Laser-driven accelerators allow to generate ultrashort (from femto- to picoseconds) high peak dose-rate (up to tens of GGy/s) accelerated particle beams. However, the radiobiological effects of ultrashort pulsed irradiation are still poorly studied. The aim of this work was to compare the formation and elimination of γH2AX and 53BP1 foci (well known markers for DNA double-strand breaks (DSBs)) in Hela cells exposed to ultrashort pulsed electron beams generated by Advanced Research Electron Accelerator Laboratory (AREAL) accelerator  and quasi-continuous radiation generated by Varian accelerator (electron energy 4 MeV) at doses of 250–1000 mGy. Additionally, a study on the dose–response relationships of changes in the number of residual γH2AX foci in HeLa and A549 cells 24 h after irradiation at doses of 500–10,000 mGy were performed. We found no statistically significant differences in γH2AX and 53BP1 foci yields at 1 h after exposure to 2 Hz ultrashort pulse vs. quasi-continuous radiations. In contrast, 20 Hz ultrashort pulse irradiation resulted in 1.27-fold higher foci yields as compared to the quasi-continuous one. After 24 h of pulse irradiation at doses of 500–10,000 mGy the number of residual γH2AX foci in Hela and A549 cells was 1.7–2.9 times higher compared to that of quasi-continuous irradiation. Overall, the obtained results suggest the slower repair rate for DSBs induced by ultrashort pulse irradiation in comparison to DSBs induced by quasi-continuous irradiation. Curis ~1 ns ), suggesFrom this perspective, the impact of ultrashort pulsed irradiation on DNA and particularly on one of the most deleterious lesions, namely double strand breaks (DSBs) formation on both qualitative and quantitative levels is of high interest. It is widely accepted that DSBs are the main trigger for the initiation of cellular processes in response to ionizing radiation ,9. AboutThe aim of the current study was the investigation of the formation and repair kinetics of DSBs, induced by ultrashort pulsed and quasi-continuous electron beam irradiation in human cancer cells.The immunofluorescence imaging of DNA repair proteins foci has been used for the quantitative study of DSBs formation. Once stained, the complex of dynamic microstructures, formed for DSBs repair and consisting of a set of proteins in many (up to few thousands) copies, can be seen as bright points called foci . The mosHuman cervix carcinoma HeLa and human lung carcinoma A549 cell lines were used in this study as the common experimental models for studying various biological effects ,22.Comparative analysis of γH2AX and 53BP1 foci yields was performed at 1 h post-irradiation and 0.25–1.0 Gy doses, following cells the irradiation on Advanced Research Electron Accelerator Laboratory (AREAL) and Varian accelerators. Cell irradiation on the AREAL accelerator was carried out using different pulse repetition and dose-rates , while irradiation on Varian machine was done at dose-rate of 5.6 Gy/min.2 = 0.99, p < 0.001) by y = 3.57 + 22.29 × x linear equation, where y is an average of foci numbers in a cell nucleus and x is an irradiation dose (Gy). Lowering the repetition rate of irradiation up to 2 Hz resulted in y = 3.62 + 19.82 × x  equation, where y is an average of foci numbers in a cell nucleus and x is an irradiation dose (Gy). In a linear equation (y = a + b × x), describing a dose-dependent relationship, the slope coefficient b reflects the increase in the effect per dose unit. The comparison of the slope coefficients of two different dose–response curves, obtained by different exposure types, reflects for how many fold one of them is more expressed compared to the other. After 2 Hz irradiation, the slope coefficient was found to be about 1.13 times lower with respect to 20 Hz, however, this difference was not statistically significant . For irradiation on the Varian accelerator, a dose-dependent relationship was found to be expressed by y = 2.64 + 17.60 × x , where y is an average of foci numbers in a cell nucleus and x is an irradiation dose (Gy). The slope coefficient was 1.27 times lower  as compared to irradiation on the AREAL accelerator with 20 Hz repetition rate.Dose-dependent changes in γH2AX foci numbers are presented in Dose-dependent changes in 53BP1 foci numbers are presented in 2 = 0.97, p = 0.002), where y is an average of foci numbers in a cell nucleus and x is an irradiation dose (Gy);(1) Irradiation on the AREAL accelerator at a 20 Hz repetition rate—y = 4.81 + 20.82 × x , where y is an average of foci numbers in a cell nucleus and x is an irradiation dose (Gy);(2) Irradiation on the AREAL accelerator at 2 Hz—y = 4.19 + 18.19 × x , where y is an average of foci numbers in a cell nucleus and x is an irradiation dose (Gy).(3) Irradiation on the Varian accelerator—y = 3.62 + 16.35 × x (Rp = 0.156). However, significant differences were observed between the irradiation effects on AREAL at 20 Hz and Varian accelerators, and particularly the linear slope coefficient differed by about 1.27 times .No statistically significant differences between the linear slope coefficients were found after irradiation on the AREAL accelerator at two repetition rates , where y is an average of foci numbers in a cell nucleus and x is an irradiation dose (Gy)). In the case of HeLa cells, the number of residual foci was changed insignificantly at the dose range of 1–10 Gy and the “saturation” effect was observed .The comparative analysis of the slope coefficients of dose–response changes in residual foci number has been performed in A549 cells. The linear slope coefficient was shown to be 1.7 times higher in the case of pulse irradiation compared to quasi-continuous irradiation  and A549 (human lung carcinoma) cell lines were obtained from the American Type Culture Collection (ATCC) and maintained in DMEM/F12  with 2.5 mM L-Glutamine , supplemented with 10% fetal bovine serum , 100 IU/mL penicillin , and 100 μg/mL streptomycin  at 37 °C in 5% CO5 cells/mL in 2.5 mL of culture medium onto coverslips  placed inside 35 mm Petri dishes  and incubated at 37 °C and 5% CO2 for 20 h.Prior to irradiation, the cells were seeded at a density of 0.4 × 10Ultrashort beam irradiation was carried out using an electron beam generated by a laser-driven radiofrequency gun-based linear AREAL accelerator. The characteristics of the AREAL accelerator have been described previously . The par10 Gy/s was estimated from the electron pulse duration of 4.5 × 10–13 s, based on the laser pulse length, acceleration process, and electron beam transport. The mean absorbed dose-rate of 1.17 ± 0.02 Gy/min (repetition rate 2 Hz sample mass of 4.2 g) and 11.70 ± 0.98 Gy/min (repetition rate 20 Hz sample mass of 4.2 g) was calculated over the period of irradiation and 1% charge fluctuation and 1% beam energy fluctuation was taken into account.The dosimetric measurements were performed with a Faraday cup , estimating the integral dose over the pulse. Cells were irradiated at doses of 0.25–10 Gy (~140 electron pulses per 1 Gy) with a repetition rate of 2 or 20 Hz. A peak dose rate of 1.6 × 10Quasi-continuous irradiation was carried out using the Varian Trilogy  electron linear accelerator. Characteristics: electron energy 4.0 MeV, dose-rate 5.6 Gy/min, electron beam size 250 × 250 mm. Dosimetry was performed by ionization method in a water phantom according to the International Atomic Energy Agency (IAEA) TRS-398 international protocol .http://focicounter.sourceforge.net/).Cells were fixed on coverslips in 2% paraformaldehyde in PBS (pH 7.4) for 20 min at room temperature, followed by two rinses in PBS and permeabilization in 0.3% Triton-X100 , supplemented with 2% bovine serum albumin (BSA) to block nonspecific antibody binding. Cells were incubated for 1 h at room temperature with primary antibody against γH2AX Y, MerckMillipore, Burlington, VT, USA) and 53BP1 , diluted in PBS with 1% BSA. Then the specimens were washed three times with PBS (pH 7.4) and incubated for 1 h at room temperature with secondary IgG (H + L) goat anti-mouse  and goat anti-rabbit  diluted in PBS (pH 7.4) with 1% BSA. The coverslips were then rinsed several times with PBS and mounted on microscope slides with ProLong Gold medium  with 4′,6-diamidino-2-phenylindole (DAPI) for DNA counter-staining. Imaging of immunocytochemical specimens were performed using a Nikon Eclipse Ni-U fluorescent microscope  equipped with a ProgRes MFcool high-resolution video camera . The filter sets: UV-2E/C (340–380 nm excitation and 435–485 nm emission), B-2E/C (465–495 nm excitation and 515–555 nm emission), and Y-2E/C (540–580 nm excitation and 600–660 nm emission). Between 300 and 400 cells were imaged for each data point. Foci were enumerated using the FociCounter software . The results are presented as the means of three independent experiments ± standard error. Statistical significance was tested using the Student


Lin28A by inducing β-catenin to compete with LSD1 for binding to TCF7L2, causing LSD1 to dissociate from the Lin28A promoter and enhancing H3K4me2 methylation in this region. Lin28A promotes PGC formation by inhibiting gga-let7a-3p maturation to initiate Blimp1 expression. Interestingly, expression of Blimp1 helped sustain Wnt5A expression by preventing LSD1 binding to the Wnt5A promoter. We thus elucidated a positive feedback pathway involving Wnt–Lin28A–Blimp1–Wnt that ensures PGC formation. In summary, our data provide new insight into the development of PGCs in chickens.The unique developmental characteristics of chicken primordial germ cells (PGCs) enable them to be used in recovery of endangered bird species, gene editing and the generation of transgenic birds, but the limited number of PGCs greatly limits their application. Studies have shown that the formation of mammalian PGCs is induced by BMP4 signal, but the mechanism underlying chicken PGC formation has not been determined. Here, we confirmed that Wnt signaling activated via BMP4 activates transcription of  Summary: We describe a Wnt–Lin28A–Blimp1–Wnt positive feedback regulation system during the formation of primordial germ cells, which is regulated by upstream BMP4 signals and H3K4me2. Primordial germ cells (PGCs) are the progenitors of sperm and ovum, which are responsible for the transmission of genetic information between germ lines . ChickenBlimp1  and Tcfap2c, differentiate into PGCs, then migrate from the bottom of the allantoic sac to the endoderm and finally settle in the genital ridge . Within 1.5 h to 3 h of laying eggs, approximately ten PGC precursors already exist in the embryo tail of Drosophila, which is completely different from that of mammals  +Blimp1] formation from mouse precursor cells in the proximal epidermis mediated via BMP4 signaling. Inhibition of BMP4 signaling markedly decreases the number of PGCs in mouse gonads (Blimp1 via the mesoderm-specific transcription factor T (Blimp1) expression in early PGCs  of chicken embryonic stem cells (ESCs) and PGCs . FunctioFig. S1A,B and Table S1), 20 genes of which were significantly enriched in the Wnt signaling pathway (P=0.0023) (Table S2). Results from quantitative reverse transcription PCR (qRT-PCR) also confirmed that the key molecules of Wnt signaling were significantly activated in the process of chicken PGC formation   of ESCs and PGCs were identified by RNA-seq A,B. Gene. S1C,D) . These rWnt5A, β-catenin and Tcf7l2 are the key Wnt signaling molecules in the formation of chicken germ stem cells   following Wnt5A interference, and significant upregulation of β-catenin and Tcf7l2 expression (P<0.01) following Wnt5A overexpression  . To furtpression D. In viv results D. Co-immch other E. These th Wnt5A F. Thus, Lin28A was the only common gene in GO and Venn diagram analyses of 7473 target genes identified in these species , suggesting that Lin28A, a highly conserved gene targeted by Wnt signal, is involved in the generation of reproductive stem cells. These results are consistent with previous reports in mammals , and the binding site for TCF7L2 was also presented in the chicken Lin28A promoter , rat  and mouse  in the Gene Transcription Regulation Database (GTRD) (no bird database available) A. Lin28A mammals . Conservpromoter C. Such rLin28A was regulated by Wnt signaling, we examined the expression of Lin28A after activation/inhibition of Wnt signaling during PGC formation in vivo. Overexpression of Wnt5A significantly increased Lin28A expression, whereas inhibition of Wnt5A expression significantly inhibited Lin28A expression  using the dual luciferase detection system (Lin28A promoter (P<0.01) (Lin28A promoter (P<0.01)  coupled with quantitative PCR (ChIP-qPCR) and found enrichment of β-catenin–TCF7L2 complexes in the Lin28A promoter (Lin28A (P<0.01), whereas inhibition of Wnt signaling significantly reduced this binding (P<0.01)  F. Howeve(P<0.01) G,H. Actiutations H, indicapromoter I. Activa(P<0.01) I. These Lin28A in PGC formation . Morphologic observations on day 2 after BMP4 induction indicated that the cells had begun to grow larger. A few embryoid bodies (EBs) appeared on day 4, and the number of EBs increased on day 6; however, no EBs appeared between days 2 and 6 after Lin28A inhibition. In contrast, small EBs began to appear on day 2 after Lin28A overexpression, and on day 4 these EBs became larger and began to break. The number of EBs increased on day 6, the cell edges began to rupture, and a few cells were released from the EBs . Lin28A overexpression significantly decreased expression of the pluripotency marker gene Nanog and increased Cvh, C-kit and Blimp1 expression. Flow cytometry analyses demonstrated that Lin28A overexpression promoted PGC formation in the BMP4 model . Periodic acid–Schiff staining (PAS) was used to monitor changes in the number of PGCs formed in the genital ridge after Lin28A overexpression/interference. Compared with the number of PGCs in the genital ridge during the normal in vivo hatching process (38±1.53), the number of PGCs in the genital ridge significantly increased following Lin28A overexpression  and significantly decreased  following Lin28A interference . Collectively, these results indicate that Wnt–β-catenin signaling promotes PGC formation by activating Lin28A expression.Next, we investigated the function of ormation A. Lin28AP4 model C,F,G. SiLin28A was one of the DEGs . Furthermore, results from qRT-PCR showed that expression of Lin28A was significantly upregulated after interference with Lsd1 in SSCs . ChIP-qPCR results also showed that the H3K4me2 enrichment level in the Lin28A promoter region was regulated by LSD1 . These results suggest that Lin28A may be the target gene of H3K4me2. To examine the effect of H3K4me2 on Lin28A regulation during PGC formation in the present study, we interfered with Lsd1 and Mll2 expression in the BMP4 induction model in vitro  formation (data not shown), we performed RNA-seq on SSCs treated with LSD1 . It was found that in vitro . Lin28A ith Mll2 A, indica results B, suggesrference C. Furthepromoter A. CollecLin28A expression, we investigated the interaction between the Wnt signal and H3K4me2 in regulating the expression of Lin28A by the dual luciferase system. Interference with Mll2 expression suppressed the response of Lin28A to Wnt signaling, whereas interference with Lsd1 significantly enhanced the response . However, in cells co-transfected with LSD1-Flag and TCF7L2-Myc, interaction between Flag and TCF7L2 was observed . Considering the correlation between TCF7L2 and β-catenin . Collectively, these results indicated that β-catenin competes with LSD1 for binding to TCF7L2, which demethylates H3K4me2 in the Lin28A promoter via LSD1 and activates Lin28A expression during PGC formation.To further elucidate the molecular mechanism regulating response A. The poly fixed , we used-catenin , we hypolet7 microRNA (miRNA) maturation. However, the let7 miRNA that interacts with Lin28A during chicken PGC formation has yet to be identified. To determine the key let7 miRNAs targeted by Lin28A, 17 gga-let7 sequences in the chicken let7 miRNA family were screened using miRDB . As Blimp1 is a PGC marker, we performed the same experiment with PGCs and iPGCs and obtained similar results . Wild-type (WT) and mutant (complete deletion of binding site) luciferase reporter vectors of the Blimp1 3'UTR were constructed separately. DF1 cells were then co-transfected with vectors for the gga-let-7a-2-3p mimic and inhibitor with Blimp1-3'UTR-WT and Blimp1-3'UTR-Mut. The gga-let-7a-2-3p inhibitor significantly increased Blimp1-3'UTR-WT luciferase activity in the double luciferase reporter assay (P<0.01), but had no significant effect on Blimp1-3'UTR-Mut (P>0.05) (gga-let-7a-2-3p mimic significantly reduced Blimp1-3'UTR-WT luciferase activity (P<0.01), but had no significant effect on Blimp1-3'UTR-Mut (P>0.05) (Blimp1 is a direct target of gga-let-7a and that gga-let-7a binds to the 3'UTR of Blimp1 to inhibit its expression.Screening the miRDB identified 1143 genes targeted by ttention . To detehe mimic E and sig results E. To fur(P>0.05) F. The gg(P>0.05) F. The sa(P>0.05) G,H. ThesBlimp1 is known to affect the level of H3K4me2  , indicating that H3K4me2 regulates key Wnt signaling molecules. We then investigated whether Blimp1 regulates H3K4me2 in the promoters of Wnt5A, β-catenin and Tcf7l2. Notably, there is a Blimp1 binding site near the Wnt5A promoter H3K4me2 enrichment site  A, which ent site B. To conactivity C. Howeveing site D, indicaF1 cells E, as waspromoter F. Intereegulated G. These Lsd1 and Mll2 expression in the in vitro BMP4 induction model revealed that Lsd1 interference via Lsd1 shRNA (shLsd1) promoted PGC formation, whereas interference with Mll2 expression inhibited PGC formation . Expression of genes that activate Wnt signaling – such as Wnt5A, β-catenin, Fzd4 and Tcf7l2 – increased significantly after interference using shLsd1 (P<0.01), whereas expression of genes that suppress Wnt signaling, such as Axin1 and Apc, decreased significantly (P<0.01) . Completely opposite results were obtained after interference with Mll2 expression  and in vivo . Collectively, these data indicated that H3K4me2 regulates PGC formation by activating Wnt5A–β-catenin–TCF7L2 signaling.Morphologic observation after interference with Lin28A–Blimp1–Wnt functions as a positive-feedback loop during PGC formation, we sought to identify the factors that activate this feedback pathway. Previously, we confirmed that BMP4 plays an important role in PGC formation. We noticed that the addition of BMP4 protein for 6 h in ESC or PGC culture medium can significantly increase the expression of signal molecules such as Wnt5A, β-catenin and Tcf7l2 (P<0.01)  A, and si and Apc A, which ficantly B, indicaficantly C,D. Flowficantly E. TherefLin28A promoter, leading to increased H3K4me2 levels and expression of Lin28A. Lin28A then activates the expression of Blimp1 by inhibiting the maturation of gga-let-7a-2-3p, thus regulating PGC formation. Notably, Blimp1 activates Wnt to initiate the Wnt–Lin28A–Blimp1–Wnt positive-feedback pathway .The present study established a new regulatory model for PGC formation. After activation of Wnt5A–β-catenin–TCF7L2 signaling, β-catenin competes with LSD1 to bind to TCF7L2 in the Lin28A expression via Wnt signaling through TCF7L2 and indirect regulation of Blimp1 expression. The entire process is also regulated by H3K4me2, indicating that although Wnt signaling plays the same biological role in the formation of mammalian and chicken PGCs, its regulation differs by species.Wnt signaling plays similar roles in the formation of mammalian and avian PGCs, but the regulatory mechanisms differ markedly . The Wntc-myc and Jun ], anti-histoneH3 , goat anti-mouse IgG (ab6786), goat anti-rabbit IgG (ab6718), and rabbit anti-rat IgG (ab6730) were obtained from Abcam. Anti-Myc  and anti-β-catenin  were obtained from Cell Signaling Technology. Anti-CVH  was obtained from Abcam.Table S3). The qPCR reaction system  was as follows: 2 μl cDNA, 10 μl TB Green Premix Ex TaqII, 0.8 μl each of upstream and downstream primers (10 μM) and 6.4 μl ddH2O. PCR reaction procedures were carried out according to the instructions provided with the Takara TBGreen™ PremixExTaq™ II.Total RNA was extracted from cells using Trizol reagent  and reverse transcribed to synthesize cDNA using a Quantscript RT kit (Tiangen). Expression of Wnt-associated signaling molecules was assessed using β-actin as an internal control (5/well chick embryo fibroblasts (CEFs)/ESCs/PGCs were seeded into a 24-well plate. CEFs/ESCs/PGCs were seeded in 24-well plates (Corning) at 1×105 cells/well. When cell density became 70%, cells were transfected with FuGENE® HD (Promega) at a 3:1 ratio of reagent  to plasmid . It should be noted that in ESCs and PGCs, transfection should be conducted continuously three times .For the overexpression vector (carrier framework pCDNA3.0), 1.5×105/well CEFs/ESCs/PGCs were seeded into a 24-well plate. When cell density became 70%, the lentiviral vector was used to infect CEFs/ESCs/PGCs under 10 multiplicity of infection and 5 ng/ml polybrene .For interference vectors , 1×102 humidified atmosphere at 37.0°C with culture medium.Chicken ESCs are derived from intracellular masses at the blastocyst stage. Fresh fertilized eggs were disinfected by 4% benzalkonium bromide and 75% alcohol. The blunt end of an egg was broken by tweezers and the egg white was removed, then blastoderm cells at stage X were collected by the spoon method in tissue culture dishes and rinsed in phosphate-buffered saline (PBS) to remove the yolks and vitelline membrane. After washing with PBS, ESCs were maintained in a 5% COg for 6 min, the supernatant discarded and the cells collected. Cell precipitation was resuspended in PGC medium. KnockOut Dulbecco's modified Eagle medium  containing 10% fetal bovine serum, 2.5% chicken serum , 100 U/ml penicillin, 100 U/ml streptolycin, 0.4μmol/l non-essential amino acid , 2 mmol/l glutamine , 0.1 mmol/l β-mercaptoethanol , 10 ng/μl mouse leukemia inhibitory factor , 10 ng/μl basic fibroblast growth factor  and 5 ng/ml human stem cell factor  was used to culture ESCs and PGCs.Fertilized eggs at 4.5 days were sterilized with 4% benzalkonium bromide and 75% alcohol to obtain chicken embryos to separate the genital bridge. After the genital bridge was cut and digested by trypsin for 3 min, the cell suspension was obtained and filtered with 400 mesh filter cloth. The cell suspension was then centrifuged at 600 2O2 for 10 min. Mayer staining was then applied for 30 s, before differentiation with hydrochloric acid and alcohol for 3 s, immersion in running water for 15 min, and treatment with acetic acid for 2 min, ethanol for 2 min and xylene for 5 min. Sections were covered with neutral gum and observed under a microscope (Nikon).Embryos were collected from hatched eggs at 2.5 days (HH stage 13–17), into which a round hole with a diameter not exceeding 0.5 cm had been made using tweezers at the blunt end. The chicken embryo was exposed, and a micro-pipettor was used to inject the processed PGCs or the encased transfection vector into the embryonic blood vessel. The embryo was then cross-sealed with medical tape for further incubation. Embryos were collected at 4.5 days to prepare paraffin sections according to a previously reported procedure . The parAll procedures involving the care and use of animals conformed to US National Institute of Health guidelines  and were approved by the Laboratory Animal Management and Experimental Animal Ethics Committee of Yangzhou University.Tcf7l2-Myc vector; and one group was transfected with the oeβ-catenin and oeTcf7l2-Myc vectors. After culture at 37°C in 5% CO2 and saturation humidity for 48 h, the cells were collected for Co-IP experiments, as previously described (DF1 cells (CEFs) and PGCs with good status were selected and divided into three groups for transfection. Cells were maintained in complete medium. One group was transfected with the overexpression (oe) β-catenin vector; one group was transfected with the oeescribed .Lsd1, one group was transfected with shMll2, and one group was left untreated. After incubation for 48 h at 37°C in 5% CO2 and saturation humidity, ChIP-qPCR was performed as follows: cell crosslinking and fragmentation, immunoprecipitation of crosslinked proteins/DNA, elution of protein/DNA complexes, and purification and recovery of DNA using centrifugal columns for ChIP-qPCR (Table S4).ESCs and PGCs with good status were selected and divided into three groups for transfection. Cells were transfected in factor medium. One group was transfected with shhttp://gtrd.biouml.org/bioumlweb/#). GO functional annotation of the predicted target genes was carried out using DAVID (https://david.ncifcrf.gov/) and KOBAS (http://kobas.cbi.pku.edu.cn/kobas3/?t=1) to identify candidate genes related to germ cell development and stem cell differentiation.Binding target genes of the Wnt signaling transcription factor TCF7L2 in three different species  were predicted using online software  and the University of California Santa Cruz (http://genome.ucsc.edu/). The promoter region and transcription start site were identified based on core promoter elements . Primers were designed with the transcription start site designated as +1 for amplification of fragments. The plasmid pLin28A-EGFP was constructed and then used to transfect DF1 cells for 24–48 h until green fluorescence was observed under a fluorescence inverted microscope. The presence/absence of green fluorescence was used to confirm that the constructed promoter fragment exhibited promoter activity. The PGL3-basic vector of different deletion fragments of the Lin28A promoter was constructed for the dual luciferase reporter gene detection system, which was used to detect promoter activity of the Lin28A target gene. The protocol was as follows: recombinant plasmids encoding different promoter fragments were co-transfected into DF1 cells with pRL-SV40 at a mass ratio of 30:1. A negative control was simultaneously prepared (co-transfection of pGL3-basic and pRL-SV40 plasmids into DF1 cells at a mass ratio of 30:1). Detailed transfection methods are available from the FuGENE product manual. Three wells of cells were transfected for each group, and transfection was repeated three times. At 48 h after initial transfection, the cells were collected and 70 μl cell lysate was added to each tube and mixed gently. Next, 70 μl fluorescent solution was added to each well and gently mixed. Renilla fluorescence was measured using a fluorescent plate reader after addition of 70 μl STOP terminating reagent followed by gentle blowing, and mixing.The 2000-bp genome sequence upstream and downstream of the coding sequence was identified based on the Myc-Tcf7l2, Lin28A promoter deletion, and the corresponding TCF7L2 binding-site mutation vector (pGL3.0-Basic+pcDNA3.1 served as the negative control). The change in promoter activity was assessed using the double luciferase reporter system, and the relative fluorescence activity is reported as the mean±s.e. of three experiments. ESCs and PGCs with good growth conditions were selected and divided into three groups each for transfection. Cells were transfected in factor medium. One group was transfected with shβ-catenin, one group was transfected with oeβ-catenin, and one group was left untreated. After incubation at 37°C and 5% CO2 for 48 h, ChIP-qPCR was performed.DF1 cells were transfected with the following vectors pcDNA3.1-β-catenin, pcDNA3.1-Lin28A overexpression and interference expression vectors and then either induced with BMP4 or injected into the blood vessels of chicken embryos. Cell samples were collected at 0, 2, 4 and 6 days after in vitro induction, and tissue samples were collected at 0 and 4.5 days during in vivo incubation. Total RNA was extracted using Trizol reagent, and cDNA was synthesized by reverse transcription. Expression of the PGC marker genes Cvh, C-kit and Blimp1, and the totipotent marker genes Nanog and Oct4, was analyzed using β-actin as an internal reference. qRT-PCR was conducted as previously described. The efficiency of PGC formation was assessed by flow cytometry and analysis of paraffin-embedded tissue sections.ESCs were transfected with Lsd1, β-catenin or Tcf7l2 vector at 37°C and 5% CO2 and saturation humidity for 48 h. Cells of each group were collected, and a lysate was prepared and subjected to immunoprecipitation (IP), after which the protein concentration was determined. Subsequent IP experiments were performed using an equal volume of lysate. Binding of LSD1 to β-catenin or TCF7L2 was confirmed by monitoring the expression of target protein (co-immunoprecipitated protein) by WB.DF-1 cells and PGCs with good status were selected, divided into six groups, and co-transfected with Lsd1-Flag and Tcf7l2-Myc. One group was co-transfected with β-catenin vector, and the other was co-transfected with pcDNA3.1 as a control. The cells were incubated at 37°C, 5% CO2 and saturation humidity for 48 h. Cells of each group were collected, and a lysate was prepared and subjected to IP, after which the protein concentration was determined. Subsequent IP experiments were carried out using an equal mass and volume of lysate, and expression of the co-precipitated target protein was evaluated by WB.DF-1 cells and PGCs with good status were selected and divided into two groups on the basis of co-transfection with http://mirdb.org/cgi-bin/search.cgi) was used to predict chicken miRNA gga-let7 sequences. DF1 cells, ESCs and PGCs with good status were selected and divided into three groups. One group was transfected with Lin28A siRNA (siLin28A), one group was transfected with oeLin28A, and one group was left untreated. DF1 cells were transfected in complete medium, and ESCs and PGCs were transfected in factor medium. After 48 h of incubation at 37°C and 5% CO2 and saturation humidity, total RNA was extracted using an miRNA extraction and isolation kit, and cDNA was synthesized by reverse transcription according to the reverse transcription kit procedure. U6 was used as an internal reference to detect changes in the relative expression of let7 miRNAs. The let7 miRNA quantitative primer sequences are shown in Table S5. qRT-PCR was carried out according to the instructions of the miRNA fluorescence quantitative detection kit. The reaction mixture was as follows: 50 ng cDNA, 10 μl 2× miRcute Plus miRNA PreMix, 0.4 μl forward primer, 0.4 μl reverse primer and ddH2O to a total volume of 20 μl. The PCR conditions were as follows: pre-denaturation at 95°C for 15 min, 94°C for 20 s, 63°C for 30 s, 72°C for 34 s (five cycles); 94°C for 20 s, 60°C for 34 s annealing/extension (40 cycles); standard dissolution curve analysis.Online software (http://mirdb.org/cgi-bin/search.cgi) was used to predict target genes related to reproductive differentiation. The targeting effect of gga-let-7a-2-3p on Blimp1 was assessed using the dual luciferase reporter gene detection system. DF1 cells were co-transfected with Blimp1-3'UTR-WT or Blimp1-3'UTR-Mut and pRL-TK at a mass to volume ratio of 10:1. Based on this, gga-let-7a-2-3p mimics or inhibitors were added, and negative controls were set up at the same time. The protocol was as follows: 2×105 DF-1 cells, PGCs and iPGCs/well were inoculated into a 24-well plate 1 day in advance of the experiment and cultured without antibiotics. When cells reached 50–60% confluence, mimic (or inhibitor) was added and diluted with 50 μl Opti-MEM to a final concentration of 50 μM. Blimp1-3'UTR-WT  and pRL-TK were gently mixed as solution A, and 4 μl FuGENE HD was diluted with 50 μl Opti-MEM and gently mixed for 5 min at room temperature to serve as solution B. After mixing solutions A and B, the mixture was gently blown three to five times and left at room temperature for 20 min, before incubation at 37°C for 10–15 min. The mixture was then slowly added to the cell culture hole, mixed with 400 μl complete medium, and incubated at 37°C and 5% CO2. Three wells were transfected at a time, each transfection was repeated three times, and the cells were collected 48 h after transfection. Next, each tube was supplemented with 70 μl cell lysate and gently mixed, after which the same volume of fluorescent solution was added to each well. Renilla fluorescence was measured using a fluorescent plate reader after addition of 70 μl STOP terminating reagent followed by gentle blowing, and mixing. Values are reported as the mean±s.e. of three repeat tests. The assay was conducted according to the instruction manual of the dual luciferase reporter gene detection kit (Promega).Online software (−ΔΔCt method. The significance of between-group differences was assessed using two-sample Student's t-tests (paired) with SPSS software, version 18.0. Data are presented as the mean±s.d. unless otherwise indicated. Significance was set at P<0.05.Hierarchical clustering of differential gene expression  was performed using Heml. Relative gene expression was calculated from PCR data using the 2


Therefore, K1.5Ba0.25CsC60with cation-vacancy sites is synthesized using a precursor method to avoid inadequate stoichiometry control and formation of impurity phases within the target composition. For this purpose, first, phase-pure K6C60, Ba6C60and Cs6C60precursors are synthesized. Stoichiometric quantities of these precursors are used for further reaction with C60to afford K1.5Ba0.25CsC60. Rietveld analysis of the high-resolution synchrotron X-ray powder diffraction data of the precursors and K1.5Ba0.25CsC60confirms that K6C60, Ba6C60and Cs6C60are single-phase and they crystallize in a body-centered-cubic structure (Im3) as reported in the literature. The analysis also shows that K1.5Ba0.25CsC60phase can be perfectly modeled using a face-centered cubic structure. No new peaks appear which could have implied the appearance of a superstructure. This suggests that there is no long-range ordered arrangement of vacant tetrahedral sites in K1.5Ba0.25CsC60.The existence of cation-vacancy sites in fullerides might lead to long-range ordering and generate a new vacancy-ordered superstructure. The purpose of this work is to search whether or not long-range ordering of vacant tetrahedral sites, namely superstructure emerges in nonstoichiometric K Synthetic efforts have been essentially focused on the synthesis of alkali fullerides with stoichiometries AxC60, due to their novel electronic properties. For instance, the observation of metallic behavior in alkali metal intercalated C60films at 300 K [2] was followed by the discovery of superconductivity for the first time in an alkali fulleride K3C60with a transition temperature,Tc, of 18 K [3], where superconducting phase is a face-centered-cubic (fcc) structure . This led to the discovery of new A3C60superconductors through varying the interfullerene separation  withTc varying between 25.9 K and 32.9 K at ambient pressure [9]). Since then, extensive research has been carried out on fcc A3C60and AxCs3−xC60fullerides to discover superconductors with higherTc and understand their molecular electronic structure [8–12]. Current understanding proves that the A3C60superconducting fullerides belong to the family of unconventional superconductivity where electron correlations play an important role for the pairing mechanism . Besides that, molecular electronic structure  and cation specific effects [12] are crucially important in producing the unconventional superconductivity in the A3C60family.The intercalated products of fullerides display unique structural, magnetic, and electronic properties which depend on the amount, size, and nature of the intercalated species, and the synthetic route employed for the intercalation of dopant into solid C60, the dopant occupies interstitial lattice positions of the solid C60and provides electrons to the host C60molecules, creating C60n- anions [1]. The charge transfer alters the properties of C60 and has thus received the most attention. In the A3C60family, intercalation of three alkali leads to a half-filledt1u band and hence a metallic behavior, excluding fcc Cs3C60[8]. The superconducting phase of the A3C60fullerides adopts the fcc structure where all tetrahedral (Td) and octahedral (Oh) holes are entirely occupied by the alkali cations [21]. In fcc A3C60structure, there are twoTd cavities (r= 1.12 Å) and oneOh (r= 2.06 Å) per C60unit. As theTd cavity is smaller than theOh one, the size of the alkali cation occupying theTd site instead of theOh one will determine the degree of lattice expansion; therefore, the volume of the A3C60fullerides can be changed systematically through substituting the alkali metals with larger ones.Upon exohedral doping of solid CxC60fullerides. The most common and effective one is the ‘solid-state direct reaction method’ where solid C60is exposed to vapor of the alkali metal which then disperses into the C60acceptor molecules at temperatures of ~100–410 °C. All the AxC60and AxA’3−xC60compounds, except Cs3C60, can be synthesized with this method [1]. The A6C60fullerides can be synthesized using the direct reaction method and used later as precursors for further reaction with C60to obtain target AxCs3−xC60compounds. The precursor method offers a significant benefit compared to the direct synthesis method because fine A6C60powders enable better stoichiometry control of the desired compound. The saturated A6C60compounds can also be prepared by a vapor-transport method [22]. In this study, direct reaction and vapor-transport methods were used to synthesize the K6C60/Ba6C60and Cs6C60precursors, respectively, which were later reacted with C60to afford the target composition.Various synthetic routes have been developed to prepare A60and Yb2.75C60exhibit long-range ordering of tetrahedral vacancies, generating a superstructure . Such structural response to tetrahedral vacancy could be established in fcc AxCs3−xC60fullerides, resulting in a different structure to the fcc. For such an exploration, in this work, 0.25K1.5Ba0.25CsC60—where  represents vacant tetrahedral sites of the fcc structure — was synthesized using stoichiometric quantities of single phase, fine, black colored K6C60, Ba6C60, and Cs6C60powders together with C60as starting materials. Here, an easy method for the synthesis of nonstoichiometric K1.5Ba0.25CsC60is presented together with the results of Rietveld analysis of high-resolution synchrotron X-ray powder diffraction data collected at ambient conditions for the structural investigation.Besides the exploration of cation related effects on the structural and electronic properties of fullerides, the effects of cation-vacancy can be investigated as well. For example, tetrahedral rare-earth metal vacancies in Sm2.75C60 was sublimed prior to synthesis. Solid C60(~500–600 mg) were first ground and then loaded in a quartz ampoule with a separating striction. This was followed by degassing for 3–4 h in a dynamic vacuum of 10−4–10−5 mbar. The sublimation process was undertaken in a tube furnace at 550 °C (ramping 10 °C/min) under dynamic vacuum. As soon as the sublimation ended, the sublimed C60was transferred to the glove box for later use. A Swagelok fitting with J. Young tap  was always used while the samples within the tubes were removed or introduced from or into the glove box. Each cycle of annealing of the intermediate and final products was ended through allowing the furnace to cool down to room temperature with a rate of 5 oC/min. Once each annealing cycle was complete, these products were ground using a pestle and mortar to improve crystallinity.All sample operations were performed in an argon-filled glove box  due to the extreme air- and moisture-sensitivity of reactants. As purchased pristine CSynthesis of K6C6060was introduced onto K in the cell. The cell was then located in a glass tube enclosed with a Swagelok fitting, removed from the glove box, evacuated for 30 min before sealing under 350 mbar of He gas pressure. The sealed sample within the glass tube was placed vertically in a box furnace and heated to 250 °C with a rate of 5 °C/min, followed by another 20 h annealing and cooling to ambient temperature with the same rate as heating. After the initial annealing was completed, the product was transferred to the glove box, removed from the reaction vessel, and ground. The ground product was then pressed into a pellet, introduced into a Ta holder for further period of 2 days annealing at 300 °C to increase the crystallinity.Stoichiometric amount of K metal was placed in a 5 mm diameter Ta cell and then stoichiometric amount of sublimed CSynthesis of Cs6C606C60was synthesized by a vapor transport method using a ~2.3× excess amount of Cs. The solid C60was first placed into a glass tube with a striction. The cesium metal was inserted in a small glass capsule (~7 mm diameter) which was lowered in the glass tube down to where the striction was. The tube enclosed with a Swagelok fitting was removed from the glove box and evacuated for 30 min before sealing under ~400 mbar of He gas pressure. A 3-zone horizontal tube furnace was used in order to create a temperature gradient between Cs and C60for vapor transport. The zone where Cs locates was ramped to 350 °C from room temperature with a rate of 2 °C/min and held there for 3 days. Simultaneously, the zone where C60locates was ramped to 330 °C (2 °C/min) for transportation of vapor from the Cs to C60zone. Before the product was cooled down to ambient temperature, the temperature gradient was reversed to get rid of any undoped Cs from the freshly formed Cs6C60. Once the cooling was complete, the product was removed from the glass tube, ground thoroughly, pelletized, and located in a Ta cell with tightened screw ends which was then sealed under He, after evacuation under dynamic vacuum, and annealed at 350 °C for another 3 days to increase the crystallinity.Phase-pure CsSynthesis of Ba6C6060were mixed and ground using a mortar and pestle, and then pressed into a pellet. The reaction mixture was loaded into a Ta holder which was placed a 15 mm diameter quartz tube then evacuated for 30 min before sealing under 500 mbar of He gas pressure. The sealed mixture was heated inside a muffle furnace using the following thermal protocol: from ambient temperature to 550 °C; held for 1 h; to 650 °C; held for 13 h; to 700 °C; held for 4 h; to 720 °C; held for 18 h. The ramping rate between each step of heating was 5 oC/min. After cooling to room temperature, the product was removed from the reaction vessel, ground, and finally pressed into a pellet and inserted into the same Ta holder for reannealing at 740 °C for 16 h.Prior to synthesis, in order to obtain oxide-free barium powder, the oxidized surface of a barium rod was first removed by filing. These initial filings were discarded, and a new diamond file was used to exfoliate the required mass of clean fine powder. Once the clean fine powder was obtained, stoichiometric amount of barium powder and CSynthesis of the target compoundK1.5Ba0.25CsC601.5Ba0.25CsC60was prepared by a solid state synthetic route according to the following stoichiometric equation describing the ideal reaction: 6K6C60+ 4Cs6C60+ Ba6C60+ 13C60→ 24K1.5Ba0.25CsC60. Stoichiometric amounts of K6C60, Ba6C60, Cs6C60, and sublimed C60precursors were mixed and ground thoroughly. The ground mixture was pelletized, introduced into a Ta cell with tightened screw ends which was then placed in a quartz tube, and evacuated for 30 min before sealing under 450 mbar of He gas pressure. The sealed pellet was then positioned in a muffle furnace at room temperature and heated at 600 °C for 16 h with an initial ramp rate of 5 °C / min, finally, the furnace was switched off to cool down to room temperature. The intermediate product was removed from the tube in the glove box, ground, and then pressed into a pellet which was introduced into the same Ta holder for further period of annealing at 300 °C for 1 h and 600 °C for 16 h with the same heating rate with 2 intermediate grindings and pelletizations to increase crystallinity.K6C60and Cs6C60, and λ = 0.399838 Å for Ba6C60) at the ESRF, Grenoble, France. SXRPD data of the target compound K1.5Ba0.25CsC60were collected on beamline BL44B2 (λ = 0.500127 Å) at the SPring-8, Japan. The samples were loaded into 0.5 mm diameter glass capillaries and sealed under ~350 mbar He pressure for the SXRPD measurements.Ambient temperature high-resolution synchrotron x-ray powder diffraction (SXRPD) data of the precursors were collected with the diffractometer on beamline ID31  were refined. Low-angle peak asymmetry rising from axial divergence was modeled with coefficientsS/L= 0.001,H/L= 0.0005 whereLis the diffractometer radius, andHandSare the sample and detectors heights, respectively [25]; a Chebyschev polynomial function (~20 terms) was applied to fit the background; the anomalous contributions to the X-ray form factors of all atoms, f’ and f’’ corrections to f, were calculated (in e/atom) using the program DISPANO [27] and implemented into GSAS as follows: for  λ = 0.4 Åf’ = 0.054, f’’ = 0.079 for K, f’ = −1.771, f’’ = 0.819 for Ba and f’ = −1.921, f’’ = 0.758 for Cs and for λ = 0.5 Å f’ = 0.094, f’’ = 0.125 for K, f’ = −1.190, f’’ = 1.228 for Ba and f’ = −1.247, f’’ = 1.138 for Cs. Intermediate refinements of lattice parameters, occupancies of the tetrahedral sites, thermal parameters, peak shape coefficients, zero correction, and background function were applied during the Rietveld analysis.SXRPD data were analyzed using the Rietveld refinement technique with the GSAS suite of the Rietveld programs [25]. The following procedure was applied for the Rietveld analysis: a complex peak shape function known as the pseudo-Voigt, which is a combination by addition of Gaussian and Lorentzian functions [26] was used to model the peak shape, and peak shape coefficients6C60, Cs6C60, and Ba6C60confirm that samples are high quality, phase-pure, and crystallize with a body-centered-cubic structure with a space group ofIm3‒and lattice parametersaK6C60 = 11.3775(2) Å,aCs6C60 = 11.7887(2) Å, andaBa6C60 = 11.1879(2) Å respectively. These values are in agreement with the previously-reported lattice parameters: 11.39 Å [28], 11.79 Å [29], and 11.1850(7) Å[30], respectively. This confirms that the synthesized A6C60fullerides can be used effectively as precursors. Fractional atomic coordinates of K6C60, Cs6C60, and Ba6C60were taken from [28], [29], and [31], respectively and were not refined. Only lattice constants and thermal displacement parameters, which were modeled isotopically, were refined together with instrumental (e.g.zero shift) and profile shape coefficients. As seen in Figure 1, a good agreement between the calculated and observed profile is obtained from the Rietveld analysis with χ2 ~1.Rietveld refinements of the SXRPD data  collected for K1.5Ba0.25CsC60readily reveals that the sample is single phase and adopts the cubic structure with fcc symmetry . All diffraction peaks can be indexed with the cubic structure, with no sign of additional reﬂections that could have indicated the existence of a vacancy-ordered superstructure. A merohedrally disordered fcc model with the space group ofFm3‒mwas employed to model the fcc phase. A cation disordered model was applied as follows. As theOh interstitial site of the fcc C60structure (r = 2.06 Å) is significantly larger than theTd one (r = 1.12 Å), larger Cs+ ions preferentially reside in theOh site. As a result, theOh cavity is only occupied by Cs+ while the smaller K+and Ba2+ions  preferentially occupy theTd site. Hence, the latter is filled by a disordered mixture of K+and Ba2+. The applicability of this method has been previously confirmed by 133Cs, 39K, and 87Rb NMR measurements .Rietveld analysis of the X-ray diffraction data of K3C60(with C60C‑C bond distances of 1.42 Å [28]). Thermal displacement parameters of the atoms (U) were modeled isotropically and allowed to refine but under the condition thatUiso of the C atoms andUiso of the K+and Ba2+ions introduced into the tetrahedral site were forced to be equivalent to each other, respectively. The K+and Ba2+occupancy in theTd site of the fcc structure was allowed to refine but total site occupancy was fixed at 1.75, and the remaining is the vacant tetrahedral site occupancy which is 0.25. The refined occupancy ratio converged to K+: Ba2+ = 0.712(10):0.163(10) leading to a refined composition of K1.42(1)Ba0.33(1)CsC60. The structural parameters of the fcc phase obtained from the Rietveld refinement are summarized in Table.The fractional atomic coordinates of the fcc phase were not allowed to refine, instead they were rescaled from fcc Rb2+for the K+cation, and the presence of theTd vacancy in fcc KxCs3−xC60led to a significant lattice contraction. The fcc lattice parameter of refined composition K1.42(1)Ba0.33(1)CsC60,a,is obtained as 14.2616(1) Å, which is smaller than those of any KxCs3−xC60ternary compositions , covering the compositional range 0.22(1) ≤ x ≤ 2 and lattice constants14.28571(7) Å ≤a≤ 14.7011(2) Å [32]. This reflects the fact that the average ionic radii of the cations residing in theTd andOh cavities,‹rA› in K1.42(1)Ba0.33(1)CsC60is smaller,‹rA›= 1.36(1) Å, than that of any KxCs3−xC60compositions (1.38 Å ≤ ‹rA› ≤ 1.64(1) Å). Refined isotropic thermal displacement parameters of the cations occupying theTd andOh cavities, and also of the C atoms are found to be in good agreement with those known from the literature[12]. Because of the smaller size of theTd site compared with theOh one, thermal displacements of the atoms residing in the former one is expected to be relatively smaller than the ones in the latter.Indeed, substitution of smaller Ba3C60fulleride family possesses remarkable physical properties  originating from their molecular electronic structure which can be easily tuned via physical/chemical pressure and temperature without altering their high fcc symmetry. The donor intercalants in molecular fulleride family possessing unique properties are not only limited to alkali and alkaline earth metals, for example, rare-earth doped Yb2.75C60fulleride becomes superconducting below 6 K and shows an exceptional crystal structure contrary to the literature [24]. In a hypothetical Yb3C60, Yb cations reside at the centers of theOh andTd sites of the fcc C60lattice. However, in Yb2.75C60,Yb cations occupy off-centered interstitial sites and leave one out of every eightTd sites vacant. These vacancy sites display long-range ordering, generating a new cation-vacancy-ordered superstructure, which leads to a unit cell with dimensions twice as large as those of the common fcc fulleride structures [24]. A similar situation is also encountered in Sm2.75C60[23]. In both cases, their complex structure arises from the long-range ordering of tetrahedral rare-earth metal vacancies. In this study, we also aimed to induce such structural response to the presence of vacancy site in K1.42(1)Ba0.33(1)CsC60, however, this could not be achieved. Inspection of the diffraction profile did not reveal any superlattice peaks at low angles that could be indexed to an enlarged unit cell, which may possibly signify the generation of a superstructure as in Sm2.75C60and Yb2.75C60. The underlying physical origin of this type of vacancy-ordering was attributed to a strong directional interaction between electron-poor, charge-deficient five-membered rings of C60and divalent ytterbium cations but not to the cation size difference [24]. However, this suggestion might not be valid in the case of using larger cations as in the present study, i.e. K+(1.38Å), and Ba2+(1.35 Å), than that of the tetrahedral hole (1.12Å) but the ionic radius of Yb2+and Sm2+are 1.02 and 1.14 Å, respectively, comparable to that of the tetrahedral hole. As it is well known, the size and amount of the dopant species has a primary effect on the crystal structure of fullerides, for instance, contrary to the literature, at low temperatures, the structure of Na2CsC60(rNa+= 1.02 Å) is primitive cubic (Pa3‒) being isostructural with pristine C60and undergoes a phase transition on heating to an fcc phase with a space group ofFm3‒m[33], and the structure of Yb2.75C60is orthorhombic with space groupPcab[24]. Therefore, it could be tentatively suggested that the size of the cations residing in the tetrahedral site should be taken into account if one aims to generate a long-range arrangement of vacant sites, namely a superstructure.The molecular, alkali doped A1.5Ba0.25CsC60fulleride using stoichiometric quantities of K6C60, Cs6C60, and Ba6C60precursors, which overcomes inadequate stoichiometry control, via solid-state synthetic route is presented. Structural characterization of the precursors and the target compound was performed with Rietveld analysis of the high-resolution synchrotron X-ray powder diffraction data. The analysis confirmed that the structure of the precursors is body-centered-cubic (Im3‒) as reported in the literature and free from impurity phases such as oxides of the metals which can be easily formed during the synthetic protocol applied.In conclusion, the preparation of the nominal K1.5Ba0.25CsC60(refined composition: K1.42(1)Ba0.33(1)CsC60) has shown no evidence for the emergence of superstructure peaks which could have resulted from the long-range ordering of the vacant tetrahedral sites. All the Bragg reflections existing in the diffraction pattern originate from the cubic structure (Fm3‒m) without any violation. This suggests that there is no long-range ordered arrangement of tetrahedral alkaline-earth metal vacancies in K1.42(1)Ba0.33(1)CsC60. The reason for this could be the use of highly symmetric Cs3C60fulleride as a parent phase and of moderately large dopant species, i.e. K+, Ba2+. This issue merits more detailed investigation through the synthesis and characterization of vacancy-doped fullerides with lower symmetry, for instance, primitive cubic Na2CsC60could be gradually substituted nonstoichiometrically by a smaller divalent cation which might generate ordering of cation-vacancy and also noninteger valence of C60.The X-ray diffraction pattern of the target compound K


Tannylated-CaO2 (TA/CaO2) nanoparticles were prepared by reacting calcium chloride (CaCl2) with hydrogen peroxide (H2O2) in ethanol containing ammonia and different amounts of TA . The prepared TA/CaO2 aggregates consisted of nanoparticles 25–31 nm in size. The nanoparticles prepared using 10 mg of TA in the precursor solution exhibited the highest efficiency for oxygen generation. Moreover, the oxygen generation from TA (10 mg)/CaO2 nanoparticles was higher in an acidic environment.This study reports a new approach for the facile fabrication of calcium peroxide (CaO The delivery of sufficient amounts of oxygen to transplanted cells and 3D-engineered tissues remains among the main challenges in the field of cellular engineering . Oxygen 2), magnesium peroxide (MgO2), and sodium percarbonate (Na2CO3·1.5H2O2) [2 is preferred, owing to its higher oxygen-generation capacity [Recent studies have focused on designing materials to supply oxygen to hypoxic regions ,5,6. Amoe Na2CO3·.5H2O2 [1capacity .2 particles by reacting Ca(OH)2 with H2O2 in an aqueous solution [4 with H2O2 in aqueous KOH [2 particles formed in aqueous solutions tend to aggregate because of hydrolysis. To overcome this, polyethylene glycol has been used as a surface modifier to stabilize the CaO2 nanoparticles [2 nanoparticles using polyvinyl pyrrolidine as a colloidal stabilizer [Several methods have been reported for the preparation of CaOsolution  or reacteous KOH . Howeverarticles ,11. Howeabilizer .2 nanoparticles using TA as a coordinate bridge between calcium ions. Additionally, the oxygen-releasing capability of the prepared CaO2 nanoparticles is examined.Tannic acid (TA), a polyphenolic biomolecule, is a versatile surface modifier due to the presence of phenolic hydroxyl groups, aromatic rings, and galloyl groups ,14. In a2·2H2O, abbreviated as CaCl2 hereafter), TA, ammonia solution , hydrogen peroxide solution , and potassium bromide (KBr) were purchased from Sigma-Aldrich . Phosphate-buffered saline (PBS) was obtained from Lonza . Absolute ethanol , methanol , and isopropanol were purchased from DUKSAN .Calcium chloride dihydrate (CaCl2 (100 mg) and TA  were dissolved in 15 mL of EtOH or MeOH using a sonicator . Under constant stirring at 600 rpm, 0.8 mL of NH4OH (1 M) was added to this mixture and reacted for 2 min. Following this, 0.16 mL of H2O2 (35 wt%) was added dropwise at a rate of 0.05 mL/min using a syringe pump . After sonication for 10 min, the mixtures were left to react overnight. The TA /CaO2 nanoparticles were subsequently obtained by centrifugation of the mixtures at 28,620 g for 10 min. All these synthetic procedures were performed at room temperature. The powders were washed with EtOH thrice and dispersed in 16 mL of EtOH for storage.CaCl2, a rotary evaporator  was used to completely remove EtOH from the samples. The TA/CaO2 powder thus obtained was characterized by Fourier transform infrared  spectroscopy. The FT-IR spectrum was acquired using the KBr pellet method at a resolution of 4 cm–1 between 4000 and 400 cm–1.To determine the mean yield of TA/CaO2 sample was removed using a rotary evaporation at 30 °C for 1 h, then further dried in a drying oven at 60 °C for 1 day to remove EtOH. After this, the dried TA/CaO2 powders were coated with platinum, then their morphologies, sizes, and elemental compositions were investigated by field-emission SEM . The average size of the randomly selected individual nanoparticles (n > 60) in each TA/CaO2 sample were analyzed using Image J software . The elemental compositions of the samples were determined by energy-dispersive X-ray spectroscopy (EDS). The thermal degradation of the samples was evaluated by thermal gravimetric analysis (TGA) . The samples were heated from 40 to 700 °C at a rate of 10 °C/min.The particle size, polydispersity index, and Z-average size of each sample (2 mL) dispersed in EtOH were measured at a scattering angle of 90° with a particle size analyzer . Frequency (%) of particle size data means the amount of each size by volume. For scanning electron microscopy (SEM) analysis, EtOH of each TA/CaO2 sample to 2 mL of each of the PBS solutions (pH 6.0 and 7.4) at room temperature. The O2 bubbles thus generated were monitored for 10 min using a smartphone camera .We added 300 μL of TA (10 mg)/CaO2 to 20 mL N2-purged PBS solutions (pH 6.0 and 7.4).The concentration of dissolved oxygen was recorded for 10 min using an Oakton DO 6+ dissolved oxygen meter  after adding 3 mL of TA (10 mg)/CaO2 against macrophages  was investigated with a cell-counting kit-8 . Cells were cultured and maintained in Dulbecco’s modified Eagle’s medium  containing 10% fetal bovine serum and 1% penicillin-streptomycin using a humidified 5% CO2 incubator. For the cytotoxicity test, macrophages (1 × 104 cells/well) were seeded into a 96-well culture plate and incubated for 24 h. Then, the cells were cultured in nitrogen (N2)-purged culture medium containing various concentrations of TA (10 mg)/CaO2  at 37 °C for 24 h, after which the cells were washed with fresh media and CCK-8 reagent solution was treated to the cells. The cells were further incubated for 2 h, and the optical density was determined at 450 nm using a microplate reader .Cytotoxicity test of the dried TA (10 mg)/CaOt-test or between many groups by one-way ANOVA . p values less than 0.05 were considered statistically significant.The data represented mean ± standard deviation, and mean values were compared between two groups by 2 nanoparticles were prepared by the reaction of calcium chloride (CaCl2) solution with H2O2 in EtOH containing ammonia solution and different amounts of TA  forming coordinate bridges with calcium ions (Equation (1), 2 nanoparticles were initially formed after adding H2O2 into the EtOH solution containing CaCl2 and TA. Through the coordination of TA with calcium ions, small-sized TA/CaO2 particles were formed at an initial reaction time and larger aggregates were subsequently obtained. The experimental yields of TA (10 mg)/CaO2, TA (25 mg)/CaO2, and TA (50 mg)/CaO2 were 43.20 ± 2.25 mg, 61.97 ± 7.80 mg, and 83.7 ± 3.24 mg, respectively, and the yield amounts increased with increasing TA amounts.TA/CaO2 was confirmed with FT-IR analysis. –1, which is assigned to the –OH stretching mode resulting from hydrogen bonding. Owing to the presence of ester groups, peaks were detected at 1640 and 1100 cm–1, arising from the C=O and C–O stretching modes, respectively. The characteristic peaks at 1492 and 1420 cm–1 are associated with the bending mode of O–Ca–O [The formation of TA/CaOf O–Ca–O .2 aggregates ranged from 220 to 263 nm, and their Z-average sizes ranged from 200 to 240 nm in diameter, with the midrange polydispersity indexes (PDIs) ranging from 0.187 to 0.383 . SEM images show that all three TA/CaO2 samples exhibited spherical shapes /CaO2, and 25.21 ± 4.06 nm for TA (50 mg)/CaO2. The particle sizes of three TA/CaO2 nanoparticles determined by dynamic light scattering appeared to be much larger than those measured by SEM in a dried state because the interactions between individual particles in EtOH might facilitate their aggregation to form much larger particles.The observed mean sizes of three TA/CaOto 0.383 . Noticeal shapes d,f. Inte2. As shown in 2 nanoparticles increased as the amount of TA was increased from 10 to 50 mg  [2 by hydrolysis, a fixed amount of each TA/CaO2 sample was added to PBS (pH 7.4) and the amount of dissolved oxygen in the solution was measured /CaO2 sample was selected for further experiments.CaOtion (2) . To detemeasured . In PBS 2 is relatively stable against decomposition but is soluble in acids [2 powder was more soluble in pH 6.0 than in pH 7.4 . The dissolved oxygen concentration was also measured in pH 6.0 and pH 7.4 PBS solutions. 2 generates more oxygen bubbles in pH 6.0 than in pH 7.4. Moreover, the oxygen generated from TA (10 mg)/CaO2 maintained a higher concentration in acidic pH 6.0 than in pH 7.4.It has been reported that solid CaOin acids . This isn pH 7.4 . To furt2 nanoparticles are synthesized in EtOH. To further investigate whether TA/CaO2 nanoparticles could be synthesized in other alcoholic solvents, we first tested the solubility of TA in MeOH, EtOH, and isopropanol. 2 nanoparticles. The mean sizes of TA (10 mg)/CaO2 nanoparticles prepared in MeOH were 236.6 ± 57.8 nm and their Z-average size was 216.5 nm in diameter with 0.209 of PDI /CaO2 (2 was much higher than that of control group. This increased cell-viability may be related to the oxygen generation from of TA (10 mg)/CaO2, and the oxygen generation may influence cell viability of macrophages.Cytotoxicity study of TA (10 mg)/CaOmg)/CaO2 . In part2 nanoparticles were synthesized using EtOH as the solvent and TA as the coordinate bridge with calcium ions. The spherical aggregates consisted of nanoparticles with sizes in the range of 25–31 nm. Among the three samples synthesized with different amounts of TA in the precursor, TA (10 mg)/CaO2 generated H2O2 and O2 most efficiently upon contact with water. Also, TA (10 mg)/CaO2 nanoparticles released more oxygen bubbles in an acidic environment. Furthermore, TA (10 mg)/CaO2 nanoparticles may improve cell viability followed by oxygen generation.TA/CaO


Octopus Kaurna-derived peptide, Octpep-1, in human BRAF(V600E) melanoma cells using proteomics and RNAseq coupled with metabolic analysis. Fluorescence microscopy verified that Octpep-1 tagged with fluorescein enters MM96L and NFF cells and distributes preferentially in the perinuclear area of MM96L cells. Proteomics and RNAseq revealed that Octpep-1 targets PI3K/AKT/mTOR signaling in MM96L cells. In addition, Octpep-1 combined with rapamycin (mTORC1 inhibitor) or LY3214996 (ERK1/2 inhibitor) augmented the cytotoxicity against BRAF(V600E) melanoma cells in comparison with the inhibitors or Octpep-1 alone. Octpep-1-treated MM96L cells displayed reduced glycolysis and mitochondrial respiration when combined with LY3214996. Altogether these data support Octpep-1 as an optimal candidate in combination therapies for melanoma BRAF(V600E) mutations.Melanoma is the main cause of skin cancer deaths, with special emphasis in those cases carrying BRAF mutations that trigger the mitogen-activated protein kinases (MAPK) signaling and unrestrained cell proliferation in the absence of mitogens. Current therapies targeting MAPK are hindered by drug resistance and relapse that rely on metabolic rewiring and Akt activation. To identify new drug candidates against melanoma, we investigated the molecular mechanism of action of the  Melanoma is the most prominent and lethal skin cancer. The main melanoma mutation is the substitution of a valine to glutamine in codon 600 (V600E) of the serine-threonine kinase BRAF(V600E) which accounts for ~50% of cases . MelanomBreakthroughs have occurred in the treatment of progressed melanoma based on comprehension of the oncogenic signaling, genetic alterations and the immunobiology of this cancer. BRAF inhibitor monotherapy (BRAFi) with the approval of vemurafenib and dabrafenib by the US Food and Drug Administration (FDA) constituted pivotal treatments against melanoma. BRAFi monotherapy has proved to be superior to MEK inhibitor (MEKi) monotherapy, with the latter linked to a narrower therapeutic window . UnfortuOctopus Kaurna + ions. Amino acids were purchased from IRIS Biotech GmbH , Bachem , or ChemImpex Inc. . Fmoc-Met-Wang resin was obtained from Peptide International .All reagents related to chemical synthesis of the compounds were obtained commercially and were used without further purification. Peptides were dissolved in water and mixed 1:1 (2O (solvent A) and 90% ACN/0.045% TFA/H2O (solvent B). Analytical HPLC quality control analysis of the investigated peptide was performed on a Shimadzu LC20AT system using an Agilent Eclipse Plus C18, 2.1 × 50 mm, 5 μm column with a flow rate of 0.3 mL/min at 45 °C. A gradient of 10 to 50% B over 40 min was used, with detection at 214 nm.Eluents for RP-HPLC consisted of 0.05% trifluoroacetic acid (TFA)/HPreparative HPLC were performed on a Waters 600E system using a gradient of 0 to 50% B over 50 min. For the prep. HPLC of the crude peptide an Agilent Eclipse XBD-C18, 21.2 × 250 mm, 5 µM column with a flow rate of 16 mL/min was used. Monoisotopic molecular masses were determined by MALDI-MS on a 4700 Proteomics Analyzer  mass spectrometer in positive ion mode.O-(6-Chlorobenzotriazol-1-yl)-N, N, N’, N’-tetramethyluronium hexafluorophosphate (HCTU)/DIPEA (1:1:1.1) relative to resin loading for 15 min. Fmoc deprotection was achieved using 50% piperidine/DMF (2 × 1 min). Final cleavage and global side chain deprotection were accomplished by stirring in 90% TFA, 5% TIPS, and 5% H2O for 90 min at ambient temperature. The suspension was filtered, washed with TFA, the filtrate concentrated under steady nitrogen flow to a minimal amount, and the peptide precipitated and washed with cold diethyl ether. The precipitate was filtered off and then dissolved in 0.05% TFA in 50% MeCN/H2O and lyophilized. The crude peptide was purified by preparative HPLC and the mass analyzed by MS in positive ion mode using an ABSciex API 2000TM. Pure fractions were combined and lyophilized (6.5 mg) (Octpep-1 (H-DPPSDDEFVSLM-OH) was assembled on a 0.25-mmol scale on a Fmoc-Met Wang resin following the Fmoc/tBu-SPPS protocol. Couplings were performed in dimethylformamide (DMF) using 4 equivalents of Fmoc-amino acid/(6.5 mg) .DPPSDDEFVSLM-OH with a modification at N-T:FITC-Ahx was chemically synthesized using FMOC-SPPS and purchased by GenScript. Pure fractions were lyophilized (20 mg) . In brie2. The melanoma cells were cultured in RPMI-1640 media supplemented with 10% FBS, and 2 mM GlutamaxTM. NFF cells were grown in RPMI-1640 containing 10% FBS. In all cells, penicillin/streptomycin (PS) (100 U/mL each) was added in the media. Cells were passaged at approximately 90% confluency and experiments were performed with passages up to 10. All cell lines were mycoplasma free.The MM96L, human melanoma and patient-derived cell line as well as the non-transformed neonatal foreskin fibroblast (NFF) cells were maintained in a humidified incubator at 37 °C and 5% COOctpep-1 tagged with fluorescein at the N-terminus was purchased from GenScript . MM96L cells were grown in coverslips and treated with 200 µg/mL Octpep-1 tagged with fluorescein at 37 °C for 48 h. Additionally, mitochondrial co-staining was performed by incubation with 100 nM MitoTracker Red  for 30 min at 37 °C followed by washes with PBS and 30 min fixation with 4% PFA at room temperature. Fluoroshield Mounting Medium with DAPI  was used to stain the nucleus and preserve fluorescence in the fixed samples. Images of the emitted fluorescence were acquired at 300, 50, and 100 ms of exposure for green, red, and blue fluorescence, respectively, using a Leica DMIL microscope with a Plan- Apochromat 63 × 1.4 NA oil immersion objective. Analysis was completed using ImageJ-Fiji software . Data are expressed as corrected total cell fluorescence using the following equation [(Cell Area × Mean Cellular Fluorescence) − (Cell Area × Mean Background fluorescence)].g for 5 min between each wash. For SWATH quantification, 100,000 adherent mm96l melanoma cells were harvested in triplicate at different time points post-stimulation  and were washed three times with Dulbecco’s PBS  with centrifugation at 800× g for 5 min between each wash. Stimulated and unstimulated MM96L cells were lysed on ice by resuspending the cells pellet in 100 mM TEAB (Thriethylammonium bicarbonate), 1% SDS (sodium dodecyl sulfate), 10 mM CHAPS, 5 mM MgCl2 supplemented with 1× Roche Complete protease inhibitors. DNA and RNA were then degraded by the addition of 50 µL and 2 µL, respectively, of 50 mM Tris, 20 mM NaCl, and 2 mM MgCl2 at 1 unit/µl of ultrapure Benzonase  followed by incubation at 4 °C for 45 min with constant agitation. Protein concentration was determined using the Pierce BCA Assay  following the manufacturer’s protocol.For the protein library generation approximately 30 million near confluent unstimulated adherent MM96L melanoma cells were trypsinised for 2 min and then harvested followed by three washes with Dulbecco’s PBS  with centrifugation at 800× g for 20 min. The protein sample was mixed with 6 volume of 8 M Urea in 100 mM TEAB, 10% isopropanol, and transferred to the filter unit and spun at 3100× g for 40 min in a 5810R  centrifuge. Detergent removal by buffer exchange was performed using a first wash of 500 µL of 8 M Urea in 100 mM TEAB, 10% (v/v) isopropanol followed by a wash with 500 µL of 100 mM TEAB, 10% isopropanol then two successive washes with 500 µL of 50 mM TEAB at 3100× g for 40 min each. The concentrated protein solution was then collected from the filter unit and transferred to an Eppendorf microcentrifuge tube and protein digestion was performed by adding Tryspin enzyme in 50 mM TEAB in an enzyme to protein ratio of 1:50 then mixed well before incubation at 37 °C in a wet chamber overnight. Peptides from the digested protein sample were desalted using a Sep-Pak Vac C18 cartridge  and the eluted fraction was lyophilized with a speed vacuum prior to OFFGELTM.600 µg of proteins in lysis buffer from the unstimulated MM96L cells were aliquoted and reduced with 0.5 M DTT stock solution added to a final concentration of 20 mM. Samples were then incubated at 75 °C for 10 min and cooled at room temperature for 10 min. Proteins were then alkylated for 30 min in darkness with 0.5 M iodoacetamide (IAA) stock solution to a final concentration of 40 mM. A 10 kDa molecular weight cut-off filter tube  was equilibrated by spinning 700 µL of 100 mM TEAB through the filter at 3100× 2O with 2% ACN (acetonitrile), 0.1% formic acid (v/v) prior to LC-MS/MS analysis.The 3100 OFFGEL Fractionator and OFFGEL kit pH 3-10  with a 24-well setup were prepared according to the manufacturer’s protocols. Lyophilized peptide mixtures were resuspended to a final volume of 3.6 mL using the OFFGEL peptide sample solution. IPG gel strips (24 cm) with a 3–10 linear pH range  were rehydrated with the Peptide IPG Strip Rehydration Solution according to the manufacturer’s protocol and 150 µL of sample was loaded in each well. Peptides were isoelectrically focused with a maximum current of 50 µA until 50 kV-h were achieved. Twenty-four fractions were recovered from each well and the wells were rinsed with 150 µL of water/methanol/formic acid (49/50/1) for 15 min. Each fraction was lyophilized and then resuspended in 30 µL of Hg. Protein samples were then transferred to different wells on the plate and spun at 3100× g for 30 min in a 5810R  centrifuge with adapted plate bucket. Detergent removal by buffer exchange was performed in two successive washes with 8 M urea, 10% isopropanol in 100 mM TEAB with centrifugation at 3100× g for 30 min between each wash. Urea was then removed by two washes with 10% isopropanol in 50 mM TEAB with centrifugation at 3100× g for 30 min between each wash. A final wash with 50 mM TEAB was performed with centrifugation as described above. Protein digestion was then performed by adding 1 µg of trypsin in 50 mM TEAB to the wells and incubating overnight at 37 °C. Peptides were recovered over the deep V-bottom plate  using an initial spin at 3100× g for 10 min followed by two centrifugations with 50 µL of 50 mM TEAB. Recovered peptides were dried in a speed vacuum for 4 h at 45 °C. Dried peptides were resolubilized in H2O, 0.1% TFA (v/v), desalted and normalized to 5 µg using ZipTipTM C18 tips  according to manufacturer’s protocol. Eluted peptides were lyophilized and then resuspended in 30 µL of H2O with 2% ACN (acetonitrile), 0.1% formic acid (v/v) spiked with the iRT calibrant  prior to LC-MS/MS analysis.The different protein samples in triplicate for each time point were prepared according to the modified FASP protocol for high throughput proteomics . Brieflym/z). An accumulation time of 100 ms was used for each fragment ion scan resulting in a total cycle time of 2.9 s. Data were acquired and processed using Analyst TF 1.7 software .All peptide samples were chromatographically separated by a 10 µL injection on an Eksigent cHiPLCTM-nanoflex system using a 15 cm long chromXP C18-CL column . A pre-concentration step (10 min) was performed employing a chromxp trap  before commencement of the gradient. A flow rate of 500 nl/min was used for all experiments. The mobile phase consisted of solvent A (0.1% formic acid [aq]) and solvent B (100 acetonitrile/0.1% formic acid [aq]) were used for the three consecutive linear gradients for peptide elution: 5–10% solvent B (acetonitrile/0.1% formic acid) over 2 min, 10–40% solvent B over 58 min and 40–50% solvent B over 5 min. A final gradient from 50% to 95% solvent B in 10 min was used to clean the column. Eluates from the RP-HPLC column were directly introduced into the NanoSpray II ionisation source of a TripleTOF 5600 MS/MS System  operated in positive ion electrospray mode. The data for OFFGEL fractions were obtained using Data Dependent Acquisition (DDA) as previously described , while tSpectral searches of processed LC-MS/MS data were performed using ProteinPilot v4.5 (AB SCIEX) using the Paragon algorithm (version 4.5.0.0). Background correction was used and biological modifications specified as an ID focus. The detected protein threshold was set as 0.5 and the false-discovery rate (FDR) was calculated using searches against a decoy database comprised of reversed sequences. Searches were conducted against the UniProt human reference proteome set comprising 70,953 protein sequences (downloaded 13th February 2017). For spectral library generation and SWATH XIC peak area extraction PeakView v2.2.0  with the SWATH acquisition MicroApp was used with ion library parameters set to 6 peptides per protein, 6 transitions per peptides, a peptide confidence threshold of 99%, and FDR threshold to 1%. The XIC time window was set to 6 min and XIC width to 75 ppm. Retention times for all SWATH experiments were normalized using the spiked iRT calibrants. To generate the quantitation table files for ions, peptides, and proteins Marker View v1.2.1.1  was used and the relative area under peaks across the different experiments was normalized based on the iRT internal calibrant. Figures and statistical analysis were generated with Python and R language and graphical packages.MM96L cells were lysed in cold RIPA buffer containing protease  and phosphatase  inhibitors and stored at −20 °C. Protein concentrations were determined using a Pierce BCA Protein assay kit . Samples were subjected to SDS-PAGE and blotted according to standard procedures. In brief, 10 μg of protein was loaded per lane. Antibodies used for Western blots are described in the Reagents section. Protein signals were visualized using enhanced chemiluminescence .Time course was performed at 30 min, 1 h, 3 h, and 6 h of MM96L cells treated with Octpep-1 at 200 μg/mL. The cells were then collected, and RNA extraction was performed by RNAsy kit  according to manufacturer’s directions. The RNA samples were sent for sequencing to the Institute for Molecular Bioscience Sequencing Facility.To determine the differentially expressed set of genes for melanoma treated with the Octpep-1 versus vehicle treated cells at different time points, we performed Ingenuity Pathway Analysis (IPA) followed by Principal Component Analysis (PCA) and enriched with GSVA and Ingenuity Z score analysis.Cell viability was evaluated by the MTT assay and measured at 570 nm absorbance on a microplate reader  after 48 h of peptides’ treatment using an MTT assay  as previously described ,35.Cellular bioenergetic measurements were performed using the Seahorse XFe96 Analyzer and XFe96 culture microplates  to investigate Oxygen Consumption Rates (OCR) and Extracellular Acidification Rates (ECAR) in MM96L. Measurements were performed after 24 h incubation with Octpep-1, ERK1/2i, mTORC1i and combination of the inhibitors with Octpep-1 in 15 × 103 melanoma cells/well in quintuplicates. OCR was tested in Seahorse XF base medium containing 15 mM glucose, 1 mM sodium pyruvate, and 2 mM L-glutamine (pH 7.4) and ECAR in Seahorse XF base medium containing 0.5 mM sodium pyruvate and 1 mM L-glutamine (pH 7.4).p-values adjusted using Kruskal–Wallis or Mann–Whitney method. The data from MTT experiments are the result of at least three independent experiments done in triplicate or quadruplicate while seahorse data are the outcome of four to six independent experiments done at least in quadruplicate. Data are represented as mean ± SEM. Significance was defined at * p ≤ 0.05 or ** p ≤ 0.01.Statistical analyses were conducted using Prism version 8.0 . Data were checked for normality to choose the appropriate test (parametric or non-parametric test). Significance for the screening of various inhibitors and their combination with Octpep-1 was calculated using a one-way ANOVA and Sidak’s multiple comparisons test. Differences between Octpep-1 and its combination with rapamycin or ERKi were examined by unpaired two-tailed t-test and Mann Whitney test, respectively. In addition, for the seahorse metabolic experiments significance was calculated using a one-way ANOVA and Dunn’s multiple comparison test with


The histone H3K36me3 mark regulates transcription elongation, pre-mRNA splicing, DNA methylation, and DNA damage repair. However, knowledge of the regulation of the enzyme SETD2, which deposits this functionally important mark, is very limited.Here, we show that the poorly characterized N-terminal region of SETD2 plays a determining role in regulating the stability of SETD2. This stretch of 1–1403 amino acids contributes to the robust degradation of SETD2 by the proteasome. Besides, the SETD2 protein is aggregate prone and forms insoluble bodies in nuclei especially upon proteasome inhibition. Removal of the N-terminal segment results in the stabilization of SETD2 and leads to a marked increase in global H3K36me3 which, uncharacteristically, happens in a Pol II-independent manner.The functionally uncharacterized N-terminal segment of SETD2 regulates its half-life to maintain the requisite cellular amount of the protein. The absence of SETD2 proteolysis results in a Pol II-independent H3K36me3 deposition and protein aggregation. The N-terminal tails of histones protrude from the nucleosome and are hotspots for the occurrence of a variety of post-translational modifications (PTMs) that play key roles in regulating epigenetic processes. H3K36me3 is one such important functionally characterized PTM. In yeast, this mark suppresses cryptic transcription from within the coding region of genes by preventing histone exchange . In mammSet2–Rpb1 Interaction) domain, and co-transcriptionally deposits H3K36me3  and Post-SET domains that are required for the catalytic activity of the enzyme, and also, the protein–protein interaction domains such as the WW and the SRI. Notably, the mammalian homolog, SETD2, has a long N-terminal segment that is not present in ySet2. The function of this region has remained obscure [In yeast, the SET domain-containing protein Set2 (ySet2) is the sole H3K36 methyltransferase . ySet2 iH3K36me3 . The delH3K36me3 . H3K36 mH3K36me3 . These h obscure , 14.Here, we show that SETD2 is an inherently aggregate-prone protein and its N-terminal region regulates its half-life. This, in turn, is important for the fidelity of the deposition of the functionally important H3K36me3 mark. Our findings reveal that the previously uncharacterized N-terminal region of SETD2 is important in governing appropriate SETD2 function and activity.To understand the regulation of the SETD2 enzyme in human cells, we introduced a construct to express Halo- or GFP-tagged SETD2 FL (full length) under the control of the CMV promoter in 293T cells. Strikingly, the expression of GFP-SETD2 FL was barely detected post-72 h of transfection although the expression of GFP in vector control transfected cells was robust  are the major pathways for protein degradation in mammalian cells [Autophagy and the ubiquitin–proteasome an cells . To inveTo test whether SETD2 is targeted for degradation by the UPS, SETD2 FL expression was checked after treating the cells with the proteasome inhibitor MG132. Proteasome inhibition led to an increase in the accumulation of SETD2. This was confirmed by western blotting of whole-cell lysate of Halo-SETD2 FL-expressing cells as well as microscopy visualization of GFP-SETD2 FL expression  has been used in human cells to rescue H3K36me3 . Hence, For this, we tested the expression level of a truncated mutant of SETD2, SETD2 C. Consistent with our hypothesis, microscopy of cells expressing the GFP-tagged proteins revealed that the expression level of SETD2 C was comparable to that of ySet2  was added to GFP-Nb (Nb’) and the expression was checked. Western blotting of the whole-cell lysates revealed a reduced expression level of Nb’ as compared to Nb with continued sensitivity to MG132 treatment  and 504–1403 (Nb), and tested their expression  [Microscopy revealed that GFP-SETD2 Na is nuclear and formed puncta Fig. c. This worm.cgi) . We deciTo test whether the full-length SETD2 protein also behaves similarly, the localization of NLS mutants of SETD2 was tested upon MG132 treatment. Strikingly, all the mutants exhibited the formation of nuclear puncta Fig. b. To chaExogenously expressed as well as the endogenous SETD2 formed puncta, especially upon MG132 treatment, that are reminiscent of inclusion bodies formed by aggregated proteins. The most striking pattern was observed with the fragment SETD2 Na that formed distinct puncta even in the absence of proteasome inhibition. As aggregates are often ubiquitinated, to confirm that the puncta formed by SETD2 are aggregated structures, the colocalization of RFP-ubiquitin with GFP- SETD2 Na was tested. Clear colocalization was observed suggesting that SETD2 Na puncta are indeed ubiquitinated aggregate structures Fig. a. GFP-SETo confirm further that SETD2 forms aggregates, we checked the solubility of Halo-SETD2 fragments Na, Nb, and C. 293T cells expressing the Halo-SETD2 fragments were lysed, their soluble and insoluble fractions separated and analyzed by western blotting with an anti-Halo antibody. Correlating with the microscopy observations, SETD2 Na that formed spontaneous puncta was highly insoluble; C was insoluble to a lesser extent Fig. c. The seCollectively, our data show that SETD2 forms aggregated ubiquitinated puncta (discussed in the Discussion section).setd2Δ (KO) 293T cells were used. Consistent with the role of SETD2 as the sole H3K36me3 depositor in humans, in the KO cells the H3K36me3 mark was not detected in the whole-cell lysates by immunoblotting  led to a marked decrease in H3K36me3 levels as compared to the FL  in SETD2 and found that strikingly, SETD2 can form nuclear puncta even without an intact NLS. This behavior is very similar to that exhibited by well-characterized aggregate-prone proteins like the mutant huntingtin. Importantly, it suggests that the robust degradation of SETD2 might be a mechanism to prevent inadvertent protein aggregation mediated by SETD2 and of SETD2 itself. This can also be a mechanism to regulate the possible liquid phase transition of SETD2 that needs to be investigated in future studies. Importantly, an increased accumulation of SETD2 also results in a reduced RNA Pol II dependency for H3K36me3 deposition, which is very uncharacteristic. SETD2 has a long N-terminal region that is missing in its yeast homolog Set2. Our work illustrates the importance of this N-terminal segment of SETD2 in maintaining the requisite intracellular amount of the protein.SETD2-HaloTag® human ORF in pFN21A was procured from Promega. Deletion mutants of SETD2 were constructed by PCR  using full-length SETD2 as a template and individual fragments were cloned. All constructs generated were confirmed by sequencing. SETD2-GFP, mRFP-Ub, pLenti puro HA-Ubiquitin, pTagRFP-C1-Fibrillarin and pCDNA3-ySet2 were procured from Addgene. siRNAs were purchased from Dharmacon and oligonucleotides were procured from Integrated DNA Technologies (IDT).l-glutamine at 37 °C with 5% CO2. MG132 (Sigma) was added at a final concentration of 10 μM for 12 h. Chloroquine (Sigma) treatment was done as indicated in the text. Cycloheximide (Sigma) was added at a final concentration of 10 μg/ml. Transfections were performed at cell confluency of 40% using Fugene HD (Promega) using a ratio of 1:4 of the plasmid (µg) to transfection reagent (µl).The cell lines used in this study  were procured from ATCC. Cells were maintained in DMEM supplemented with 10% FBS and 2-mM n = 4000) were plated in triplicate in 60-mm tissue culture plates and were allowed to grow as a monolayer for 14 days. Cells were incubated in the complete culture medium, with media changes every 2–3 days. After 14 days, the cells were fixed with 4% paraformaldehyde for 1 h. The colonies were stained with 0.5% crystal violet (0.5% in 70% ethanol) for 1 h at room temperature, rinsed and air-dried. Images were captured and the quantification of the number and size of the colonies was done using ImageJ.The cells  instructions. It was further treated with DNaseI (NEB) for 30 min at 72 °C to degrade any possible DNA contamination. RNA (2 μg) was subjected to reverse transcription using the QScript cDNA synthesis mix according to the manufacturer’s instructions. cDNAs were then amplified with the corresponding gene-specific primer sets. For RT-PCR, PCR was conducted for 24 cycles using the condition of 30 s at 94 °C, 30 s at 60 °C and 30 s at 72 °C. The PCR products were analyzed on a 1% agarose gels containing 0.5 μg/ml ethidium bromide. The sequence of oligos is in Additional file Histones were isolated and analyzed as described previously . For immH3K36me3 , H3K36me2 , H3K36me1 , H3 , Halo , β-actin , SETD2 , FLAG , Pol II .To prepare soluble and insoluble extracts, 293T cells were washed with 1xPBS, collected by centrifugation, and resuspended in lysis buffer . The lysed cells were centrifuged at 13,000 rpm for 20 min. The supernatant was collected as the soluble fraction. The pellet was washed with lysis buffer containing 600-mM NaCl). The remaining insoluble pellet following another centrifugation was resuspended in Laemmli buffer (Biorad) and solubilized by sonication on ice.293T cells expressing the protein of interest were harvested in 1xPBS and collected by centrifugation. The cells were lysed by resuspending in lysis buffer . The lysed cells were centrifuged at 13,000 rpm for 20 min. The supernatant was collected and diluted 1:3 by adding dilution buffer . The diluted lysate was added to 100 µl of pre-equilibrated Magne® HaloTag® Beads  and incubated overnight on a rotator at 4 °C. The beads were then washed with wash buffer  protease was used per 100 µl of elution buffer.293T cells were plated onto glass coverslips in a 6-well plate. Cells were washed with 1× PBS and fixed in 4% paraformaldehyde for 20 min at 37 °C. Cells were then washed three times with cold 1 × PBS and permeabilized for 5 min with 1× PBS containing 0.2% Triton-X 100. Permeabilized cells were then blocked for 30 min with blocking buffer (3% BSA and 0.1% Triton-X in 1× PBS). Cells were stained with primary antibodies against SETD2  for 1 h at room temperature. A secondary antibody conjugated with AlexaFluor 568 was applied for 1 h at room temperature. All the images depicted in the same panel were captured using the same settings.Additional file 1: Additional figures.


Peficitinib is an oral pan-Janus kinase inhibitor for the treatment of rheumatoid arthritis. Co-administration of peficitinib with metformin, a type 2 diabetes therapy, can occur in clinical practice. Hepatic and renal uptake of metformin is mediated by organic cation transporter 1 (OCT1) and OCT2, respectively, and its renal excretion by multidrug and toxin extrusion 1 (MATE1) and MATE2-K. This study investigated the effect of peficitinib on metformin pharmacokinetics in vitro and in healthy volunteers.Inhibitory effects of peficitinib and its metabolite H2 on metformin uptake into human OCT1/2- and MATE1/2-K-expressing cells were assessed in vitro. In an open-label, drug–drug interaction study, 24 healthy volunteers received a single dose of metformin 750 mg on Days 1 and 10, and a single dose of peficitinib 150 mg on Days 3 and 5–11. Blood and urine samples were collected pre-dose on Days 1 and 10, and at intervals ≤ 48 h post-dose. Metformin concentration was determined by liquid chromatography–tandem mass spectrometry and its pharmacokinetic parameters calculated.inf) by 17.4%, maximum plasma concentration (Cmax) by 17.0%, and renal clearance (CLR) by 12.9%. Co-administration of peficitinib with metformin was generally well tolerated.Peficitinib, but not H2, inhibited metformin uptake into OCT1- and MATE1/2-K-expressing cells. Repeated-dose administration of peficitinib reduced metformin area under the concentration–time curve from 0 h extrapolated to infinity  contains supplementary material, which is available to authorized users. Rheumatoid arthritis (RA) is an autoimmune disease that is associated with chronic, painful joint inflammation , 2. RA c14C-labelled peficitinib ([14C]peficitinib) suggested that approximately 64% of the peficitinib dose was absorbed metformin in the presence of either peficitinib or its metabolite H2 for 2 or 5 min. Cells were then washed and lysed, and the amount of radioactivity in the cell lysate was measured using liquid scintillation counting. The uptake of [14C]metformin into the cells in the presence of peficitinib or H2 was expressed as a percentage of the control (see online Human embryonic kidney cells (HEK293) expressing OCT1, OCT2, MATE1 or MATE2-K were incubated with  drugs) within 7 days prior to Day 1; received peficitinib or metformin hydrochloride previously.The main inclusion criteria are as follows: age 20 to 44 years; body mass index (BMI) of ≥17.6 kg/mSubjects were admitted to hospital on Day 1 and were discharged on Day 12. As food increases peficitinib exposure , subjectConcomitant use of medications and therapies, including OTC drugs, was prohibited during the study period.Blood samples for assessment of metformin concentration in plasma were collected pre-dose and at 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 6, 8, 10, 12, 24, 36 and 48 h post-dose on Days 1 and 10.Urine samples for assessment of metformin concentration were collected pre-dose (spot urine) on Days 1 and 10, and at intervals up to 48 h post-dose.inf), area under the plasma concentration–time curve from the time of dosing to the last measurable concentration (AUClast), maximum plasma concentration (Cmax), apparent oral clearance (CL/F), time to maximum plasma concentration (tmax) and terminal elimination half-life (t1/2). Urinary PK parameters estimated for metformin included the percentage of the drug dose excreted into urine from the time of dosing to the collection time of the last measurable concentration (Aelast%), and renal clearance (CLR).Bioanalysis of metformin concentrations in plasma and urine was conducted at LSI Medience Corporation, Itabashi-ku, Tokyo , clinical laboratory tests  and 12-lead electrocardiograms (ECGs).Treatment-emergent adverse events (TEAEs) that occurred after Day 1 of study drug administration to immediately before Day 3 of study drug administration were those in the ‘metformin alone’ phase; those that occurred after Day 3 of study drug administration to immediately before Day 10 of study drug administration were those in the ‘peficitinib alone’ phase; and those that occurred after Day 10 of study drug administration were those in the ‘peficitinib + metformin’ phase. TEAEs were graded using the National Cancer Institute’s Common Terminology Criteria for Adverse Events (NCI CTCAE) guidelines (version 4.0) .The planned sample size was 24 subjects, based on the precedent set by other PK studies of a similar nature , 22. Themax, AUCinf, AUClast and CLR of metformin were used in analysis of variance (ANOVA) using the SAS MIXED procedure with treatment as a fixed effect and subject as a random effect. The least-square (LS) geometric mean ratio (GMR) and its 90% confidence interval (CI) were calculated using the exponential-transformed LS geometric mean difference and CI. Missing data were not imputed or used for analyses.Statistical analyses were performed using SAS(R) software, version 9.4 . To assess the effect of peficitinib on the PK of metformin, natural log-transformed plasma CClinicalTrials.gov identifier: NCT02760342), informed consent form and information for distribution to subjects were reviewed and approved by the CPC Clinical Trial Hospital Institutional Review Board. This study was conducted in accordance with the study protocol, the International Council on Harmonization (ICH) guideline for Good Clinical Practice, applicable regulations and guidelines governing clinical study conduct, and the ethical standards as laid down in the 1964 Declaration of Helsinki and its subsequent amendments. Informed consent was obtained from all individual participants included in the study.The study protocol  on the uptake of [14C]metformin into OCT1-, OCT2-, MATE1- and MATE2-K-expressing cells, with IC50 values of 0.247 μmol/L, 71.4 μmol/L, 10.0 μmol/L and 20.8 μmol/L, respectively  remained 70.4%, 111.1%, 76.1% and 84.0%, respectively, of uptake in the absence of inhibitors. H2 was therefore not considered to be an inhibitor of any of the transporters tested.Peficitinib inhibited the uptake of [ly Figs. –S4. Alth2, and mean (SD) BMI was 21.2 (1.9) kg/m2  age was 32.5 (5.9) years. BMI ranged from 18.1 to 25.2 kg/mFigure inf, 0.831 (0.785–0.879) for AUClast and 0.830 (0.786–0.876) for Cmax  were 0.826 (0.784–0.870) for AUCR was 0.871 (0.822–0.924)  for CL0.871 0.82–0.924  subjects experienced a total of six TEAEs. Three (12.5%) subjects experienced a total of four mild (NCI CTCAE Grade 1) TEAEs after receiving peficitinib alone, and all four TEAEs were considered to be related to peficitinib administration. The four TEAEs were increased alanine aminotransferase , soft faeces  and increased aspartate aminotransferase . One (4.2%) subject experienced two mild TEAEs that were considered not to be drug related after receiving a combination of peficitinib and metformin. All TEAEs resolved without treatment.There were no clinically significant mean changes from baseline in vital sign measurements or 12-lead ECG variables during the study.R by 12.9%.Patients with RA have an increased risk of type 2 diabetes , and con50 value of 0.247 μmol/L; no meaningful inhibitory effect was observed with H2. In a previous clinical study, the Cmax of peficitinib following a single oral dose of 150 mg, under fasted conditions, was 524.5 ng/mL [Prior to conducting the clinical study, we investigated the interactions of peficitinib and H2 (the metabolite of peficitinib with the highest exposure) with the human metformin transporters OCT1, OCT2, MATE1 and MATE2-K in vitro. Peficitinib inhibited OCT1-mediated metformin transport with an IC.5 ng/mL ). Pefici.5 ng/mL –18. Inst.5 ng/mL . Further.5 ng/mL . Inhibit50 value of 10.0 μmol/L, while H2 had no meaningful inhibitory effect on either MATE1 or MATE2-K. This effect of peficitinib on MATE1 might account for the slight decrease in CLR of metformin observed in the clinical study. However, the concurrent reduction in metformin exposure suggests that this modest decrease in CLR had a negligible impact on the other PK parameters of metformin. Our findings indicate that peficitinib would not be categorised as an OCT1 or MATE1 inhibitor, since the Japanese Ministry of Health, Labour and Welfare (MHLW) guideline on DDIs defines a weak inhibitor as a drug that changes mean exposure to a concomitantly administered medication ≥ 1.25-fold [The in vitro findings also showed that peficitinib inhibited MATE1-mediated metformin transport with an IC.25-fold . MoreoveVerapamil, which may be used in type 2 diabetes mellitus patients with heart problems, is a known OCT1 inhibitor , 29. Co-A single daily dose of peficitinib (150 mg) was used in our clinical study, which represents the standard dosage used in Japan  and is tIn conclusion, this study demonstrated that co-administration of peficitinib and metformin slightly reduced both exposure and renal clearance of metformin in healthy male subjects, but peficitinib 150 mg administered alone and in combination with metformin was generally well tolerated. Consequently, the interaction between peficitinib and metformin was considered not to be clinically relevant, and no dose adjustment is required when peficitinib and metformin are used concomitantly.ESM 1(DOCX 2.43 mb)ESM 2(PNG 1276 kb)ESM 3(PNG 1276 kb)ESM 4(PNG 1276 kb)ESM 5(PNG 1276 kb)ESM 6(PNG 1926 kb)ESM 7(PNG 1926 kb)