Neospora caninum, an obligate intracellular protozoan, is the major cause for neosporosis and brings serious economic losses to cattle breeding industries worldwide. After invasion, dense granules proteins are abundantly secreted and being important components of parasitophorous vacuole and intravacuolar network where N. caninum survives and replicates. The aim of the present study was to evaluate the protective immunity induced by DNA vaccines with genes encoding dense granules proteins 1 (GRA1), GRA4, GRA9, GRA14, GRA17, and GRA23 against N. caninum tachyzoites in BALB/C mice. Eukaryotic expressing plasmids of pcNcGRAs were constructed and the mice were intramuscularly immunized with pcNcGRAs followed by challenging infection with lethal doses of N. caninum. Immune responses were evaluated through monitoring the levels of serum antibodies, measurement of lymphocyte proliferation, and secretion of cytokines. Immune protection assays were carried out through monitoring survival time, body weight, and parasite burden in the brains. Results showed that all the pcNcGRA DNA vaccines could trigger remarkably specific humoral and cellular responses, with higher levels of IgG and IgG2a antibodies as well as obviously increased secretion of Th1-type IFN-γ cytokines. The immune protective efficacy revealed that pcNcGRA4, pcNcGRA14, and pcNcGRA17 DNA vaccines could individually increase the survival rate to 50, 37.5, and 25% in comparison with 0% in the control group; prolong the survival time more than 20.88 ± 11.12, 18.88 ± 10.83, and 16.63 ± 10.66 days compared with the control group of 4 ± 1.31 days; and decrease parasite burden in the brains to 297.63 ± 83.77, 471.5 ± 110.74, and 592.13 ± 102.2 parasites/100 ng comparing with 1221.36 ± 269.59 parasites/100 ng in the control group. These findings indicated that NcGRA4, NcGRA14, and NcGRA17 are potential vaccine candidates; NcGRA4 displayed better performance in immune protective efficacy and could be further combined with other advantageous antigens applied to the development of safe and effective DNA vaccines against N. caninum. Neospora caninum, classified as an obligate intracellular protozoan parasite of phylum Apicomplexa, is the major pathogen for neosporosis in cattle and dog breeding industries  1-type immune response dominated by secretion of gamma interferon (IFN-γ), interleukin (IL)-12 cytokines, and production of IgG2a antibody is not only vital for acute fections , 9. Alsosporosis , 11.Toxoplasma gondii, a closely related parasite to N. caninum, multiples DNA vaccines have already been developed and verified to be promising in the prevention of toxoplasmosis, such as dense granules proteins 1 (GRA1), GRA4, GRA7, and GRA14, for they could activate high levels of humoral and cellular protective immunity for relative long periods  and SAG4  and then inhalation of CO2 (10–15 s) when they exhibited signs of illness and lost 20% body weight.All the animal experiments were approved by the Animal Welfare and Research Ethics Committee of Jilin University (permission number: pzpx20190929065). Six- to eight-week-old female BALB/C mice were maintained with SPF-level food and water N. caninum tachyzoites were propagated by continuous passage in monolayer bovine kidney epithelial  cells, which were cultured in Dulbecco's modified Eagle's medium  and supplemented with 10% fetal bovine serum , 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in 5% CO2 conditions. To obtain enriched and purified tachyzoites for the following experiments, Nc-1 strain infected cells were collected at the stationary growth phase, released with a 27-gauge needle , purified with Percoll (Millipore), and enriched in the phosphate-buffered saline to a final concentration of 1 × 108 parasites/ml.The Nc-1 strain of N. caninum tachyzoite lysate antigen (NLA) was prepared as follows: purified tachyzoite suspension was treated with Protease Inhibitor Cocktail  and then lysed going through three cycles of freezing at −80°C and thawing at 4°C followed by ultrasonicaton on ice. Then, the mixture was centrifuged at 10,000 × g at 4°C for 30 min and supernatants were collected. After filtering through a 0.22-μm sterilized membrane , NLA was stored at −80°C for further use.BamHI and EcoRI were individually introduced into the 5′ ends of forward and reverse primers of NcGRA1. Restriction enzyme cutting sites of EcoRI and XholI were individually added to the 5′ ends of forward and reverse primers of NcGRA4, NcGRA14, NcGRA17, and NcGRA23. Restriction enzyme cutting sites of HindIII and BamHI were individually added to the 5′ ends of forward and reverse primers of NcGRA9. Meanwhile, sequences of HA tag (TACCCATACGATGTTCCAGATTACGCT) were added into reverse primers after enzyme cutting sites. Target PCR products were obtained using PCR assays with Pfu DNA Polymerase  and then cloned into pMD18-T vector  according to the manufacturer's manual. After sequencing, positive recombinant plasmids were cleaved with individual restriction enzymes  and linked with linearized eukaryotic expression vector pcDNA 3.1(+) cleaved with same enzymes using T4 DNA ligase (Takara). After identification by restriction analysis, the positive recombinant expression plasmids of pcNcGRAs were purified using HiPure Plasmid EF Mega Kit  and concentrations were measured on a Nanodrop ND-2000 apparatus . The eukaryotic recombinant plasmids of pcNcGRAs were diluted in sterile endotoxin-free PBS to a final concentration of 1 mg/ml and stored at −20°C for further use.Total RNA was extracted from purified tachyzoites using RNAiso Plus , genomic DNA was removed, and cDNA was synthesized by the MonScript RTIII Super Mix with dsDNase (Two-Step) (Monad) according to the instructions. Specific amplification primers of NcGRAs were designed and listed in Plasmid of pcNcGRAs were transiently transfected into RAW264.7  cells previously prepared on coverslips in 24-well plates using Lipofectamine 2000 Transfection Reagent , and empty pcDNA 3.1(+) vector was used as negative control. After 72 h, cells were washed three times with PBS and fixed in 4% paraformaldehyde at room temperature (RT) for 10 min. After permeabilization in 0.1% Triton X-100 at RT for 20 min, cells were then washed with PBS three time times and blocked in 5% BSA at RT for 2 h. Next, cells were incubated with mouse anti-HA-Tag mAb  at 4°C overnight and fluorescein (FITC)-conjugated Affinipure Goat Anti-Mouse IgG(H + L)  at 37°C for 1 h, respectively. After washing unconjugated antibodies, cells were viewed on a fluorescence microscopy .7N. caninum tachyzoites at weeks 9 through intraperitoneal injection while monitoring the survival time for 30 days, and brain tissues were isolated for parasite burden assays.As shown in 2SO4 per well and OD450nm values were measured on a microplate reader .The specific antibody levels against NLA were monitored at weeks 2, 4, 6, and 9 in each group. NLA (100 μg/ml) was coated in 96-well plates (100 μl per well) at 4°C overnight. The plates were then blocked with 5% skim milk at RT for 2 h. Then, 100 μl of serum (100-fold dilution in PBS) was added into wells and incubated at 37°C for 2 h. Then, unconjugated antibodies were removed from wells through washing three times with PBST buffer and the remaining antibodies were detected with 100 μl of detection secondary antibodies of HRP-conjugated goat anti-mouse IgG, IgG1, or IgG2a  at 37°C for 1 h. After washing three times with PBST, 100 μl of TMB substrate solution  was added into each well and incubated at 37°C for 15 min. The reaction were stopped by 100 μl of 2 M H5 cells per well and cultured in RPMI 1640 medium with 10% fetal bovine serum . Cells were then stimulated with NLA at a final concentration of 10 μg/ml, concanavalin A  as positive control, or non-treatment as negative control at 37°C/5% CO2 for 72 h. The supernatants were discarded and replaced with fresh medium containing 10 μl of Cell Counting Kit-8 solution . After incubation at 37°C/5% CO2 for 3 h, the OD450nm values were measured on a microplate reader (BioTek).Lymphocyte proliferation assay was carried out by isolation of spleens at weeks 6 and 9 (four mice per time-point and per group). Single-cell suspensions were prepared as previously described . Splenoc450nm values were read and converted to picograms per milliliter. Concentrations were calculated based on the standard curves generated in each plate.Cytokine levels of IL-4 and IFN-γ in the splenocyte culturing surpernatants were measured using IFN gamma or IL-4 Mouse ELISA Kit  according to the manufacturer's instructions. The ODN. caninum tachyzoite Nc-1 strain DNA samples (1.0 × 102-1.0 × 107 parasites/ml). The parasite burden was measured by amplifying the Nc5 gene (Parasite burden was measured in brain tissues from immunized and infection mice at weeks 9 (eight mice per group) using real-time quantitative qPCR method. Genomic DNA was extracted using TIANamp Genomic DNA Kit  according to instructions and standard curve was established with serially diluted Nc5 gene  and para*p < 0.05, **p < 0.01, or ***p < 0.001. Graphs were generated in GraphPad Prism 7.00 . All the experiments were performed three times with three technical replicates.Statistical analyses were conducted using SPSS . All data represented mean ± SDs. A one-way ANOVA followed by Levene's test was used to analyze the difference between immunized groups and control groups or among different immunized groups. Correlation analysis was evaluated using non-parametric Spearman correlation with two-tailed tests. Kaplan–Meier survival curves were analyzed using log-rank (Mantel–Cox) tests. Differences were regarded as significant at in vitro (p > 0.05), whereas each pcNcGRA-immunized group triggered remarkably increased levels comparing with the control group. The pcNcGRAs significantly triggered high IgG antibodies at week 2 (*p < 0.05 or **p < 0.01), the levels were increased at week 4 (**p < 0.01 or ***p < 0.001), and the OD450nm values peaked at week 6 (***p < 0.001). Moreover, the high antibody levels lasted for 5 weeks after the last immunization (***p < 0.001).After sequencing, positive recombinant plasmids of pcNcGRAs were individually transfected into RAW264.7 cells and proteins were successfully expressed in vitro . To expl450nm values of IgG1 and IgG2a (p > 0.05). The IgG1 levels in pcNcGRA-immunized groups slightly increased (*p < 0.05), whereas the IgG2a levels remarkably upregulated (**p < 0.01 or ***p < 0.001) in comparison with that in the control groups. These data indicated that immunization with pcNcGRA DNA vaccines mainly induced Th1 immune responses in mice.To identify the Type 1 T helper (Th1) and Type 2 T helper (Th2) cells stimulated by pcNcGRA DNA vaccines, the IgG1 and IgG2a antibody levels in serum were determined at week 6. As shown in p > 0.05), by contrast, the pcNcGRA-immunized mice caused obviously high levels of lymphocyte proliferation both at week 6 and 9 after stimulation with 10 μg/ml of pcNcGRAs  or 5 μg/ml of ConA (***p < 0.001). Overall, these results indicated that mice immunized with pcNcGRAs induced cell immune response.To assess the cell-mediated immune responses, spleens were isolated from mice after 2 and 5 weeks since the last immunization. Lymphocyte proliferation assays were conducted using ConA as positive control, NLA as stimulators, and medium as negative control. As shown in **p < 0.01), on the contrary, insignificant changes of IL-4 levels (p > 0.05). Furthermore, correlation analysis was performed to explore the relationship between humoral immune response and cellular immune response. As shown in p = 0.0012, r2 = 0.3390) but also appeared between IFN-γ secretion and IgG2a antibody levels . There was a negative correlation between IL-4 and IgG1, but was not significant . Overall, these data illustrated that all the pcNcGRAs triggered Th1 response.The culturing supernatants were harvested from NLA-stimulated splenocytes for cytokine measurement. The secretion levels of Th1 (IFN-γ) and Th2 (IL-4) were determined and results are displayed in 7N. caninum tachyzoites and monitored the survival time for 30 days. As shown in p = 0.0074; pcNcGRA4: 20.88 ± 11.12 days, p = 0.0001; pcNcGRA9: 7.75 ± 4.65 days, p = 0.0056; pcNcGRA14: 18.88 ± 10.83 days, p < 0.0001; pcNcGRA17: 16.63 ± 10.66 days, p = 0.0003; pcNcGRA23: 10.25 ± 7.03 days, p = 0.0074) displayed significantly longer survival time than the PBS-treated control groups (4 ± 1.31 days). During the monitoring periods, pcNcGRA4 and pcNcGRA14-immunized mice prolonged 10 more days of life and increased survival rate to 50 and 37.5%, respectively. In contrast, the death rates in pcNcGRA1 and pcNcGRA9-immunized groups were both 100%. For the pcNcGRA17 group, the death rate decreased to 75%. In addition, further analysis was made between different immunized groups. The pcNcGRA4-immunized mice showed significantly higher survival percentage than that in the pcNcGRA1 or pcNcGRA23 (*p < 0.05) and pcNcGRA9 (**p < 0.01). Moreover, pcNcGRA4-immunized mice showed significantly increased survival percentage than pcNcGRA1, pcNcGRA9, or pcNcGRA23 (*p < 0.05). Comparisons between other immunized groups manifested no obvious significance (p > 0.05).To assess the protective function of pcNcGRA vaccines, eight mice per group were infected with lethal doses of 2 × 10p < 0.001; pcNcGRA9: 738.36 ± 159.42 parasites/100 ng, p = 0.029; pcNcGRA14: 471.5 ± 110.74 parasites/100 ng, p = 0.001; pcNcGRA17: 592.13 ± 102.2 parasites/100 ng, p = 0.005; pcNcGRA23: 656 ± 144.3 parasites/100 ng, p = 0.009) except for pcNcGRA1-immunized group . Comparisons between immunized groups with low parasite burden presented that pcNcGRA9, pcNcGRA17, or pcNcGRA23 shared similar levels with pcNcGRA1 group (p > 0.05). It is worth mentioning that the pcNcGRA4-immunized mice shared the lowest levels of parasite burden, which were remarkably decreased than other immunized groups (**p < 0.01 or ***p < 0.001) except for pcNcGRA14 (p = 0.096). Meanwhile, body weight was also recorded throughout the whole survival period. As shown in N. caninum infection, and pcNcGRA4 DNA vaccine elicited the best protection effects on infected mice and could improve the survival rate to 50% as well as decrease the parasite burden in brain tissues to 24.38%.The parasite burden in infected mice brain tissues was determined in Neospora vaccine of Bovilis Neoguard composed of tachyzoite lysate available in partial countries  and dramatically increase the IgG2a antibody levels (**p < 0.01 or ***p < 0.001) in comparison with the control group. These data were in accordance with other DNA immunizations that trigger mixed Th1/Th2 immune response, and Th1 immune response occupies the predominant function with a higher level of IgG2a . Correlation analysis was also made and a significant difference occurred when comparing IgG total with IFN-γ or IgG2a with IFN-γ. The high levels of IFN-γ would contribute to enhance the macrophage function, disrupt parasitophorous vacuole through GTPases and p65 guanylate-binding proteins, and further eliminate the intracellular parasites (p > 0.05). As expected, the p-values calculated from correlation analysis showed that no significant difference was observed between IgG and IL-4. These data indicated that immunization with pcNcGRA DNA vaccines could not cause sufficient B-cell proliferation and mast cell responses. This may be the main reason for the incomplete protection of mice from challenging with lethal dose of N. caninum.The Th1-type immune response was closely related with the secretion of IFN-γ cytokine , 53. To arasites , 55. Mea*p < 0.05); obviously upregulated levels existed in the pcNcGRA9, pcNcGRA14, and pcNcGRA23 groups (**p < 0.01); and an extremely significant increase occurred in the pcNcGRA4 and pcNcGRA17 groups (***p < 0.001).T cell–mediated immunity mainly controls intracellular parasite infection. Splenocyte proliferation assays were carried out to determine the difference between immunized groups and the control group. A slightly increased level occurred in the pcNcGRA1 group .JD and WL designed the research. GY, WL, QY, and JW conducted the research. GY, YW, TZ, XZ, and HF analyzed the data. PZ, QY, and WL wrote the article. JD and LC directed the project. All authors have read and approved the article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ligand-receptor docking simulations have suggested that the electrostatic interaction between the carboxyl group of second-generation antihistamines and the amino group of Lys179ECL2 and Lys1915.39 of human H1 receptors might contribute to increased affinity of these antihistamines to H1 receptors. In this study, we evaluated the roles of Lys179ECL2 and Lys1915.39 in regulating the electrostatic and hydrophobic binding of bilastine to H1 receptors by thermodynamic analyses. The binding enthalpy and entropy of bilastine were estimated from the van ’t Hoff equation using the dissociation constants. These constants were obtained from the displacement curves against the binding of [3H] mepyramine to membrane preparations of Chinese hamster ovary cells expressing wild-type human H1 receptors and their Lys179ECL2 or Lys1915.39 mutants to alanine at various temperatures. We found that the binding of bilastine to wild-type H1 receptors occurred by enthalpy-dependent binding forces and, more dominantly, entropy-dependent binding forces. The mutation of Lys179ECL2 and Lys1915.39 to alanine reduced the affinity of bilastine to H1 receptors by reducing enthalpy- and entropy-dependent binding forces, respectively. These results suggest that Lys179ECL2 and Lys1915.39 differentially contribute to the increased binding affinity to bilastine via electrostatic and hydrophobic binding forces.Bilastine, a zwitterionic second-generation antihistamine containing a carboxyl group, has higher selectivity for H L. L1 receKd) of ligands is determined by the thermodynamic binding forces of ligands, and these are the binding enthalpy (∆H°) and entropy (∆S°)  a  mepyramine, a radioligand for H1 receptors, to evaluate actual roles of Lys179ECL2 and Lys1915.39 in regulating the binding affinity of bilastine via its thermodynamic binding forces.Docking simulation was performed to simply estimate the configuration of bilastine at the ligand-binding pocket of human Heceptors b. We fou1 receptors by mutations of Lys179ECL2 and/or Lys1915.39, the IC50 for bilastine was first obtained from the displacement curves for the binding of 3 nM [3H] mepyramine to membrane preparations of CHO cells expressing wild-type human H1 receptors (WT), Lys179ECL2 or Lys1915.39 mutants of WT to alanine (K179A and K191A), and both Lys179ECL2 and Lys1915.39 mutants of WT to alanine (K179A + K191A) at 4 °C–37 °C  for WT, K179A, K191A, and K179A + K191A at a standard temperature of 25 °C (298.15 K). The Ki values of bilastine were 1.92 ± 0.08 nM for WT (n = 4), 5.20 ± 1.18 nM for K179A (n = 7), 2.57 ± 0.16 nM for K191A (n = 4), and 7.96 ± 0.76 nM for K179A + K191A (n = 4). Thus, the affinity of bilastine for H1 receptors was significantly reduced by approximately 1.3 to 4.1 times following the mutation of Lys179ECL2 and/or Lys1915.39. These results suggest that Lys179ECL2 and Lys1915.39 contribute to the increased affinity for bilastine.Ki = ∆H°/RT − ∆S°/R. T∆S° versus ∆H° for bilastine obtained from the van ’t Hoff plots. In the binding of bilastine to WT (G° (= ∆H° − T∆S°) for bilastine were obtained by the binding enthalpy (∆H°) and more dominantly the binding entropy (−T∆S°). These results are consistent with our previous findings that entropy-dependent binding forces of second-generation antihistamines were significantly higher than those of first-generation antihistamines -mepyramine was purchased from PerkinElmer . Bilastine was purchased from Tokyo Chemical Industry . Chinese hamster ovary cells  were purchased from the RIKEN Bioresource Center . The expression vectors (3×HA hH1R/pcDNA3.1(+)) for human H1 receptors tagged with three molecules of hemagglutinin (YPYDVPDYA) at the N terminus were purchased from the Missouri S&T cDNA Resource Center . Other materials were purchased from Sigma-Aldrich . mepyramine, a radioligand for H1 receptors, was performed in accordance with the methods described previously [3H] mepyramine in the presence or absence of various concentrations of bilastine for 3 h at 37 °C, 24 h at 25 °C and 14 °C, and 7 days at 4 °C in normal HEPES buffer . The reaction mixture was filtered through glass fiber filters, and the radioactivity trapped on the filters was determined by scintillation counting. All determinations were made in quadruplicate. The protein content in the membrane preparations was determined using a BCA protein assay kit .CHO cells stably expressing WT, K179A, K191A, and K179A + K191A were cultured, and membrane preparations were obtained as described previously ,26,27. Teviously ,26,27. Bt-test or analysis of variance with Bonferroni correction. Results with a value of p < 0.05 were considered significant.All data were presented as means ± standard errors of means of at least three measurements performed in quadruplicate. Statistical significance was evaluated using Student’s 50IC for bilastine was determined by fitting the displacement curves to the one-site model :B is the amount of [3H] mepyramine bound, C is the free concentration of bilastine, and P is the percentage of the binding sites of bilastine.The Ki values for bilastine were estimated from the Cheng and Prusoff equation, as follows  mepyramine, and Kd is the dissociation constant for [3H] mepyramine.The  follows ,26,27,34


G-quadruplex (G4) structures formed by guanine-rich nucleic acids are implicated in essential physiological and pathological processes and serve as important drug targets. The genome-wide detection of G4s in living cells is important for exploring the functional role of G4s but has not yet been achieved due to the lack of a suitable G4 probe. Here we report an artificial 6.7 kDa G4 probe (G4P) protein that binds G4s with high affinity and specificity. We used it to capture G4s in living human, mouse, and chicken cells with the ChIP-Seq technique, yielding genome-wide landscape as well as details on the positions, frequencies, and sequence identities of G4 formation in these cells. Our results indicate that transcription is accompanied by a robust formation of G4s in genes. In human cells, we detected up to >123 000 G4P peaks, of which >1/3 had a fold increase of ≥5 and were present in >60% promoters and ∼70% genes. Being much smaller than a scFv antibody (27 kDa) or even a nanobody (12–15 kDa), we expect that the G4P may find diverse applications in biology, medicine, and molecular devices as a G4 affinity agent. G-quadruplexes (G4s) are four-stranded secondary structures formed by guanine-rich nucleic acids. Putative G-quadruplex forming sequences (PQSs) are abundant in the genomes of animal cells with particular enrichment near transcription start sites (TSSs), implying an essential role of G4s in transcription . AccordiRecently, G4s have been detected in chemically fixed human cells by immunostaining  or immunTo overcome the aforementioned difficulties, we engineered a 64 amino acids (64-aa) 6.7 kDa protein based on a 23-aa segment (RHAU23) of the G4-binding domain of the RHAU , an RNA helicase able to bind and unwind G4s . This G4DNA coding the RHAU23-(GTGSGA)-3xFLAG (RHAU23) or RHAU23-(GTGSGA)3-RHAU23-(GTGSGA)-3xFLAG were synthesized by Generay Biotechnology  and inserted into pET28b between the Nde I and EcoR I sites to obtain pET28b-RHAU23 and pET28b-G4P. The former coding sequence was also inserted into pIRES2-EGFP between the Nhe I and EcoR I sites to obtain pG4P-IRES2-EGFP. The DNA fragment containing a nuclear localization signal (NLS) of the SV40 large antigen (PKKKRKV) was synthesized by Sangon  and inserted into pG4P-IRES2-EGFP at the Nhe I site to obtain plasmid pNLS-G4P-IRES2-EGFP. The construction of AAVS1 donor and PX330 plasmids for knock-in was as described . BrieflyA549, NCI-H1975, 293T, HeLa-S3, 3T3 and DF-1 cells were kindly provided by Stem Cell Bank, Chinese Academy of Sciences. Cells were grown in DMEM supplemented with 10% FBS and 1 × penicillin-streptomycin.For transient transfection, cells were cultured in 15 cm dishes to 70–80% confluence and transfected with 30 μg plasmid pNLS-G4P-IRES2-EGFP using lipofectamine 3000 (Thermo Scientific) according to the manufacturer's instructions. Cells were cultured for an additional 24 h before harvesting. For G4P knock-in, AAVS1 donor and PX330 plasmid containing G4P and AAVS1 gRNA were co-transfected into 293T cells using lipofectamine 2000 (Thermo Scientific). After 24 h, GFP positive single cell was sorted by a flow cytometer  into a 96-well plate. Cells were cultured for two weeks and the cell lines with stable expression of G4P were verified by PCR, western blot, and immunofluorescence.The plasmid pET28b-G4P and pET28b-RHAU23 were transformed into the E.coli strain BL21 (DE3). Cells were grown in LB medium supplemented with 0.05 mg/ml kanamycin at 37°C for 3 h. G4P and RHAU23 expression were induced with 1 mM isopropyl thiogalactoside for 4 h at 37°C. The two peptides were purified using the Capturem HIS-tagged Purification Miniprep Kit  and diluted in buffer containing 20 mM Tris–HCl (pH 7.4), 150 mM NaCl, 0.1 mM EDTA, 50% glycerol and stored at −20°C.CD spectroscopy was conducted as described . The incY) in a 1:1 stoichiometry against the free G4P concentration (X) to the equation: Y = F1 × X/(Kd1 + X)+(1 – F1) × X/(Kd2 + X), where F1 denoted the fraction of the subpopulation associated with the Kd1.DNAs  were dishttp://genome.ucsc.edu/). Global searching of four types of PQS motifs  was performed using home-made Perl scripts. 4G PQS motifs were identified using a regular expression Chromosome files  in fasta format were downloaded from the UCSC website , 40 U RNase inhibitor (Thermo Scientific), 2 mM NTP, 0.5 μg pEGFP-N1 plasmid, and 0.5 μg PQS or control plasmid. The reaction was stopped by addition of EDTA to 16 mM. 1 μl of samples were used as input and diluted in 50 μl water. The remaining samples were mixed with 0.2 μM G4P and incubated at 4°C for 30 min.Plasmids containing the indicated PQS or a mutant control sequence  on the nFor the pull-down assay, 12 μl anti-FLAG M2 magnetic beads (Sigma-Aldrich) were washed three times with binding buffer  and then resuspended in 200 μl binding buffer. The beads were then incubated with the DNA samples on a rotating mixer at 4°C for 2 h. After the removal of the supernatant, the beads were washed ten times with the binding buffer accompanied with three times of transfers to new tubes. The precipitated plasmids were released from the beads by incubating with 0.3 mg/ml 3xFLAG peptide (Sigma-Aldrich) in a 50 μl binding buffer at 4°C for 0.5 hours. The released plasmid and the input samples were treated with RNase A at 37°C for 15 min and proteinase K at 60°C for 20 min, followed by inactivation at 95°C for 10 min.qPCR was conducted on the CFX Connect thermocycler (Bio-Rad) for the PQS or control plasmid using a PCR primer pair of 5′-TCCGACACTATGCCATCCTGA-3′ and 5′-TGCAGTCGTTTCGTATCGTTGA-3′. The EGFP gene on the pEGFP-N1 plasmid was amplified using a primer pair of 5′-GCAGAAGAACGGCATCAAGG-3′ and 5′-CGGACTGGGTGCTCAGGTAG-3′. After calibrating with the internal control, the enrichment was calculated using input DNA as a reference.8 transiently or stably transfected cells expressing G4P were crosslinked with 1% formaldehyde for 20 min at room temperature. Fixation was quenched by 0.125 M glycine for 15 min. The fixed cells were washed twice with PBS, suspended in NP-40 buffer  and incubated on ice for 10 min. After centrifugation at 800 × g for 5 min, the cell pellet was resuspended in a CHAPS buffer . The suspension was incubated on ice for 30 min and centrifuged at 800 × g for 5 min. The pellet was resuspended in 1 ml 1× dsDNase digestion buffer supplied with 50 μl dsDNase  and incubated at 37°C for 20 min with constant agitation. A final concentration of 20 mM EDTA was added to terminate the reaction. The samples were pelleted by centrifugation at 15 000 × g at 4°C and the resulting supernatant was collected and incubated on ice. The pellet was resuspended in 500 μl wash buffer  and sonicated for 30–60 s in an ice-cold water bath. After centrifugation at 15 000 × g for 5 min, the supernatant of chromatin fragment was collected and combined with the supernatant from the previous step.Approximately 0.5–1 × 10For library preparation, 50 μl of anti-FLAG M2 magnetic beads (Sigma-Aldrich) were washed with washing buffer  and blocked in the same buffer containing 75 μg/ml single-stranded sperm DNA and 1 mg/ml BSA. 1% chromatin fragment was saved as input and the remaining was incubated with blocked anti-FLAG magnetic beads in rotation at 4°C for 3 h. The beads were sequentially washed ten times with washing buffer and transferred to new tubes three times. The chromatin was eluted with 300 μg/ml 3xFLAG peptide (Sigma-Aldrich) at 4°C for 1 h. The eluted chromatin and the input samples were incubated with proteinase K at 65°C overnight. After sequential RNase A and proteinase K digestion, DNA fragment was cleaned by extraction with phenol:chloroform:isoamyl alcohol, followed by ethanol precipitation. Libraries were constructed from the recovered DNA fragment using the NEBNext Ultra II DNA LibraryPrep Kit from Illumina (NEB) according to the manufacturer's instructions. The next-generation sequencing was performed with Illumina HiSeq X Ten by Genewiz .http://genome.ucsc.edu/) unless otherwise indicated. Coordinate duplicates in the bed files were removed. Peaks of reads enrichment were identified with the macs2 software  or Encode (https://www.encodeproject.org/) database and processed as described above whenever applicable.Clean paired-end sequencing data in fastq format were mapped to the human genome (hg19) using the Bowtie2 software  with thesoftware  using –qTotal RNA from cells expressing G4P was prepared using the SV Total RNA Isolation System (Promega). mRNA was purified from total RNA using the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB). RNA library was constructed using the NEBNext Ultra II Directional RNA Library Prep Kit from Illumina (NEB) according to the manufacturer's instructions. The next-generation sequencing was performed with Illumina HiSeq X Ten by Genewiz .Primers for amplifying PQS-proximal and distal regions were given in Expression and purification of nucleolin (NCL) were conducted as described .Kd) of ∼1 μM  , G-vacanKd. High-affinity Kd at low-nM was obtained for each G4 DNA with various fractional quantities  were subsequently obtained using the Samtools (≥3(N1–7G≥3)≥3  and a 3xFLAG tag at the N- and C-terminal of the G4P core, respectively, without compromising the binding to G4s . We thenSamtools  and thenSamtools . The bin–7G≥3)≥3 ,54 ≥3 . We term–7G≥3)≥3  and the –7G≥3)≥3 . The tin–7G≥3)≥3 . Similar–7G≥3)≥3 .in vitro studies in which transcription efficiently induced G4s at both sides of a TSS , intragenic, TES ± 2 kb, and intergenic regions Figure , where Tof a TSS ,48,53,57In vitro transcriptional formation of G4s positively correlates with transcription activity . Most importantly, it overcomes the problem of impermeability and disulfide bonds associated with antibodies such that the G4P is readily applicable in living cells. With the removal of >90% of the amino acid residues from the original RHAU, the G4P is unlikely to interact with other proteins as the RHAU and other proteins might do, therefore, ensuring direct target recognition and specificity. While native G4-binding proteins can also locate G4 formation, they may result in false-positive detection because of potential interaction with other macromolecules. For instance, the hnRNP A2* protein can bind the RNA component of telomerase in addition to telomere DNA G4s  and processed  G4P-ChIP datasets, and PQS motifs (bigbed) have been deposited in and can be downloaded from the NCBI Gene Expression Omnibus (GEO) under accession code GSE133379.gkaa841_Supplemental_FileClick here for additional data file.


E. coli transcription terminator factor Rho that was crystallized as an impurity present in preparations of an overexpressed bacterial membrane-transport protein.A structure is reported of the  Escherichia coli, a contaminant protein was crystallized. The identity of the contaminant was revealed by mass spectrometry to be the Escherichia coli transcription terminator factor Rho, structures of which had been previously determined in different conformational states. Although Rho was present at only ∼1% of the target protein , it preferentially crystallized in space group C2 as thin plates that diffracted to 3.30 Å resolution. The structure of Rho in this crystal form exhibits a hexameric open-ring staircase conformation with bound ATP; this characteristic structure was also observed on electron-microscopy grids of the NaAtm1 preparation.During the crystal structure analysis of an ATP-binding cassette (ABC) transporter overexpressed in  This can consequently lead to the inadvertent purification of contaminant proteins that might otherwise be present at negligible levels when the target protein expresses at high levels. In unfortunate cases, these impurities may crystallize more readily than the target protein, leading to misplaced enthusiasm until the contaminant is recognized. As examples, the multi-drug efflux pump AcrB is a well known crystallization contaminant in membrane-protein preparations owing to its relatively high expression level during recombinant protein expressions with antibiotic selection and its nonspecific binding to Ni–NTA columns  from Novosphingobium aromaticivorans, using an E. coli expression system and a modified version of a previously published protocol  were lysed in lysis buffer consisting of 100 mM NaCl, 20 mM Tris pH 7.5, 40 mM imidazole pH 7.5, 10 mM MgCl2, 0.5%(w/v) n-dodecyl-β-d-maltopyranoside , 0.5%(w/v) octaethylene glycol monododecyl ether  with the addition of lysozyme, DNase and protease inhibitor. After stirring for 3 h at 4°C, the lysate was subjected to ultracentrifugation at ∼113 000g for 45 min at 4°C. The supernatant was collected, loaded onto a pre-washed Ni–NTA column in buffer consisting of 100 mM NaCl, 20 mM Tris pH 7.5, 50 mM imidazole pH 7.5, 0.05% DDM, 0.05% C12E8 and eluted with the same buffer containing 350 mM imidazole pH 7.5. The protein was further purified by size-exclusion chromatography (SEC) using a HiLoad 16/60 Superdex 200 column  with SEC buffer consisting of 100 mM NaCl, 20 mM Tris pH 7.5, 0.05% DDM, 0.05% C12E8. Peak fractions were collected and concentrated to ∼20 mg ml−1 using a 100 kDa cutoff Amicon Ultra 15 concentrator (Millipore). Macromolecule-production information is summarized in Table 1Rho copurified with the bacterial ABC exporter 2.2.NaAtm1 under optimized conditions based on MemGold (Molecular Dimensions) condition No. 68 at 20°C by hanging-drop vapor diffusion. The final crystallization condition consisted of 100 mM NaCl, 100 mM Tris pH 8.3, 28% polyethylene glycol 2000 monomethyl ether (PEG 2000 MME), 0.2 M nondetergent sulfobetaine 221 (NDSB-221), 20 mM ATP pH 7.5. The crystallization sample was prepared in 1 mM ATP pH 7.5, 5 mM EDTA pH 7.5 in the presence and absence of 5 mM oxidized glutathione (GSSG) pH 7.5, which is a transport ligand for NaAtm1. Thin plate-shaped crystals appeared in about two weeks. The crystals were harvested in cryoprotectant solution consisting of 100 mM NaCl, 100 mM Tris pH 8.3, 28% PEG 2000 MME with PEG 400 at 10%, 15% and 20% before flash-cooling in liquid nitrogen. Crystallization information is summarized in Table 2Rho crystallized during the crystallization trials of 2.3.JBluIce–EPICS  and on beamline 12-2 at the Stanford Synchrotron Radiation Laboratory. The final data set was collected on the GM/CA beamline 23-ID-B with an EIGER 16M detector (Dectris) using 2.4.CCP4 program MOLREP  with MSP1D1 was carried out with 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) at a 1:2:130 molar ratio of NaAtm1:MSP1D1:POPC. This reconstituted sample was incubated overnight at 4°C. After two hours of incubation, BioBeads were added at 200 mg ml−1 for detergent removal. The sample was then subjected to size-exclusion chromatography on a Superdex 200 Increase 10/300 column . The peak fractions were pooled and concentrated to ∼8 mg ml−1.The expression plasmid for the membrane-scaffolding protein MSP1D1 was purchased from Addgene (plasmid No. 20061). The expression and purification of MSP1D1 were carried out using published protocols with minor modifications (Ritchie −1 in the presence of 5 mM GSSG and 5 mM AMPPNP. 3 µl protein sample was applied onto freshly glow-discharged QuantiFoil Cu R2/2 300 mesh grids and blotted for 4 s with a blot force of 0 and 100% humidity at room temperature using a Vitrobot Mark IV (FEI). Data were collected on a 200 keV Talos Arctica with a Falcon III detector at a magnification of 92 000 and a total dose of 81 e Å−2 at the Caltech CryoEM Facility.EM grids were prepared using a protein concentration of 4 mg mlcryoSPARC2 .Data processing was performed with 3.NaAtm1 from N. aromaticivorans. NaAtm1 was recombinantly expressed in E. coli BL21-Gold (DE3) cells with a C-terminal 6×His tag following a previously established protocol . SDS–PAGE gels indicated a high degree of purity, although in subsequent analysis of overloaded gels a small amount of another protein was present at a molecular weight of ∼40 kDa .Crystals of Rho were unexpectedly obtained during studies of the bacterial ABC transporter hy Fig. 1a. SDS–PDa Fig. 1b.C2, with unit-cell parameters a = 161.8, b = 101.9, c = 184.0 Å, β = 107.8°. The asymmetric unit volume was of sufficient size to accommodate an NaAtm1 dimer (molecular weight of 133 kDa). Analysis of the self-rotation function calculated from the diffraction data revealed a noncrystallographic symmetry (NCS) sixfold axis offset ∼3–4° from the crystallographic a axis. Interaction of the perpendicular twofold and sixfold axes generates a set of noncrystallographic twofold rotation operations separated by 60° in the plane perpendicular to the NCS sixfold axis . Given the unit-cell dimensions, this apparent NCS was incompatible with dimeric NaAtm1, which raised the possibility that a contaminant had crystallized. With an estimated molecular weight of ∼40 kDa based on the observation of a faint impurity band in the gel and a Matthews coefficient analysis, we performed molecular replacements with known crystallization contaminants .The identification of Rho (molecular weight 47 kDa) was established by a mass-spectrometric analysis of the peptides prepared by trypsin digestion of the protein in the SDS–PAGE bands. Using this information, we were able to obtain a molecular-replacement solution using Rho in the AMPPNP-bound state (PDB entry y Table 4. The eley Table 4a.b). The r.m.s.d. between the present structure and the molecular-replacement input model (PDB entry 1pvo) is 2.5 Å using the Cα positions for the superposition and 3.3 Å when using all atoms of the six subunits in the hexamer. The r.m.s.d.s between individual subunits in the present structure and PDB entry 1pvo are 0.59–0.73 Å, reflecting their similar tertiary structures. The relationship between adjacent subunits in the broken staircase of the present structure may be approximated by a screw operation with a rotation around the screw axis of 60.5° per subunit and a corresonding translation along the axis of −7.7 Å. In comparison, the corresponding values for PDB entry 1pvo are 57.9° and −8.2 Å, respectively. Reflecting the larger rotation angle per subunit, the present structure exhibits a more closed ring in comparison to the original broken-staircase structure . At the level of subunit–subunit interactions, however, the differences are subtle and we could not identify the specific interactions responsible for these differences in hexamer structure. Of note, the nucleotide-binding site is at the interface between subunits and small changes in subunit–subunit interactions may reflect the presence of different nucleotides: ATP in this structure and either no nucleotide (apo), ATPγS or AMPPNP in other open-ring structures . The open-ring conformation that Rho adopts in solution  nanodiscs. The NaAtm1 nanodisc sample was prepared by incubating detergent-purified NaAtm1 with MSPs and lipids, and was further purified by size-exclusion chromatography. Similar to the purification in detergent, the peak fractions were collected for single-particle cryoEM sample preparation . The 2D classification reported one class of Rho in the broken hexameric state , again suggesting that Rho has a similar hydrodynamic radius and plausibly a similar molecular weight to our reconstituted NaAtm1 in nanodiscs.We have also observed Rho in single-particle cryoEM studies of on Fig. 4a. The 2te Fig. 4b, againIn a structural analysis of a prokaryotic chloride channel, a single peptide of Rho was identified in a mass-spectrometric analysis of the gel band (Abeyrathne & Grigorieff, 2017E. coli transcription termination factor Rho, 6wa8PDB reference:


Takakura 3B ankle arthritis is featured as obliteration of ankle space with subchondral bone contact. Among these patients, some have medial distal tibial platform erosion. It is hard to treat this kind of patients. The purpose of this study was to evaluate the therapeutic outcomes of intra-articular opening osteotomy combined with lateral ligament reconstruction for Takakura 3B ankle arthritis with medial distal tibial platform erosion.From September 2009 to May 2016, 17 patients with Takakura 3B ankle arthritis were reviewed, including 3 male and 14 female patients. All underwent the operation of intra-articular opening osteotomy combined with lateral ligament reconstruction. All patients were available for analysis. The main outcome measurements included TT angle, AOFAS score, VAS score, SF-36 scale, and AOS scale.All patients were followed for a mean follow-up of 87.2 months . The VAS scale improved from 5.5 ± 1.6 to 2.3 ± 1.9. The mean AOFAS score improved from 47.7 ± 15.7 to 75.8 ± 12.0. The SF-36 scale improved from 41.6 ± 14.0 to 67.7 ± 14.6. The AOS improved from 60.9 ± 13.9 to 28.2 ± 17.7. The TT angle improved from 14.3 ± 5.0° to 5.3 ± 4.0°. The TAS and TLS changed from 83.4 ± 2.6° and 77.5 ± 2.3° to 90.7 ± 2.3° and 78.6 ± 2.2°. However, the LTAS was not corrected significantly.Intra-articular opening osteotomy combined with lateral ligament reconstruction is an effective method to treat varus ankle arthritis with medial distal tibial platform erosion. According to Takakura and Tanaka, varus ankle arthritis was classified into four types: stage 1, early sclerosis and formation of osteophytes without changing of ankle joint space; stage 2, narrowing of medial joint space without subchondral bone contact; stage 3, obliteration of ankle space with subchondral bone contact; and stage 4, varus ankle joint with complete bone contact. Stage 3 was further classified into stages 3A and 3B. Stage 3B ankle arthritis was defined as obliteration of ankle space extended to the roof of the dome of the talus with subchondral bone contact [For young adults and those who do not want to sacrifice the native ankle joint, joint-sparing methods instead of total ankle replacement or ankle arthrodesis are very important. Supramalleolar osteotomy is an effective joint-sparing surgical treatment for varus-type ankle arthritis, especially those with small TAS angles –7.In our practice, we do notice positive outcomes after supramalleolar osteotomy for stage 3B ankle arthritis . HoweverThe purpose of this study was to evaluate the therapeutic outcomes of intra-articular opening osteotomy combined with lateral ligament reconstruction for Takakura 3B ankle arthritis with medial distal tibial platform erosion.The current study was approved by our institutional review board. From September 2009 to May 2016, 17 patients  with Takakura 3B ankle arthritis were reviewed, including 3 male and 14 female patients. All underwent the operation of intra-articular opening osteotomy combined with lateral ligament reconstruction. The mean age was 52.35 ± 8.05 years. The inclusion criteria were (1) Takakura stage 3B ankle arthritis, (2) medial distal tibial platform erosion, (3) normal lateral tibial surface, and (4) painful ankle arthritis undergoing at least 1-year conservative treatment.The exclusion criteria were (1) end-stage ankle arthritis, (2) patients with neuropathic arthropathy or rheumatoid arthritis, (3) patients with regional infection around the ankle joint or had other ankle surgeries, and (4) patients with severe osteoporosis or large bone loss.Weightbearing X-rays of ankle joint were performed for every patient preoperatively and postoperatively, including anteroposterior ankle views (AP) and lateral ankle views. In this study, we defined the lateral tibial articular surface angle (LTAS) as the angle between the axis of the tibia and the lateral tibial surface. The talar tilt angle (TT) was regarded as the angle between the distal articular surface of the tibia and the upper surface of the talus. The general tibial articular surface angle (TAS) was defined as the angle between the axis of the tibia and the whole tibial surface. The whole tibial surface was a line drawn from the medial apex of the mortise to the lateral apex of the mortise. The tibial lateral surface angle (TLS) is designated as the angle between the axis of the tibia and the surface of the distal tibia in the lateral view. The hindfoot alignment view, which was recommended by Saltzman and el-Khoury, was also performed to evaluate hindfoot alignment condition . The tibIn this study, all ankle arthritis was varus type. The osteotomy approach was through a medial longitudinal incision. A K-wire was placed as a guide wire of osteotomy. Two or three K-wires were placed parallel to the ankle joint surface portion of the tibial platform within the subchondral bone just under the articular cartilage at the apex of the plafond angulation. They help the prevention of destruction of the talus cartilage from the saw blade during the osteotomy. It also acted as a hinge during deformity correction. Then the intra-articular osteotomy was performed. The intraoperation fluoroscopic assessment was performed to evaluate whether the tibial articular surface was normal. If the varus talus could not return to normal, then a release procedure of medial ankle ligament including the superficial and deep deltoid ligament was performed. Debridement of osteophytes was performed if there was impingement around the ankle joint such as the anterior distal tibial osteophyte to improve ankle motion through the medial incision and lateral incision. A wedged allograft was shaped and inserted into the osteotomy site and was fixed by a locking plate. The ankle joint was flexible, and we fixed it into a neutral position with 1–2 K-wires, which were removed 3–4 weeks postoperatively. In this position, the medial ligaments were sutured and lateral ligaments were reconstructed.Lateral ligament reconstruction was performed via a minimally invasive method . FifteenA cast was used for 6 weeks. Part weight bearing was allowed 6 weeks postoperatively with the cast. Full weight bearing was allowed 2 to 3 months postoperatively. Patients came to the hospital 2 weeks, 6 weeks, 3 months, and 1 year postoperatively. All the patients were followed up annually.t test was used for assessing differences between preoperative and postoperative measurements. A p value less than 0.05 was considered to indicate statistical significance.All analyses were performed with the SAS software version 8.1 . The results were given as means and standard deviation. The paired All patients were followed. Patients were followed for a mean follow-up of 87.2 months . There was no loss of follow-up. The VAS scale improved from 5.5 ± 1.6 to 2.3 ± 1.9. The mean AOFAS score improved from 47.7 ± 15.7 to 75.8 ± 12.0. The SF-36 scale improved from 41.6 ± 14.0 to 67.7 ± 14.6. The AOS improved from 60.9 ± 13.9 to 28.2 ± 17.7. The TT angle improved from 14.3 ± 5.0° to 5.3 ± 4.0°. The TAS and TLS changed from 83.4 ± 2.6° and 77.5 ± 2.3° to 90.7 ± 2.3° and 78.6 ± 2.2°. However, the LTAS was not corrected significantly.In this study, 2 patients rated their outcomes as “excellent” and 7 patients rated their outcomes as “good.” Four patients rated their results as “fair.” On the other hand, a “poor” outcome was observed in 4 patients because of the consistent discomfort. However, none of the patients underwent ankle joint arthroplasty or arthrodesis because of the cost involved or other reasons.Ten patients underwent calcaneal osteotomy. Thirteen patients recalled an old ankle sprain history. All of them had a long-term ankle sprain history 5–38 years). One patient had a lateral malleolar fracture history. Three patients had no incentive , and 4 [One of the causes of varus-type ankle arthritis was chronic ankle instability , 15. In For varus ankle arthritis, especially Takakura stage 3 or 4 ankle arthritis, previous studies have reported good results of ankle arthrodesis or ankle replacement –20. HowePrevious studies reported that it was contraindication if the varus ankle was rigid and could not be corrected to normal under fluoroscopic examination before the surgery , 21. HowWe think the most important aspect of this kind of procedure is that there is enough residual articular cartilage. Ankle joints with more than 50% residual articular cartilage tend to get better prognosis than those that are not. So, we did an osteotomy procedure for this kind of ankle arthritis. We do not perform osteotomy surgery for stage 4 ankle arthritis. Joint cartilage of stage 4 ankle arthritis was always extensively destructed. For this kind of patients, even though realignment of the ankle joint may alleviate symptoms, joint damage will progress soon.For Takakura 3B ankle arthritis with medial distal tibial erosion, intra-articular opening osteotomy combined with lateral ligament reconstruction is an effective method to treat this kind of varus ankle arthritis.


The natural compound baicalin (BA) possesses potent antiviral properties against the influenza virus. However, the underlying molecular mechanisms of this antiviral activity and whether macrophages are involved remain unclear. In this study, we, therefore, investigated the effect of BA on macrophages.via flow cytometry, qRT-PCR, immunofluorescence, a cell culture transwell system, and GC-MS–based metabolomics both in vivo in H1N1 A virus-infected mice and in vitro.We studied macrophage recruitment, functional phenotypes (M1/M2), and the cellular metabolism +, F4/80+) by approximately 90% while maintaining the proportion of M1-polarized macrophages in the bronchoalveolar lavage fluid of infected mice. This BA-stimulated macrophage M1 phenotype shift was further verified in vitro in ANA-1 and primary peritoneal macrophages by measuring macrophage M1 polarization signals . Meanwhile, we observed an activation of the IFN pathway (upregulation of IFN-β and IRF-3), an inhibition of influenza virus replication (as measured by the M gene), and distinct cellular metabolic responses in BA-treated cells.BA treatment drastically reduced macrophage recruitment (CD11bBA triggered macrophage M1 polarization, IFN activation, and other cellular reactions, which are beneficial for inhibition of H1N1 A virus infection. Novel mutant influenza A viruses, such as avian H7N9, do not respond well to current antiviral therapies and can cause substantial excess mortality during pandemics and the annual flu season  and act as critical modulators of influenza virus infection severity and the development of lethal pulmonary injury . Their pvia modulation of macrophage functions.Chemicals with anti-influenza activity derived from natural products are rapidly emerging, some of which may constitute possible novel drug options . BaicaliReprogramming of the host cellular metabolism triggered by the influenza virus is highly associated with inflammation and immune cell activation , and metBA  was dissolved in DMSO to generate a 50-mM stock solution and stored at 4°C. It was subsequently diluted in cell culture medium to different working solutions. For animal experiments, BA and ribavirin solutions were prepared with 0.5% CMC-Na.® 488 molecular probe was purchased from Santa Cruz Biotechnology . All other chemicals were obtained from Beyotime Biotechnology .Antibodies against mouse CD11b, F4/80, CD86, CD206, and cleaved interleukin-1β (IL-1β) were obtained from Biolegend . Secondary antirabbit IgG Fab2 Alexa Fluor Male BALB/c mice (16–18 g) were purchased from the Shanghai SLACCAS Laboratory Animal Co., Ltd. . Mice were housed under specific pathogen-free (SPF) conditions and given free access to sterile water and standard mouse chow. All experimental protocols were approved by the Animal Experiment Committee of Fudan University  .The influenza virus A/FM/1/47 (H1N1) was supplied by the Shanghai Center for Disease Control & Prevention  and was stored in aliquots at -70°C. For each experiment, an aliquot was thawed to ensure the use of a fresh preparation.50 of A/FM/1/47 (H1N1). Two hours postinfection, mice were treated with 80 mg/kg of BA (BA group) , virus-infected mice (Virus group), BA-treated infected mice (BA group), and ribavirin-treated infected mice (Ribavirin group). All mice except control mice were anesthetized under 1% pentobarbital and infected intranasally (i.n.) with 5×LDA group) , 20 mg/kA group) , and 0.5in vitro experiments, the H1N1/PR/8/34 TC adapted strain was obtained from ATCC  and stored in aliquots at -70°C. For each experiment, an aliquot was thawed to ensure the use of a fresh preparation.For Murine ANA-1 macrophages and epithelial BEAS-2B cells were obtained from the Cell Bank of the Shanghai Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences .5 cells/ml) with 10% (v/v) heat-inactivated fetal bovine serum (Gibco), 0.3 mg/mL l-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C under 5% CO2.Murine ANA-1 macrophages  and prim2.BEAS-2B cells  were cul50) of H1N1 virus  for 2 h and then transferred to BA solution (diluted in 1640 medium for macrophages and DMEM for BEAS-2B cells) for 24 or 48 h.The cells were infected with 10× the median tissue culture infectious dose  of different grouped mice was collected three times by lavage with 0.5 mL ice-cold PBS under pentobarbital anesthesia at 4 days postinfection. The collected BALF was centrifuged at 700×g at 4°C for 5 min, and the harvested cells were resuspended in 200 μL PBS. The cells were stained with fluorescently labeled antibodies against the following mouse proteins: CD11b+, F480+ (macrophages), CD11b+, F4/80+, CD86+ (M1 phenotype macrophage), and CD11b+, F4/80+, CD206+ (M2 phenotype macrophage) (For the rophage) .in vitro study (24 and 48 h postinfection), infected ANA-1 macrophages were stained with fluorescently labeled antibodies against the following mouse proteins: CD86+ (M1 phenotype) and CD206+ (M2 phenotype).For the Flow cytometry analysis was performed on a BD FACS Calibur . All plots shown on populations were gated on lymphocytes by forward and side scattering, collecting 100,000 cells per sample. Compensation settings were adjusted using isotype controls. Data analysis was performed using the FlowJo software .High levels of cellular iNOS and TNF-α are often observed in M1 polarized macrophages. Thus, we quantified cellular iNOS  and TNF-α (using a TNF-α ELISA kit) to evaluate the macrophage polarization.iNOS, Arg-1, influenza M gene, INF-β, IL-10, IRF-3, and IRF-7 were normalized to the geometric mean of GAPDH mRNA levels by using a ΔΔCT method  according to the manufacturer’s instructions. The RNA was converted into cDNA using a Reverse Transcription Kit . qRT-PCR was performed using the StepOne Plus RT-PCR System (Applied Biosystems) using the following thermocycling parameters: 94°C for 5 min, followed by 40 cycles of 94°C for 5 s and 60°C for 30 s. The mRNA levels of T method . The pri4 cells/well). ANA-1 or BEAS-2B cells were grown in the lower room (105 cells/mL). The cells in lower chambers were infected with the influenza PR/8/34 virus (10×TCID50) for 2 h and then transferred to fresh culture medium or BA dilutions.ANA-1 macrophages  were cultivated in the upper chamber of 24-well Transwell plates (10Macrophage recruitment was observed under a Leica EL6000 microscope (Leica Microsystems CMS GmbH).® 488 molecular probes (1:2000 dilution). After counterstaining cell nuclei with DAPI (1:1000 dilution of 100 µg/mL stock solution) for at least 3 min, immunofluorescent images were obtained using a Leica EL6000 microscope (Leica Microsystems).Cells were fixed with 4% paraformaldehyde buffer for 30 min, permeabilized with 0.5% Triton X-100 for 20 min, and blocked with QuickBlock™ Blocking Buffer for 30 min at room temperature. The cells were probed with IL-1β antibodies (1:1000 dilution) at 4°C overnight and subsequently detected with antirabbit IgG Fab2 Alexa Fluor The cell samples were frozen and thawed at room temperature. Three hundred μL methanol were added to 100 μL of the homogenate to precipitate the protein, and the mixture was shaken vigorously for 30 s. Ribitol (10 µL at 0.02 mg/mL) was added to the sample as the internal standard. The mixture was then ultrasonically extracted for 10 min, followed by centrifugation (3000 × g) for another 10 min. Then, 150 μL of the supernatant was transferred to the GC vial and evaporated to dryness under a stream of nitrogen gas. The chemical derivatization of the cell metabolites was carried out using the combination of methoxymation and silylation. A 30-μL aliquot of methoxyamine pyridine solution (20 mg/mL) was added to the vial. The methoxymation was performed at 37°C for 90 min. Then, 30 μL of bis-(trimethylsilyl)–trifluoroacetamide plus 1% trimethylchlorosilane were added to each sample. The samples were mixed on a vortex for 1 min and incubated at 70°C for 60 min. After incubation, samples were again vortexed for 1 min and transferred to vials for GC/MS analysis (Agilent 7890A GC system).The GC/MSD Chem Station Software  was used for the autoacquisition of GC total ion chromatograms and fragmentation patterns. For each peak, the software generates a list of similarities comparing with every substance within the National Institute of Standards and Technology (NIST) mass spectra library. Peaks with a similarity index of more than 70% were assigned compound names, and those having less than 70% similarity were listed as unknown metabolites. Each sample was characterized by the same number of variables, and each of these variables was represented across all observations with the same sequence. Thus, a data matrix was generated by the intensities of the commensal peaks from all samples to characterize the biochemical pattern of each sample .z-score, and then set the minimum value to -2 (dark blue), the median to 0 (white), and maximum value to 2 (dark red).To create the metabolite heat map, we normalized data for each metabolite [/standard deviation], obtaining a t-test (comparison of two groups) or one-way ANOVA (for comparisons with more than two groups sample). In all cases, probability levels less than 0.05 (P < 0.05) were considered to indicate statistical significance.All statistical analyses were performed using GraphPad Prism for Windows (Version 6.0). The Gehan-Breslow-Wilcoxon test was used to analyze the survival of mice, and other experimental data were evaluated by using the Student’s 50 influenza virus A/FM/1/47 caused 75% mortality within 14 days postinfection, accompanied by visible body weight loss. Treatment with 80 mg/kg BA attenuated death and body weight loss of infected mice (As shown in +-F4/80+ macrophages per 105 cells) to the lungs. BA treatment (80 mg/kg) significantly reduced virus-induced macrophage recruitment (1% of 105 cells). Furthermore, we then analyzed the polarization of recruited macrophages from the virus and BA groups and found that BA treatment resulted in a higher proportion of M1 macrophages than in virus mice (97% CD11b+-F4/80+-CD86+ triple positive vs. 95% triple positive) as shown in We collected the BALF of infected mice  at 4 days postinfection and analyzed macrophage recruitment and phenotype. As shown in We, therefore, concluded that recruitment and polarization of macrophages might play a critical role in the antiviral effects of BA.in vitro (50) with or without BA treatment. As shown in ANA-1 macrophages are a commonly used murine macrophage cell line , which win vitro . TherefoIn iNOS and Arg-1) in ANA-1 macrophages and primary peritoneal macrophages. In ANA-1 cells, BA (10 µM) markedly upregulated the iNOS transcription, increased the iNOS/Arg-1 ratio, and suppressed influenza M gene replication at 24 h postinfection  between untreated infected and BA-treated infected cells. The results indicate that BA treatment upregulates IFN-β and IRF-3 transcription and downregulates transcription of IL-10 as shown in We further evaluated the effects of BA (5 µM) on the IFN signaling pathway in ANA-1 cells at 24 and 48 h postinfection. Influenza virus replicated in ANA-1 cells in a time-dependent manner, which was inhibited by BA treatment. We further compared These results suggest that BA exerts its suppression of viral replication at an early stage by triggering macrophage M1 polarization and activating IFN signaling.The IL-1 cytokine family cytokines are cytosolic proteins that exhibit inflammatory activity upon release into the extracellular space. The mature, cleaved form of IL-1β is closely related to the hyperactivated macrophage .We assessed the activation of IL-1β in ANA-1 cells at 24 h postinfection by immunofluorescence. As shown in + ANA-1 macrophages and human BEAS-2B cells  in infected BEAS-2B and ANA-1 cells.To explore the influence of BA on macrophage migration, we used wild-type and GFP2B cells  in the TTo evaluate the role of TNF-α in BA-treated macrophages, we then used Etanercept, which is a commercially available soluble TNFR fusion protein inhibiting TNF-α at 70 µg/mL. TNF-α inhibition appreciably abolished IL-1β cleavage and cell–cell adhesion triggered by BA . Partial least squares discriminant analysis (PLS-DA) was undertaken to identify the differential metabolites of different groups . BA treatment resulted in a noticeable deviation of metabolic responses in infected ANA-1 cells compared with normal and virus-infected samples . This M1 polarization by BA after infection echoed similar M1-promoting effects of BA in the tumor microenvironment  can be found in the article/supplementary material.The animal study was reviewed and approved by The Animal Experiment Committee of Fudan University .PG and HZ carried out all in vitro and in vivo experiments in this study. WZ and HH carried out the immunoassays. CS, MC and CX performed the data analysis. CH participated in the design of the study. YC and XS conceived the study, participated in its design and coordination and helped to draft the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the Key Program of Nation Nature Science Foundation of China (No.U1604283), Shanghai Science and Technology Funds (Nos. 17ZR1401700), and Shanghai Municipal Health Commission project (Nos.201740200).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Eukaryotic replication origins are licensed by the loading of the replicative DNA helicase, Mcm2-7, in inactive double hexameric form around DNA. Subsequent origin activation is under control of multiple protein kinases that either promote or inhibit origin activation, which is important for genome maintenance. Using the reconstituted budding yeast DNA replication system, we find that the flexible N-terminal extension (NTE) of Mcm2 promotes the stable recruitment of Dbf4-dependent kinase (DDK) to Mcm2-7 double hexamers, which in turn promotes DDK phosphorylation of Mcm4 and −6 and subsequent origin activation. Conversely, we demonstrate that the checkpoint kinase, Rad53, inhibits DDK binding to Mcm2-7 double hexamers. Unexpectedly, this function is not dependent on Rad53 kinase activity, suggesting steric inhibition of DDK by activated Rad53. These findings identify critical determinants of the origin activation reaction and uncover a novel mechanism for checkpoint-dependent origin inhibition. Cdc45-MCM-GINS), which encircle single-stranded DNA (ssDNA) during unwinding  are quantitatively and specifically cleaved at the engineered TEV site by TEV protease, resulting in Cdt1⋅MCM complexes containing the Mcm2-Δ127 N-terminal truncation, whereas wildtype Cdt1⋅MCM complexes are resistant to TEV protease cleavage  and AAA+ ATPase domains are conserved between archaeal and eukaryotic MCM proteins, the long unstructured NTEs of Mcm2, −4, and −6 are unique to eukaryotes. The NTEs of Mcm4 and −6 play a fundamental role during DNA replication by serving as phospho-acceptors for DDK kinase during origin activation . In contcleavage .2-TEV (ORC-Cdc6-Cdt1-MCM)  , DNA-boudt1-MCM) . While Mdt1-MCM) . Using t) /100 mM NaCl/1 mM DTT/1 x protease inhibitor cocktail (Pierce). The cell suspension was pipetted dropwise into liquid nitrogen to generate frozen popcorn and stored in −80°C. Cells were lysed by crushing the popcorn in a Spex freezer mill, using 10 cycles of 2 min run + 1 min cooldown at 15 CPS. Resulting whole cell lysate was thawed and supplemented with 1 volume of 45 mM buffer A/100 mM NaCl/1 mM DTT/1 x protease inhibitor cocktail. 5 M NaCl was added to the lysate to a final concentration to 300 mM. After 20 min of gentle agitation at 4°C, cell lysate was centrifuged in a T-647.5 rotor (Thermo Fisher) at 40,000 rpm for 1 hr at 4°C. The clear soluble phase was recovered and DDK pulled down with 1 mL packed FLAG affinity agarose beads (Sigma) for 4 hr at 4°C with gentle rocking. Beads were collected by centrifugation and washed with 10 volumes of buffer A/300 mM NaCl/1 mM DTT. Beads were resuspended in 1 volume of buffer A/300 mM NaCl/2 mM MnCl2/1 mM DTT and incubated with λ protein phosphatase (NEB) at 50 U/mL for 1 hr at 23°C with agitation. Beads were collected and protein eluted in five volumes of buffer A/300 mM NaCl/1 mM DTT supplemented with 0.25 mg/mL 3xFLAG peptide. Eluates were analyzed by SDS-PAGE. The FLAG pulldown was repeated until DDK was depleted from the extract. Fractions containing DDK were pooled, and the volume reduced to 0.5 mL using an Amicon spin concentrator (Millipore). The pooled, concentrated eluate was fractionated on a 24 mL Superdex 200 Increase 10/300 GL  gel filtration column in buffer A/300 mM NaCl/1 mM DTT. Fractions were analyzed by SDS-PAGE and peak fractions containing DDK were pooled and concentrated using Amicon spin concentrator before dialysis against buffer A/100 mM KOAc/2 mM β-mercaptoethanol. The concentration of the purified DDK was determined by SDS-PAGE and Coomassie stain using BSA standards. Purified DDK was stored in aliquots a −80°C.The ybbR-FLAG-tagged DDK was purified from strain YSA35. Cells were grown in 48L YP/2 % glycerol/2 % lactic acid pH 5.5 (YPLG) at 30°C to a density of 2 × 10E. coli BL21-CodonPlus (DE3)-RIL cells (Agilent). A colony of cells freshly transformed with plasmid p1035 was grown in 3 L of LB supplemented with 50 μg/mL ampicillin and 34 μg/mL chloramphenicol at 37°C. At OD600 ~0.6, 1 mM IPTG was added and the temperature reduced to 4°C. After 1 hr, the temperature was raised to 20°C and the cells were incubated for an additional 16 hr. Cells were collected by centrifugation, rinsed twice with dH2O, once with buffer B (50 mM Tris-HCl pH 7.5/1 mM EDTA/10 % glycerol/10 mM benzamidine/150 mM NaCl), and resuspended in buffer B supplemented with 1x protease inhibitor cocktail (Pierce) and 1 mM DTT. Cells were lysed by addition of 10 mg lysozyme (Thermo Scientific) and incubation for 30 mins at 4°C followed by sonication. The clear, soluble phase was isolated after centrifugation of the whole-cell lysate in a T-647.5 rotor (Thermo Fisher) at 40,000 rpm for 30 min at 4°C. Nhp6 was pulled down with 0.5 mL packed Ni-NTA agarose beads (Qiagen) for 3 hr at 4°C with gentle agitation. Beads were collected and washed with 20 volumes of Buffer B/1 mM DTT. Protein was eluted with five volumes of buffer B/1 mM DTT/100 mM imidazole, and eluates were analyzed by SDS-PAGE. Eluate fractions containing Nhp6 were pooled and the volume reduced to 0.5 ml using an Amicon spin concentrator (Millipore). The pooled concentrate was fractionated by gel filtration chromatography using a Superdex 200 10/300 GL  column in buffer B/1 mM DTT. Elution fractions were analyzed by SDS-PAGE and Nhp6 peak fractions were pooled and concentrated using a spin concentrator. Purified Nhp6 was aliquoted, snap-frozen, and stored at −80°C.Nhp6 was expressed as a N-terminal 6x His-tag fusion protein in SPT16 and N-terminally CBP-tagged POB3 under control of the GAL1,10 promoter (strain YDR 125). Cells were grown in 12 L YPLG at 30°C up to a density of 2 × 107 cells/mL. Protein expression was induced with 2 % galactose for 4 hr. Cells were collected by centrifugation, washed with 25 mM HEPES-KOH pH 7.6/1 M sorbitol, and resuspended in 0.5 volumes of Buffer C (25 mM Tris-HCl pH 7.5/0.02 % NP-40 substitute/10 % glycerol)/100 mM NaCl/1 mM DTT/1 x protease inhibitor cocktail (Pierce). The cell suspension was pipetted dropwise into liquid nitrogen to generate frozen popcorn and stored at −80°C. Cells were lysed by crushing in a Spex freezer mill with 10 cycles of 2 min run and 1 min cooldown at 15 CPS. Thawed whole cell lysate was supplemented with 1 volume of Buffer C/100 mM NaCl/1 mM DTT/1 x protease inhibitor cocktail and the final concentration of NaCl adjusted to 300 mM using a 5 M NaCl stock solution. After 20 min of gentle agitation at 4°C, the cell lysate was centrifuged in a T-647.5 rotor (Thermo Fisher) at 40,000 rpm for 1 hr at 4°C. The clear soluble phase was recovered and supplemented with 2 mM CaCl2. FACT was pulled down from the extract with 0.5 mL packed calmodulin affinity resin (Agilent) for 4 hr at 4°C with gentle rocking. Resin was collected by centrifugation and washed with 10 volumes of Buffer C/300 mM NaCl/2 mM CaCl2/1 mM DTT. Protein was eluted from the resin with 7 volumes of Buffer C/300 mM NaCl/1 mM EDTA/2 mM EGTA/1 mM DTT, and eluates were analyzed by SDS-PAGE and Coomassie stain. The calmodulin pulldown was repeated until FACT was depleted from the extract. Fractions containing FACT were pooled and incubated for 16 hr at 4°C with 400 μg TEV protease to remove the CBP tag. The digest was diluted threefold with buffer D (25 mM Tris-HCl pH 7.5/1 mM EDTA/10 % glycerol)/1 mM DTT to reduce the final NaCl concentration to 100 mM, and fractionated on a MonoQ 5/50 GL  column in buffer D/1 mM DTT using a gradient of 0.1–1 M NaCl over 20 column volumes. Fractions containing FACT were pooled and concentrated to a volume of 0.5 ml using an Amicon spin concentrator (Millipore). The concentrate was gel-filtered on a 24 ml Superdex 200 10/300 GL  column equilibrated in 25 mM HEPES-KOH pH 7.5/1 mM EDTA/10 % glycerol/300 mM KOAc/1 mM DTT. Elution fractions were analyzed by SDS-PAGE and Coomassie stain. FACT-containing peak fractions were pooled, spin-concentrated, aliquoted, snap-frozen and stored at −80°C.FACT complex was purified after overexpression in yeast cells harboring codon-optimized copies of 2-TEV complex was supplemented with 22.5-fold molar excess of TEV protease and incubated for 1 hr at 30°C. The reaction was fractionated on a Superdex 200 Increase 10/300 GL  gel filtration column in 45 mM HEPES-KOH pH 7.6/5 mM Mg(OAc)2 / 0.02 % NP-40 substitute/10 % glycerol/100 mM KOAc/1 mM ATP/1 mM DTT, and Cdt1·MCM2Δ127-containing peak fractions pooled, aliquoted, snap-frozen in liquid nitrogen and stored at −80°C.Purified Cdt1·Mcm2-72 / 0.5 mM EDTA/0.5 mM EGTA/0.01 % NP-40 substitute/10 % glycerol/1 mM AMP-PNP/1 mM DTT for 30 min at 30°C. The sample was fractionated on a Superdex 200 Increase 3.2/300  gel filtration column in 25 mM HEPES-KOH pH 7.6/10 mM Mg(OAc)2/0.02 % NP-40 substitute/5 % glycerol/185 mM KOAc/1 mM DTT/1 mM AMP-PNP. Elution fractions were analyzed by SDS-PAGE and Coomassie stain or western blotting.1.2 μM purified DDK was incubated with 1.4 μM purified Rad53 variant in 32 mM HEPES-KOH pH 7.6/200 mM KOAc/10 mM Mg(OAc)MCM loading, MCM phosphorylation, and MCM-DDK binding assays were performed on a linear 3 kbp ARS305-containing DNA covalently linked on one end to HpaII methyltransferase (M.HpaII) and immobilized on paramagnetic beads via a 5’ photocleavable biotin on the other end. The template was PCR-amplified from p470 using oligo DR772, which contains a photocleavable 5’ biotin moiety, and oligo DR2417, which contains a M.HpaII-binding sequence modified with 5-fluoro-2′-deoxycytidine (BioSynthesis). The purified PCR product was coupled to Dynabeads M280 streptavidin magnetic beads (Invitrogen). M.HpaII (NEB) was conjugated to bead-bound DNA in 50 mM Tris-HCl pH 7.5, 10 mM EDTA, 100 μM SAM at a ratio of 4 units M.HpaII per 90 fmol of DNA for 16 hr at 37°C with agitation. M.HpaII-conjugated bead-bound DNA was washed and stored in 10 mM HEPES-KOH pH 7.6/50 mM KOAc/1 mM DTT at 4°C.in vitro DNA replication assays were performed on ARS1-containing p1017 (4.8 kbp) or ARS305-containing p470 (10 kbp) DNA. Plasmid DNAs were initially isolated using a maxiprep kit (Qiagen). To remove nicked plasmid species, purified plasmid DNA was fractionated on a 10–40% sucrose gradient in 20 mM Tris-HCl pH 7.5/1 mM EDTA/1M NaCl using an AH-629 swinging bucket rotor (Thermo Scientific) at 27,000 rpm for 20 hr at 20°C. 0.5 ml fractions were collected and analyzed by agarose gel-electrophoresis in the absence of ethidium bromide. The gel was stained post-run with ethidium bromide. Supercoiled DNA-containing fractions were pooled, dialyzed against 10 mM Tris pH 7.5/2 mM EDTA, concentrated using an Amicon spin concentrator (Millipore) to 1 to 2 mg/ml, and stored in aliquots at −20°C.The plasmid unwinding assay was performed on circular 3 kbp ARS1-containing p79, while the 2/0.5 mM EGTA/10 % glycerol/0.1 mg/mL BSA for 30 min at 4°C. 0.5 μg of purified supercoiled plasmid DNA was subsequently added along with 3 mM ATP, 20 mM creatine phosphate, and 50 ng/μL creatine kinase in a total volume of 10 μL for 1 hr at 30°C. The chromatin template was immediately used for in vitro replication.To generate chromatinized templates for DNA replication assays, 1.5 μg Nap1, 0.5 μg Histone octamer, 0.2 μg Isw1a, and 0.8 pmol ORC were mixed and incubated in 10 mM HEPES-KOH pH 7.5/50 mM KCl/5 mM MgCl2/0.02 % NP-40 substitute/5 % glycerol/100 mM KOAc/1 mM DTT/5 mM ATP or ATPγS, using 88 nM ORC, 86 nM Cdc6, 420 nM Cdt1·Mcm2-7 (wildtype or mutant as indicated), and 0.3 μg of bead-bound DNA. After the reaction, beads were magnetically separated from the supernatant, and washed once with Wash Buffer (45 mM HEPES-KOH pH 7.6/5 mM Mg(OAc)2/1 mM EDTA/1 mM EGTA/0.02 % NP-40 substitute/10 % glycerol)/300 mM KOAc, once with Wash Buffer/500 mM NaCl, and once with Binding Buffer (25 mM HEPES-KOH pH 7.6/10 mM Mg(OAc)2/0.02 % NP-40 substitute/5 % glycerol/100 mM KOAc). Beads were resuspended in Binding Buffer/1 mM DTT and supplemented with either 9.45 μM TEV protease (a 22.5-fold molar excess over Cdt1·Mcm2-7) or an equal volume of TEV protease storage buffer (50 mM Tris-HCl pH 7.5/1 mM EDTA/10 % glycerol/100 mM NaCl/1 mM DTT) as a mock control in a total volume of 40 μL. The reactions were incubated for 1 hr at 30°C with agitation. Beads were magnetically separated from the supernatant and washed once with Wash Buffer/300 mM KOAc, once with Wash Buffer/500 mM NaCl, and once with Binding Buffer. Beads were resuspended in 20 μL Wash Buffer/50 mM KOAc/1 mM DTT, and the DNA eluted from the beads by exposure to UV312 nm for 10 min using a hand-held UV lamp. The supernatant, containing the DNA and DNA-bound proteins, was analyzed by SDS-PAGE followed by silver staining and immunoblot. Replicate experiments were performed two to three times to ensure reproducibility.MCM loading reactions were carried out with agitation for 30 min at 30°C in a 40 μL reaction volume in 25 mM HEPES-KOH pH 7.6/10 mM Mg(OAc)312 nm for 10 min. The supernatant, containing the DNA and DNA-bound proteins, was analyzed by SDS-PAGE followed by silver staining and immunoblot.MCM loading was carried out as described above. Following the TEV protease or mock cleavage and wash steps, beads were resuspended in Binding Buffer/5 mM ATP/1 mM DTT and supplemented with purified DDK at 150 nM or indicated concentrations in a total volume of 40 μL. The reaction was incubated for 20 min at 30°C with agitation. Beads were magnetically separated from the supernatant, and washed once with Wash Buffer/300 mM KOAc, once with Wash Buffer/500 mM NaCl, and once with Binding Buffer. Beads were resuspended in 20 μL Wash Buffer/50 mM KOAc/1 mM DTT, and the DNA was eluted from the beads by exposure to UV2 and 5 mM ATP or AMP-PNP for 20 min at 30°C before adding to the resuspended beads. For sequential addition, the binding reaction was carried out as above for 10 min instead of 20 min, at which point 250 nM Rad53 was added and the reaction carried out for another 10 min. Beads were magnetically separated from the supernatant and washed once with Wash Buffer/100 mM KOAc. For 312 nm for 10 min. The supernatant, containing the DNA and DNA-bound proteins, was analyzed by SDS-PAGE followed by silver staining and immunoblot. Experiments were repeated two to three times to ensure reproducibility.MCM loading was carried out as described above. For 2 / 0.02 % NP-40 substitute/5 % glycerol/100 mM KOAc/1 mM DTT in a reaction volume of 10 μL for 1 hr at 30°C. MCM loading was subsequently carried out in 25 mM HEPES-KOH pH 7.6/10 mM Mg(OAc)2/0.02 % NP-40 substitute/5 % glycerol/100 mM KOAc/1 mM DTT/3.5 mM ATP using 65 nM ORC, 104 nM Cdc6, 130 nM Cdt1·Mcm2-7, 20 mM creatine phosphate, and 50 ng/μL creatine kinase in a reaction volume of 20 μL for 30 min at 30°C. Where indicated, 2.9 μM TEV protease was added (a 22.5-fold molar excess over Cdt1·Mcm2-7) for 1 hr at 30°C. 60 nM DDK was then added for 20 min at 30°C. For CMG assembly and activation the reaction was supplemented with 34 nM CDK, 0.5 mg BSA, 40 nM Sld3·7, 40 nM Cdc45, 50 nM GINS, 34 nM Pol ε, 54 nM Dpb11, 40 nM Sld2, 100 nM RPA, 20 nM Top1 and 14 nM Mcm10 in a total volume of 50 μL with the salt concentration adjusted to 185 mM KOAc for 30 min at 30°C. The reaction was quenched with 17 mM EDTA, 0.2 % SDS, and 0.8 U Proteinase K (NEB) for 30 min at 37°C. DNA was purified by phenol:chloroform extraction and centrifugation through a G-25 spin column . Samples were analyzed by agarose gel-electrophoresis and post-run ethidium-bromide stain. The experiments were repeated twice.0.5 μg of supercoiled p79 plasmid DNA was first relaxed with 100 nM purified Top1 in 25 mM HEPES-KOH pH 7.6/10 mM Mg(OAc)2/0.02 % NP-40 substitute/5 % glycerol/100 mM KOAc/1 mM DTT/3.5 mM ATP/20 mM creatine phosphate/50 ng/μL creatine kinase. Where indicated, 1.8 μM TEV protease or an equivalent volume of TEV storage buffer was added for 1 hr at 30°C. 60–140 nM DDK was then added for 20 min at 30°C. For 32P-dATP or α-32P-dCTP in a total volume of 50 μL with the salt concentration adjusted to 185 mM KOAc for 30 min at 30°C. Reactions on chromatin templates were additionally supplemented with 2 μM Nhp6 and 200 nM FACT. The reactions were quenched with 17 mM EDTA, 0.2% SDS, and 0.8U Proteinase K (NEB) for 30 min at 37°C. DNA was isolated by phenol:chloroform extraction and centrifugation through a G-25 spin column . Samples were run on 0.8 % denaturing agarose gel in 30 mM NaOH/2 mM EDTA, dried, and exposed to a phosphorimager screen. Images were scanned on a Typhoon FLA-9500 and analyzed with ImageJ.MCM loading was performed for 30 min at 30°C in a reaction volume of 20 μL including 40 nM ORC, 64 nM Cdc6, 80 nM Cdt1·Mcm2-7, 0.5 μg supercoiled plasmid DNA and a reaction buffer containing 25 mM HEPES-KOH pH 7.6/10 mM Mg(OAc) In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:The paper reports an interesting role for the DDK (Cdc7-Dbf4) protein kinase in activating the Mcm2-7 double hexamer after formation of pre-Replictive Complexes are formed on origins of DNA replication. The activity is mediated by DDK binding to an essential region within the Mcm2 amino-terminus and DDK then phosphorylates Mcm4 and Mcm6 subunits. The paper also demonstrates a new role for the checkpoint kinase Rad53 that blocks DDK activation of the Mcm2-7 double hexamer. The observations point to a new mechanism for control of DNA replication initiation.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, including Bruce Stillman as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by a Reviewing Editor and Kevin Struhl as the Senior Editor.Thank you for submitting your article "Antagonistic control of DDK binding to licensed replication origins by Mcm2 and Rad53" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife papers without additional data, even if they feel that they would make the manuscript stronger. Thus the revisions requested below only address clarity and presentation.We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, we are asking editors to accept without delay manuscripts, like yours, that they judge can stand as Summary:The authors have studied how the DDK (Cdc7-Dbf4) protein kinase activates the initiation of DNA replication. They show, using an Mcm2 subunit in which the amino-terminus can be removed, that this region of Mcm2 is not required for Mcm2-7 double hexamer (DH) formation, but is required for DNA replication. The Mcm2 amino terminus recruits the DDK and promotes phosphorylation of Mcm4 and Mcm6 subunits, but after that it is not required for the Sld3-dependent step in the initiation of DNA replication. Finally, the authors address the role of Rad53 in the inactivation of DDK phosphorylation. They find that Rad53 inhibits DDK binding to loaded Mcm2-7 double hexamers or that it promotes DDK removal from the Mcm2-7 DH and find that this inhibition is lessened by mutating the Rad53 kinase domain but does not require ATP hydrolysis or phosphorylation of Mcm2-7 by DDK or Rad53.This is a nice paper whose strength are the studies identifying the N-terminal extension of Mcm2 as a DDK binding site on the Mcm2-7 double hexamer. The evidence that removing this binding site inhibits Mcm2-7 phosphorylation, DNA unwinding, and DNA replication initiation is robust and compelling. The use of a protease cleavable form of the Mcm2 protein is a nice touch that allows the removal of the tail at various times in any reaction. This provides clear evidence that the 1-127 region of Mcm2 is important at the DDK step of the initiation reaction. Although this finding does not eliminate additional roles for this region that act later during replication, the authors point out that the distribution of products is not changed suggestion no role during elongation. It is interesting that there is relatively little effect of including chromatinized DNA in the reaction, given that part of what is removed is the binding site for histones.The last part of the work regarding the involvement of Rad53, and specifically that Rad53 dimerization is needed for DDK displacement by Rad53, is more speculative and would need more evidence to validate the model presented in Figure 7. Thus, toning down the discussion on this point would be valuable.The revised paper does not require new experimental data, but addressing the issues raised below in a revised manuscript would strengthen the report.Revisions:1) The level of DDK required for maximal binding to the Mcm2-7 DH and the phosphorylation of Mcm4 and Mcm6 is 75nM under the conditions employed, but Figure 5—figure supplement 1 shows that 300nM of DDK does not saturate the amount of DNA synthesis. Perhaps the authors could comment on this difference. Of course, DDK might perform multiple functions in the initiation of DNA replication.2) In Figure 1, does the NTE truncation affect MCM loading (as well as DH stability)? This issue is actually addressed in Figure 5B, but the issue would usefully be covered at the start, either by including data in this figure or by making reference in the text to where the experiment will be presented.3) Figure 3: Why does the efficiency of replication appear increased when TEV protease is added to MCM2-wt ? Also, in Figure 3B, it would be useful to show a “-TEV protease” negative control within the Mcm2-TEV set of reactions.4) Figure 4: Figure 4C: What concentration of DDK was used ? Figure 4E: A band shift suggests that Dbf4 seems to be phosphorylated/modified when ATP is added. Could the authors explain, and discuss possible reasons and interpretation?Could the authors explain in the text for what reason ATP/an ATP analogue might be needed to promote full levels of DDK association?5) Figure 5: Figure 5C: The text states that there is no evidence of asymmetric or uni-directional replication coming from the activation of a single hexamer, but the reasons given for this interpretation are difficult to understand. This conclusion needs to be better explained.6) The weakest aspect of the paper revolves around the model that DDK bound Mcm2 is required for both of the two Mcm2-7 complexes for efficient initiation to occur. Although the authors propose a model that the 1:1 complex is inactive, the data is not sufficiently different from what would be expected if this complex were just slightly less active (e.g. 2-fold). More importantly, the model of that DDK bound to one kinase phosphorylates the tails of the other kinase has no support in the paper and should be removed or additional experiments added. If this trans model is right, then there are clear experiments to demonstrate this is the case that are well within the authors' abilities. They should be added to support the model or the model should be left out of the paper so that if or when they demonstrate this interesting idea it can be a clear new result. This is not an essential add but if such experiments are not added then the model should be removed. The authors would need more replicates of Figure 6 C, D, and E to be a convincing quantitative argument. Even then, there are clearly other explanations for the data.7) Figure 6: It is shown that like wt Rad53, a kinase-dead mutant Rad53 is largely able to repress DDK binding to MCM, but the kinase-dead mutant Rad53 is unable to displace DDK from pre-formed DH. The authors suggest that their result would be explained if Rad53 dimerization is the essential requirement to inhibit DDK binding to Mcm2-7, mediated by Rad53 trans-autophosphorylation. While an interesting suggestion, they do not really test this idea. It is therefore suggested that the authors modulate the conclusions and model arising from Figure 6, to avoid the false impression that the necessity for Rad53 trans-auto-activation and dimerization has been demonstrated. One possibility would be to remove the data on the kinase-dead mutant altogether. Alternatively, the authors should considerably tone down their interpretation and discussion concerning the phenotype of the kinase-dead mutant . Revisions:1) The level of DDK required for maximal binding to the Mcm2-7 DH and the phosphorylation of Mcm4 and Mcm6 is 75nM under the conditions employed, but Figure 5—figure supplement 1 shows that 300nM of DDK does not saturate the amount of DNA synthesis. Perhaps the authors could comment on this difference. Of course, DDK might perform multiple functions in the initiation of DNA replication.We assume the reviewer was referring to the MCM DH-DDK binding data in Figure 4A, which demonstrate saturation of DDK binding to wildtype MCM DHs at 75 nM DDK. Please note that the experiment in Figure 5—figure supplement 1B has been carried out with the Mcm2-D127 truncation in order to demonstrate that increased DDK concentrations can partially suppress the replication defect of Mcm2-D127, consistent with the notion that the Mcm2 NTE is an affinity determinant for DDK. This experiment, therefore, corresponds to the MCM DH-DDK binding experiment in Figure 4B, lanes 5-8, which also does not reach saturation of DDK binding to MCM DHs even at 300 nM DDK.Nonetheless, we do indeed observe that DNA synthesis does also not saturate at 75 nM DDK in replication assays with Mcm2-wt . We believe this is due to the fact that in the replication assay DDK is added to a complex mixture of MCM DHs and free Cdt1·MCM complexes present at the MCM loading step of the replication reaction, whereas free Cdt1·Mcm2-7 complexes are removed by washes of the DNA beads in the MCM DH-DDK binding experiments of Figure 4 prior to addition of DDK. It is, therefore, likely that the excess of free Cdt1·MCM competes with DNA-bound MCM DHs for DDK in the replication assay.2) In Figure 1, does the NTE truncation affect MCM loading (as well as DH stability)? This issue is actually addressed in Figure 5B, but the issue would usefully be covered at the start, either by including data in this figure or by making reference in the text to where the experiment will be presented.We have moved panels A and B from the original Figure 5 to Figure 1 as new panels D+E and have made according changes to the main text.3) Figure 3: Why does the efficiency of replication appear increased when TEV protease is added to MCM2-wt ? Also, in Figure 3B, it would be useful to show a “-TEV protease” negative control within the Mcm2-TEV set of reactions.The “-TEV protease” replication control experiment for Mcm2-TEV is shown in the original Figure 2—figure supplement 2.4) Figure 4: Figure 4C: What concentration of DDK was used ?DDK was used at 150 nM. While previously noted in the Materials and methods section, we now indicate this also in the figure legend to Figure 4C for clarity.Figure 4E: A band shift suggests that Dbf4 seems to be phosphorylated/modified when ATP is added. Could the authors explain, and discuss possible reasons and interpretation?DDK is known to undergo autophosphorylation. This has been shown for both yeast and human DDK. We now provide appropriate references in the main text.Could the authors explain in the text for what reason ATP/an ATP analogue might be needed to promote full levels of DDK association?We can only speculate about the reasons, but now consider in the Discussion the possibility that by analogy to other protein kinases ATP binding either induces a conformational change or stabilizes a conformation in Cdc7 that is more conducive to substrate engagement.5) Figure 5: Figure 5C: The text states that there is no evidence of asymmetric or uni-directional replication coming from the activation of a single hexamer, but the reasons given for this interpretation are difficult to understand. This conclusion needs to be better explained.An expanded explanation is now included in the main text.6) The weakest aspect of the paper revolves around the model that DDK bound Mcm2 is required for both of the two Mcm2-7 complexes for efficient initiation to occur. Although the authors propose a model that the 1:1 complex is inactive, the data is not sufficiently different from what would be expected if this complex were just slightly less active (e.g. 2-fold). More importantly, the model of that DDK bound to one kinase phosphorylates the tails of the other kinase has no support in the paper and should be removed or additional experiments added. If this trans model is right, then there are clear experiments to demonstrate this is the case that are well within the authors' abilities. They should be added to support the model or the model should be left out of the paper so that if or when they demonstrate this interesting idea it can be a clear new result. This is not an essential add but if such experiments are not added then the model should be removed. The authors would need more replicates of Figure 6 C, D, and E to be a convincing quantitative argument. Even then, there are clearly other explanations for the data.cis- or trans-phosphorylation of Mcm4/6 by DDK. In response to the reviewer’s comments we have, therefore, removed the interpretation of the data of Figure 5 with regard to DDK stoichiometry and refocused the description of the data in Figure 5 on the lack of uni-directional origin firing in the presence of both Mcm2-WT- and Mcm2D127. In addition, we now point out in the Discussion that it will be important in future studies to determine the stoichiometry of DDK in the DDK-MCM DH complex. At this point we do not make any assumptions in the paper regarding the cis-/trans-phosphorylation mechanism of Mcm4/6 by DDK.We agree with the reviewer that additional evidence is needed to address the stoichiometry of the DDK-MCM DH complex and to resolve the issue of 7) Figure 6: It is shown that like wt Rad53, a kinase-dead mutant Rad53 is largely able to repress DDK binding to MCM, but the kinase-dead mutant Rad53 is unable to displace DDK from pre-formed DH. The authors suggest that their result would be explained if Rad53 dimerization is the essential requirement to inhibit DDK binding to Mcm2-7, mediated by Rad53 trans-autophosphorylation. While an interesting suggestion, they do not really test this idea. It is therefore suggested that the authors modulate the conclusions and model arising from Figure 6, to avoid the false impression that the necessity for Rad53 trans-auto-activation and dimerization has been demonstrated. One possibility would be to remove the data on the kinase-dead mutant altogether. Alternatively, the authors should considerably tone down their interpretation and discussion concerning the phenotype of the kinase-dead mutant .We agree with the reviewer that the role of Rad53 autophosphorylation and dimerization needs to be affirmed by additional data. This is complicated by the fact that activation/autophosphorylation depends on Rad53 dimerization. In light of the reviewer’s comments we have removed the statement on the roles of Rad53 autophosphorylation and dimerization from both the Abstract and the conclusions in the main text. Instead, we raise the possibility in the Discussion that the Rad53 oligomeric state or its phosphorylation state, or both, may control the ability of Rad53 to sequester DDK.Moreover, we now present additional data demonstrating that Rad53-wt, but not Rad53-kd, can form a stable complex with DDK, which supports our model for the steric inhibition of DDK by Rad53. In addition, while our previous experiments have focused on the effect of Rad53 on DDK binding to MCM DHs, we now include data that extend the effect of Rad53 on DNA synthesis. We consider our observation of a steric mechanism for Rad53 function a major novel finding and consider the data involving Rad53-kd important to support this observation.