existed between the data of two comparable experimental groups. In order to choose a proper test for analyzing significant statistical difference between two groups, a Shapiro–Wilk test was performed for all the datasets to determine if they followed normal distribution. If the normality test was passed, the data were presented as mean ± standard error of the mean, and the significance of intergroup statistical difference was analyzed by two-tailed unpaired n = 8 per group, n = 8 per group).We examined whether activation of SFKs increases CGRP release and IL-1β gene expression in the TG. When treated the TG with 1 mM pYEEI, the SFKs activator, the CGRP level was 32.8 ± 1 pg/mL, which was not significantly different from the CGRP level at 35.1 ± 1.9 pg/mL in the YEEI group , the SFKs inhibitor, slightly reduced the CGRP level from the TG to 19.8 ± 4 pg/mL, which was not significantly different from that in the H2O2 group (n = 7), a significant reduction in the CGRP level to 15.9 ± 1 pg/mL was seen compared to that in the H2O2 group  also significantly reduced the level of CGRP to 13.4 ± 0.9 compared to that in the H2O2 group , CCL2 to 8.9 ± 1.7 pg/mL , and CXCL1 to 2.6 ± 0.7 pg/mL  in the TG culture medium , CCL2 to 2.5 ± 0.5 pg/mL (p = 0.0069), and CXCL1 to 1 ± 0.3 pg/mL (p = 0.0476) when compared to the respective data in the CGRP group  and one anti-inflammatory cytokine (IL-10), 3 µM CGRP promoted the levels of IL-1β to 4.6 ± 0.4 pg/mL  and CCL2 to 1.5 (4.5) (vs. 0.9 (1.2) in the Kreb’s group, p = 0.0207) in the TG  and CCL2 to 0.8 (0.3) (p = 0.0002) in comparison with that in the CGRP group  and 3 µM BIBN4096 (n = 8) reduced the protein level of phosphorylated SFKs at Y416 to 0.07 ± 0.01 compared to that in the CGRP group  and BIBN4096 (n = 8) reduced the protein level of phosphorylated SFKs at Y416 to 0.06 ± 0.01 and 0.05 ± 0.01 in comparison with that in the CGRP alone group  Amide and BIBN4096, respectively. When the protein level of phosphorylated SFKs at Y416 was normalized to that of β-actin, 3 µM CGRP markedly increased the protein level of phosphorylated SFKs at Y416 to 0.13 ± 0.01 compared to that at 0.06 ± 0.01 in the Kreb’s group .We then carried out further analysis to explore whether the SFKs activity and cytokine release enhanced by CGRP are correlated. A positive relationship between the increased levels of phosphorylated SFKs at Y416 and IL-1β release -activated CGRP release and IL-1β gene expression in the mouse TG . We ther2O2, the SFKs inhibitor, saracatinib, markedly reduces CGRP release from TG in a concentration-dependent manner (2O2 in the mouse TG (We then explored whether modulating SFKs activity may be only effective when the TG is pre-primed. Indeed, in the mouse TG that is pre-sensitized by Ht manner . Consistmouse TG . These dSimilar to the release of CGRP, the SFKs inhibitor saracatinib also reduces the release of IL-1β, CCL2, and CXCL1 from the mouse TG pre-sensitized by exogenous CGRP , which cOne question to ask from our data is how the SFKs-mediated TG sensitization is sustained. In our study, besides cytokine release by CGRP being reduced by saracatinib at 20 min post treatment , the ind2O2 (The molecular mechanism underlying the SFKs-mediated crosstalk between CGRP and cytokines in sensitizing TG has yet to be fully defined. Interestingly, SFKs activation induced by either H2O2  or exoge2O2  can be r2O2 , we can 2O2 . Taken t2O2 . Given tIn this study, only male mice are used to explore the role of SFKs in TG sensitization so that the effect of hormonal fluctuation in females can be minimized. Similarly, the previous studies on investigating the role of SFKs in the CSD-induced migraine with aura model  and the The present study demonstrates that SFKs activity plays a pivotal role in facilitating the crosstalk between CGRP and cytokines by transmitting CGRP receptor/PKA signaling to potentiate TG sensitization and ultimately trigeminovascular sensitization. These findings shed light on the SFKs-mediated peripheral mechanism of migraine pathogenesis and support the promising efficacy of drugs targeting SFKs for migraine therapy.


We studied the effects of ruthenium complexes 1–3 at a low concentration of 50 µM on the viability and the cell cycle of peripheral blood mononuclear cells (PBMCs) and HL-60 leukemic cells exposed to oxidative stress induced by hydrogen peroxide (H2O2). Moreover, we examined the influence of these complexes on DNA oxidative damage, the level of reactive oxygen species (ROS), and superoxide dismutase (SOD) activity. We have observed that ruthenium complexes 1–3 increase the viability of both normal and cancer cells decreased by H2O2 and also alter the HL-60 cell cycle arrested by H2O2 in the sub-G1 phase. In addition, we have shown that ruthenium complexes reduce the levels of ROS and oxidative DNA damage in both cell types. They also restore SOD activity reduced by H2O2. Our results indicate that ruthenium complexes 1–3 bearing succinimidato and phthalimidato ligands have antioxidant activity without cytotoxic effect at low concentrations. For this reason, the ruthenium complexes studied by us should be considered interesting molecules with clinical potential that require further detailed research.In these studies, we investigated the antioxidant activity of three ruthenium cyclopentadienyl complexes bearing different imidato ligands: (η It was shown that these complexes reduced lipid peroxidation and decreased intracellular ROS levels with comparable effectiveness to the standard steroidal drug dexamethasone or α-tocopherol  and [Ru(p-cym)(thiochrysin)Cl]—showed good anti-aggregating activity in washed platelet samples [p-cym)(thiochrysin)Cl] has been able to inhibit in vitro thrombus formation and it was shown to affect haemostasis in mice [Studies have also shown that some ruthenium complexes have a protective effect on the cardiovascular system . For exaynthesis . It was  samples . The com in mice . It was  in mice .5-cyclopentadienyl)Ru(CO)2-N-methoxysuccinimidato (1), (η5-cyclopentadienyl)Ru(CO)2-N-ethoxysuccinimidato (2), and (η5-cyclopentadienyl)Ru(CO)2-N-phthalimidato (3)   showed n1–3 (2O2) to induce oxidative stress. Incubation with H2O2 was preceded by pre-incubation of cells with ruthenium complexes 1–3. For comparison, we also pre-incubated the cells with ruthenium chloride (RuCl3) and succinimide ligand. We investigated the influence of ruthenium complexes 1–3 on viability, cell cycle, and DNA oxidative damage. We also determined their effect on the level of reactive oxygen species (ROS) and superoxide dismutase (SOD) activity.Our previous results prompted us to investigate the antioxidative activity of ruthenium complexes bearing succinimidato and phthalimidato ligands 1–3  in norma1–3 were synthesized as previously described (1) [2) [3) [3), 2′,7′-dichlorofluorescein (H2DCFDA), Hank’s balanced salt solution (HBSS), dimethyl sulfoxide (DMSO), and hydrogen peroxide (H2O2) were purchased from Sigma-Aldrich . Succinimide was purchased from Fluka. All other chemicals were of the highest commercial grade available. A stock solution of complexes and succinimide (10 mM) was dissolved in DMSO.Ruthenium complexes ibed (1) , (2) [22 [2) [3) . IMDM me2 and sub-cultured every 2–3 days to maintain exponential growth.HL-60 (human promyelocytic leukemia) cell line was obtained from the American Type Culture Collection (ATCC) and cultured in Iscove’s Modified Dulbecco’s Medium (IMDM) with 15% fetal bovine serum, streptomycin/penicillin solution (100 μg/mL and 100 U/mL). HL-60 cells were cultured in flasks at 37 °C in 5% COPeripheral blood mononuclear cells (PBMCs) were isolated from the buffy coats obtained from healthy donors from Central Blood Bank, Lodz. Each donor agreed to donate blood. The first step of isolation of PBMCs was a mix of fresh blood from buffy coats with PBS at ratio 1:1. In the next step, the mixture was centrifuged in a density gradient of Lymphosep  at 2200 RPM for 20 min with the lowest values of acceleration and deceleration. Then the cells were washed three times by centrifugation with 1% PBS. After isolation cells were suspended in RPMI 1640 medium. The study protocol was approved by the Committee for Research on Human Subjects of the University of Lodz (17/KBBN-UŁ/III/2019).6 cells/mL. Cells were pre-incubated with ruthenium complexes 1–3, succinimide, and RuCl3 at a concentration of 50 µM for 24 h at 37 °C in 5% CO2. Next, cells were washed with warm PBS. Then, cells were seeded on a 96-well plate in the count of 50,000 for PBMCs and 15,000 for HL-60 cells. Next, cells were incubated with H2O2 at concentrations of 0.2, 0.4, and 0.6 mM for 4 h at 37 °C in 5% CO2. Next, 10 μL of resazurin salt was added to each well, and the plates again were incubated at 37 °C in 5% CO2 for 2 h. After that, fluorescence was measured with HT microplate reader Synergy HT  using λex = 530/25 nm and an λem = 590/35 nm. The effects of ruthenium complexes, RuCl3, and succinimide on cell viability were quantified as the percentage of control fluorescence.The cell viability resazurin assay was performed similarly to the method described by O’Brien et al. . Resazur6 cells/mL. The cells incubated with 100 ng/mL nocodazol (NOC) for 24 h at 37 °C were the positive control. The cells were incubated with ruthenium complexes 1–3, succinimide, and RuCl3 at a concentration of 50 µM for 24 h at 37 °C in 5% CO2. Next, cells were washed with warm PBS. Then, cells were again seeded on 6-well plates. Next, cells were incubated with H2O2 at concentrations of 0.1 and 0.2 mM for 24 h at 37 °C in 5% CO2. Then, cells were collected and washed twice with PBS. After that, cells were resuspended in PBS and put on ice for 15 min. Then, one volume of −20 °C absolute ethanol was added and the samples were stored at 4 °C. Before the analysis, samples were resuspended in 300 µL of staining solution containing 40 µg/mL PI (propidium iodide) 200 µg/mL RNase A. Samples were incubated for 30 min at 37 °C in the dark until analysis. DNA content was analyzed using an LSRII flow cytometer .HL-60 cells were seeded on 6-well plates at a density of 0.5 × 101–3, succinimide, and RuCl3 at a concentration of 50 µM for 24 h in 37 °C in 5% CO2, then cells were washed with PBS and incubated with H2O2 at 0.025 or 0.05 mM for 15 min on ice. Cell viability after incubation with H2O2 was in the range of 95–100% (data not shown). After incubation with H2O2 cells were centrifuged, suspended in LMP agarose, and spread onto a microscope slide. The slides were processed as described below in the We pre-incubated cells with complexes The comet assay was performed under alkaline conditions according to the procedure of Tokarz et al. . A freshElectrophoretic separation was performed in the solution containing 30 mM NaOH and 1 mM EDTA, pH > 13 at an ambient temperature of 4 °C (the temperature of the running buffer did not exceed 12 °C) for 20 min at an electric field strength of 0.73 V/cm (28 mA). Then, the slides were washed in water, drained, stained with 2 µg/mL DAPI, and covered with coverslips. To prevent additional DNA damage, the procedure described above was conducted under limited light or in the dark.The comets were observed at 200× magnification in an Eclipse fluorescence microscope  attached to a COHU 4910 video camera  equipped with a UV-1 A filter block and connected to a personal computer-based image analysis system Lucia-Comet v. 6.0 . Fifty images (comets) were randomly selected from each sample and the mean value of DNA in the comet tail was taken as an index of DNA damage (expressed in percent).2DCFDA) probe was used. H2DCFDA is a cell membrane-permeable non-fluorescent probe. 2′,7′-dichlorofluorescein diacetate is de-esterified intracellularly and turns into highly fluorescent 2′,7′-dichlorofluorescein upon oxidation. PBMCs and HL-60 cells were seeded at a density of 2.5 × 106 cells/mL and 0.5 × 106 cells/mL, respectively, and were incubated with ruthenium complexes 1–3, succinimide, and RuCl3 at a concentration of 50 µM for 24 h at 37 °C in 37 °C in 5% CO2. Next, the cells were washed twice with HBSS containing Ca2+ and Mg2+ and stained with 20 µM H2DCFDA  for 30 min at 37 °C in darkness. Then, the cells were washed twice with HBSS and incubated with 1 mM and 5 mM H2O2 at 37 °C in darkness. The intensity of fluorescence was measured after 30 min with λex  =  495 nm and λem  =  530 nm using a microplate reader Synergy HT . The data were analyzed according to the following formula: (Tx  −  T0/T0)  ×  100, where Tx is the DCF fluorescence measured at the indicated time and T0 is the DCF fluorescence measured at the beginning of the analysis.To measure reactive oxygen species (ROS), a 2′,7′-dichlorofluorescein diacetate  to obtain cell lysates. SOD activity was measured using the SOD Assay Kit-WST  by following the manufacturer’s instructions. Cells lysates served as the sample solution. The mixture was placed into each well with 200 µL of WST working solution. Twenty microliters of enzyme working solution were added into each well and mixed thoroughly. The plate was incubated at 37 °C for 20 min. The absorbance was read at 450 nm using a microplate reader Synergy HT .PBMCs and HL-60 cells were seeded at density 3 × 10t-test . Differences were considered statistically significant when the p-value was <0.05.Data of cell viability, ROS measurement, cell cycle, and SOD activity are presented as the mean values ± standard deviation (SD) of at least three replicates. Values in the comet test are expressed as mean values + standard error of the mean (SEM) of three experiments; data from three experiments were collected and statistical parameters were calculated. Statistical analysis was performed using the Mann–Whitney test  and the Student 1–3 at a concentration of 50 µM. We chose this concentration based on our previous studies in which we showed an increase in cell viability for both PBMCs and HL-60 after 24 h incubation with ruthenium complexes at 50 µM [All experiments were performed with ruthenium complexes 2O2 for 4 h and the viability was determined with a resazurin reduction assay. We observed a significant increase (p < 0.001) in the viability of normal and cancer cells pre-incubated with all ruthenium complexes compared to the cells that were not pre-incubated (3 under the same conditions showed a further decrease (p < 0.001) in viability following incubation with H2O2, except for the PBMCs which were incubated with H2O2 at 0.4 and 0.6 mM. We also observed a decrease (p < 0.001) in the viability of the HL-60 cells that were pre-incubated with succinimide. In the case of the normal cells, we detected the opposite effect of pre-incubation with the ligand—an increase in the viability of PBMCs compared to the cells that were only incubated with H2O2 at 0.4 and 0.6 mM (p < 0.001).After pre-incubation, the cells were incubated with Hncubated . The cel2O2, we observed an increase in the number of cells in the sub-G1 (p < 0.001), S (p < 0.001) and G2/M (p < 0.01) phases of the cell cycle and a decrease in the G0/G1 phase (p < 0.001) compared to the control cells (2O2 at a higher concentration causes a further growth of HL-60 cells in the sub-G1 phase (p < 0.001) and a significant decrease in the number of cells in the phases G0/G1 (p < 0.01) and G2/M (p < 0.01).After incubation of HL-60 cells with 0.1 mM Hol cells . H2O2 at1–3 compared to the cells incubated only with H2O2, both at 0.1 and 0.2 mM (p < 0.001). A similar decrease in the number of cells was observed in the S phase (p < 0.001). In addition, we demonstrated cell arrest in the G2/M phase by pre-incubation of HL-60 cells with complexes 1–3 and incubation with 0.2 mM H2O2.We showed a decrease in the number of cells in the sub-G1 phase after pre-incubation with ruthenium complexes 3 or succinimide and then incubated with H2O2 showed similar changes in the course of the cell cycle as the cells pre-incubated with ruthenium complexes 1–3 (HL-60 cells that were pre-incubated with RuClexes 1–3 .2O2 at two concentrations of 0.025 and 0.05 mM. Then we investigated the influence of pre-incubation with ruthenium complexes 1–3 on oxidative DNA damage (1 (p < 0.001) and complex 2 (p < 0.05), but only in the case of incubation with H2O2 at 0.05 mM .We induced DNA oxidative damage in PBMCs and HL-60 cells using HA damage . In the  0.05 mM . After i2O2 at 0.025 and 0.05 mM for all ruthenium complexes (p < 0.001) .In the experiment with HL-60 cells, we detected a significant decrease in oxidative DNA damage induced by H< 0.001) . We show2DCF-DA probe to determine the effect of ruthenium complexes 1–3 on reactive oxygen species (ROS) induced by H2O2 at concentrations of 1 and 5 mM (p < 0.001) in the level of ROS for complexes 1 and 2 and 1 mM H2O2 (3 increased the level of H2O2 induced ROS (p < 0.001). A similar effect to the one found in complex 3 was observed in the PBMCs pre-incubated with succinimide and then incubated with 5 mM H2O2 (p < 0.001). Complex 2 has the greatest potential to scavenge ROS in normal cells; it also reduced endogenous ROS levels (p < 0.001).We used an Hand 5 mM . In the  mM H2O2 . In contp < 0.001) in ROS levels by all ruthenium complexes (2O2 at 1 mM (p < 0.001) and 5 mM (p < 0.01). All ruthenium complexes and succinimide significantly reduced endogenous ROS levels in contrast to RuCl3.In HL-60 cells, we clearly showed a significant reduction  after the incubation of PBMCs .In this experiment, we investigated the influence of ruthenium complexes on SOD activity under oxidative stress conditions . First, of PBMCs  and HL-6of PBMCs  with H2Oresearch ,26. It sp < 0.001) in HL-60 cells treated with H2O2 (p < 0.001). In the case of HL-60 cells pre-incubated with 50 µM RuCl3, we did not detect any changes in SOD activity compared to the cells that were not pre-incubated with RuCl3. In the case of PBMCs, we observed an increase in SOD activity (p < 0.001) after pre-incubation with complexes 1 and 2. Complex 3 as well as RuCl3 and succinimide did not increase SOD activity (p > 0.05)  .2O2. A correlation and gene ontology pathway analysis identified a rigid association with genes intertwined in cell cycle progression and proliferation after cell incubation with H2O2 [2O2 IC25. It was also shown that the top genes related to ROS production or antioxidant defense were only in modest correlation [2O2 is regulated tightly, enabling its use as a cellular signaling molecule while minimizing its potential to cause cellular damage [2O2 induces necrosis and a low concentration induces apoptosis. In HL-60 cells H2O2-induced apoptosis is regulated by decreased BCL-2 [2O2 evoked apoptotic events through the increase of Ca2+ in HL-60 cells, inducing caspase -9 and -3 activation, induction of the mitochondrial permeability transition pore (mPTP), and activation of proapoptotic proteins [Here, we investigated the antioxidant activity of three ruthenium complexes with succinimidato and phthalimidato ligands against normal PBMCs and HL-60 leukemic cells. For this purpose, we used 24 h pre-incubation of cells with the complexes and then induced oxidative stress by incubating cells with Hith H2O2 . The tenrelation . The intr damage . A high ed BCL-2 . Moreoveproteins .2O2 at a concentration range between 0.2 and 0.6 mM reduced the viability of normal and cancer cells  through the interaction with metal ions near DNA, induces DNA damage such as modified bases, apurinic/apyrimidinic (AP) sites, and single-strand breaks (SSBs). The addition of OH● at position C8 within the guanine ring generates the oxidative product, 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG). Similarly, the addition of OH● at position C8 of deoxyadenosine generates the oxidative product 8-oxo-7,8-dihydro-2′-deoxyadenosine (8-oxodA). These radicals are capable of further reduction or oxidation forming 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) or 8-oxo-7,8-dihydroguanine (8-oxoG), in deoxyguanosine or 4,6-diamino-5-formamidopyrimidine (FapyA) or 7,8-dihydro-8-oxoadenine (8-oxoA) in deoxyadenosine. Another prevalent oxidative product is thymine glycol, produced by the insertion of OH● at position C5 of the thymine rings. Similarly, another oxidation product of cytosine is cytosine glycol, which upon deamination leads to the formation of uracil glycol [Hydrogen peroxide, by producing hydroxyl radicals (OHl glycol .1 (p < 0.001) and ruthenium complex 2 (p < 0.05) (p < 0.001) (2O2-induced oxidative DNA damage by tricarbonyldichlororuthenium (II) dimer (CORM-2) and the CO-depleted molecule (iCORM-2) [2O2 in both types of cells (2DCFDA) probe to detect ROS. The acetate groups on H2DCF allow for diffusion across the plasma membrane, after which both groups are cleaved by intracellular esterases to form H2DCF. H2DCF is reactive toward many types of oxidants, including nitrogen dioxide (●NO2), the carbonate radical anion (CO3●–), the hydroxyl radical (OH●), Fe2+, Cu+, thiyl radicals , and peroxidases  [Here, we observed a statistically significant decrease in oxidative DNA damage in PBMCs for ruthenium complex  < 0.05) . In the < 0.001) . PreviouiCORM-2) . This maof cells . We usedoxidase) ,34. Impo2O2 and other oxidants. The oxidative stress-induced downregulation of MnSOD activity causes superoxide radical accumulation and superoxide can be converted to OH● in the presence of transition metals  [hsEC-SOD is inhibited and fragmented by H2O2. The enzyme was inhibited by H2O2  in a dose-dependent manner, with an IC50 value of 0.8 mM and complete inhibition at ~2 mM. The mechanism of inhibition was similar to that of bovine Cu,Zn-SOD, including the oxidation of proline (Pro 112) and histidine  residues proximal to the active site Cu [Superoxide dismutases (SODs) including MnSOD, Cu/Zn-SOD, and extracellular SOD along with catalase are composed as the first line of defense against ROS. Some studies also showed that oxidative stress downregulated MnSOD in several oxidative stress models induced by Heaction) . Researceaction)  showed t site Cu ,36.p < 0.001) in HL-60 cells treated with H2O2 (p < 0.001). In the case of PBMCs, we observed an increase in SOD activity (p < 0.001), decreased by H2O2, after pre-incubation with complexes 1 and 2  energy gap was lower by 1.36 eV when compared with succinimide [3 indicate that the succinimide ligand, rather than the ruthenium metal center, is responsible for the antioxidant activity of complexes 1–3. We plan to undertake further electrochemical investigations of succinimide and complexes 1–3 to shed some light on this problem. Taking into account the fact that oxidative stress is involved in many human diseases, including cancer, antioxidant treatment involving new molecules that exhibit antioxidant properties in biological systems could be of potential interest from a clinical point of view.Our results show that ruthenium complexes


Experimental results confirm the ultimate sample possess a 3-dimensional chain-like structure which are constructed by numerous ferrite’s nanoparticles with ~60 nm in diameter. Their electromagnetic parameters can be also manipulated by such a chain structure, especially the dielectric loss, where a sharply increases can be observed on within a lower filling ratio. It greatly benefits to the EM absorbing property. In this article, the electromagnetic absorption layer made with a lower content of ferrite possess the excellent electromagnetic absorption ability, where the optimized effective absorption band was nearly 6.4 GHz under a thickness of 1.8 mm. Moreover, the filling ratio is only 30 wt%. Our method for designing of chain-like magnetic material can be helpful for producing wideband electromagnetic absorption in a low filling ratio.The magnetic nanochain-like material has been regards as one of the most promising electromagnetic (EM) absorbing material but remains a challenging. Herein, magnetic chain-like ferrite (included Fe How ability ,14. For f 2.8 mm . Zhu andf 2.5 mm . Accordif 2.5 mm . More imf 2.5 mm ,20. The f 2.5 mm ,22. To sf 2.5 mm ,24. Thesf 2.5 mm ,26,27,28of Jason . This me3O4, NiFe2O4, CoFe2O4) were easier to form a 3D network structure after dispersing into the matrix. Once 3D network structure is formed, the dielectric loss ability can be increased sharply, based on the conductive percolation threshold. Such a chain-like sample possess a wideband EM absorption ability under a thin thickness. The method of utilizing structural strategy to formation of chain-shaped ferrite has great significance in making wideband high-performance absorber.In this article, herein, we designed a chain-shaped ferrite which using the structural strategy to increase the dielectric loss ability. The as-prepared chain-shaped ferrite (Fe2), ferric chloride (FeCl3), ferrous chloride (FeCl2), Nickel acetate (NiCl2) were obtained from Shandong Chemical Reagent Co., Ltd. . Polyvinylpyrrolidone , ethylene glycol (EG), cyclohexane and glycerol were purchased from Sinopharm Chemical Reagents Co. . All of the chemical reagents were analytically pure and used without further purification.Cobalt acetate (Co(Ac)3, 1.0 mmol FeCl2 are dissolved into solution, containing ethylene glycol (20 mL), and then ultrasonic for 1.0 h. Subsequent, the solution was transferred into a Teflon-lined stainless-steel autoclave and kept at 200 °C for 20 h. After cooled to room temperature, the precipitation can be collected by centrifuge , washed with isopropanol, cyclohexane, and ethanol for 6~10 times. Finally, the dried sample was continuous to heat at 300 °C for 2 h, aiming to removal of PVP. The NiFe2O4 and CoFe2O4 were prepared via replacing the FeCl2 with Co(Ac)2) and NiCl2.The chain-shaped ferrite was prepared by a solvothermal-process. Typically, 1.5 g PVP, 2.0 mmol FeClThe phase compositions of these hybrids are confirmed by powder X-ray diffraction (XRD) patterns, using Cu Kα radiation (λ = 0.154178 nm). Morphologies, especially the chain-like structure are observed by a Field emission scanning microscope . Fourier transform infrared spectra (FT-IR) was characterized by the Fourier transform infrared spectrometer . Magnetization hysteresis loops (M-H) curve was recorded on a vibrating sample magnetometer  at 298 K.RL) curve could be gained by inputting the electromagnetic parameters into the below formulas [inZ relates to input impedance of the absorber, f is the frequency of electromagnetic wave, d is the thickness of the absorber, while c is the velocity of light. εr (εr= ε′−jε″) and rµ (rµ = µ′−jµ″) are the relative complex permittivity and permeability of the absorber.To obtain the electromagnetic parameters, the as-prepared ferrites were homogeneously blended with paraffin wax in the weight ratios of 10~40 wt%. Subsequent, the mixture was pressed into a ring-shaped structure with outer diameter of 7.0 mm and inner diameter of 3.04 mm, respectively. The electromagnetic parameters were analyzed on an E5080A vector network analyzer at 2–18 GHz. Finally, the frequency dependency of reflection loss  and FeO6 (670 cm−1) [3O4. In addition, another two peaks at 1389 and 1620 cm−1 are belonging to C–O and C–C, which are original from the PVP. After annealed at 300 °C, C-based peaks are entirely disappeared, which may be due to the decomposition of PVP.To make sure the successfully removal of PVP, FT-IR spectra of Fe70 cm−1) ,34. It so are corresponding to (200), (311), (222), (400), (331), (422), (511) and (440) crystal planes of spinel Fe3O4 (JCPDS: card no: 75–1609). In comparison with Fe3O4, the diffraction peaks of CoFe2O4 and NiFe2O4 present a slight right shift, which is due to varied unit cell volume after dotted by Co or Ni.The magnetization properties were compared by the VSM at room temperature. 3O4 nanoparticles closely contact well with each other and resulting in distinct chain-shaped structure. The average sizes of Fe3O4 nanoparticles are approximately 60 nm, as statists in 2O4 and NiFe2O4 both possess the same chain-like structure, as depicted in The chain-like structures were investigated by the TEM images, as showed in ε′, ε″), which are account for the electrical storage and dielectric loss capability, respectively [ε′ values exhibit the decreased tendency without any remarkable fluctuation. Meanwhile, ε′ linearly increases significantly as enhancing the weight ratio of ferrite. It is interesting that exceeding 30 wt%, ε′ values become slowly increases. Among these ferrites filling absorption layers, Fe3O4 has the largest ε′ value at whole 2~18.0 GHz. For example, the absorption layer containing 10 wt% of Fe3O4 has a ε′ value about 4.3~3.9, which is greater than CoFe2O4 (3.8~3.1) and NiFe2O4 (3.4~3.1), respectively. At 30 wt%, Fe3O4 still reaches the largest ε′ value of 9.4–7.4. The ε″ value as a function of frequency are shown in ε″ value, similar phenomenon can be observed, and two conclusions are summarized as follows:ε″ can be observed at ferrite weight regions of 10~30 wt%, but slowly increases between 30~40 wt%.A higher content of ferrite would lead to the strong dielectric loss ability. Besides, the distinct enhancement of 3O4 is easier to present the strongest dielectric loss behavior than CoFe2O4 and NiFe2O4.FeSubsequent, the as-prepared ferrites were homogeneous mixed with paraffin in various certain weight ratio, which was used to test the EM parameters. ectively ,36. In Fε′ and ε″ are actually highly associated with their relaxation polarization and conductive loss ability [ε′ and ε″ value both, which two typically physical phenomena can be observed, that is, sharply decreased ε′ value since the frequency dispersive effect, and dielectric resonance peak in ε″ [ε′ versus ε″ will turn to be a single semicircle, normally denoted as the Cole-Cole semicircle, according to the classic Debye-theory. In details, the relative complex permittivity can be drawn as follow [εs, ε∞, τ are static permittivity, relative dielectric permittivity at high-frequency limit, and polarization relaxation time, respectively. After the separation of real and imaginary parts, gives:To reveal the varied permittivity, the conductive percolation has been used in this article. As we known,  ability ,38. Of p ability ,40. Eithak in ε″ . Concerns follow ,43:(3)εrε′-ε″ can be expressed as above:Based on the Equations (4) and (5), the ε′ and ε″ both. Usually, the weight value for fabricating continuous conductive network was denoted as percolation threshold [ε′ and ε″ values increases as rising the weight ratio. Once beyond the percolation value, ε′ and ε″ possess slowly increasing tendency. In our case, the percolation threshold value of ferrite is estimated to be 30 wt%, which are almost a half of current advances [3O4 with the highest ε″ value which may due to the strongest electron hoping between Fe3+ and Fe2+ [According to Equation (6), each Cole-Cole semicircle is corresponding to one Debye relaxation process. Taking the 30 wt% of ferrites as cases, they did not present obviously semicircles, as shown in hreshold . Before advances ,46. The and Fe2+ .μ′ and μ″). Generally, the μ′ and μ″ values are standing for the storage and dissipation capability of magnetic field, respectively [μ′ values of these ferrites filling absorption layer are only a tiny bigger than 1.0. After increasing to 30 wt%, these μ′ values are ranging in 1.25~1.15 and simultaneous possessing a decreased tendency. Up to 40 w%, μ′ values have a distinct improvement and all of μ′ values are larger than 1.25. But overall, μ′ of either Fe3O4, CoFe2O4 or NiFe2O4 has a little difference in μ′ values, which were attributed to the nearly magnetization behaviors. Because of similar crystal structure and magnetization, their present approximately magnetic loss ability.The permeability values are investigated in ectively . At 10–2The reflection loss values (RL) values of these samples can be obtained via coaxial-line method. One can see that ferrite filling absorption layer shows the poor electromagnetic absorption, which the reflection loss value is as bigger as −10 dB  at entirely thickness region . But con3O4, the minimum RL value of CoFe2O4 absorption layer equals to −57.8 dB with a matched thickness of 2.0 mm. Meanwhile, the maximum effective absorption region is estimated to be 6.2 GHz under identical 1.8 mm. As for the NiFe2O4, the optimized RL value and effective absorption region respective −17.1 and 4.2 GH, and corresponding thickness are 2.0 and 1.8 mm. But continues increases to 40 wt%, minimum RL value and effective absorption band do not enhance significantly. To comprehensively consider the bandwidth, thickness and filling ratio, the absorption layer made with 30% of Fe3O4 would be the optimized electromagnetic performance. Compared with the ferrite-based EM materials in recent advances  constructed by 60 nm of nanoparticles have been prepared by a facile solvothermal route. The as-prepared chain-like structure are highly favoring for the permittivity values under a relatively low filling ratio. The results indicate that ε″ value of the absorption layer filling with 30% were beyond 2.5. Meanwhile, such a magnetic ferrite possessed moderately magnetic loss ability. Owing to the dielectric and magnetic loss behavior, a wideband absorption region of 6.4 GHz under a thinner thickness of 1.8 mm. The EM absorption mechanism can be obtained at a lower filling ratio has been in-depth investigation, which are greatly benefits to the chain-like structure.To summary up, magnetic chain-like ferrites (such as Fe


Streptomyces sp. strain Gö40/10 has the potential to produce a range of secondary metabolites, one of which is collinolactone, a compound with neuroprotective properties and potential for pharmaceutical applications. The genome was sequenced with Oxford Nanopore Technologies MinION and Illumina MiSeq systems and consists of a single 9,635,564-nucleotide linear chromosome.The actinomycete  Streptomyces sp. strain Gö40/10 was isolated from soil collected in Bolivia in 1991 and grown in medium NL 8711, which is used routinely for agar slopes  medium with 3.4% sucrose at 30°C with shaking at 250 rpm  with the setting --nano-hq and a total of five polishing iterations  and run with a FLO-MIN106D (R9.4.1) flow cell on a MinION device. Base calling was done using Guppy base caller (v5.0.11+2b6dbff) in high-accuracy mode followed by demultiplexing by the Guppy barcoder. Adaptors were trimmed from Nanopore reads using Porechop (v0.2.4) . After tv2.9-b176 with theIllumina MiSeq data were generated from a KAPA HyperPlus library , with size selection of approximately 450 to 600 nt, resulting in 2.3 million clusters of read length 2 × 151 bp. Illumina reads were trimmed using Trim Galore with Cutadapt (v2.10), applying the settings --length 100 and --quality 20 .Illumina data were aligned to the Nanopore assembly using bowtie2-align (v2.3.4.1), with an overall alignment rate of 97.08% . FinallyStreptomyces cinerochromogenes, and the closest autoMLST (v231601f) (Streptomyces reticuli (GenBank accession number LN997842.1) at 92.8% average nucleotide identity (ANI). The benchmarking universal single-copy orthologs (BUSCO) score   as a mem231601f)  match waO) score  was 100%Analysis with antiSMASH   with thePRJNA721311. Raw reads were deposited in the SRA under BioSample accession numbers SAMN18970196 (Illumina) and SAMN18970196 (Nanopore). The genome sequence can be found under GenBank accession number CP084203.1.The data are available under NCBI BioProject accession number