HPAI H7N9 bearing NA222L-119V had good replication ability with peak value of more than 6log10 TCID50/ml in MDCK cells. H7N9/PR8 virus bearing NA222L-119V substitutions leaded to diffuse pneumonia, significant weight loss and fatality in mice. NA E119V made H7N9/PR8 virus resistant to oseltamivir, and I222L-E119V had synergistic resistance to oseltamivir in mice. Due to the good fitness of drug resistant strains of HPAI H7N9 virus, it is necessary to strengthen drug resistance surveillance and new drug research.That the high frequency and good replication capacity of strains with reduced susceptibility to neuraminidase inhibitors (NAIs) in highly pathogenic avian influenza H7N9 (HPAI H7N9) virus made it a significance to further study its drug resistance. HPAI H7N9 viruses bearing NA I222L or E119V substitution and two mutations of I222L-E119V as well as their NAIs-sensitive counterpart were generated by reverse genetics for NA inhibition test and replication capability evaluation in vitro. The attenuated H7N9/PR8 recombinant viruses were developed to study the pathogenicity and drug resistance brought by the above substitutions to mice. The IC As the mutant of H7N9, HPAI H7N9 virus is characterized by the insertion of polybasic amino acids at the cleavage of its hemagglutinin (HA) protein5. Although the mortality of HPAI H7N9 virus in laboratory confirmed human cases is not significantly higher to that of its low pathogenic (to birds) counterpart, the former seems to have a shorter course of disease and more severe clinical symptoms6.Human infection with highly pathogenic avian influenza (HPAI) H7N9 virus appeared at the end of 2016 and re-emerged at 20197. The approved NAIs in China, included oseltamivir, zanamivir and peramivir, and most H7N9 patients received oseltamivir10. Laninamivir, as well as the polymerase inhibitor baloxavir marboxil, which were recently approved by some countries/areas, are possible antiviral options against influenza infection13. Due to the poor error correction ability of the single stranded RNA virus14, influenza virus is prone to adaptive mutation for survival15. Early use of appropriate NAIs can prevent mice infected with lethal dose of H7N9 virus from severe weight loss and death16. However, amino acid mutation at the key site of neuraminidase (NA) may lead to reduced susceptibility of influenza virus to NAIs and thus weaken the therapeutic effect17. There are 19 highly conserved residues in the NA active site of all influenza A and B viruses. These include eight catalytic residues  that directly contact the sialic acid (SA) and 11 framework residues  that support the enzymatic binding pocket19. When the conserved amino acids in the NA active site of influenza virus mutated, the susceptibility to NAIs may be reduced. NA R292K substitution could lead to resistance among multiple NA subtypes of influenza virus, but other resistance mutation sites may be subtype-specific21. Recombinant N9 proteins bearing substitutions showed that R292K, H274Y conferred high increase in oseltamivir half-maximal inhibitory concentration (IC50), and E119D conferred high zanamivir IC50. Additionally, R152Kof A (H7N9) virus conferred reduced inhibition by laninamivir22.Neuraminidase inhibitors (NAIs) are the only way to treat influenza infection in most countries in the world currently23. HPAI H7N9 viruses with reduced susceptibility to neuraminidase inhibitors  showed good replication capacity in mammalian cells24. Interestingly, NA 119V or D substitutions in HPAI H7N9 showed reduced susceptibility to oseltamivir and zanamivir respectively24. Like E119 amino acids, I222 amino acids are also located in the frame region of NA, and it has been reported that I222 substitutions combined with other frame region resistance sites have been detected in seasonal influenza cases26. In this study, we want to test if the two framework mutations (E119V and I222L) of NA could coexist in HPAI H7N9 virus, and study its effect on viral fitness and drug resistance effect in mammalian cells and mice.Our previous research found that the rate of strains isolated from human with reduced susceptibility to NAIs in HPAI H7N9 virus was highIn this study, A/Guangdong/17SF006/2017 (H7N9) was used as a backbone. The NAIs-sensitive HPAI H7N9 virus constructed by reverse genetics containing NA 119E, 222I, was called rg006NA. The HPAI H7N9 reassortment viruses bearing substitutions were called rg006NA119V, rg006NA222L and rg006NA222L-119V according to their mutations.–1–10–6, and none were lethal to 9–11 day embryonated chicken eggs. The results of trypsin dependence test showed that all the four H7N9/PR8 recombinant viruses could not replicate without TPCK-trypsin, suggesting that the virus was dependent on TPCK-trypsin. The results showed that the recombinant H7N9/PR8 virus was attenuated successfully.The H7N9/PR8 reassortment virus and its NA mutants were called rg006NA/PR8, rg006NA119V/PR8, rg006NA222L/PR8 and rg006NA222L-119V/PR8 respectively. The sequencing of H7N9/PR8 recombinant viruses showed that 339–342KRTA polybasic amino acids in HA had been knocked out correctly, and all the six internal genes were from PR8. The four recombinant H7N9/PR8 viruses of rg006NA/PR8, rg006NA119V/PR8, rg006NA222L/PR8 and rg006NA222L-119V/PR8 were cultured at 35 °C for 48 h at a dilution of 10The recombinant viruses used in this study were confirmed by sequencing and listed in Supplementary Table 50 of oseltamivir (mean IC50 (nM) ± SD, 141.73 ± 10.52) and zanamivir (mean IC50 (nM) ± SD, 11.81 ± 0.32) respectively. The IC50 fold change of oseltamivir or zanamivir to rg006NA222L were 4.50 and 2.46 times respectively, which did not meet the phenotype resistance standard. However, when I222L and E119V substitutions coexist in the NA protein of HPAI H7N9 virus, the IC50 fold change of oseltamivir to rg006NA222L-119V is 306.34 (mean IC50 (nM) ± SD, 493.20 ± 29.10) times than that of its susceptible strain, and 3.5 times than that of the E119V single point mutant virus. The result showed that NA I222L-E119V substitutions had synergistic resistance effect to oseltamivir for HPAI H7N9 virus. On the other hand, rg006NA222L-119V induced a mean 7.53-fold increase in the IC50 of zanamivir, which is similar to the value of E119V single point mutant virus. Besides, the fold change of IC50 value of all the three mutant viruses to peramivir and laninamivir was less than 10, suggesting normal inhibition.The susceptibility of HPAI H7N9 reassortment viruses to NAIs was assessed Table . The sub#P < 0.05), but they showed similar replication level after 48hpi in MDCK cells. Replication peak value of rg006NA222L-119V reached more than 6log10 TCID50/ml in 48 h although its replication level was lower than that of rg006NA(*P < 0.05). The results showed that rg006NA222L-119V, the two sites mutant virus with synergistic drug resistance effect to NAIs, had good replication ability in mammalian cells though lower than that of the sensitive strain.To test the effect of the NAIs resistance mutations on the growth characteristics of the HPAI H7N9 virus, we determined infectious titers in MDCK cells. MDCK cells were infected with rg006NA, rg006NA119V, rg006NA222L and rg006NA222L-119V virus by 0.001 MOI. At time points 0, 18, 24, 48, 72 and 96hpi, viral titers were determined on MDCK cells. Rg006NA and rg006NA119V virus showed similar replication level in MDCK cells at different time points (P > 0.05) , all mice infected with these four strains survived except one mouse in the 104 TCID50 rg006NA222L/PR8 group  . There was no significant difference of hazard ratio between the mutant virus (rg006-NA119V/rg006NA222L/PR8/rg006NA119V-222L/PR8) and rg006NA/PR8 (P > 0.05).The survival ratio of rg006NA222L-119V/PR8 virus at 105–106 TCID50 was 0% (the same as rg006NA222L/PR8), which was even lower than that of rg006NA  and rg006NA119V   of rg006NA/PR8, rg006NA119V/PR8, rg006NA222L/PR8 and rg006NA222L-119V/PR8 virus to C57 mice was 105 TCID50, 104.75 TCID50, 104.38 TCID50 and 104.5 TCID50, respectively.The mouse lethal dose of 50% animals (MLDP > 0.05) except that in lung tissue 1dpi (#P < 0.05) of the 105 TCID50 group , which indicated viral replication, and on 5dpi, no viral load was detected in trachea. When administered with 105 TCID50, the viral load in trachea of the mice decreased from 1 to 3 dpi, and on 5dpi it could only be detected in one mouse in the rg006NA222L-119V/PR8 and the rg006NA222L/PR8 group respectively. And on 7 dpi, no viral load was detected in trachea of all the mice. When administered with 104 TCID50 or 105 TCID50 of the 4 viruses, the viral load in lungs of mice was high at 1 dpi and 3 dpi, and then decreased from 5 dpi. On 7 dpi, viral load in lung could only been detected in one mouse infected with rg006NA/PR8, rg006NA222L/PR8 or rg006NA222L-119V/PR8 virus in the 105 TCID50 dose group respectively. On 14 dpi, no virus load was detected in lungs of all the mice. In general, the viral load in the respiratory tract of mice infected with rg006NA/PR8, rg006NA119V/PR8, rg006NA222L/PR8 and rg006NA222L-119V/PR8 virus bore out similar trend.The viral load of rg006NA/PR8, rg006NA119V/PR8, rg006NA222L/PR8 and rg006NA222L-119V/PR8 in the respiratory tract of mice were shown in Fig. oup Fig. B. When a50) of rg006NA/PR8, rg006NA119V/PR8, rg006NA222L/PR8 and rg006NA222L-119V/PR8 was 0.83 TCID50, 1 TCID50, 0.83 TCID50 and 1.38 TCID50 respectively. The results showed that the infectivity of the four viruses to mice was similar and all of them can infect mice at a low dose.Through the detection of HA antibody in mouse serum . There was no statistical difference between the survival day of rg006NA119V/PR8 and rg006NA222L-119V/PR8 infected mice in oseltamivir or non-treatment group (P > 0.05). There was no significant difference of hazard ratio between the oseltamivir-treated and non-treated group of all the four viruses (P > 0.05) (TableWe infected mice with 2MLD05) Table. The rg0P < 0.05, Fig. P < 0.05, Fig. P < 0.05, Fig. P < 0.05, Fig. The viral load of rg006NA/PR8, rg006NA119V/PR8, rg006NA222L/PR8 and rg006NA222L-119V/PR8 in lungs of mice with oseltamivir administration are shown in Fig. Four days after infection, lungs of rg006NA/PR8, rg006NA119V/PR8, rg006NA222L/PR8 and rg006NA222L-119V/PR8 infected mice with oseltamivir administration were taken for pathological observation. By HE staining Fig. A, withouThe distribution of NP antigen was consistent with the above result, that the use of oseltamivir reduced the lung antigens in mice infected with rg006NA/PR8 and rg006NA222L/PR8 Fig. B, but diIn all, E119V substitution in NA made the H7N9/PR8 reassortment virus resistant to oseltamivir in mice. What’s more, NA substitutions of I222L and E119V had synergistic resistance effect on the H7N9/PR8 reassortment virus to oseltamivir in mice.27. After a single site mutation of R292K, the enzyme activity of HPAI H7N9 virus decreased significantly and its replication ability decreased to some extent19. According to the substitution with reduced susceptibility to NAIs detected in HPAI H7N9 cases24, we used reverse genetic technology to develop double sites mutant HPAI H7N9 viruses carrying I222L-119V, H274Y-R292K and E119V-R292K, but only the enzyme activity of rg006NA222L-119V virus was enough to perform the NA inhibition test.Generally, when influenza viruses possess drug resistant mutations, their replication level in cells or their pathogenicity to animals may be reduced to varying degrees, especially when mutations occur in catalytic residues that directly contact the sialic acid (SA)26. Besides, prophylactic treatment with oseltamivir had failed in two H1N1 with H274Y-I222M mutations infected patients25. These results show that the two site mutations in the framework region can coexist in influenza viruses, and some of them have synergistic drug resistance effect. However, these studies are limited to clinical discovery or cell level studies of seasonal influenza viruses, and there are few studies of influenza virus with double resistance sites in a mouse model.Seasonal influenza viruses bearing two mutations in the frame region of NA had been report before. A H3N2 virus possessing the mutations E119V-I222V, isolated from human cases and induced a synergistic effect on oseltamivir resistance test in vitro28 and with the amino group on C-4 of oseltamivir29. E119A/D/G/I/V substitutions have been examined conferring resistance to NAIs in influenza viruses of various subtypes34. And in this study, E119V substitution in HPAI H7N9 virus induced a mean 88.03-fold increase in the IC50 of oseltamivir. The I222 residue is a conserved residue in NA of influenza viruses and I222V/M/L/R/T substitutions had been reported with the N1, N2, and B backgrounds conferring moderate resistance to oseltamivir36. However, NA I222L substitution of HPAI H7N9 did not lead to drug resistance to oseltamivir, zanamivir, peramivir and laninamivir according to WHO-AVWG criteria. In this study, the two mutations of E119V-I222L were chosen to study the synergistic drug resistance of HPAI H7N9 virus in mice. The IC50 fold change of oseltamivir to rg006NA222L-119V is 306.34 times than that of its susceptible strain, and 3.5 times than that of the E119V single point mutant virus, which showed synergistic resistance effect. A crystal structure model of the NA monomer of the A/Anhui/1/2013 H7N9 virus (PDB ID: 4MWJ) was used to display and label the two mutations (E119V and I222L) in H7N9 virus  is taken orally and then converted to its active form (oseltamivir carboxylate) to disseminate systemically. The recommended oral dose for adolescents and adults is 75 mg oseltamivir twice daily for 5 days40. Excessive concentration of oseltamivir is not conducive to antiviral treatment43. The concentration of oseltamivir selected in this study is 20 mg/kg/day, which is feasible and effective in this study. Consistent with the result of NA inhibition test in vitro, E119V-I222L in N9 from HPAI H7N9 virus have synergistic drug resistance effect to oseltamivir in mice  compared with rg006NA119V/PR8, rg006NA222L/PR8 and their sensitive strain  virus even lead to more weight loss and fatality rate than that of rg006NA/PR8 and rg006NA119V/PR8 in mice , lung/trachea virus load and lung pathological changes among the four virus infection mice groups. The N9 E119V-I222L virus which had synergistic drug resistance effect did not seriously damage of its fitness in mice.NAIs development is structure-based drug designce Figs. . When inIn terms of bio-safety and practical conditions, the recombinant viruses used in the mouse experiment were attenuated. Compared with their corresponding HPAI H7N9 viruses, the replication ability on MDCK cells of the attenuated H7N9/PR8 recombinant viruses decreased to some extent (data not shown). The pathogenicity of the H7N9/PR8 viruses was weaker than their corresponding HPAI H7N9 viruses in mice. This is one of the limitations of this study. However, the H7N9/PR8 recombinant viruses developed in this study could replicate in respiratory tract of C57 mice and lead to pneumonia, weight loss and even death, indicating the C57 mice infected with H7N9/PR8 recombinant viruses with reduced susceptibility to NAIs were feasible model for the study on the mechanism of HPAI H7N9 NAIs resistance and the preliminary selection of alternative drugs.In summary, although NA I222L did not lead HPAI H7N9 virus to drug resistance itself, it could increase the resistance of HPAI H7N9 virus with NA E119V without severely impaired fitness in vitro. Consistent with results in vitro, substitutions of I222L and E119V in neuraminidase from HPAI H7N9 influenza virus exhibited a synergistic resistance effect to oseltamivir without significantly reduced pathogenicity in mice. Due to the good fitness of drug resistant strains of HPAI H7N9 virus, it is necessary to strengthen drug resistance surveillance, as well as research and application of new drugs.All experiments including any relevant details were approved by designated institution and/or licensing committee, and all experiments were performed in accordance with the relevant guidelines.24. In short, HPAI H7N9 A/Guangdong/17SF006/2017 (abbreviated as 006) isolated from a human case was used as backbone virus. All eight gene segments of 006 strain were artificially synthesized (Sangon Biotech) and cloned into the pHW2000 vector. The NAIs sensitive HPAI H7N9 reassortant virus encoded 222I and 119E in the NA protein. A single-point mutation was introduced to convert 119E to 119V or 222I to 222L in NA. Then, in the 119V background, single-point mutations I222L in the NA gene were carried out. E119V or I222L substitutions were generated with the following primers:NA E119V Forward, gtcgcatgaaacatagggtactcttgtgactaaaacatc,NA E119V Reverse, gatgttttagtcacaagagtaccctatgtttcatgcgac,NA I222L Forward, acacatgggcccgaaacttactaagaacacaggaa,NA I222L Reverse, ttcctgtgttcttagtaagtttcgggcccatgtgt.The generation of HPAI H7N9 recombinant viruses was described in Tang et al.Mutations were introduced into the NA plasmid using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). HPAI H7N9 reassortment viruses were used to examine the effect of NA amino acid substitutions on NAIs susceptibility and viral replication efficiency in vitro.HAdel Forward, caaataggcctcttctctttggaacctcaggaacattc,HAdel Reverse, gaatgttcctgaggttccaaagagaagaggcctatttg.Considering the bio-safety and actual conditions, attenuated H7N9 reassortment viruses were prepared to evaluate the pathogenicity and drug resistance in mice. The internal six genes of the attenuated virus were from A/PR/8/34 (H1N1). The HA gene was from 006strain after deletion of its highly pathogenic molecular markers (KRTA in 339–342 residue). The recombinant H7N9/PR8 attenuated virus was prepared by transfection of the above seven plasmids and 006 NA plasmids. HA 339–342KRTA deletion was generated with the following primers:46. Supernatants in culture flask were inoculated in embryonated eggs at 35 °C. Hemagglutination test was used for detection of positive viruses. Virus stocks were sequenced for verification, and virus titrations were determined with MDCK cells. The log10 TCID50/ml was calculated by Reed and Muench method.The recombinant virus was generated by reverse genetics. In general, the eight plasmids were co-transfected into 293T/MDCK co-cultured monolayer for 72 hVirus sequencing: confirm whether each gene is correct and knock out of polybasic amino acids on HA.Lethal dose of H7N9/PR8 recombinant virus on chicken embryo.Trypsin dependent test: confirmed that the replication of recombinant H7N9/PR8 virus depends on TPCK trypsin.The recombinant H7N9/PR8 virus was evaluated as follows:24. In short, cells were grown and maintained in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum , HEPES , penicillin (100 units/ml), and streptomycin . The NA inhibitors used in vitro experiment were oseltamivir carboxylate (Hoffman-La Roche), zanamivir (GlaxoSmithKline), Peramivir (MCE company) and Laninamivir (MCE company). The pro-drug oseltamivir phosphate (oseltamivir) used in mice was purchased from MCE company. The NA inhibitors were prepared in sterile distilled water and stored in aliquots at − 20 °C.The grown and maintainance of MDCK cells and 293 T cells were described before47. IC50 values, defined as the concentration of drug required to reduce enzyme activity by 50%, were calculated using GraphPad prism5 software. Interpretation of IC50 was performed using the WHO Antiviral Working Group (WHO-AVWG) criteria49: the testing virus was compared with the drug-sensitive reference virus, for influenza A viruses, a < tenfold increase in IC50 represents normal inhibition, and a ten to 100-fold increase represents reduced inhibition, while a > 100-fold increase is highly reduced inhibition.Inhibition of NA enzyme activity of the 4 NA inhibitors was assessed in the fluorescence-based NA inhibition assay, using the NA-Fluor Influenza Neuraminidase Assay kit  as previously described24. Briefly speaking, MDCK cells were infected with the recombinant virus at an MOI of 0.001 at 37 °C 5% CO2. Cells were washed twice with PBS after 1 h of incubation, and infection medium containing TPCK-trypsin was added. At time points 0, 18, 24, 48, 72 and 96 h post-infection (hpi), supernatants were collected, and 3 biological repeats were set up for each sample. Viral titers were determined on MDCK cells.The method of growth kinetics of HPAI H7N9 viruses in MDCK cells was described before1–106 TCID50 recombinant H7N9/PR8 virus under isoflurane anaesthesia50. The weight change from 1 to 14 days post-infection (dpi) was calculated as a percentage of weight on 0 dpi (n = 5/group). Mice that showed severe disease and lost ≥ 20% of initial weight were euthanized. On 1, 3, 5, 7 and 14 dpi after inoculation, the lungs and trachea were removed from 3 mice of each group infected with 104–105 TCID50 virus, rinsed with sterile phosphate-buffered saline (PBS), homogenized, and resuspended in 1 ml of PBS. The suspensions were cleared by centrifugation at 3000×g for 20 min, and virus yield was determined by TCID50 in MDCK cells. On 3dpi, Lungs of mice infected with 104–105 TCID50 virus (n = 3/group), were fixed in 10% paraformaldehyde and then for pathological study.All mouse experiments were conducted after the approval of the Ethics Committee of the National Institute for Viral Disease Control and Prevention, China CDC (20191106039). For pathogenic study, specific-pathogen-free (SPF), female 8-week-old C57BL/6 mice were intranasal inoculated with 50 μl of 1050) of recombinant H7N9/PR8 viruses under isoflurane anaesthesia. Starting 0 dpi, mice in the drug treatment group were orally administered 100 μl oseltamivir at the dosage of 20 mg/kg/day BID  for 5 days16. Untreated mice orally received 100 μl PBS twice daily for 5 days. Body weights and survival of infected mice were monitored for 14 days daily (n = 5/group). Mice that lost more than 20% of their initial weight were euthanized. The body weight change of mice was calculated and shown as a percentage of its initial weight. On 2, 4 and 6 dpi, the lungs were removed from 3 mice per group for viral load titration and pathological study.SPF, female 8-week-old C57BL/6 mice were intranasal inoculated with 50 μl of two 50% mouse lethal doses . The distribution of antigens (influenza A nucleoprotein) was studied by immunohistochemistry (IHC).Pathological research methods were described in previous studies50)49. In short, at 14 dpi, serum samples were obtained from mice that survived from the pathogenic study. Sera were collected by retro-orbital bleed, treated with receptor-destroying enzyme for 18 h, and then heat-inactivated at 56 °C for 1 h, and tested by HI assay with 1% turkey red blood cells. HA titer ≥ 40 is positive; HA titer < 40 is negative.Hemagglutination inhibition (HI) assay test was done to calculate the mouse infective dose of 50% animals (MID37 in Protein Data Bank (PDB) was used, based on which a crystal structure model of the NA monomer was obtained. DeepViewv4.1.0 software was used to display and label drug resistance mutations.The existing NA protein crystal structure of A/Anhui/1/2013 H7N9 virus (PDB ID: 4MWJ)P value of < 0.05 was considered significant.NAIs susceptibility and viral replication were analysis by the GraphPad Prism 5.0 software. Student’s t test was used to evaluate differences between two groups. ANOVA test and Kruskal–Wallis H test were used to evaluate differences among four groups. Cox regression was used to analyze hazard ratio by SPSS software. A All experiments involving HPAI H7N9 viruses were operated by qualified person in BSL-3 laboratory of National Institute for Viral Disease Control and Prevention, Chinese center for disease control and prevention.We confirmed the study was carried out in compliance with the ARRIVE guidelines.Supplementary Figure S1.Supplementary Table S1.Supplementary Table S2.


Pseudomonas aeruginosa are involved in the control of biofilm formation and virulence factor production. The current study evaluated the ability of halogenated dihydropyrrol-2-ones (DHP) (Br (4a), Cl (4b), and F (4c)) and a non-halogenated version (4d) to inhibit the QS receptor proteins LasR and PqsR. The DHP molecules exhibited concentration-dependent inhibition of LasR and PqsR receptor proteins. For LasR, all compounds showed similar inhibition levels. However, compound 4a (Br) showed the highest decrease (two-fold) for PqsR, even at the lowest concentration (12.5 µg/mL). Inhibition of QS decreased pyocyanin production amongst P. aeruginosa PAO1, MH602, ATCC 25619, and two clinical isolates (DFU-53 and 364707). In the presence of DHP, P. aeruginosa ATCC 25619 showed the highest decrease in pyocyanin production, whereas clinical isolate DFU-53 showed the lowest decrease. All three halogenated DHPs also reduced biofilm formation by between 31 and 34%. The non-halogenated compound 4d exhibited complete inhibition of LasR and had some inhibition of PqsR, pyocyanin, and biofilm formation, but comparatively less than halogenated DHPs.Quorum-sensing (QS) systems of  Pseudomonas aeruginosa is an opportunistic Gram-negative bacterium that is acknowledged as a critical pathogen by the World Health Organisation (WHO) .,29.2O5),P. aeruginosa strains used in this study included P. aeruginosa MH602 lasB::gfp(ASV) for LasR analysis and PAO1 pqsA::gfp for PqsR analysis, ATCC 25619, and clinical isolates DFU-53  and 364707 . All clinical isolates were patient de-identified before being gifted to us from the hospitals. Clinical bacteria from patients were previously isolated and characterized by qualified pathologists from the respective hospitals based on procedures detailed in ASM Clinical Microbiology Procedures Handbook—4th Edition, ASM Press(edited by Amy L. Leber). None of the authors were involved in the isolation or characterization of clinical isolates from patients. Bacterial isolates, once received in our research laboratory, were immediately grown in a full-strength (100%) Tryptone Soy Broth  in a test tube at 37 °C, 150 rpm for 24 h. Following 24 h of growth, the bacterial stock was prepared by mixing 1.2 mL of bacterial culture with 400 µL of DMSO (100%), and stored in 2 mL of the cryogenic vials  at −80 °C. To note: the final concentration of DMSO in the bacterial stock is 25%. P. aeruginosa isolates from the frozen stock were plated as required onto Tryptone Soy agar (Oxoid) and grown for 24 h by incubating at 37 °C. Further growth was initiated by striking a colony from the Tryptone Soy agar plate into the 5 mL of Muller–Hinton Broth  for 24 h at 37 °C, with orbital shaking at 150 rpm for all experiments.P. aeruginosa strains used in this study was tested aganist ciprofloxacin. A stock of ciprofloxacin (100 µg/mL) was prepared by dissolving it in MHB. P. aeruginosa strains were grown for 24 h at 37 °C, 150 rpm in 5 mL MHB, as mentioned above. After 24 h of growth, each P. aeruginosa culture was diluted (by resuspending in MHB to a density of 0.15 ± 0.04 (OD600nm)), either in the presence or absence of ciprofloxacin . Then, 200 µL of the dilute P. aeruginosa culture (in the presence or absence of ciprofloxacin) was placed in the 96-well plates and the growth was recorded by measuring the absorbance at OD600nm at 0 after 24 h incubation at 37 °C. Any increase in growth at 24 h was determined by subtracting the absorbance readings obtained at 0 h.The antibiotic susceptibility of all P. aeruginosa MH602 lasB::gfp(ASV) and PAO1 pqsA::gfp isolates were grown overnight in MHB, supplemented with 30 µg/mL gentamicin at 37 °C, 150 rpm [600nm, measured using a plate reader . M9 minimal media was chosen due to its low autofluorescence in comparison to MHB and Tryptone soy broth. Finally, 200 µL of diluted M9 minimal salt medium was dispensed into 96-well plates  and incubated at 37 °C with orbital shaking at 150 rpm. Production of GFP via the receptor proteins LasR and PqsR and bacterial growth was monitored every hour for up to 10 h by measuring fluorescence at Ex485nm and Em535nm and absorbance at OD600nm for LasR and PqsR and bacterial growth, respectively. Bacterial growth (OD600nm) was further measured after 24 h and 48 h of treatment. To quantify LasR and PqsR expression, the GFP fluorescence values measured at every time point were divided by their respective OD600nm absorbance. A graph was plotted as GFP/OD vs. time in hours. 150 rpm . AdditioP. aeruginosa was grown as described above. The bacterial culture (200 µL) was aliquoted in 96-well plates at a density of 0.1 ± 0.025 (OD600nm). The bacteria were incubated for 48 h at 37 °C with 150 rpm orbital shaking in M9 minimal media containing 10% MHB, in the presence or absence of DHP. After 48 h, the supernatant was removed and the biofilm was washed once with phosphate-buffered saline  to remove any loosely bound bacterial cells. The attached biofilms were then treated with 0.01% crystal violet dye and incubated for 60 min at 37 °C and 150 rpm. After 60 min, excess dye was removed by washing with PBS three times. The plates were then dried for 20 min at 37 °C, followed by the addition of 200 µL of 80% ethanol and incubation for 30 min at 37 °C, with orbital shaking at 150 rpm to dissolve the dye from the biofilm biomass. Aliquots (100 µL) of dye were transferred to a new 96-well plate, to which 100 µL of PBS was added to make up a total volume of 200 µL in each well. The plates were then analyzed for biofilm biomass by measuring absorbance at OD550nm using a plate reader. As mentioned in For assessing biofilm biomass, P. aeruginosa strains were grown overnight in MHB, except for PAO1 and MH602, which were grown in MHB supplemented with 30 µg/mL gentamicin, at 37 °C with orbital shaking at 150 rpm. Bacterial cultures grown in MHB were diluted to a total of 5 mL of M9 minimal salt media in presence or absence of DHP 12.5, 25, and 50 µg/mL and PBS or DHP ethanol solvent to a density of 0.1 ± 0.025 (OD600nm) and incubated for 48 h at 37 °C with 150 rpm orbital shaking. After 48 h, cultures were transported in a sterile 15 mL Falcon tube and centrifuged at 4500 g for 10 min at 10 °C. After centrifugation, the supernatant was removed and pyocyanin was extracted as described previously [g at 10 °C. The bottom blue layer was pipetted into sterile 15 mL Falcon tubes and treated with 0.1 M HCl at a ratio of 1:0.5 . The mixture was vortexed for 5 s, followed by centrifugation for 10 min at 10 °C and 4500× g. An aliquot (200 µL) of the final pink top layer composed of acidified pyocyanin was pipetted into wells of a 96-well plate and quantified for pyocyanin by measuring absorbance at OD520nm using a Tecan Infinite M1000 Pro plate reader.All eviously . In brie4a and 4c showed good activities in all assays and thus were chosen to assess their impact on human fibroblast cell lines HFF-1. Cytotoxicity was assessed based on the British Standard ISO 10993-5:2009—direct contact assay [v/v), penicillin (100 IU/mL), and streptomycin (100 μg/mL). HFF-1 cells were grown at 37 °C in a 5% (v/v) CO2 and harvested at 90% confluence using 0.12% v/v trypsin–EDTA. Cells were collected by first quenching Trypsin 1:1 v/v with supplemented media and transferred to conical centrifuge tubes, followed by centrifugation . The supernatant was aspirated, and the cell pellet was suspended in supplemented DMEM media for further experiments.After analysis of the results, compounds ct assay . HFF-1  and allowed to incubate at 37 °C in a 5% (v/v) CO2 atmosphere, with the medium being exchanged every alternative day until a confluence of 95–100% of cells (verified using a light microscope) was achieved. At 95–100% confluence, the medium was discarded and cells were supplemented with 1 mL of the growth medium, followed by placement of a freshly made filter disc containing 50 µL of 4a  or 4c (25 and 50 µg/mL) in the center of the wells. The filter disc was prepared by adding 50 µL of test compounds directly onto 6 mm antibiotic filter paper discs and incubating them at room temperature for 2 h to allow the discs to absorb test compounds. The plates containing HFF-1 and the filter disc were then incubated for 24 h at 37 °C in a 5% (v/v) CO2 atmosphere. After 24 h, the filter disc and the cell growth media were removed and replaced with 1 mL sterile PBS. The cell morphology of the HFF-1 cells and their confluence were assessed using a light microscope.v/v) CO2. After 24 h, the fluorescence of the HFF-1 cells was measured at Ex544nm and Em590nm using a plate reader. For the controls, a filter disc containing 50 µL 0.9% w/v NaCl and another containing 50 µL 100% DMSO (a positive control that causes cells death) were used. Based on ISO 10993-5:2009 [Quantification of HFF-1 cell metabolic activity (a cell viability assay) was analyzed by adding to the cells 50 µL of 0.05% resazurin solution  to 1 mL PBS and incubating them for 24 h at 37 °C in a 5% ) was used for conducting two-tailed unpaired t-tests in order to determine the p-values. The data presented are of the mean ± standard deviation (SD), with p-values < 0.05 deemed as significant.For all statistical analysis, GraphPad Prism (P. aeruginosa by inhibiting virulence factor (pyocyanin) production and biofilm formation whilst having low cytotoxicity to human fibroblasts. This current study needs to be extended to elucidate the resistance potential of P. aeruginosa after repeat exposure to DHP and whether exposure to DHPs can change their antibiotic susceptibility profile. Further, an exploration of how DHPs affect transcription of the genome of P. aeruginosa would provide valuable additional information.Developing compounds that inhibit QS is a strategy to reduce the production of virulence factors as well as biofilm formation (and hence limit antibiotic use) to combat bacterial infections. DHPs have a significant impact on isolates of


However, the specific roles and regulation of the four different Notch receptors have remained elusive. We have previously reported that the oncogenic PIM kinases phosphorylate Notch1 and Notch3. Phosphorylation of Notch1 within the second nuclear localization sequence of its intracellular domain (ICD) enhances its transcriptional activity and tumorigenicity. In this study, we analyzed Notch3 phosphorylation and its functional impact. Unexpectedly, we observed that the PIM target sites are not conserved between Notch1 and Notch3. Notch3 ICD (N3ICD) is phosphorylated within a domain, which is essential for formation of a transcriptionally active complex with the DNA-binding protein CSL. Through molecular modeling, X-ray crystallography, and isothermal titration calorimetry, we demonstrate that phosphorylation of N3ICD sterically hinders its interaction with CSL and thereby inhibits its CSL-dependent transcriptional activity. Surprisingly however, phosphorylated N3ICD still maintains tumorigenic potential in breast cancer cells under estrogenic conditions, which support PIM expression. Taken together, our data indicate that PIM kinases modulate the signaling output of different Notch paralogs by targeting distinct protein domains and thereby promote breast cancer tumorigenesis  The Notch signaling pathway orchestrates tissue development and homeostasis, but when dysregulated, it can also promote tumorigenesis and support cancer progression , 2, 3, 4The ICDs of all four Notch receptor paralogs are composed of a CSL/RBPJ-associated molecule (RAM) domain, ankyrin repeats flanked by two nuclear localization sequences, a transactivation domain, and a C-terminal domain rich in proline, glutamic acid, serine, and threonine residues , 3. The PIM1 and NOTCH1 mRNAs in patients with breast cancer  cannot bind CSL to induce the canonical CSL-dependent transcriptional program. Surprisingly however, it supports NOTCH1 and NOTCH3 mRNA was connected to poor survival, although only NOTCH3 showed a statistically significant correlation (A and B).In our previous study, we showed that PIM kinases phosphorylate Notch1 to promote tumorigenicity of estrogen receptor (ER)–positive breast cancer cells . In addirelation , A and BNOTCH1 and NOTCH3 on breast cancer growth in vivo, we used the CRISPR-/Cas9-based genomic editing to prepare MCF-7 breast cancer cell-based KO cell lines lacking endogenous expression of either NOTCH1 or NOTCH3 genes and the corresponding proteins (A–C). WT or KO cells were orthotopically inoculated into the mammary glands of athymic nude mice. The parental MCF-7 cells efficiently formed tumors as expected, but the growth of tumors derived from the NOTCH1 or NOTCH3 KO cells was significantly decreased (C), indicating that both genes are essential for estrogen-dependent mammary tumorigenesis.In order to evaluate the oncogenic potential of proteins , A–C. WTecreased C, indicaPIM1 mRNA upregulation is rather protective .We were then interested in testing whether there is a similar protumorigenic cross talk between PIM1 and Notch3 as previously shown for Notch1 . SurprisPIM1 and NOTCH3 mRNA expression levels in different breast cancer stages, whereas the PIM1 levels were reduced in most subtypes as compared with control samples (A–D). When the expression levels of PIM1 and NOTCH3 mRNA levels were compared with each other, positive correlations were observed in all stages, except stage IV (E). Moreover, a weak positive correlation was observed in the luminal A subtype but not in other subtypes . To determine whether one or both serine residues are true PIM target sites, we used site-directed mutagenesis to replace them with alanine residues. When recombinant WT N3ICD protein or phosphodeficient serine to alanine (SA) mutants were subjected to radioactive in vitro kinase assays, the S2064A mutation did not reduce phosphorylation of N3ICD (A), indicating that S2064 is not a PIM target site. By contrast, all three PIM family members targeted S1673, as demonstrated by 70 to 80% decrease in phosphorylation of the S1673A mutant.Based on our previous results , we expeof N3ICD A, indicaB).To confirm phosphorylation of Notch3 in a cellular context, FLAG-tagged N3ICD was transiently overexpressed in MCF-7 cells with or without His-tagged PIM1, and the samples were treated with the PIM-selective inhibitor 1,10-dihydropyrrolocarbazole-3-carbaldehyde (DHPCC-9) or its solvent dimethyl sulfoxide (DMSO) as a control. The phosphorylation status of immunoprecipitated N3ICD was analyzed with the phospho-AKT substrate (PAS) antibody, which recognizes not only the AKT-targeted sequence RXXpS/pT but also the PIM-targeted consensus sequence RXRHXpS/pT . This anFig. 2C). To further demonstrate that endogenous N3ICD is phosphorylated in a PIM-dependent fashion, we immunoprecipitated NOTCH3 from MCF-7 cells cultured in the presence or the absence of the PIM inhibitor DHPCC-9. Analysis with the PAS antibody showed that PIM inhibition reduces the phosphorylation status of N3ICD (D), indicating that NOTCH3 is an endogenous PIM kinase target.Coimmunoprecipitation of PIM1 and N3ICD also indicated that these proteins are able to interact in cells C. To furof N3ICD D, indicaA), indicating that they are likely to interact regardless of whether N3ICD is phosphorylated or not. Overexpressed PIM1 and N3ICD were predominantly localized in the nucleus irrespective of the phosphorylation status of N3ICD (A and B). Fluorescence lifetime-imaging data confirmed the interactions of PIM1 with both the WT and mutant N3ICDs (C). However, when interactions of endogenously expressed human PIM1 and N3ICD proteins were analyzed by proximity ligation assays, strong positive signals were mostly detected in the cytoplasmic compartments (D), suggesting differences in the localization of overexpressed versus endogenous proteins.We next used fluorescence microscopy to examine the interactions between fluorescently labeled forms of PIM1 and Notch3. N3ICD was tagged with green fluorescent protein (GFP), and the PIM-targeted S1673 residue was mutated to alanine to create a phosphodeficient mutant (SA) or to glutamic acid to create a phosphomimicking mutant (SE). When PIM1 tagged with red fluorescent protein (RFP) and GFP-tagged N3ICD were transiently overexpressed in MCF-7 cells and imaged by confocal microscopy, PIM1 colocalized to the same extent with the WT N3ICD as with the phosphomutants A, indicaof N3ICD , A and Bt N3ICDs C. Howeveartments D, suggesThe PIM target residue in Notch3 is in the RAM domain, which is required for canonical Notch signaling activity as it mediates binding with the DNA-binding protein CSL , 15. To A–C). These structural data also suggested that binding between N3RAM and CSL would be disrupted by phosphorylation at S1672. To confirm this, we performed isothermal titration calorimetry (ITC) on mouse CSL with human RAM peptides, using either N3RAM, phosphorylated N3RAMpS1672, or N1RAM (residues 1754–1781), which was used as a positive binding control. Binding between naïve CSL and N3RAM was weaker (Kd = 0.187 μM) as compared with binding between CSL and N1RAM (Kd = 0.022 μM) and, in line with our predictive model, no binding could be detected between CSL and the phosphorylated N3RAMpS1672  in complex with the human N3RAM peptide (residues 1665–1682) , A–C. ThA).To assess the importance of N3ICD phosphorylation status on CSL binding in cells, we overexpressed FLAG-tagged CSL and GFP-tagged N3ICD proteins and analyzed their interactions by immunoprecipitation. In line with our ITC experiments, CSL coprecipitated with both the WT N3ICD and the phosphodeficient SA mutant, whereas hardly any coprecipitation was observed with the phosphomimicking SE mutant A.Figure NOTCH3 or CSL genes had been knocked out by the CRISPR/Cas9 technique (NOTCH3 (B). By contrast, transactivation by the phosphomimicking SE mutant was severely compromised. As expected, no activity was detected in the KO cells lacking CSL protein. Similar results were obtained in T47D cells, except that WT N3ICD was more active than the SA mutant (C). This may be partly explained by the observation that there was less PIM protein expression as compared with MCF-7 cells (B) to regulate N3ICD activity in a phosphorylation-dependent fashion.We next used CSL-dependent luciferase reporter assays to analyze the effect of N3ICD phosphorylation on transcriptional activity. The WT N3ICD, the phosphodeficient (SA) mutant, or the phosphomimicking (SE) mutant were transiently overexpressed in MCF-7 and T47D luminal A breast cancer cells as well as in the derivatives of MCF-7 cells, in which the echnique . In MCF- (NOTCH3 B. By conA mutant C. This m-7 cells B to reguD). However, PIM inhibition by the pan-PIM inhibitor DHPCC-9 reduced activity of N1ICD, but increased that of N3ICD, which was in line with the data on the overexpressed N3ICD phosphomutants (B). We then analyzed endogenous NOTCH activity in MCF-7 cells or their KO derivatives lacking either NOTCH1 or NOTCH3. When these cell lines were cultured on plates coated with the DLL1 Notch ligand and treated with either DMSO, the Notch inhibitor PF03084014, or another pan-PIM inhibitor, AZD-1208, lack of either NOTCH1 or NOTCH3 abrogated NOTCH activity nearly to the same negligible level as inhibition of NICD cleavage by PF03084014 (E). As expected from our previous data  mRNA expression on NOTCH1, as demonstrated by its responsiveness to PIM inhibition as well as its remarkably reduced levels in the absence of NOTCH1 (F). By contrast, HES1 (hairy and enhancer of split 1) mRNA expression was more dependent on NOTCH3 activity, as there was no major difference between WT and N1KO MCF-7 cells. Interestingly, overexpression of the SA mutant upregulated both HEY1 and HES1 expression more efficiently than WT N3ICD. Taken together, these data demonstrate that PIM kinases support the CSL-dependent transcriptional activity of Notch1 but reduce the activity of Notch3.To determine whether phosphorylation affects the expression of CSL-dependent Notch target genes, MCF-7 cells or their f NOTCH1 F. By conin vivo, WT or phosphomutant forms of N3ICD were transiently overexpressed in MCF-7 or T47D cells, followed by transplantation of the cells onto the chorioallantoic membranes (CAMs) of fertilized chicken eggs, and the growth of the xenografted cells was followed for 5 days. The CAM model was chosen for this purpose as it offers a fast and easy in vivo system to analyze the tumorigenic potential of transiently transfected cells. As the growth of MCF-7 and T47D cells is estrogen-dependent, larger tumors were obtained when the xenografts were treated with 100 μM estradiol . In the presence of E2, overexpression of WT N3ICD or the phosphomimicking SE mutant supported tumor growth, whereas the phosphodeficient SA mutant abrogated it to the same extent as lack of E2. By contrast, in the absence of E2, all N3ICD variants reduced tumor growth.To assess the physiological role of Notch3 phosphorylation diol E2; , A and BC).As additional controls, cycloheximide pulse-chase experiments followed by Western blotting were conducted to demonstrate that the expression levels or intrinsic stability of the FLAG-tagged Notch3 proteins were not influenced by PIM-mediated phosphorylation C.A and C), we xenografted WT and SAKI cells on CAM and assessed tumor growth. The SAKI cells expressing the phosphodeficient SA mutant generated smaller tumors in response to E2 than parental MCF-7 cells (D), suggesting that phosphorylation of N3ICD supports its oncogenic activity.To further confirm the relevance of PIM-mediated phosphorylation, we used the CRISPR/Cas9 technique to prepare MCF-7–based knock-in (KI) mutant cells, where the PIM-targeted serine 1672 of the endogenously expressed human NOTCH3 was mutated to alanine to create a cell line named SAKI  . After v-7 cells D, suggesE). These data suggest that in addition to affecting the nuclear transactivation activity of N3ICD, phosphorylation may also play a role in its stabilization in the cytoplasm.As an additional control experiment, we fractionated WT and SAKI cells and analyzed by Western blotting the endogenous NOTCH3 protein levels. The levels of the full-length NOTCH3 protein were similar in both cell lines. Unexpectedly, the cytoplasmic levels of N3ICD were decreased in the SAKI cells as compared with WT cells, whereas the nuclear N3ICD levels remained relatively similar in both cell lines E. These A). Phosphorylated N3ICD cannot bind CSL and therefore remains transcriptionally inactive but nonetheless promotes cell survival and tumor growth under estrogenic conditions . Conversely, the stronger transactivation potential observed with the SA mutant as compared with WT N3ICD in MCF-7 cells suggests that nonphosphorylated N3ICD is the driver of CSL-dependent canonical Notch3 signaling, whereas PIM kinases act as a brake to inhibit Notch3 transcriptional output in the nucleus.We have previously shown that PIM kinases phosphorylate mouse Notch1 on serine 2152 (corresponding to human NOTCH1 S2162) within the second nuclear localization sequence, promoting its nuclear translocation and CSL-dependent transcriptional activity . Here, wFig. 7The high structural and sequence similarity between NOTCH1 and NOTCH3 allowed us to model the human NOTCH3 transcriptional complex superimposed on the NOTCH1 complex . Based oPIM upregulation has been associated with malignant breast cancer  and clinical data  were obtained from cBioPortal database from Cancer Genomics, Breast Invasive Carcinoma  dataset. Expression and clinical data were merged and analyzed in R. The packages plyr , ggpubr , and Hmisc (rcorr function for Pearson's correlation coefficiency) were used.kmplot.com Kaplan–Meier Plotter mRNA gene chip, Breast Cancer. Probe Id 209193_at was selected for PIM1 and 203237_s_at for NOTCH3. Patients were split by Auto select best cutoff, and analyses were performed to all data or restricted to subgroups. Data were exported as txt, and plots were prepared in R using packages survival and survminer (functions survfit and ggsurvplot).Correlation of gene expression levels to overall survival was analyzed by S-transferase fusion protein production and radioactive in vitro kinase assays were carried out as previously described  were cultured and transfected by electroporation or Fugene HD as previously described . To inhiThe stable MCF-7–based KO and KI cell lines were created by the CRISPR-/Cas9-based genome editing technique, as described in 3VO4. Protein concentrations were determined using the Bio-Rad Protein Assay Dye Reagent according to manufacturer's protocol . For immunoprecipitation of FLAG-tagged N3ICD, 500 μg of protein was combined with 50 μl of anti-FLAG M2 affinity agarose gel  in 1 ml of lysis buffer. After 1 h rotation at +4 °C, the agarose gel was washed four times with the lysis buffer. For immunoprecipitation of His-tagged PIM1, 500 μg of protein was combined with 50 μl of HisLink Protein Purification Resin (Promega) in 500 μl of lysis buffer supplemented with 10 mM imidazole . Samples were incubated in rotation at +4 °C for 30 min, after which resin was washed four times with 20 mM imidazole in 10 mM Tris–HCl, pH 7.5. His-linked protein elution was performed in rotation at +4 °C for 30 min in 10 mM Tris–HCl, pH 7.5 buffer with 300 mM imidazole, and 250 mM NaCl. For immunoprecipitation of GFP-tagged proteins, GFP-trap IP system  was utilized according to manufacturer's protocol. Samples were prepared for Western blotting by addition of preheated 2× Laemmli Sample Buffer, vortexing and heating for 5 min at +95 °C.Cells were lysed in 50 mM Tris–HCl, pH 7.5 buffer containing 10% glycerol, 100 mM NaCl, 1 mM EDTA, 1% NP-40, Mini-EDTA Free Protease inhibitors , 50 mM NaF, 0.5 mM natriumpyrophosphate, and 1 mM Nag for 40 min. About 60% w/v ammonium sulfate was added to the supernatant to precipitate the protein and then centrifuged at 11,000g for 45 min. The protein pellet was resuspended in binding buffer  and incubated overnight with nickel affinity resin. His-tagged protein was eluted from the nickel affinity resin and then cut with ubiquitin-like protease to remove the SMT3 tag. A sulphopropyl ion exchange column was used to separate the cut SMT3 from the CSL protein. CSL was then sized on an S200 16/60 sizing column into buffer containing 20 mM Tris pH 8, 0.5 M NaCl, 1 mM EDTA, 1% ethylene glycol, and 0.1 mM Tris(2-carboxyethyl)phosphine. CSL was then concentrated and flash frozen in liquid nitrogen and stored at −80 ˚C until further use.Mouse CSL amino acid residues 53 to 474 corresponding to the conserved and structurally ordered core domain were cloned into the pSMT3 expression vector. BL21(DE3) cells transformed with pSMT3-mCSL were grown in LB to an absorbance of 1.5 followed by IPTG induction. Cell pellets were resuspended in lysis buffer, sonicated, and centrifuged at 15,000Human NOTCH1 residues 1754 to 1781 (VLLSRKRRRQHGQLWFPEGFKVSEASKK) and NOTCH3 residues 1665 to 1682  corresponding to the RAM domains were synthesized with 95% purity by Peptide2.0 and further purified by vacuum centrifugation before ITC.GTGGGAAAGA-3’ and the reverse complementary sequence 5’-AATCTTTCCCACGGT-3’ showing the CSL-binding site underlined. Single-stranded oligonucleotides were further purified on a Resource Q ion exchange column and then buffer exchanged into a buffer containing 10 mM Tris pH 8.0, 500 mM NaCl, and 1 mM MgCl2. Single-stranded oligonucleotides were added together in equal molar amounts and boiled for 10 min and then slowly cooled to room temperature to allow for proper annealing.The following 15-mer oligonucleotide sequences were ordered from Eurofins Scientific (Luxembourg): 5’-TTACC12121, and had unit cell dimensions of a = 66.45 Å, b = 97.66 Å, and c = 104.62 Å.Crystallization experiments were set up under paraffin oil with CSL at 150 μM, N3RAM at 170 μM, and 15-mer DNA at 165 μM. The mother liquor solution contained 0.2 M ammonium fluoride and 14% PEG 3350. Crystals were slowly transferred into the mother liquor supplemented with 20% xylitol for cryoprotection before freezing in liquid nitrogen. Diffraction data were collected at the Advanced Photon Source, beamline 24-ID-C-NE. The CSL/N3RAM/DNA crystals diffracted to 2.4 Å, belonged to the space group P 2HES1 DNA site (Protein Data Bank [PDB]: 3IAG) combined with the Caenorhabditis elegans structure of the Lin-12 RAM peptide (PDB: 3BRF) as the search model. Refinement was initially performed in Phenix , and protein interfaces were analyzed with the PISA server   .CSL and NOTCH RAM peptides were dialyzed overnight in 50 mM sodium phosphate and 150 mM sodium chloride buffer. Experiments were conducted using the VP-ITC MicroCalorimeter manufactured by MicroCal. All experiments were performed at 20 °C with CSL in the cell at 10 μM and RAM peptides in the syringe at 100 μM. Each experiment was performed in triplicate with 20 injections, 14 μl per injection. Heat of dilution experiments were performed by injecting syringe samples into a cell containing only buffer, and all analyses were performed with the heat of dilution subtracted before fitting.Western blotting was performed as previously described , while dNotch activity was measured from cells transiently transfected with the 12xCSL-luciferase (RBPJ)  and the 2O controls were included to ensure that RNA preparations and PCR mixtures were not contaminated. The primer sequences for detection of NOTCH1, NOTCH3, HEY1, HES1, and UBC (a house-keeping control) mRNAs have been described in RNA was extracted with the NucleoSpin RNA kit (Macherey-Nagel) according to the manufacturer's instructions. Complementary DNA samples were prepared using the SensiFAST cDNA Synthesis Kit (Meridian Bioscience) according to the manufacturer's instructions from equal amounts of RNA. The reaction mixtures for quantitative PCR were prepared with 5× HOT FIREPol EvaGreen qPCR Mix Plus (Solis BioDyne) according to the manufacturer's instructions (Sigma–Aldrich). RNA and H10438/04.10.07/2016). Athymic Nude-Foxn1nu female mice (Envigo) were housed under controlled conditions and supplemented with estradiol pellets as previously described (6 MCF7 cells or their derivatives mixed 1:1 with Matrigel (BD Biosciences). At the time of xenotransplantation, mice were 7 weeks old. Tumor measurements and animal sacrifice were performed as previously described , strong (R2 = 0.600–0.799), moderate (R2 = 0.400–0.599), or weak (R2 = 0.2–0.399). Figures were prepared by CorelDRAW Graphics Suite 2020 (Corel Corporation) or Adobe Illustrator CS5 15.0.0 (Adobe).Bar graphs and scatter plots were produced by R, version 3.6.3. (The R Foundation), Microsoft Excel 2016 (Microsoft Corporation), or GraphPad Prism 4.00 (GraphPad Software), and results were analyzed by Student's https://github.com/nmsantio/Gene-expression-analysis).The completed refined structure of CSL/N3RAM/DNA was deposited into the PDB with accession code 6WQU. Detailed code for R is available in Github (This article contains U. L. holds research grants from Merck KGaA, no personal remuneration. All other authors declare that they have no conflicts of interest with the contents of this article.


The catalytic evaluation of γ-Fe2O3@MBD/Pd-Co showed promising results in the Mizoroki–Heck and Buchwald–Hartwig amination reactions of various iodo-, bromo- and challenging chloroarenes in aqueous media. The synergetic cooperative effect of both Pd and Co and dispersion of the catalyst in water due to the encapsulation of γ-Fe2O3 by melamine-based dendrimer lead to high catalytic performance compared with the monometallic counterparts. The dispersion of the magnetic catalyst also facilitates the recovery and reuse of the catalyst by ten consecutive extraction and final magnetic isolation with no loss of catalytic activity, keeping its structure unaltered.A Pd-Co bimetallic alloy encapsulated in melamine-based dendrimer supported on magnetic nanoparticles denoted as γ-Fe Concerning the catalytic metals employed, palladium is the most versatile element in industrial and academic research playing the most prominent part in the removal of halogen atom from halogenated organic compounds. Therefore, since the pioneering works, the homogeneous palladium complexes have attracted the interest of scientists to address coupling reactions due to their excellent functional group tolerance, as well as their excellent catalytic efficiency5. However, application of homogeneous palladium catalysts has some limitations such as non-reusability, high cost, poisoning and losing activity, which are important from economic and environmental viewpoints. These problems can be largely overcome by supporting Pd nanoparticles (NPs) or complexes on varied solid supports6. Moreover, considering the consumption of expensive Pd, the development of bimetallic nanoparticles of non-noble metal elements with Pd can not only reduce costs, but also stabilizes active Pd species, and improves its resistance to poisoning7. The supported bimetallic nanoparticles with two special advantages of using of a lower quantity of Pd and the recovery of the heterogeneous NPs by filtration from the reaction media, could be used as suitable catalysts in cross-coupling reactions. Moreover, because of the synergistic effect, the catalytic activity and selectivity obtained over bimetallic nanoparticles were much higher than that obtained over the monometallic counterparts. Along this line, Pd-based bimetallic nanoparticles including Pd–Ag, Pd-Cu, Pd-Co, Pd-Au or Pd–Ni, supported on different solid substances have been reported11. Although heterogeneous catalysts can be recovered by filtration or centrifugation methods, these methods are time consuming, and result the impurities in the product due to the loss of the catalyst particles.Haloarenes are frequently transformed into a variety of valuable compounds catalyzed by transition metals such as palladium, copper, nickel, and cobalt with various ligands through cross-coupling reactions13. This separation is operationally very simple, economic and promising for industrial applications. Moreover, MNPs possess exclusive physical properties, for example, high surface area, surface modification ability and extraordinary thermal and chemical stabilities. However, MNPs suffer from aggregation during the catalytic processes, resulting in a sensible decrease in their catalytic efficiency. Therefore, encapsulation of MNPs using noble metals, carbon, silica and biopolymers stabilize and protect them from oxidation and agglomeration15.In recent decades, magnetic nanoparticles (MNPs), which have been widely surveyed towards many medical and biological uses, have emerged as modern and attractive materials for the supporting of catalytically active species. The magnetically immobilized catalysts can be isolated from the reaction mixture employing magnets16. In the dendrimers, the existence of several inner and outer functional groups allows the coordination with transition metals contributing to their stabilization. Because of this property, dendrimers are principally suitable host for metal catalysts. Nanometals are encapsulated within the dendrimer cavities, so that their agglomeration is circumvented and the well-dispersed nanoparticles are obtained. Moreover, the existence of a large number of cavities in dendritic polymers, could absorb and concentrate reactants, and makes the reaction proceed more efficiently. Importantly, the substrates can still simply contact with the encapsulated catalytically active nanoparticle in the dendrimers. As the melamine is rich with nitrogen and contains triazine rings with high stability, melamine-based dendrimers (MBD) with abundant metal-binding nitrogen groups have aroused a lot of interest recently for the incorporation of metal nanoparticles for using in catalytic reactions19.Dendrimers are a relatively novel class of polymers with a well-defined highly branched, three-dimensional structure and are being used for encapsulation of MNPs28, herein, a melamine-based dendrimer was built to the 1.5-generation onto the γ-Fe2O3 surface employing a divergent method to synthesize γ-Fe2O3@melamine-based dendrimer (γ-Fe2O3@MBD). Bimetallic Pd-Co alloy nanoparticles were then attached to γ-Fe2O3@melamine-based dendrimer via a co-complexation method followed by reduction with sodium borohydide. This novel catalyst was fully analyzed by several instrumental techniques and applied as a water-dispersible/magnetically reusable palladium-cobalt catalyst for the Mizoroki–Heck and Buchwald–Hartwig amination reactions in aqueous media. The synergistic cooperative effect of both Pd and Co in the catalyst lead to high catalytic performance in the cross-coupling reactions in aqueous media.In our continuous interest in developing a greener catalyzed reaction3 and DMSO-d6 as solvent and TMS as internal standard. The purity of the products and the progress of the reactions were accomplished by TLC on silica-gel polygram SILG/UV254 plates. TEM analysis was performed using TEM microscope (Philips EM 208S). EDS mapping were found using a TSCAN MIRA3. FT-IR spectra were recorded on a Shimadzu Fourier Transform Infrared Spectrophotometer (FT-IR-8300). Thermal gravimetric analysis (TGA) was performed using a Shimadzu thermos-gravimetric analyzer (TG-50). X-ray diffraction (XRD) was done on a Bruker D8-advance X-ray diffractometer with Cu Kα (λ = 0.154 nm) radiation. XPS analyses were performed using a VG-Microtech Multilab 3000 spectrometer, equipped with an Al anode. The deconvolution of spectra was carried out by using Gaussian–Lorentzian curves. The Pd and Co content on the catalyst was determined by OPTIMA 7300DV ICP analyzer. Qualitative elemental analysis was determined and analyzed by CHNO elemental analyzer .Chemicals were purchased from Merck Chemical Company. NMR spectra were recorded on a Bruker Avance DPX-400 and 300 using deuterated CDCl2O320 (1.5 g) was sonicated with Et3N (10 mL) and melamine  for 30 min. The mixture was refluxed for 48 h at 60 °C. The resulting light-brown solid was isolated by an external magnet and washed several times with distilled H2O (3 × 20 mL) and EtOH (3 × 20 mL). It was dried in a vacuum oven at 50 °C. Elemental analysis of γ-Fe2O3-melamine for nitrogen (9.88%) showed that 1.2 mmol of melamine was loaded on 1 g of γ-Fe2O3-melamine.Chloro-functionalized γ-Fe2O3-melamine from the previous step (1 g) and Et3N (10 mL) was sonicated about 30 min at room temperature. ECH (1 mL) was added to this stirring mixture drop wise. The mixture was heated to a temperature of 60 °C and kept for 24 h. Then, melamine  was added and further stirred for 48 h at the same temperature. The resulting solid was isolated by an external magnet and washed several times with distilled H2O (3 × 20 mL) and EtOH (3 × 20 mL). It was dried in a vacuum oven at 50 °C. Elemental analysis of γ-Fe2O3-MBD for nitrogen (17.5%) showed that in total, 2 mmol of melamine was loaded on 1 g of γ-Fe2O3-MBD.The synthesized γ-Fe2O) and γ-Fe2O3-MBD (0.2 g in 10.0 mL) sonicated for 20 min. A solution of CoCl2⋅6H2O  was added to the resulting mixture and sonicated for further 20 min. The pH of the sonicated mixture was controlled between 8 and 10 using sodium hydroxide solution (0.3 M). Then, a solution of sodium borohydride  was added to the reaction mixture and stirred for 24 h at ambient temperature. The resulting black solid was separated from aqueous media using an external magnet, washed well with distilled H2O (3 × 20 mL) and EtOH (3 × 20 mL) and dried under vacuum at 50 °C for 4 h.A mixture of palladium acetate (0.1 g in 3.0 mL H2O3-MBD/Pd-Co (0.05 mol% based on Pd) was added to a stirred suspension of haloarene (1 mmol), Et3N (2 mmol), alkene (1.3 mmol) in water (1 mL). The resulting mixture was heated at 60 °C. The reaction was monitored by TLC and, after the times shown in Table 4 and filtered. Organic solvent evaporated under vacuum to give the crude product, which was purified by column chromatography (silica gel) using 50:1 volume ratio of n-hexane:EtOAc as eluent. The aqueous phase, containing the bimetallic catalyst, was again used for a new identical process.γ-Fe2O3-MBD/Pd-Co (0.05 mol% based on Pd) was added to a stirred mixture of iodoarene (1 mmol), t-BuONa (2 mmol), and the arylamine (1.2 mmol) in H2O (1 mL). The resulting suspension was heated at 50 °C and the reaction was monitored by TLC. After the times depicted in Table 4 and the solvent evaporated under vacuum to give the crude product, which was purified by column chromatography (silica gel) using 10:1 volume ratio of n-hexane:EtOAc as eluent. The aqueous phase, containing the bimetallic catalyst, was again used for a new identical process.γ-Fe2O3-MBD/Pd-Co (0.07 mol% based on Pd) was added to a stirred suspension of chloroarenes or bromoarenes (1 mmol), t-BuONa (2 mmol), and arylamines (1.2 mmol) in H2O (1 mL). The resulting mixture was heated at 70 °C. The reaction was monitored by TLC and, after the times shown in Table 4 and the solvent was evaporated under vacuum to give the crude product, which was purified by column chromatography (silica gel) using 10:1 volume ratio of n-hexane:EtOAc as eluent. The aqueous phase, containing the bimetallic catalyst, was again used for a new identical process.γ-Fe2O3@MBD/Pd-Co) is illustrated. In the first step, γ-Fe2O3 was functionalized by the reaction with 3-chloro-trimethoxypropylsilane and subsequent treatment with melamine to produce γ-Fe2O3-melamine (Scheme G0.5). The nucleophilic reaction of γ-Fe2O3-melamine with epichlorohydrin (ECH), as a bifunctional molecule for growing of dendritic branches afforded γ-Fe2O3-melamine-ECH . The last step involves the intercalating of metal complexes into the interior cavity of γ-Fe2O3@MBD followed by reduction with NaBH4 as a reducing agent.In Scheme e Scheme , G0.5. TH Scheme , G1. Rin2O3, γ-Fe2O3-melamine, γ-Fe2O3-melamine-ECH, γ-Fe2O3@MBD and γ-Fe2O3@MBD/Pd-Co) were determined via FT-IR spectroscopy  per 1 g of the catalyst. Elemental analysis of γ-Fe2O3-melamine and γ-Fe2O3@MBD showed that the loadings of melamine on the catalyst were 9.88 and 17.50%, respectively, based on the nitrogen content.The metal content of γ-Fe2O3@MBD/Pd-Co established the crystalline structure of the bimetallic Pd-Co alloy nanoparticles. The sample exhibited characteristic diffraction peaks at 2θ = 30.3, 35.7, 43.4, 53.7, 57.2 and 62.9°, which corresponded to the (2 2 0), (3 1 1), (4 0 0), (4 2 2), (5 1 1), and (4 4 0) reflections of the cubic maghemite29. The peaks at 2θ values of 40.5, 46.4 and 67.90° are due to the (1 1 1), (2 0 0), and (2 2 0) diffraction peaks corresponding to the face centered cubic Pd (JCPD-46-1043)10. The XRD pattern of the sample showed two diffraction signals were placed around 2θ = 44.8 and 47.5° due to the Co species (JCPD-15-0806)30. However, those peaks mentioned above slightly shift to larger angles compared with the single metal counterpart31. The shift could be related to the bimetallic alloy formation.As shown in Fig. 2O3@MBD. In this plot the first weight loss of 1.18% (< 171 °C), corresponded with the loss of physically adsorbed water. The second weight elimination of 11.61% (171–446 °C) occurred as a consequence of degradation and decomposition of the organic material. These data justified that the melamine-based dendrimers are conveniently grafted on the magnetic nanoparticles. Magnetic properties of γ-Fe2O3@MBD/Pd-Co and γ-Fe2O3 were surveyed employing a vibrating sample magnetometer (VSM) at room temperature  was related to the coating of γ-Fe2O3 by melamine-based dendrimer. The magnetization curves did not show a hysteresis loop, which justifiyed the superparamagnetic nature of the resulting NPs. High magnetic properties of NPs were appropriate for their further recovery from the reaction media by the simple magnetic separation using a conventional magnet.Figure .61% 171–46 °C occ2O3@MBD/Pd-Co surface , 286.0 (C–O and C=N), and 288.1 (C–N) eV33. Deconvolution of N1s region showed two peaks at 398.0 and 399.5 eV corresponding to C–N=C and N–H, respectively  and 340.4 eV (3d3/2), corresponded to Pd in the zero oxidation state. The peaks at 336.6 (3d5/2) and 341.8 eV (3d3/2) indicated that a small amount of Pd presents in (II) oxidation state36. The typical peaks located at 780.6 (2p3/2) and 796.4 eV (2p1/2) revealed the presence of cobalt (0) in the catalyst  and 798.4 eV (2p1/2) corresponding to cobalt (II) species. Different weak satellite peaks at 785.8, 788.6, 801.3 and 803.0 eV30, showed the existence of Co3O4 on the catalyst surface37. The displacement of the 2p3/2 signal of cobalt to a lower energy and a positive shift in the 3d5/2 peak of Pd indicated the alloying of Co with Pd38. Moreover, the atomic distribution of palladium versus cobalt in the surface of γ-Fe2O3@MBD/Pd-Co is 1:8.45, which is notably higher than the stoichiometric value (1:4.2) calculated by ICP analysis. This larger value demonstrates that the bimetallic Pd-Co nanoparticles generates a core–shell structure with a cobalt-rich shell and a palladium-rich core40, and consequently, a larger cobalt surface is exposed.A deep XPS analysis was done to characterize the chemical composition of γ-Feace Fig. . The peaace Fig. a. The C1ace Fig. b showed 2O3 magnetic nanoparticles was shown. Comparing TEM image of γ-Fe2O3@MBD/Pd-Co  Fig. . In Fig.-Co Fig. b with γ-ace Fig. c. The siace Fig. f, illust2O3@MBD/Pd-Co are presented in Fig. 2O3@MBD/Pd-Co surface. As it is observed in the Fig. The EDS elemental mapping images of the γ-Fe46. These reactions, involving the generation of new carbon–carbon bonds formation, have found several commercial applications for the synthesis of fine chemicals such as herbicide prosulfuron, anti-inflammatory naproxen, or anti-asthma agent Singulair in the multi-ton scale in each year47. Generally, the Mizoroki–Heck cross-coupling reaction is catalyzed by palladium complexes48. However, another transition metals such as Ni49, Co50 and Cu51 have been recently reported as catalysts for this purpose. Due to the advantageous of bimetallic catalysts such as enhanced catalytic activity which comes from the synergistic effect of the monometallic counterparts, in the past decades, some bimetallic catalysts such as Pd/Cu52, Pd/Pt53, Pd/Co11, Pd/Au54, Pd/Ni55, Pd/Fe56 and etc. have been developed for the Mizoroki–Heck cross-coupling reaction. The synthetic routes published in the literature have certain limitations due to the necessary high temperature, the large amount of catalyst loading, the introduction of additives, the employment of organic solvents and the use of an unrecyclable catalyst. In addition, most of the reported methods suffered from lack of generality for the coupling reactions employing chloroarenes. Continuing with our research on the design and the preparation of novel and attractive catalytic systems to conduct environmentally friendly cross-coupling reactions4, herein, we have surveyed the catalytic activity of a novel γ-Fe2O3@MBD/Pd-Co as the first magnetically recyclable Pd/Co bimetallic catalyst in the Mizoroki–Heck coupling reactions.Mizoroki–Heck cross-coupling reactions are applied for the preparation of natural products, pharmaceuticals and biologically active moleculesn-butyl acrylate (1:1.3 molar ratio) in water was investigated as a bench reaction to optimize the effect of the catalyst loading, type of the base and temperature on the reaction completion (Table 2O3@MBD/Pd-Co using Et3N as the base (2 equiv.) at 60 °C  under optimized reaction conditions was also studied in the presence of the catalyst containing different ratios of Pd:Co  produced the desired product with the same yield as γ-Fe2O3@MBD/Pd-Co containing Pd:CO (1:4.2). Therefore, the optimum molar ratio of Pd:Co in the catalyst should be 1:4.2.The coupling reaction of iodobenzene with Co Table . As the 2O3@MBD/Pd-Co was investigated by employing various aryl halides to react with olefins in water  was used as a scavenger for the metal. Under this condition, any considerable change in the progress of the reaction was not observed.The heterogeneous nature of the catalyst was checked by the hot filtration and poisoning tests. In the hot filtration test, after approximate 40% of the coupling reaction of chlorobenzene with ure Fig. . In the 59. Arylamines are extremely important ligand for the coordination to transition metals60. The Buchwald–Hartwig coupling of amines and aryl halides in the presence of Pd is a great method for the preparation of arylamines63. Since the discovery of Buchwald–Hartwig amination reaction64, research efforts have concentrated on this reaction and significant development have been achieved on the improvement of the conventional reaction conditions such as using diverse transition metals, ligands, and solvents as well as extending substrate scope65. The importance of bimetallic catalysts in organic synthesis has recently encouraged organic chemists to use these kinds of catalysts in coupling reactions. Along this line, Fe@Pd nanowire66, Pd/Ni nanoparticles67 and Pd/Cu complexes68 have been used as bimetallic catalysts for the C-N coupling reactions. Encouraged by the facile Mizoroki–Heck cross-coupling reaction catalyzed by γ-Fe2O3@MBD/Pd-Co, we have tried our catalyst for C–N coupling reaction as well. Surprisingly, to the best of our knowledge, there is not any report on the Buchwald–Hartwig amination reaction catalyzed by Pd/Co bimetallic nanoparticles.Arylamines and their derivatives possess a paramount importance as intermediates for pharmaceuticals and natural products, agrochemicals, conducting polymers (PANI), and dyes in the chemical industry2O3@MBD/Pd-Co toward the Buchwald–Hartwig amination reaction, the cross-coupling reaction of chlorobenzene as a poor reactive aryl donor, with aniline in aqueous media, was selected as a bench reaction. Several reaction factors such as the catalyst loading, base and temperature were screened. The results are collected in Table t-BuONa was found to be the most effective base. When any bases was not used, the product was isolated in 54% yield after 24 h  and the corresponding products were obtained in 82–93% yields 2, γ-Fe2O3@MBD/CoCl2, physical mixture of γ-Fe2O3@MBD/Pd and γ-Fe2O3@MBD/Co, Pd(OAc)2, CoCl2.6H2O, and Fe2O3@MBD/Co3O4 were also investigated and the results were compared with the bimetallic catalyst (γ-Fe2O3@MBD/Pd-Co)  Fig. . Obvious69, we tentatively proposed a plausible mechanisms for Mizoroki–Heck and Buchwald–Hartwig amination reactions in the presence of γ-Fe2O3@MBD/Pd-Co  for the efficient Mizoroki–Heck and Buchwald–Hartwig amination reactions in water. A variety of iodo-, bromo- and chloroarenes successfully reacted with acrylates, styrene and anilines to yield the corresponding products. Using this protocol, products were achieved in good to high yields in water as an ecofriendly solvent and without using any additives. The synergistic cooperative effect of both Pd and Co in the encapsulated catalyst leads to high catalytic performance in the cross-coupling reactions in aqueous media. The dispersion of the magnetic catalyst facilitates the catalyst recovery and reuse by ten consecutive extraction and at the end magnetic isolation. The experiments based on isolation of the catalyst in the hot filtration test and using S8 in the poisoning test, showed that the observed catalysis was heterogeneous in nature. Use of water as an ecofriendly solvent, simple catalyst recovery and reuse, ease of work-up and not needing any additive make this method an environmentally benign procedure for the carbon–carbon and carbon–nitrogen cross-coupling reactions (In summary, in this work, melamine-based dendrimer supported on γ-Feeactions .Supplementary Information.


Curcuma longa that has been shown to exhibit antioxidant and anti-inflammatory potency in metabolic disorders. However, the underlying mechanisms of curcumin in adipocytes remain largely unexplored. We studied the effects of curcumin on adipogenic differentiation and mitochondrial oxygen consumption and analysed the possible mechanisms. 3T3-L1 preadipocytes were used to assess the effect of curcumin on differentiation of adipocytes. The Mito Stress Test measured by Seahorse XF Analyzer was applied to investigate the effect of curcumin on mitochondrial oxygen consumption in 3T3-L1 adipocytes. The effect of curcumin on the morphology of both white and brown adipose tissue (WAT and BAT) was evaluated in a high-fat diet-induced obese mice model. We found that curcumin dose-dependently  induced adipogenic differentiation and the intracellular fat droplet accumulation. Additionally, 10 µM curcumin remarkably enhanced mature adipocyte mitochondrial respiratory function, specifically, accelerating basic mitochondrial respiration, ATP production and uncoupling capacity via the regulation of peroxisome proliferator-activated receptor γ (PPARγ) (p < 0.01). Curcumin administration also attenuated the morphological changes in adipose tissues in high-fat diet-induced obese mice. Moreover, curcumin markedly increased the mRNA and protein expressions of mitochondrial uncoupling protein 1 (UCP1), PPARγ, peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) and PR domain protein 16 (PRDM16) in vivo and in vitro. Collectively, the results demonstrate that curcumin promotes the adipogenic differentiation of preadipocytes and mitochondrial oxygen consumption in 3T3-L1 mature adipocytes by regulating UCP1, PRDM16, PPARγ and PGC-1α expression.Accumulating evidence suggests that mitochondrial dysfunction and adipocyte differentiation promote lipid accumulation in the development of obesity and diabetes. Curcumin is an active ingredient extracted from  Countermeasures to control the enlargement of white adipose tissue (WAT) and induction of white adipocyte browning may offer a new solution in fighting obesity and its associated diseases ,2. MethoMitochondria are considered the cellular ‘powerhouse’ since they provide the site and essential materials for various metabolic processes during which ATP is generated from the chemical energy contained in three major energy sources . UsuallyCurcuma longa), which is not only a traditional Chinese herb but also a dietary spice. Accumulating evidence has demonstrated that it could prevent and treat a wide range of diseases by its antioxidant and anti-inflammatory properties in preclinical trials, for instance, arthritis, cancer, neurodegenerative disorders, metabolic syndrome, cardiovascular diseases, diabetes and so on [κB (NF-κB), tumor necrosis factor-α (TNFα), and monocyte chemoattractant protein-1 (MCP-1), which might contribute to its therapeutic potential in obesity and diabetes [Curcumin -1,6-heptadien-3,5-dione) is an active ingredient extracted from turmeric  for 2 days and further culturing in DMEM with 10 µg ml−1 insulin for an additional two days. Subsequently, the DMEM was renewed every 48 h until the cells were fully differentiated.3T3-L1 preadipocytes were acquired from the CAMS & PUMC Cell Center and were then cultured in DMEM containing 10% FBS and 1% penicillin-streptomycin at the temperature of 37°C and 5% COOil Red O was adopted to determine differentiated intensity of 3T3-L1 cells, which has been used in our previous studies . And dif. 2.3Curcumin was dissolved in dimethyl sulfoxide (DMSO) and prepared as a 10 mM stock solution. Then the stock was diluted in the medium to the indicated concentration for the experiment. The differentiated 3T3-L1 adipocytes were cultured with the medium supplemented with curcumin of several concentrations for 24 h, and then the relative cell viability was calculated as the percentage of untreated cells applying the CCK-8 assay. The doses of curcumin used in this study were determined based on previous investigations ,16. Brie. 2.4Oil Red O staining was applied to measure the lipid accumulation intracellularly. Briefly, after the cells were incubated with or without curcumin  for 24 h, the culture medium was removed, and the cells were rinsed in phosphate buffer saline (PBS) for 3 × 5 min and then fixed in 4% formaldehyde for half an hour. Next, a dilute solution of Oil Red O (3:2) was filtered using a 0.45 µm filter followed by incubating the fixed cells for 1 h. All above procedures were carried out at room temperature. Then, the stained lipid droplets in cytoplasm were visualized by microscopy. The Oil Red O-stained 3T3-L1 cells were dissolved in 200 µL of isopropyl alcohol and transferred to a 96-well microplate. The absorbance of the dissolved Oil Red O was detected at 510 nm by a microplate reader.. 2.54 cells) were seeded in each well of a 24-well XF Cell Culture Microplate. When the cells were differentiated into mature adipocytes, curcumin was added at various concentrations  for 48 h. Then the growth medium was exchanged with XF basic medium (pH = 7.4), which contained 4.5 g l−1 glucose, 4.0 mM glutamine and 1.0 mM sodium pyruvate. OCR measurements were performed following the sequential addition of 2.0 µM oligomycin, 1.0 µM FCCP and 1.0 µM rotenone/antimycin. All above experimental procedures were carried out at 37°C. The following mitochondrial parameters were determined: basal respiration, ATP production , maximal respiratory capacity  and spare respiratory capacity . The results are expressed in pmol (O2).min−1 and normalized to the cell number.The oxygen consumption rate (OCR) in 3T3-L1 adipocytes was measured to evaluate oxidative phosphorylation using the Seahorse Biosciences XF Extracellular Flux Analyzer  following the manufacturer's operating instructions. In brief, 100 μl of 3T3-L1 cell suspension (2 × 10. 2.6n = 10) was administered at 50 mg kg−1 d−1 by oral gavage to the obese mice for an additional eight weeks. The same volume of saline was then given by gavage to the obese (diet-induced obesity (DIO), n = 10) and normal mice . During this period, all the mice were maintained on the original diet. After eight weeks of intervention, the mice were sacrificed under pentobarbital anaesthesia (60 mg kg−1) after fasting for 12 h. Adipose tissue was removed and stored at −80°C for histological and molecular biological analysis. Six samples were selected randomly from each group for further analysis of histological evaluation, and gene expression and protein expressions. And for each type of molecular biology analysis, the adipose tissues were from the same mice.Thirty-six male C57BL/6 J mice  were purchased from Beijing HFK Bioscience Co., Ltd.  and divided into two groups that received two different diets. Ten mice in the control group were fed a standard chow diet comprising 10% kcal (MD12031) of fat, while the remaining mice in the other group were given a high-fat diet containing 60% kcal (MD12033) of fat for 12 weeks. Twenty mice in the high-fat diet group were confirmed as obese as their body weight was 20% greater than their peers. The food was purchased from Mediscience Co., Ltd. , and the diet compositions of MD12031 and MD12033 are listed in electronic supplementary material, 1 (S1). Next, curcumin  samples were fixed in 10% paraformaldehyde and then dehydrated in a gradient ethanol series . The samples were defatted in xylene, embedded in paraffin, dyed with haematoxylin-eosin and photographed under a microscope . The sizes of WAT and BAT cells were measured by counting 50 cells using I. 2.83VO4, 1 mM PMSF and 10 mM NaF (pH 7.5), and the proteins were quantified applying the Micro BCA™ Protein Assay Reagent Kit. Protein separation was performed using SDS-PAGE gel electrophoresis with 40 mg protein per well. After electrophoresis, the proteins were subsequently transferred onto a PVDF membrane, which were next incubated with UCP1, PRDM16, PGC-1α and PPARγ antibodies (1 : 1000 dilution) overnight at 4°C. The membrane was then incubated with a corresponding secondary antibody (1 : 2000). Blots were generated using an ECL kit under the guidance of the manufacturer's instructions. The greyscale of protein bands was analysed by ImageJ software. GAPDH was used as a loading control.Adipose tissues and cells were lysed using RIPA buffer with a protease inhibitor cocktail, 20 mM Tris, 1 mM EDTA, 140 mM NaCl, 1% Nonidet P-40 (NP-40), 50 U of aprotinin/ml, 1 mM Na. 2.9−ΔΔCT relative quantification method according to the manufacturer's instructions.Total RNA was isolated from adipose tissue and 3T3-L1 cells applying TRIzol reagent in accordance with the operating manual provided by the manufacturer. Reverse transcriptases (RTs) from the isolated RNA into cDNA were then carried out following by quantitative real-time PCR which was performed in accordance with the protocol recommended by the manufacturer . The primers used in the current study were listed in . 2.10p-value is less than 0.05.SPSS software (v. 20.0) was applied for statistical analyses. The results are presented as the means ± standard errors (Mean ± s.e.m.). One-way analysis of variance was applied for comparisons among groups, followed by the LSD or SNK test for comparisons between two groups. The difference was considered significant if the . 3. 3.1a, curcumin exhibited a subtle influence on preadipocyte viability at 10, 20 and 35 µM compared to that in the control group (p > 0.05). However, the viability of the preadipocytes decreased by approximately 55.0% and 74.7% compared to that in the control group upon incubation with 50 µM and 75 µM curcumin, respectively (p < 0.01). The results indicated that high concentration of curcumin may have cell toxicity.As shown in figure 2b). In addition, curcumin could increase the triglyceride level in differentiated 3T3-L1 cells (c). These results suggest that curcumin could influence lipid profiles though promotion of adipocyte differentiation.Next, the 3T3-L1 cells were incubated with different concentrations  of curcumin for 8 days to induce differentiation. The results of Oil Red O staining revealed that different concentrations of curcumin accelerated adipocyte differentiation and increased the numbers of mature adipocytes b. In addL1 cells c. These . 3.2a, a typical bioenergetic profile, involved in a four-step analysis: (1) basal OCR, 3T3-L1 cells were incubated in normal medium; (2) ATP synthesis turnover, oligomycin (2.0 mM) was supplemented to the medium to inhibit ATP synthase; (3) maximal mitochondrial respiratory capacity, cells were motivated with FCCP (1.0 mM); and (4) non-mitochondrial respiration, rotenone (1.0 mM) was introduced to inhibit complex I.As shown in figure 3b, the OCR of curcumin-treated (10 µM) adipocytes remained higher than that of adipocytes in the control group. Notably, different doses of curcumin (20 and 35 µM) exerted opposite effects on the mitochondrial respiratory function of mature adipocytes. Specifically, the basal mitochondrial respiration in the curcumin-treated (10 µM) group was elevated by 16.5% compared to that of the control, but 20 and 35 µM curcumin reduced basal mitochondrial respiration by 13.6% and 49.5%, respectively . This difference resulted in a 27.9% increase and 22.6% and 37.5% decreases in the ATP production of cells treated with curcumin at 10, 20 and 35 µM, respectively, compared to that in control cells . Moreover, interventions with different doses of curcumin altered the maximal mitochondrial respiratory capacity of adipocytes. As shown in figure 3e, 10 µM curcumin enhanced the maximal mitochondrial respiratory capacity by 20.5%. However, the maximal mitochondrial respiratory capacity was reduced by 16.7% and 32.4% in cells treated with 20 µM and 35 µM curcumin, respectively, compared to that of the control (p < 0.05). There was no significant difference in the spare capacities of cells treated with 20 and 35 µM curcumin (p > 0.05), but the spare capacity of the 10 µM curcumin treatment group was increased by 26.4% compared to that in the control group (figure 3f). These results indicate that 10 µM curcumin significantly promotes mitochondrial respiratory function. By contrast, curcumin at doses of 20 and 35 µM inhibits mitochondrial respiratory function.As shown in figure 3. 3.3a). Specifically, the PPARγ antagonist significantly reduced the basal mitochondrial respiration (figure 4b), ATP production (figure 4c), maximum respiration (figure 4d) and spare capacity (figure 4e) after supplementation with curcumin (p < 0.01). Taken together, these data indicate that curcumin significantly improves the mitochondrial respiratory capacity in part through PPARγ induction in mature adipocytes.Next, we examined the PPARγ alterations, a key factor in adipocyte differentiation induction , in respfigure 4. 3.4p < 0.05). Furthermore, GW9662 (a PPARγ inhibitor) treatment attenuated the curcumin-induced upregulation of PPARγ activity to a level near that achieved with GW9662 alone. These results indicate that curcumin could partly enhance PPARγ RNA and protein expressions, and then exert further pharmacological functions. Additionally, GW9662 blocked the increased protein expressions of UCP1 and PRDM16 in response to curcumin treatment (p < 0.05), but it did not decrease the PGC-1α expression. These results show that curcumin induces the browning of white adipocytes through triggering PPARγ.As demonstrated in . 3.5a) and the volume of fat cells was significantly larger in DIO group mice compared to those in normal group mice (figure 6c). After curcumin (50 mg kg−1 d−1) treatment, the cell sizes were markedly decreased. The nuclei (blue) and cytoplasm (pink) of adipose cells were squeezed to one side because of the emergence of lipid droplets in the DIO group compared with those in normal BAT (figure 6b). Moreover, the BAT architecture in obese mice demonstrated a higher number of lipid droplets. Curcumin treatment significantly decreased the fat cell size and attenuated lipid droplet accumulation compared with those in the DIO group (figure 6d). Interestingly, more visible cavity beige fat cells were observed in the curcumin treatment group than in the control group, which indicated that curcumin can inhibit the hypertrophy of white fat cells and promote the transition of white fat cells to brown fat cells.As shown in figure 6. 3.6p < 0.05). After curcumin (50 mg kg−1 d−1) treatment, the WAT mRNA expression levels of UCP1, PRDM16, PGC-1α and PPARγ were increased by 4.7-, 3.5-, 6.0- and 7.2-fold, respectively, compared with those in the DIO group (p < 0.05). The mRNA expression levels of UCP1, PRDM16, PGC-1α and PPARγ in the BAT were increased by 2.9-, 4.4-, 3.0- and 2.3-fold, respectively, compared with mice in the DIO group.As shown in . 3.7p < 0.05). Additionally, the expression levels of these proteins in BAT were higher in the curcumin treatment group than in the vehicle treated DIO group (p < 0.05).As shown in . 4It is generally accepted that abdominal obese individuals exhibit a higher trend to develop metabolic diseases and cardiovascular complications . The reain vivo results revealed that curcumin attenuated pathological damage and enhanced the browning process of adipose tissues in DIO mice, which corresponds with the results of previous studies [Adipose tissues are important insulin-sensitive peripheral tissues that are traditionally considered to primarily function as an energy store. When fuel is required, triglycerides in adipocytes are broken down into free fatty acids (FFAs), released into circulation, and further oxidized to provide energy for utilization . Neverth studies .2+ homeostasis [Mitochondria are important organelles participating in the regulation of numerous cellular activities, including apoptosis, thermogenesis, reactive oxygen species production, redox and Caeostasis ,32. Mitoeostasis ,34. And eostasis . To exameostasis . And we eostasis , which mPPARγ, a vital nuclear receptor, exerts a principal function in the regulating adipogenic differentiation and lipid metabolism ,37. So fβ (C/EBPβ), phosphoenolpyruvate carboxykinase (PEPCK) and so on [+ adipocytes. PGC-1α is also an important nuclear transcription factor that controls the expression of UCP1 [PPARγ is a chief regulator that can promote the differentiation process of white adipocytes collaborating with multiple other regulators, such as PGC-1, CCAAT/enhancer binding proteinnd so on . Accordind so on . During  of UCP1 ,44 and a of UCP1 . PRDM16, of UCP1 . Moreove of UCP1 . In our . 5Taken together, the results of the present study demonstrated that curcumin may provide a new solution for controlling and managing obesity and other metabolic diseases. The underlying mechanism may be attributed to an improvement in mitochondrial function and promotion of adipogenesis via regulation of PPARγ expression, which further led to increased expression levels of the relevant transcription factors PGC-1α, UCP1 and PRDM16 in adipocytes. In the future, corresponding clinical trials are necessary to confirm these results.Click here for additional data file.