CYP1A1 may be associated with an increased susceptibility to cervical cancer.The study aimed to evaluate four common polymorphisms of the CYP1A1 and cervical cancer susceptibility among Northeast Thai women. CYP1A1 is an enzyme in phase I of the cytochrome P450 (CYP) superfamily, and plays a key role in detoxification of carcinogens. Host genetic predisposition in the CYP1A1 m1, m3, and m4 genotypes were detected using PCR-RFLP, whereas the CYP1A1 m2 genotype was investigated using real-time PCR. Haplotype analysis was performed using PHASE algorithm version 2.1.1. A case-control study was conducted involving 204 patients with squamous cell cervical cancer (SCCA) and 204 age-matched healthy controls. DNA was extracted from peripheral blood leucocytes. CYP1A1 m3 was monomorphic. Association between the common CYP1A1 polymorphisms, m1 and m2, and cervical cancer risk was not observed (p>0.05), nor was any association found between the m1–m2–m4 haplotype and cervical cancer risk (p>0.05). Interestingly, the CA genotype of CYP1A1 m4 was observed in 30.88% of the cervical cancer patients but was absent in healthy controls. CYP1A1 m4 polymorphism but no other common polymorphisms  in the risk for cervical cancer.This finding may be useful when screening for risk of cervical cancer among Northeast Thai women.Our results demonstrated a possible involvement of the  CYP1A1 polymorphisms are associated with an increased susceptibility to cancer ; due to alterations to mRNA expression and enzymatic activity . These changes may influence the balance between electrophilic molecules and reactive metabolites, leading to DNA adduct formation and the initiation of carcinogenesis .Cytochrome P450 1A1 (CYP1A1), an important enzyme in phase I of cytochrome P450 superfamily (CYPs), plays a key role in the detoxification of several xenobiotics  and endogenous substances  . In addition to these substances, CYP1A1 has potential for catalyzing environmental procarcinogens such as polycyclic aromatic hydrocarbons (PAHs), leading to generation of reactive metabolites that are highly reactive towards DNA . Accumulating evidence shows thatCYP1A1 is located on 15q22-24 . It is generally expressed in extrahepatic tissues, especially epithelial tissues . Among CYP1A1 variant alleles, common variants include four single nucleotide polymorphisms (SNPs): T3801C (m1), A2455G (m2), T3205C (m3), and C2453A (m4) . Ordinary, m1 and m3 are located in the 3’ noncoding region, giving rise to a MspI restriction site; whereas m2 and m4 are located in exon 7, leading to the amino acid transition of isoleucine to valine on codon 462 and threonine to asparagine on codon 461, respectively . To date, genetic polymorphisms of human CYP1A1 have been widely studied for the susceptibility to various cancers  . Notwithstanding, to our knowledge there are no recent reports of any association between the four polymorphic loci of the CYP1A1 and cervical cancer susceptibility . Our aim was thus to evaluate these four polymorphic loci of CYP1A1 for the cervical cancer susceptibility among Northeast Thai women.Comprising seven exons and six introns, the Subjects0 = 0.05 . Each subject was informed of the methodology and objectives of the research and signed an informed consent form. The study was reviewed and approved by the Ethics Committee of KhonKaen University (HE 571482).This was a case-control study, including 408 female volunteers between 26 and 81 years of age-conducted at Srinagarind Hospital and KhonKaen Hospital in KhonKaen Province, Northeast Thailand, between February 2009 and August 2011. The subjects included 204 cases of pathologically-defined squamous cell carcinoma of the cervix and 204 age-matched healthy women  confirmed by cytological and histological examination.The sample size was obtained from the case-control sample size table where the level of significance, α = 0.05; the power = 0.9; β = 0.2; relative risk, R = 3.0; and proportion of mutant type, PDetection of CYP1A1 polymorphismsCYP1A1 were analyzed. The m1, m3, and m4 SNPs were examined using the polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) method , while the m2 SNP was examined using TaqMan probe real-time PCR method . Three milliliters of EDTA-blood samples were collected from all subjects. The genomic DNA was extracted from the buffy coat using GF-1 Blood DNA Extraction Kits . Four polymorphisms  of the The PCR condition and PCR reaction mixture are as followm2, m3, m4 SNP detection– The method and sequence of primer pairs follow Hussein et al., (2014). The PCR reaction mixture contained 12.5 µL of 2X Taq Master Mix, 0.2 µL of each primer, 11.1 µL of distilled water, and 1 µL of genomic DNA (80 ng/µL), reaching a final volume of 25 µL. The following condition was used to amplify: a holding stage at 95°C for 9 minutes, followed by 35 cycles of denaturation at 94°C for 60 seconds, annealing at 57°C for 45 seconds, and extension at 72°C for 45 seconds, and the final elongation step at 72°C for 4 minutes. The PCR product was digested using the MspI enzyme  in 1 UL of l0x NE buffer (New England. USA), incubated at 37°C for 2 hours confirmed by 2 % agarose gel electrophoresis, followed by ethidium bromide staining, and photographed under UV light. m2 SNP detection® probe with primers  was designed for a specific SNP . The respective FAM and VIC fluorescence dyes on the probes were used for the wild type  and the mutant type  .The PCR reaction of 10 µL included 5 µL of TaqMan Universal PCR Master Mix, 0.5 µL of TaqMan probe with primers, 3.5 uL of distilled water, and 1 µL of genomic DNA. The holding stage was at 95°C for 10 minutes, followed by 40 cycles of denaturation at 95°C for 15 seconds, annealing at 60°C for 60 seconds, and extension at 60°C for 60 seconds. The TaqManm3 SNP detectionMspI enzyme  was used to distinguish the m3 genotype, incubated at 37°C for 2 hours and confirmed by 2 % agarose gel electrophoresis with GelRed .The method and sequence of primer pairs followed Hirata et al., (2008). The PCR reaction mixture contained 12.5 µL of 2X Taq Master Mix, 0.2 µL of each primer, 9.1 µL of distilled water, and 3 µL of genomic DNA, reaching a final volume of 25 µL. The holding stage was at 94°C for 9 minutes, followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 54°C for 40 seconds, and extension at 72°C for 40 seconds, with the final elongation step at 72°C for 4 minutes. The m4 SNP detectionBsaI enzyme  was used to distinguish the m4 genotype, incubated at 37°C for 4 hours, and confirmed by 2.5% agarose gel electrophoresis with GelRed .The method and sequence of primer pairs followed Cascorbi et al., (1996). The PCR reaction mixture contained 12.5 µL of 2X Taq Master Mix, 0.5 µL of each primer, 9.5 µL of distilled water, and 2 µL of genomic DNA, reaching a final volume of 25 µL. The holding stage was at 94°C for 5 minutes, followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 61°C for 30 seconds, and extension at 72°C for 30 seconds, with a final elongation step at 72°C for 4 minutes. The Statistical analysesCYP1A1 polymorphisms and cervical cancer risk was assessed using uni- then multivariate logistic regression. The Hardy-Weinberg equilibrium was tested using Pearson’s chi-square. The haplotype analysis of the CYP1A1 was performed using PHASE algorithm version 2.1.1. The strength of association between the CYP1A1 polymorphisms and cervical cancer risk was measured using odds ratios (ORs) with 95% conﬁdence intervals (CIs). A p-values of <0.05 was considered as statistically significant.Statistical analyses were performed using STATA software V.14. The association between each SNP of the CYP1A1 are summarized in CYP1A1 m3, was monomorphic. The A allele of m4 was detected only in the cases  but the AA genotype was not detected (p>0.05). The genotype distributions of the m1 and m2 polymorphisms among the controls and cases were consistent with the Hardy-Weinberg equilibrium (HWE) (p>0.05); however, that of the m4 polymorphism among the cases deviated from the HWE (p<0.01). The presence of the A allele carrying chromosomes being only among the cases was an unexpected finding (Fisher’s exact test p<0.0001). We then evaluated the characteristics of the m4 A allele carriers. Among the 204 cases, the A allele carriers did not show (a) age dependency, (b) different HPV positivity, or (c) difference in age of menarche.Genotype distributions and allele frequencies of the four loci of the detected . No assoCYP 1A1 m1–m2–m4 is shown in p>0.05).The haplotype analysis of CYP1A1 and development of cervical cancer among Northeastern Thai women. In this study, the m3 locus was monomorphic as in other reports for Indians  and Americans (San Francisco) . The polymorphic status in the m3 locus may be specific for persons of African-American descent . Our results show a lack of significant association between m1 and m2 polymorphisms and cervical cancer risk, which as in several others tudies-done among Japanese, Israeli Jewish, Polish, Chinese, and Indian populations . To contrast, significant associations between m1 and/or m2 polymorphism and increased cervical cancer risk have been documented in several populations , and a meta-analysis indicated that the m1 (CC) genotype was associated with an increased risk for cervical cancer among Asians and Mixed populations . It is thus premature to conclude the role of m1 and m2 polymorphisms in cervical cancer development.We evaluated the association between four common polymorphisms in the human p>0.05). At present, only a few studies have reported an association between CYP1A1 haplotypes and cancer susceptibility. Wright et al.,(2010) and Shah et al., (2008) showed that when using the T–A–C haplotype as the reference, the C–G–C and T–G–C haplotypes were associated with an increased risk of lung cancer. To compare, Chang et al., (2003) showed that the C–A–C haplotype was associated with a decreased risk of prostate cancer via the role of estrogen metabolism. These inconsistent findings may indicate a specific role of the CYP1A1 haplotypes in different types of cancer.No significant association was found between the m1–m2–m4 haplotypes and cervical cancer risk in the current study (p<0.0001) strongly suggests participation of the CYP1A1 m4 A allele in the development of cervical cancer. We attempted to determine the cause of the high concentration of the A allele among cases ; by analyzing personal parameters.We then considered with respect to CYP1A1 function in the metabolic activation of carcinogens. As for m4 polymorphism, possible associations have been documented between the A allele and risk for lung cancer , laryngeal squamous cell carcinoma  and thyroid cancer . No association has been observed between m4 polymorphism and breast, colorectal, and gastric cancer . Moreover, none of the previous studies have documented any association between m4 polymorphisms and cervical cancer; notwithstanding, our finding that the A allele existed only among cases ; however, high up-regulation of the CYP1A1 mRNA, protein, and activity can be detected by inducers  . While CYP1A1 activity trends to be highest for the m4 variant in benzo[a]pyrene metabolism , the m4 variant exhibits the greatest catalytic efficiency for producing diol species and significantly increases formation of diol epoxide-2 (BPDE2)—the potent mutagenic species associated with increased cancer risk . Smoking is a major risk factor for development of cervical cancer among Northeast Thai women . Although our smokers were passive smokers, those with the A allele may be more vulnerable to the carcinogenicity of smoke than those with the CC genotype. There is thus a possibility that the m4 variant of the A allele plays a key role in the development of cervical cancer.Benzo[a]pyrene-an environmental procarcinogen-is a specific substrate for CYP1A1 activation leading to generation of highly reactive intermediates-such as B[a]P-7,8-diol-9,10-epoxide (BPDE), which is classified as the potent carcinogenic metabolite . BPDE-DNA adducts have been identified in human epithelial cervical tissue, especially among smokers . In human tissues,CYP1A1 m4 polymorphism over against other common polymorphisms  in the risk for cervical cancer among Northeast Thai women.In summary, our results demonstrated the possible involvement of the


The as-prepared ternary Ag2O/AgBr–CeO2 composite with double Z-scheme construction was analyzed by various analytical techniques. Ag nanoparticles (NPs) used as the electron medium could reduce the recombination of photoelectrons and holes, thus leading to the improvement of photocatalytic performance of these catalysts. Due to the unique structure and composite advantages, the optimal Ag2O/AgBr–CeO2 photocatalysts exhibit the superior tetracycline (TC) degradation efficiency of 93.23% and favorable stability with near-initial capacity under visible light irradiation. This ternary Z-scheme structure materials will be the well-promising photocatalysts or the purification of antibiotic wastewater.In this work, CeO Recently, a variety of methods such as physical absorption, biodegradation, et al. have been developed to eliminate pollution of TC6. Among the above methods, photocatalytic degradation is one of the most potential because of its low mammalian toxicity and high-efficiency. Nonetheless, the great mass of semiconductors such as TiO2, ZnO et al. can only degrade the contaminants under the UV light, which reduces the utilization effect of visible light7. Therefore, the quest for an ideal visible light absorption photocatalyst has become the focus on attention.With the unreasonable use of antibiotics in human production and life, antibiotics which come from the wastewater of medical treatment2) is a potential photocatalyst because of its outstanding catalytic activities, lower production costs, and excellent optical and chemical properties8. However, its widespread application under visible light irradiation is hindered owing to its over quick recombination of photoelectron hole pairs and the narrow range of light responses9. Recent years, a variety of coping methods such as morphological control9, doping other elements10, heterojunctions engineering11 have been adopted to remedy the shortcomings, thus improving the catalytic activity of CeO2. Among them, constructing heterojunctions can effectively improve catalytic performance due to the enhanced light absorption ability and effective charge transfer rate. And the increasing number of heterojunctions such as C3N4/CeO212, BiOI/CeO213, AgI/CeO214 and Co3O4/CeO215, are reported to apply for photocatalytic degradation of TC. Although the photocatalytic activity of binary composite has a great improvement, it is still some distance from practical application.Cerium oxide  and strong oxidization of PS II. The difference is that oxidation reaction and reduction reaction of traditional heterojunctions occur in the valence band and conduction band of PS I and PS II, respectively, although they can effectively improve the photoelectron-hole separation. However, due to the relationship between PS II and PS I position, although the range of light response is widened, the ability of REDOX will be weakened. Compared with the traditional heterojunction, Z-scheme photocatalytic materials can not only ensure the expansion of the optical response range, but also improve the oxidation and reduction capacity of PS I and PS II. The Z-scheme photocatalytic materials will have an excellent application prospect.The Z-scheme photocatalytic system originates from the light reaction stage of plant photosynthesis in nature18. And an increasing number of ternary photocatalytic materials have been reported, such as Fe3O4/Bi2S3/BiOBr19, Ag3PO4/TiO2/Fe3O420, Bi2WO6/Ag2S/ZnS21, and V2O5/BiVO4/TiO222. In spite of some ternary composites reported, there are few reports about cerium dioxide ternary materials. Therefore, it is expected to design a preeminent three-way CeO2-based photocatalyst with favorable catalytic properties.In recent years, more and more researchers have paid attention to the preparation of ternary composites. In the whole ternary catalytic reaction process, photo-generated carriers can be transferred in multiple steps through the induction mechanism to achieve the purpose of electron and hole separation, thereby achieving photocatalytic activity beyond the binary catalytic system2-based photocatalysts. Ag2O has excellent visible light absorption properties, while AgBr has excellent photocatalytic activity24. Compared to the existing materials, the two may be the better choice. However, pure Ag2O and AgBr also have problems such as lower photo-generated carrier yield, unstable photocatalytic activity and high carrier recombination rate. To address these shortcomings, researchers often use them as co-catalysts to improve their stability. For example, Wen et al. fabricated Ag2O–CeO2 photocatalyst to degrade enrofloxacin effectively by Ag2O nanoparticles embellishing CeO2 spindles25. Huang et al. synthesized flower-like AgBr/Bi2WO6, which degraded 87.5% TC solution within 60 min under visible light irradiation6. Thus, we attempt to modify cerium dioxide with silver oxide and silver bromide to construct Ag2O/AgBr–CeO2 ternary composites.It’s necessary to select suitable doping materials which could directly affect the efficiency of whole ternary composites, to design the ternary CeO2, Ag2O, and ACA-2 were shown in Fig. 2 nanosheets are about 1–5 μm and 50–100 nm, respectively. The size of Ag2O NPs is about 206 nm  crystal plane of CeO2 was 0.343 nm26. The observed lattice spacing of 0.247 and 0.237 nm in HRTEM image are separately consistent with the (220) crystal plane of AgBr27 and (200) crystal plane of Ag2O28, respectively. Meanwhile, Selected Area Electron Diffraction (SAED) Patterns could also confirm the theory , (200), (220), (311), (222), (400), (331) and (420) planes of the cubic phase CeO2 crystal (JCPDS: 34-0394)27. Moreover, other crystal phases cannot be caught in the XRD pattern, indicating that the crystallinity of synthesized CeO2 is favorable. The sample of pure Ag2O shows the typical diffraction peaks at 2θ of 32.76°, 38.10°, 54.74°, 65.41°, and 68.69°, which corresponds to the (110), (111), (200), (220), (331) and (222) planes (JCPDS: 41-1104)25. The diffraction peak of AgBr standard cards (JCPDS: 06-0438)29 correspond to XRD patterns of the pure AgBr. Respecting the ACA-2 composites, the typical diffraction peaks of CeO2, AgBr, and Ag2O can be clearly observed. According to Supplementary Fig. 2O due to the shading effect of the close peaks of CeO225. According to the above analysis, the crystal phase of Ag2O/AgBr–CeO2 was not affected by loading the Ag2O and AgBr.The XRD patterns of pure Ag30. The positions of Ag 3d5/2 and Ag 3d3/2 peaks are at 367.44 and 373.45 eV  and Br 3d3/2 (68.67 eV) in Fig. 31. As presented in Fig. 2O27. Therefore, the consequence of XPS analysis indicates that Ag2O and AgBr connect with CeO2 via chemically bound interface rather than the physical contact.X-ray photoelectron spectroscopy (XPS) tests were carried out to determine the elemental composition and the chemical state of ACA-2. The XPS survey spectrum in Fig. 2 and AgBr exhibited visible light absorption with absorption band edges at 436 and 519 nm, respectively33. Meanwhile, pure Ag2O revealed an evident absorption in completely visible light scope, which corresponds with the previous reports34. As shown in Fig. g) could be obtained by Kubelka–Munk function: αhv = A (hv—Eg)n/2, where Eg, α, A, h, ν correspond to energy gap, absorption coefficient, a constant, Planck’s constant and light frequency. CeO2, Ag2O and AgBr were indirect bandgap, and four was the value of n35. The Eg values of CeO2, AgBr and Ag2O were about 2.98, 2.56 and 1.34 eV, respectively.UV–Vis spectra were illustrated in Fig. ECB =X − EC − 0.5Eg and EVB = ECB + Eg, where X, EC and Eg represented the electronegativity of crystalline semiconductors, the energy of free electrons on the hydrogen scale (~ 4.50 eV NHE) and the energy gap of semiconductors37. The ECB of CeO2, Ag2O and AgBr were calculated to be − 0.39, 0.13 and 0.02 eV. Then their EVB was corresponded to 2.59, 1.47, and 2.58 eV.According to the empirical formulas 2 was only 16.99%. For Ag2O/AgBr–CeO2 composites, the ACA-2 sample showed the best photocatalytic activity, which can remove 95.59% RhB. It could be discovered that the quantity of AgBr affected their ability of degradation. The degradation rate was increased because the purity of AgBr was from 7.92 to 15.37 wt%. When the content of AgBr was exceeded 15.37 wt%, its shading effect had an impact on the photocatalytic activity of composites. As shown from the photocatalytic degradation reaction kinetics of RhB in Fig. −1) based on the muri-Hinshelwood model was higher than other samples38. However, the kinetic constant of CeO2 was only 0.00182 min−1. According to the analysis in Fig. The photocatalytic performances of the samples over RhB were studied under visible light. The details of the dark reaction experiments were shown in Supplementary Fig. 2 and AOC after 60 min of visible light irradiation, respectively. As expected, when AgBr nanoparticle put into composites, the photocatalytic activity of ACA-2 (93.68%) was more outstanding than other materials. The catalyst exhibited evidently degradation effect was analogous to its catalytic behavior of removal RhB. Meanwhile, the degradation effect kinetic of TC for all products were studied in Fig. −1, while CeO2 was only 0.00149 min−1. The information could prove that the photocatalytic activity of CeO2 could be improved by Ag2O and AgBr modification.Moreover, TC was selected as a typical antibiotic pollutant, and the obtained samples were degraded to eliminate the influence of dye self-sensitization. The details of the dark reaction experiments were shown in Supplementary Fig. 2O/AgBr–CeO2. In Fig. 36. (2) As the concentration of pollutants increases, the number of intermediates produced in the reaction increases, which led to the competition with TC39. Meanwhile, for TOC degradation, ACA-2 also shows the best removal rate  on photocatalytic degradation of TC solution using ACA-2. As shown in Fig. 2SO4 decreased slightly40. However, when NaCl appended to the solution, the removal efficiency of TC was reduced to some extent, which might put down to competitive adsorption of Cl- with other substances38. The addition of NaHCO3 has a significant effect on the degradation of TC because HCO3- has the function of a free radical scavenger, which can consume some active free radical27. Therefore, the reduction of radicals decreased the photocatalytic degradation of TC by ACA-2.It is well known that there are many kinds of anions in practical wastewater, which may affect the degradation efficiency of polluted solution by photocatalysts. Therefore, we studied the effects of various anions , sodium oxalate  and benzoquinone 42 were added to the solution of TC before photodegradation. As presented in Fig. ·O2− played an important role in reaction systems. Meanwhile, when Na2C2O4 and IPA were added into the TC solution, the degradation rate of TC was 63.54% and 65.99% respectively, less than the removal of no scavenger. Therefore, holes and ·OH also played a pivotal role in the catalysis system. It can be deduced that the h+, ·OH and ·O2− were the main active radicals affecting the elimination of TC solutions.To research the photocatalytic mechanism of Ag2O/AgBr–CeO2 catalyst is composed of p-type semiconductor Ag2O and n-type semiconductor CeO2 and AgBr. The p–n heterojunction is effectively constructed in the composite catalyst, which can effectively separate the electron-hole43. On the side, photocurrent density could be an efficient method of evaluating the transfer properties of the photogenerated electrons as well. And the higher the photocurrent response the higher separation efficiency. From Fig. 2, AOC, CAB and ACA-2 composites under illuminating visible light were measured. These composites and CeO2 showed the stable photocurrents under visible light irradiation respectively generating electrons and holes44. And the result of EIS spectra of ACA-2 , indicating that ·O2− are generated in the reaction system and participate in the photocatalytic degradation reaction. According to Fig. ·OH participate in the photocatalyst reaction. It is consistent with the results of active species trapping experiments.In order to verify the activity of free radicals, the samples were tested by Electron Paramagnetic Resonance (EPR)2 is excited to produce photoelectrons under visible light irradiation. The electrons on the CeO2 (CB) jump to the CB of AgBr and continue to transfer to silver oxide (CB), which follows the law of conservation of energy. Meanwhile, h+ on the surface of CeO2 (VB) should transfer to VB of AgBr. Holes don’t rest on AgBr (VB) and jump to the valence band of Ag2O. Holes could combine with H2O to form hydroxyl radicals (H2O/OH: 2.40 eV vs. NHE). However, e− on the conduction band of Ag2O can’t combine with O2 to form ·O2− due to the higher conduction band of Ag2O (1.30 eV) than superoxide radical potential (O2/·O2−: − 0.046 eV vs. NHE)47. Therefore, photocatalyst doesn’t produce ·O2− to oxidize organic matter that is inconsistent with those of free radical capture experiments. Based on the above analysis, the conjecture of Scheme 0. Thus, the Z-scheme heterojunction system with Ag NPs as the bridge between Ag2O, AgBr and CeO2 can be carried out under visible light. As presented in Fig. 2O and AgBr are excited to produce photogenic electrons (e−) and holes (h+) under visible light irradiation. In addition, the electrons in the conduction band of Ag2O are transferred to the Ag NPs working as the electronic medium. So in such a situation, an electron of Ag2O is attracted by the electron trap constructed by Ag NPs. In a similar way, e- of AgBr (CB) also transfers to Ag NPs. In the meantime, the holes in the valence band of Ag2O are transferred to the Ag NPs that are combined with e− of Ag NPs. Holes in the AgBr (VB) are in the same way. The electrons flowing into the Ag NPs rapidly recombine with the holes, leading to an accelerated charge transfer rate. Moreover, Ag NPs realize the spatial isolation of photoelectric pairs, which greatly limits the undesirable recombination. Electrons of CeO2 (CB) combine with oxygen in H2O to form superoxide radicals (O2/·O2−: − 0.046 eV vs. NHE) and ·O2− can transform TC to CO2 and H2O. Holes in the valence band of Ag2O (1.47 eV) reacts with TC to form CO2 and H2O. h+ in the AgBr (VB) reacts with H2O to form ·OH because of the higher valence band of AgBr (2.58 eV) than H2O/·OH potential (H2O/·OH: 2.40 eV vs. NHE)48. And then hydroxyl radicals oxidize organic matter into degradation products. The Scheme 2O/AgBr–CeO2 composites could point out a particular mechanism guess, which not only improves the efficiency of carrier separations but also consolidates its stability.The mechanism of photodegradation of TC over the catalyst is shown in Fig.  of Ag2O .30 eV th48  (Scheme m/z) of 445 is due to the TC molecule. In addition, the m/z values produced during the photocatalytic degradation of TC in the presence of ACA-2 were 450, 406, 362, 334, 318, 290, 274, 246, 230 and 202. Based on the previous reports and the analysis of the results, two possible degradation pathways were proposed. For the first route, the double bond on TC are reduced to TC-2 (m/z = 450). And then TC-2 (m/z = 450) deaminates to form TC-3 (m/z = 334). TC-4 (m/z = 290) was obtained by removing one of the hydroxyl groups from TC-3 (m/z = 334). The second pathway was from the TC (m/z = 445) to TC-5 (m/z = 406) via fracture of the double bond. Afterwards, TC-5 deaminates to form TC-6 (m/z = 362), and TC-6 removed methyl group to form TC-7 (m/z = 318). Hydroxide radical of TC-7 was changed into TC-8 (m/z = 274) after oxidation. As the same time, TC-8 removed a formyl group to form TC-9 (m/z = 246). TC-9 continued to break off a carbonyl group to get TC-10 (m/z = 230). Meanwhile, TC-10 break the ring and removed a hydroxymethyl to form TC-11 (m/z = 202). Finally, a part of intermediates can ultimately mineralized into H2O and CO2.In order to further understand the degradation process of TC, the spectroscopy of the catalytic process of the intermediate products was detected by liquid chromatography-mass spectrometry) Scheme . Many in2O/AgBr–CeO2 photocatalyst was successfully prepared by the acid corrosion process of Ag2O/CeO2 that was gained through in situ loading of Ag2O onto CeO2 nanosheets. Thus, a double Z-scheme heterojunction degradation mechanism featuring Ag NPs as the bridge between the photogenerated electrons and holes was proposed, which can degrade 93.23% TC solution. Transient photocurrent response indexes that ternary Ag2O/AgBr–CeO2 composites demonstrate the ability to efficiently separate photo-generated electrons and holes. Free radical capture experiments indicate that the degradation of TC mainly relies on three factors . We believe that the novel three-way Ag2O/AgBr–CeO2 catalyst could have great potential in the field of energy and environmental protection. This work could provide a different Z-scheme heterojunction system to construct ternary catalysts improving the degradation efficiency.All in all, a novel ternary Ag3)3·6H2O), hydrobromic acid (HBr), silver nitrate (AgNO3), sodium hydroxide (NaOH) and Sodium Bicarbonate (NaHCO3) were all provided from Sino pharm chemical reagents Co., which were all analytical reagents. Rhodamine B and Tetracycline were provided from Alighting reagent Co.Cerium nitrate hexahydrate 3·6H2O was added to 30 ml H2O and stirred for 30 min at 0 ℃, which is solution A. Moreover, 0.75 g NaHCO3 also dissolved in 30 ml H2O, which is labeled as solution B. Solution B is dropwise added to solution A, the mixed solution stirred for 30 min. Then, the solution aged for 15 h. The powders were washed and dried at 60 °C for 12 h.CeO2 microsheets  were dispersed in 40 ml of ultrapure water (solution A) with the ultrasound equipment, then 5.00 ml AgNO3 (0.1 M) was added slowly to solution A under vigorous stirring. The mixed solution stirred for 30 min at room temperature. Meanwhile, 5.00 ml NaOH (0.1 M) was added drop by drop to the above mixed solution with stirring for 2 h at dark environment. The products were washed five times with ultrapure water and ethyl alcohol, and collected after being dried at 60 °C for 12 h. The sample was marked as AOC.CeO2–Ag2O (2.30 g) samples were ultrasonically dispersed in 50 ml of distilled water. Then a certain volume of HBr (0.1 M) was added in the above solution with stirring for 4 h in a dark environment. Afterward, the products were washed five times by ultrapure water and dried at 60 °C for 12 h. The synthetic flowchart of Ag2O/AgBr–CeO2 is illustrated in Scheme 2O/7.92 wt% AgBr–CeO2, 14.23 wt% Ag2O/15.37 wt% AgBr–CeO2, 9.21 wt% Ag2O/22.39 wt% AgBr–CeO2, 4.48 wt% Ag2O/29.03 wt% AgBr–CeO2, 35.31 wt% AgBr–CeO2 were marked as ACA-1, ACA-2, ACA-3, ACA-4 and CAB.CeOof HBr 0. M was ad4 as reference material. Electrochemical measurements were performed at Zahner electrochemical workstation using a standard three-electrode system. Detailed instructions are attached to the supporting information. Electron paramagnetic resonance (EPR) was used to verify the existence of ·O2− and ·OH.It is convenient to analyze and study the crystal structure and material type of samples through X-Ray Diffraction  and the field emission scanning electron microscopy . Field Emission Transmission Electron Microscope  achieved TEM and HRTEM photographs with an acceleration voltage of 200 kV. X-ray photoelectron spectroscopy  performed an elemental analysis of the samples. UV–Vis diffuse reflectance spectra (DRS) were recorded on Cary 5000 using with BaSOThe photocatalytic activity of composites was studied by the degradation of Rhodamine B (RhB) and tetracycline (TC) under the visible light irradiation (λ > 420 nm). The catalyst (0.025 g) was dispersed in the 50 ml solution with RhB or TC (the concentration of RhB or TC was 10 mg/l). The mixed solution stirred for 30 min in a dark environment to reach adsorption equilibrium. Afterward, the suspension was illuminated in visible light . 4.00 ml suspensions that were achieved with every 15 min were centrifuged to remove catalysts. The concentration of the RhB (554 nm) and TC (357 nm) was received by the UV-1200 spectrophotometer at its maximum absorption wavelength.Supplementary Information.


Aging is accompanied by the accumulation of senescent cells that alter intercellular communication, thereby impairing tissue homeostasis and reducing organ regenerative potential. Recently, the administration of mesenchymal stem cells (MSC)-derived extracellular vesicles has proven to be more effective and less challenging than current stem cell-based therapies. Extracellular vesicles (EVs) contain a cell-specific cargo of proteins, lipids and nucleic acids that are released and taken up by probably all cell types, thereby inducing functional changes via the horizontal transfer of their cargo. Here, we describe the beneficial properties of extracellular vesicles derived from non-senescent MSC, cultured in a low physiological oxygen tension (3%) microenvironment into prematurely senescent MSC, cultured in a hyperoxic ambient . We observed that senescent MCS, treated with EVs from non-senescent MCS, showed reduced SA-β-galactosidase activity levels and pluripotency factor  overexpression and increased glycolysis, as well as reduced oxidative phosphorylation (OXPHOS). Moreover, these EVs’ cargo induced the upregulation of miR-302b and HIF-1α levels in the target cells. We propose that miR-302b triggered HIF-1α upregulation, which in turn activated different pathways to delay premature senescence, improve stemness and switch energetic metabolism towards glycolysis. Taken together, we suggest that EVs could be a powerful tool to restore altered intercellular communication and improve stem cell function and stemness, thus delaying stem cell exhaustion in aging. This senescent phenotype includes morphological alterations, reduced proliferation rate [INK4a mRNA expression and high senescence associated-β-galactosidase activity levels [2), also called physioxia [Cellular senescence is the consequence of several stresses by which cells lose their ability to proliferate . Our groy levels . Furtherhysioxia . The acchysioxia . Similarhysioxia ,7.www.microvesicles.org”). Logically, depending on the characteristics of the cell of origin, EVs may trigger different responses on target cells. These vesicles can transfer signals capable of altering cell fate, cell function and the reprogramming of recipient cells through epigenetic modifications [Extracellular vesicles (EVs), which include exosomes and microvesicles, are released into the extracellular microenvironment by almost all cell types and act as delivery carriers of intercellular communication through the transfer of their contents. Among them, several proteins, lipids, mRNAs, miRNAs and DNA have been described   and 1% antibiotic) under the same conditions of temperature and oxygen. Dental pulp stem cell characterization was performed as previously described [Intact third molars were collected from men and women (aged from 15 to 35 years old). All patients were informed and agreed freely to participate and signed the informed consent by contributing the extracted tooth, which was always extracted for reasons independent of this study. The study was approved by the institutional review board of the University of Valencia. Cells cultured from dental pulps did not exhibit any clinical and/or radiological sign or symptom of inflammation and/or infection. To isolate the cells, the pulps were firstly fragmented by trituration, then chemically digested with 2 mg/mL ethylenediaminetetraacetic acid (EDTA) in Krebs–Henseleit buffer, and finally digested with a combination of type I collagenase and type II dispase at a final concentration of 4 mg/mL during 30 min in a humid incubator at 37 °C, 5% COAfter the first passage, the DPSCs were divided in two groups—one group was moved to a humid incubator with an oxygen pressure of 21%, while the other group was kept in the same incubator used for the isolation at 3% oxygen. To perform the experiments, we used hDPSCs at passage 4, referred to as “young”, and hDPSCs at passage 25, referred to as “old”. All of the reagents were purchased from Gibco, Invitrogen, Madrid, Spain.g for 10 min, then at 2000× g for 10 min and then at 10,000× g for 30 min to remove whole cells, cell debris and aggregates. The supernatant was then ultracentrifuged at 100,000× g for 70 min. Pelleted vesicles were suspended in phosphate-buffered saline (PBS), ultracentrifuged again at 100,000× g for 70 min for washing, resuspended in PBS and prepared for treatment, transmission electron microscopy and flow cytometry analysis. Acceptor cells were treated with EVs for 48 h . In order to quantify the amount of EVs used for treatment, we measured total vesicle protein. On average, an amount of 7 µg of vesicle protein was used for the treatment of 1 million cells.We based our characterization of the EVs population on the definition proposed by the “International Society of Extracellular Vesicles” . hDPSCs For transmission electron microscopy (TEM), extracellular vesicles were included in agar and fixed with 2.5% glutaraldehyde, washed with 0.1 M phosphate buffer pH 7.2, and post-fixed with 2% osmium tetroxide in phosphate buffer. After washes with water, they were sequentially dehydrated in 30% EtOH, 50% EtOH, 70% EtOH, 90% EtOH and 100% EtOH. Samples were included in resin and polymerized at 60 °C for 48 h. Ultrathin slides (60 nm) were finally stained with 2% uranil acetate and lead citrate, prior to viewing by TEM, using a Jeol JEM1010  microscope at 60 kV. The images were acquired with a digital camera MegaView III with Olympus Image Analysis Software 2.4 .For flow cytometry analysis, EV pellets obtained from 10 mL supernatant were resuspended in 100 µL PBS. The EVs were stained with 4 µg/mL of APC-antihuman CD63  for 30 min at 4 °C in darkness. After incubation, positive events were read by fluorescence-activated cell sorting (FACS)–Verse flow cytometry. One unstained sample of the EVs and only one sample with antibody at 4 µg/mL in PBS were used as negative controls. The calibration of the cytometer was assessed using fluorescent particles of standardized size .® LacZ Kit  following the manufacturer’s instructions. hDPSCs were washed twice with warm PBS and treated with trypsin . Following 1000× g centrifugation for 2 min, pelleted cells were resuspended in Staining Medium. Then, 50 µL aliquots of resuspended cells (1 × 106 cells/mL) were placed into an appropriate flow cytometer tube and stained with 50 µL prewarmed fluorescein Di-β-D-Galactopyranoside (FDG) working solution for exactly one minute at 37 °C. FDG loading was stopped by adding 900 µL ice-cold staining medium containing 1.5 µM propidium iodide. The FDG values were read by FACS–Verse flow cytometry until 20,000 events were recorded.Senescence-associated-β-Gal staining was performed using the FluoReporterg and 2 min centrifugation, pelleted cells were resuspended in 1 × Annexin-Binding Buffer. Then, 100 µL aliquots of resuspended cells (1 × 106 cells/mL) were placed into an appropriate flow cytometer tube and stained with 5 µL Annexin V-FITC and 5 µL propidium iodide for exactly 15 min at 37 °C in darkness. After incubation, 400 µL of 1 × Annexin-Binding Buffer were added. The values were read by FACS–Verse flow cytometry  until 20,000 events were recorded.Apoptosis was assessed using ANNEXIN V FITC Apoptosis detection kit , following the manufacturer’s instructions. hDPSCs were washed twice with warm PBS and treated with trypsin . Following a 1000× g and 2 min centrifugation, pelleted cells were resuspended in serum-free DMEM containing 1 g/L glucose and 1% P/S. Then, 500 µL aliquots of resuspended cells (5 × 105 cells/mL) were placed into an appropriate flow cytometer tube and stained with 5 µL DHR123  at a final concentration of 1 µg/mL. The cells were then incubated for 30 min at 37 °C in darkness. After incubation, the intracellular peroxide values were read by FACS–Verse flow cytometry until 20,000 events were recorded.hDPSCs were washed twice with warm PBS and treated with trypsin . Following a 1000× 2 incubator overnight.To assess mitochondria respiration in cells, an XFe96 extracellular flux analyzer  was used to measure the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Then, 7000 h DPSCs were seeded into each well of a 96 XFe Cell Culture Microplate. One day prior to assay, the XFe Sensor Cartridge was hydrated using XFe Calibrant at 37 °C in a non-CO2 incubator for 30 min. On the day of assay, 1 µM oligomycin, 2 µM FCCP, and a 0.5 µM mix of rotenone/antimycin A  were used to measure ATP production, maximal respiration and nonmitochondrial respiration, respectively. Proton leak and spare respiratory capacity were then calculated using these parameters and basal respiration.The XFe Cell Mito Stress Test measures key parameters of mitochondrial function by sequential compound injections. One hour prior to assay, the culture medium was changed into unbuffered XFe Base Medium pH 7.4, supplemented with 1 mM pyruvate, 2 mM glutamine and 10 mM glucose, and the culture plate was incubated at 37 °C in a non-CO2 incubator for 30 min. On the day of assay, 10 mM glucose, 1 µM oligomycin and 50 mM 2-Deoxy-D-glucose (DG)  were used to measure glycolysis, glycolytic capacity, glycolytic reserve and non-glycolytic acidification.The XFe Glyco Stress Test measures glycolytic function in cells by sequential compound injections. One hour prior to assay, the culture medium was changed into unbuffered XFe Base Medium pH 7.4, supplemented with 2 mM glutamine, and the culture plate was incubated at 37 °C in a non-COAfter both assays, all measurements were normalized to total protein concentration using Lowry–Folin assay.Total RNA was isolated from hDPSCs by using TRIzol reagent , according to the manufacturer’s instruction. RNA was quantified by measuring the absorbance at 260 nm with NanoDrop 2000. The purity of the RNA preparations was assessed by the 260/280 ratio.cDNA was synthesized from 2 µg total RNA using the High-Capacity cDNA Reverse Transcription Kit . Each reaction was set for 20 µL: 10 × RT Buffer (2 µL), 25 × dNTP Mix 100 mM (0.8 µL), 10 × RT Random Primers (2 µL), Reverse Transcriptase (1 µL), RNase Inhibitor (1 µL), Nuclease-Free Water (3.2 µL) and RNA sample (10 µL). Following the manufacturer’s recommendations, incubation was established for 10 min at 25 °C, followed by 120 min at 37 °C, and then at 85 °C for 5 min and finally cooled to 4 °C to collect the cDNA on a T100 Thermal Cycler .PCR was performed using the Maxima SYBR Green Master Mix kit . Each reaction was set for 10 µL: 2 × Maxima SYBR Green/ROX qPCR Master Mix (5 µL), Forward Primer (0.3 µL), Reverse Primer (0.3 µL), Nuclease-Free Water (3.4 µL) and Product from RT reaction (1 µL). Target and control were run in separate wells following the procedure: 10 min at 95 °C and then 40 cycles of denaturation at 95 °C for 15 s and annealing and extension at 60 °C for 1 min per cycle using the detection system 7900HT Fast Real-Time PCR System . All the experiments were repeated at least three times for each sample.SOX2 , OCT4 (3′-GATCCTCGGACCTGGCTAAG-5′ and 5′-GACTCCTGCTTCACCCTCAG-3), KLF4 (3′-CCCACATGAAGCGACTTCCC-5′ and 5′-CAGGTCCAGGAGATCGTTGAA-3′) and cMYC (3′-CGCCCTCCTACGTTGCGGTC-5′ and 5′-CGTCGTCCGGGTCGCAGATG-3′) were normalized against the GAPDH (3′-TGAACGGGAAGCTCACTGG-5′ and 5′-TCCACCA- CCCTGTTGCTGTA-3′) housekeeping gene. Relative expression was analyzed using the standard curve method.Total RNA was isolated from hDPSCs by using TRIzol reagent , according to the manufacturer’s instruction. RNA was quantified by measuring the absorbance at 260 nm with NanoDrop 2000. The purity of the RNA preparations was assessed by the 260/280 ratio.cDNA was synthesized from 2 µg total RNA using the TaqMan MicroRNA Reverse Transcription Kit . Each reaction was set for 20 µL: 100 mM dNTPs (0.4 µL), MultiScribe RT (4 µL), 10 × RT Buffer (2 µL), RNase Inhibitor (0.25 µL), Nuclease-free water (3.35 µL), 5 × Pool RT Primers (8 µL) and RNA sample (2 µL). Following the manufacturer’s recommendations, incubation was established for 30 min at 16 °C, followed by 30 min at 42 °C, and then at 85 °C for 5 min and finally cooled to 4 °C to collect the cDNA on a T100 Thermal Cycler .PCR was performed using the TaqMan Universal Master Mix kit . Each reaction was set for 10 µL: 20 × TaqMan Small RNA Assay (0.5 µL), 2 × TaqMan Universal PCR Master Mix II no UNG (5 µL), Nuclease-free water (3.5 µL) and Product from RT reaction (1 µL). Target and control were run in separate wells following the procedure: 2 min at 50 °C, 10 min at 95 °C and then 45 cycles of denaturation at 95 °C for 15 s and annealing and extension at 60 °C for 1 min per cycle using the detection system 7900HT Fast Real-Time PCR System . All the experiments were repeated at least three times for each sample.hsa-miR-302b (TaqMan™ MicroRNA Assay) was normalized against the RNU66  housekeeping gene. Relative expression was analyzed using the ∆∆CT method.Total protein was harvested by lysing the cells in a lysis buffer containing a protease inhibitor cocktail . Protein content was determined by a modified Lowry method . In totan was not the same in all the groups, the comparison of Scheffé was used. All values are means ± SD of measurements in at least three different cultures (three replicates each). Significance was defined as * p < 0.05, ** p < 0.01, and *** p < 0.001.Quantitative variables are expressed as means and SD. Qualitative data are expressed as total number and percentage. Statistical analysis consisted of Student’s T-test for two means. If Human dental pulp stem cells (hDPSCs) shed a diverse population of extracellular vesicles (EVs). To obtain them, we collected supernatants from hDPSCs after 48 h of culture and we performed differential ultracentrifugation, as shown in 2) impairs cell proliferation, leading to premature senescence. However, during long term culture under physiological oxygen levels (3% O2), hDPSCs only suffer replicative senescence. To determine whether EVs could modify senescence, we analyzed both replicative and premature senescence. We first isolated EVs from young hDPSCs (passage 5) cultured at 3% O2. We used sEVs from these cells to treat premature senescent cells (young hDPSCs at passage 5 cultured at 21% O2). As shown in 2). As we can observe in In previous studies, we demonstrated that the long term culture of hDPSCs under standard culture conditions , which is developed after genotoxic stress in culture. SASP is characterized by the secretion of myriad factors, as well as the release of small extracellular vesicles, including exosomes and microvesicles ,18. As cAn additional feature of the senescent phenotype is a high metabolic rate , and EV In contrast to somatic cells, pluripotent stem cells show higher rates of glycolysis and lower levels of mitochondrial metabolism, marked by a reduced mitochondrial content . Thus, iThe exchange of genetic information through EVs may explain mechanisms involved in the maintenance of stemness or differentiation, as well as in stem cell-mediated tissue repair after injury . Accordi2, suggesting that miR-302b expression is influenced by oxygen tension. Furthermore, we detected increased expression levels of miR-302b in hDPSCs cultured at 21% O2 after 12 h of treatment with EVs, as shown in To evaluate whether the impact of physioxia on pluripotency factors is associated with changes in the miRNA pattern, we analyzed the expression of the ESC/pluripotency specific miR-302b. hDPSCs, cultured at physioxia, showed higher expression levels of miR-302b compared to those cultured at 21% O2. Interestingly, we did not observe any changes in ROS levels, as shown in 2 showed reduced levels at the G0/G1 phase, and higher levels at the S and G2/M phases, suggesting an overall increased cell proliferation.It is also suggested that miR-302b increases cell proliferation and protects cells from oxidant-induced cell death in human mesenchymal stem cells . TherefoExtracellular vesicle formation allows for the vehiculation of proteins, nucleic acid and lipids, all of them specific to the cell of origin. Therefore, EVs are potentially capable of different biological activities, depending on their content. The transfer of bioactive molecules may change the phenotype and function of the recipient cells. As our results show, introducing EVs derived from a young and healthy micro ambient cell can delay senescence progression. However, not all recipient cells may change phenotypically, as this is the case of replicative senescent cells. These cells have been submitted to serial passaging, thus accumulating damages. On the contrary, our premature senescence model is associated with oxidative stress, which makes young DPSC look older . EV trea2 in vitro condition, or physioxia, maintains the stable expression of OSKM pluripotency factors during serial passages. More specifically, SOX2 and OCT4 are upregulated at early passages and their expression decreases upon passages, suggesting their involvement in stemness induction, whereas cMYC and KLF4 expression at early passages is low and increases with passages, which correlates with their implication in stemness maintenance. On the contrary, 21% O2 induces premature senescence, which is accompanied by the rapid downregulation of all of these factors’ expressions in early passages [As we described in previous studies, 3% Opassages . Additiopassages ,27,28.According to our results, EVs derived from physioxia-cultured cells might be enriched in miR-302b, and its internalization by prematurely senescent cells unleashed the upregulation of OSKM factors. Supporting our findings, it has been proven that miR-302b silences AOF1/2 and DNMT1 activities and, in conjugation with the co-suppression of MECP1/2 and HDAC2, results in global DNA demethylation and chromosomal H3K4me2/3 modifications and subsequently, these epigenetic events induce ESC-specific gene expression, such as SOX2, OCT4 and Nanog . Further2, may be influenced by the EV-encapsulated miR-302b directly, or through HIF-1α modulation, as shown in 2. Thus, the exogenous and endogenous upregulation of miR-302b in acceptor cells could be happening at the same time to induce and maintain the stemness properties.Interestingly, vesicle-encapsulated miR-302b from hDPSCs, cultured under physioxia, also caused an overexpression of HIF-1α in hDPSCs cultured under oxidative stress conditions. In accordance with our results, it has been suggested that miR-302b may induce HIF-1α expression through the ERK pathway ,33. Furt2 upon vesicle internalization. It is known that HIF-1α promotes glycolysis and HIF-1α-dependent pyruvate dehydrogenase kinase (PDK) activation. PDK, in turn, prevents pyruvate oxidation by suppressing the pyruvate dehydrogenase (PDH) complex [2 could be due to HIF-1α overexpression following miR-302b enriched EV internalization.Most stem cells rely on anaerobic glycolysis to maintain their undifferentiated state and for faster ATP generation and the production of cellular building blocks to meet the anabolic demands of high proliferative growth . As they complex . TherefoSummarizing, microenvironment modulation, through vesicle-encapsulated miR-302b, delays the premature senescence of hDPSCs, and probably reduces SA-β-galactosidase activity by modulating the p53–p21 and p16–pRb signaling pathways. At the same time, miR-302b improves cell stemness by increasing OSKM factor expression, mainly through 2NR2F2 inhibition. Furthermore, miR-302b ameliorates cell function and switches energy metabolism by increasing HIF-1α levels in target cells, possibly through ERK pathway regulation. Taken together, EVs enable micro ambient modulation and cell-to-cell communication via the transfer of functionally relevant biomolecules that regulate senescence, stemness and metabolism. Furthermore, the isolation of EVs derived from young and healthy mesenchymal stem cells is easy, sustainable, reproducible and has a high output, which significantly shortens the time required for cell-based therapies . Thus, t


It has been reported that the lncRNA SNHG16 has significantly increased expression in pancreatic adenocarcinoma (PC). However, the functions and mechanisms of SNHG16 are not clear. The aim of this study was to explore the effects of SNHG16 on PC.qRT-PCR analysis was applied to detect the expression levels of SNHG16, miR-302b-3p and SLC2A4 in PC tissues and cells. CCK8 and EdU assays were used to evaluate the proliferation of PC cells. Transwell assays were used to assess PC cell migration and invasion. Apoptosis was evaluated by flow cytometry, and the expression of apoptosis-related proteins  was tested by western blotting. The interactions between miR-302b-3p and SNHG16 or miR-302b-3p and the 3’UTR of SLC2A4 mRNA were clarified by a dual luciferase reporter assay and RNA immunoprecipitation.SNHG16 expression was significantly elevated in PC tissues and cell lines and was associated with poor prognosis of PC patients. Knockdown of SNHG16 reduced PC cell proliferation, migration and invasion. SNHG16 acted as a sponge to regulate miR-302b-3p expression in PC cells. In addition, miR-302b-3p targeted SLC2A4 directly.SNHG16 promoted the progression of PC via the miR-302b-3p/SLC2A4 axis and was expected to be a potential target for the early diagnosis and treatment of PC. Pancreatic adenocarcinoma (PC) is one of the most severe gastrointestinal malignancies and is the fourth most common cause of cancer-related death . Due to It has been proven that only 2% of the genome sequence is capable of coding proteins, whereas more than 95% of transcripts are identified as noncoding RNAs . Long noRecently, small nucleolar RNA host gene 16 (SNGH16) was gradually discovered to be involved in tumorigenesis in many cancer types , 11. SNHIn this study, we aimed to explore the roles of the lncRNA SNHG16 with functional and mechanistic impact PC. By detecting SNHG16 expression in paired PC tissues and cell lines, we found that the lncRNA SNHG16 was significantly overexpressed in PC. In addition, elevated SNHG16 played an oncogenic role in PC cell proliferation, migration, invasion and apoptosis. Mechanistic investigations revealed that SNHG16 acted as a sponge of miR-302b-3p, which reversed SNHG16-induced proliferation by targeting SLC2A4 in PC.PC samples and corresponding adjacent noncancerous tissues were collected from PC patients who underwent surgery at the First Hospital of Lanzhou University. The study was approved by the ethics committee of the First Hospital of Lanzhou University, and all included patients signed informed consent forms. Clinical tissue specimens were collected and the clinicopathologic characteristics were presented in 2 at 37 °C.The human normal pancreatic duct epithelial cell line (HPY-Y5) and pancreatic adenocarcinoma cell lines  were purchased from the Chinese Academy of Sciences, Shanghai, China. All PC cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum  in a sterile incubator with 5% CO2O. The thermal cycling protocol was as follows: 37 °C for 15 min, 85 °C for 5 s and 4 °C for 15 min. The expression of SNHG16 and SLC2A4 was detected by using a SYBR Green Real-Time PCR Kit . miR-302b-3p expression was analyzed with a Hairpin-it™ miRNA qPCR kit . Amplification of the transcripts involved an initial denaturation step at 95 °C for 2 min followed by 40 cycles at 95 °C for 5 s, 55 °C for 30 s, and 72 °C for 30 s. The mRNA and miRNA expression data are presented as fold changes in the mRNA/miRNA abundance normalized to β-actin or U6 snRNA expression. The primer sequences used in this study are presented in Table Total RNA was extracted from tumor tissues and cells with TRIzol reagent  according to the manufacturer’s instructions. cDNA was generated with a reverse transcription kit from Takara . Amplification reactions included 2 µl of Mix, 2 µl of RNA and 6 µl of ddHThe miR-302b-3p mimics, miR-302b-3p inhibitors, small hairpin RNA of SLC2A4 (sh-SLC2A4) and corresponding negative controls (NC mimic or sh-NC) were purchased from RiboBio, Guangzhou, China. The inhibitors and shRNAs were transfected into BxPC3 and Panc-1 cells using Lipofectamine 3000  according to the manufacturer’s directions. The transfection efficiencies were detected by qRT-PCR at 48 h post transfection. Transfected cells were combined for the subsequent experiments.The lentiviral particles LV-shSNHG16 and LV-sh-NC were purchased from GenePharma, China. The lentiviral particles were used to directly infect BxPC3 and Panc-1 cells in the presence of 8 µg/ml polybrene (Sigma-Aldrich) to construct the stably transfected cells. Puromycin  was added to the medium 48 h after infection and maintained for 2 weeks to select stably transfected cells (BxPC3/Panc-1-sh-SNHG16 or BxPC3/Panc-1-sh-NC).5. Then, 4% paraformaldehyde  was used to fix the cells for 10 min. Then, 1% Triton  was used to clear the cells for 5 min after three washes with PBS. Subsequently, the cells were incubated with dyeing agent for 30 min in the dark, stained with DAPI  and incubated for 5 min at 37 °C. Images were acquired using a microscope at a magnification of 400×.The reagents used in the EdU assay were purchased from RiboBio . PC cells were seeded in confocal dishes at a density of 3 × 10PC cells were seeded in 6-well plates and grown to 70–80% confluence. A wound was created by scraping with a 100 µl pipette tip. The remaining cells were cultured in serum-free DMEM for 48 h. Images of migration were acquired at 0 and 48 h after scraping.Cells were suspended in serum-free DMEM. One hundred microliters of the cell suspension was seeded in the upper compartment of 8 µm transwell chambers in a 24-well culture plate  for 48 h, and 500 µl of DMEM supplemented with 10% FBS was added to the lower compartment of the chambers. Chambers with inserts uniformly coated with Matrigel (BD Biosciences) were used for the invasion assay. The transwell chambers were fixed with 4% paraformaldehyde for 15 min and stained with 0.1% crystal violet for 30 min at room temperature. Then, cells on the upper surface of the inserts were wiped off with a cotton swab. Stained cells were washed with PBS and observed with a microscope. The capacities for cell migration and invasion were assessed by determining the average numbers of stained cells in 5 regions.Full-length SNHG16 (WT or Mut) was synthesized and cloned into the pGL3-–Promoter vector . Dual luciferase reporter assays were conducted in BxPC3 and PANC-1 cells cotransfected with pGL3-Promoter-SNHG16 (WT or Mut) and NC mimics or miR-302b-3p mimics. Luciferase activity was analyzed with a dual luciferase reporter assay system (Promega) according to the manufacturer’s instructions.Total protein was extracted from cells with RIPA buffer . The concentration of total protein was determined with a BCA protein assay kit . Thirty micrograms of total protein was separated on 6%, 8% or 10% SDS-PAGE gels and was then transferred to PVDF membranes . After blocking with 5% skim milk for 2 h, membranes were incubated overnight with a specific primary antibody at 4 ℃. The specific primary antibodies  were purchased from Proteintech Group, and the concentration of these antibodies applied in this study was 1:500. Specific primary antibodies  were purchased from Abcam, and the concentration of these antibodies applied in this study was 1:1000. Next, membranes were incubated with the corresponding secondary antibodies for 2 h at room temperature. The protein signals were visualized with ECL western blotting substrate .t-test was performed to compare the means of two groups. One-way ANOVA and the Bonferroni test for multiple comparisons were applied to analyze differences among two or more groups. Spearman correlation analysis was performed to detect correlations between SNHG16, miR-302b-3p and SLC2A4 levels. Significant differences were defined as those with P < 0.05.All statistical analyses were performed with SPSS 22.0 software  and Prism version 7.0 software . Data are presented as the mean ± SEM values. An unpaired In this study, the expression level of SNHG16 in human PC tissues was first evaluated by qRT-PCR. Compared with that in pair-matched adjacent normal samples, SNHG16 expression was significantly increased in PC tissues  were used to screen seven targets of miR-302b-: SLC2A4, RSBN1, ELK4, NFIA, CELF1, WEE1 and NTN4. Among these targets, SLC2A4 (solute carrier family 2 member 4) was the gene with the most significantly increased expression in PC cells Fig. a. The puTo confirm the correlation of the SNHG16/miR-302b-3p/SLC2A4 axis with PC progression, PC cells with stable silencing of SNHG16 were transfected with the miR-302b-3p inhibitor, miR-302b-3p inhibitor plus sh-SLC2A4, or NC inhibitor, and the biological phenotypes of these PC cells were analyzed. First, after transfection of Bx-PC3 and Panc-1 cells with sh-SNHG16, transfection of the miR-302b-3p inhibitor significantly inhibited miR-302b-3p expression, and transfection of sh-SLC2A4 decreased the expression of SLC2A4 Fig. a. CCK-8 PC is a highly aggressive solid tumor that frequently causes local invasion and early metastasis, which causes more than 300,000 deaths annually. The prognosis of PC patients is very poor, and the overall 5-year survival rate is less than 5% . TherefoSmall nucleolar RNA host gene 16 (SNHG16) has been reported as an oncogenic lncRNA in multiple cancers, such as colorectal cancer , breast The pivotal conclusion of this study is that SNHG16 plays a critical role in PC. The results demonstrated that SNHG16 is significantly upregulated in human PC tissues and PC cells, suggesting that this elevated lncRNA plays crucial roles in PC progression. Moreover, it was found that increased SNHG16 expression was associated with overall survival and indicated poor prognosis in patients with PC. This finding suggests that SNHG16 might be a prognostic marker for PC.Functional experiments indicated the inhibitory effects of SNHG16 silencing on PC cell proliferation, migration and invasion and the promotive effect on apoptosis. Mechanistically, the target gene, miR-302b-3p, was predicted and identified by a luciferase reporter assay and RIP. MiR-302b-3p was highly expressed in both PC tissues and cells. Subsequent functional experiments confirmed that miR-302b-3p promoted the proliferation, migration and invasion of PC cells, effects opposite those of SNHG16. These data implied that SNHG16 acts as a sponge to negatively regulate miR-302b-3p expression in PC cells. Moreover, we investigated whether miR-302b-3p can directly target the 3’UTR of SLC2A4 and inhibit SLC2A4 expression in PC cells.SLC2A4 is the gene encoding the insulin-sensitive glucose transporter GLUT4, which is an insulin-sensitive glucose transporter that plays a key role in glucose homeostasis . SLC2A4 Expression of the lncRNA SNHG16 is elevated in PC tissues and cell lines.Knockdown of SNHG16 suppresses PC cell migration and invasion.SNHG16 acts as a sponge to regulate miR-302b-3p expression in PC cells.Overexpression of miR-302b-3p inhibits the proliferation, migration and invasion of PC cells and promotes their apoptosis.MiR-302b-3p targets SLC2A4 and inhibits its expression directly.SNHG16 promotes the progression of PC via the miR-302b-3p/SLC2A4 axis.SNHG16 is expected to be a potential target for the early diagnosis and treatment of PC.Additional file 1: Table S1. Clinical pathologic features.


All derivatives produced vasorelaxant effects with IC50 values ranging from 3.41 ± 0.65 µM to 39.72 ± 6.77 µM. Compounds 2c, 2e and 2f demonstrated the highest potency in endothelium-intact aorta rings , and they achieved around 90% relaxation (30 μM). In aorta rings without an endothelium, the effect of compound 2f was abolished. Using the MTT assay to test for cell viability, only compound 2b induced cytotoxicity at the maximum concentration employed (30 µM). The results show that vasorelaxation by 4-phenylquinazolin-2(1H)-one derivatives might depend on the activation of a signalling pathway triggered by endothelium-derived factors.Quinazolinones have pharmacological effects on vascular reactivity through different mechanisms. We synthesized 4-phenylquinazolin-2(1H)-one derivatives under microwave irradiation and tested them on the rat thoracic aorta. The prepared compounds  Heterocyclic drugs make up over 60% of major retail medicines for the pharmaceutical market ,2. ConsiQuinazoline derivatives have also demonstrated antihypertensive effects, such as the class of the alpha 1-adrenergic (α1) receptor antagonists used in the treatment of hypertension ,7. DrugsThereby, considering all this knowledge, the existence of several reports on vascular activity attributed to quinazolines and, notably, few studies that focus on the biological effects of the isomer quinazolin-2(1H)-one, we decided to investigate the putative effects of its derivatives on the rat thoracic aorta. In the present investigation, we synthesized 4-phenylquinazolin-2(1H)-one derivatives synthetized under microwave irradiation and tested them for their vasorelaxation properties using experimental protocols on the rat thoracic aorta.Several methodologies for the 4-phenylquinazolin-2(1H)-one scaffold have been described in the literature; however, they require prolonged reaction times ,17 or ex2a–f, except compound 2b, were obtained in less than 1 h and obtained with good yields (63–92%). Compound 2b, which has an electron withdrawing group (nitro group), had very poor yield with the traditional methodology (16%), but this was increased using the microwave methodology (31%).50 ranging from 3.41 ± 0.65 µM to 39.72 ± 6.77 µM) in endothelium-intact aorta arterial rings pre-contracted with Phe 1 µM (2f was found to be the most potent in endothelium-intact vessels (IC50 = 3.41 ± 0.65 µM), while compounds 2a, 2b and 2d showed the lowest potency in the endothelium-intact aorta at the concentrations employed in the present study. Additionally, compounds 2c, 2e and 2f demonstrated the highest potency with an IC50 of 4.31 ± 0.90 µM, 4.94 ± 1.21 µM and 3.41 ± 0.65 µM, respectively, and they achieved around 90% relaxation at the highest concentration of 30 μM in endothelium-intact aorta rings. Thus, we decided to test compound 2f at lower concentrations (30–300 nM) in endothelium-intact and endothelium-denuded aorta rings.All tested compounds induced concentration-dependent relaxation  on aortic rings endothelium-intact and endothelium-denuded pre-contracted with Phe 1 μM are shown in Preliminarily, we decided to investigate the effect of compound 2a–2f) showed a significant difference at the highest concentration of 30 µM, it is important to note that the percentage of living cells in this case was above 70%, except for compound 2b (69.05%). According to ISO 10993: 5 (2009), samples that reduce cell viability values below 70% should be considered cytotoxic. Therefore, only compound 2b could be considered cytotoxic at the concentration of 30 µM. In addition, we highlight that these compounds in general had IC50 values below 30 µM.Based on the results of the MTT assay , it was 2a–2f were able to relax the aorta. Furthermore, the results indicate an effect directly related to the presence of the endothelium for the 2f analogue. Therefore, it is possible that vasorelaxation is dependent on the modulation of some signalling pathway triggered by endothelium-derived factors (EDFs) 1a–f (1 m1a–f (1mmol) and urea (15 mmol) in glacial acetic acid (10 mL) was irradiated in a sealed tube at 140 °C (200 w) for 30–45 min in a CEM Discover microwave reactor. The reaction mixture was filtered, and the precipitate was washed with water.A mixture of 2-aminobenzophenone All compounds were dissolved with DMSO 100% to obtain a stock solution of 10 mM. In all experiments, the stock solution was employed to obtain a final concentration diluted in Krebs Henseleit solution (vascular reactivity assay) and DMEM supplemented with 10% FBS (cytotoxicity assay).Rattus norvegicus) weighing between 200 and 300 grams were supplied by the Central Animal Facility of Fluminense Federal University (NAL-UFF) . The animals were kept in standard environmental conditions of temperature (22 ± 1 °C) and 12 h light/dark cycle, with free access to food  and water. All experiments were conducted in accordance to the guidelines established by National Council for Animal Experimentation Control . This study was approved by the Ethics Committee in the Use of Animals of the Fluminense Federal University (CEUA-UFF), certificate under protocol number: 795/2017.Male Wistar normotensive rats (L-phenylephrine hydrochloride (Phe), acetylcholine chloride (Ach) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich . Reagents employed in the preparation of Krebs Henseleit solution were all obtained from Merck .2 1.1; CaCl2 2.5; NaH2PO4 1.2; N-[2-hydroxyethyl] piperazine-N’-[2-ethane-sulfonic acid] (HEPES) 10; NaHCO3 15; and glucose 11, bubbled constantly with a gas mixture of 95% O2 and 5% CO2 (pH 7.4). Each arterial segment was suspended between two steel hooks connected to an isometric transducer to measure tension through a data acquisition system . After a stabilization period of 60 min at a rest tension of 1.0 g with periodic changes of solution (every 15 min), a stable contraction was achieved with 1 µM Phe. Functional endothelial integrity was assessed by the ability of 10 µM ACh to induce ≥70% relaxation in vessels pre-contracted with 1 µM Phe.Animals were anesthetized by isoflurane (1% mg/kg) inhalation and, after the loss of motor control, rats were euthanized by cervical dislocation. Subsequently, the thoracic aorta was dissected and cleaned of connective tissue to obtain rings 4 mm in length that were suspended in a 15 mL organ bath . Tissues were kept in Krebs Henseleit solution at 37 °C with the following composition (mM): NaCl 120; KCl 5.0; MgClCercopithecus aethiops) kidney cell line) were used and obtained from the Rio de Janeiro Cell Bank  and grown in Roswell Park Memorial Institute medium  supplemented with 10% heat-inactivated fetal bovine serum , without antibiotics. Cells were plated in plastic Petri dishes and incubated at 37 °C under a 95% O2/5% CO2 atmosphere. Cell culture was maintained up to 15 days until cells reached 90% confluence.A 3--2,5-diphenyl-2H-tetrazoliumbromide (MTT) assay was employed to assess the general cytotoxicity for 4-phenylquinazolin-2(1H)-one derivatives. VERO cells  or compounds  were added to the culture medium and VERO cells were incubated for an additional 48 h. At the end of the treatment period, the medium was replaced with complete fresh medium containing 5 mg/mL tetrazolium dye (MTT) and incubated for 3 h; then, the medium was removed and 100 μL of DMSO was added. Absorbance was measured with a plate reader  at a wavelength of 550 nm and expressed as a percentage of the control (vehicle).The MTT cell viability assay investigates the number of viable cells by measuring the mitochondrial-dependent reduction of water-soluble tetrazolium into an insoluble formazan product. The absorbance obtained is directly proportional to the activity of functional mitochondria and represents the number of viable cells. Briefly, VERO cells were seeded at a density of 1 × 10P-value less than 0.05 was termed statistically significant. The IC50 values  were obtained by actual concentration-response curve fitting using GraphPad Prism 6.0 software . Statistical significance was determined using Bonferroni´s multiple comparison for vascular reactivity experiments and using Turkey´s multiple comparison for cytotoxicity evaluation following two-way analysis of variance (ANOVA).Values are expressed as mean ± standard error of mean (SEM). A 2a–f exhibited significant concentration-dependent effects that were dependent on the presence of an intact endothelium, suggesting a mechanism of action mediated by endothelium-derived factors (EDFs). Additionally, only one of the active compounds exhibited significant cytotoxicity at the highest concentration employed, suggesting low toxicity. Nevertheless, further toxicity investigations are required to ensure these findings. In conclusion, this series of molecules displayed the potential to serve as templates for the development of new drugs for diseases involving vascular smooth muscle, especially in disorders in which endothelial dysfunction is a relevant pathophysiological feature.All tested compounds were obtained through simple synthetic reactions using simple methodologies. The reaction methodology using microwave irradiation provided better yields and lower reactional times when compared with the traditional methodology. In vitro screening for a relaxant effect on aorta rings isolated from rats showed that compounds


However, molecular Eu2+ complexes with 5d-4f transition are thought to be too air-unstable to explore their applications. In this work, we synthesized four Eu2+-containing azacryptates EuX2-Nn  and systematically studied the photophysical properties in crystalline samples and solutions. Intriguingly, the EuX2-N8 complexes exhibit near-unity photoluminescence quantum yield, good air-/thermal-stability and mechanochromic property (X = I). Furthermore, we proved the application of Eu2+ complexes in organic light-emitting diodes (OLEDs) with high efficiency and luminance. The optimized device employing EuI2-N8 as emitter has the best performance as the maximum luminance, current efficiency, and external quantum efficiency up to 25470 cd m−2, 62.4 cd A−1, and 17.7%, respectively. Our work deepens the understanding of structure-property relationship in molecular Eu2+ complexes and could inspire further research on application in OLEDs.Divalent europium 5 Though divalent-europium-based complexes are promising materials for next-generation light-emitting devices, their poor air stability limits their applicability. Here, the authors report the design of air stable divalent-europium-based complexes for efficient organic light-emitting diodes. For the well-established luminescence of lanthanide ions, f–f transition can be observed under ambient conditions, while the 5d–4f transition is usually absent due to thermally quenching by fast intersystem crossing from 4fn−15d1 to 4fn configuration. In divalent lanthanide systems, 5d–4f transition is much more prominent for its spin-allowed nature and the stabilization of the 5d orbitals8.The 52+ ions, Eu2+ ions exhibit strong 5d–4f transition and great applications for two reasons: (1) the 5d level is near or below 6P7/2, decreasing the multiphoton relaxation6, (2) the reduction potential of Eu3+/Eu2+ is not too negative. The research on luminescent properties of Eu2+ ions can be roughly divided into two categories: Eu2+ dopants in inorganic matrix and molecular Eu2+ complexes. The first one has been extensively studied while the latter remains unexplored in many aspects. The physicochemical properties of Eu2+ complexes are mainly studied using cyclopentadienyls, hydrotris(pyrazolyl)borates, silylamides and their derivatives as ligands11. Recently, Allen et al. reported series of Eu2+-containing azacryptate complexes which have aroused growing interest for their attractive luminescent properties, photoredox catalytic performance and magnetic resonance imaging15.Among all Lnd–4f luminescent mechanism enables Eu2+ complexes to have great potential in high-performance organic light-emitting diodes (OLEDs), a technology has successfully been commercialized in cutting-edge displays and is under developing in solid-state lighting. To reach 100% theoretical exciton utilization efficiency (EUE), which is the key parameter to enhance the energy efficiency, phosphorescence17, thermally activated delayed fluorescence (TADF)19 and organic radical materials20 were discovered in succession and applied as emitters in OLEDs. Comparing with the traditional f–f transition and other currently used emitters, divalent europium compounds have the following significant advantages: (i) short decay lifetime: the f–f transition is spin-forbidden with long lifetimes up to milliseconds, strongly limiting their maximum luminance, while 5d–4f transition is spin-allowed with typical lifetimes in nanosecond scale, which significantly reduce the excited-state quenching to reach higher luminance and lower efficiency roll-off, (ii) high EUE: Eu2+ ion exhibits a unique transition for the open-shell electron from 4f65d1 to 4f7, which can harvest 100% exciton energy theoretically21, (iii) easily tunable emission by varying coordinate environment: the 5d orbitals are sensitive to the ligand field while the 4f orbitals, effectively shielded by 5s5p, are not sensitive to surroundings25. (iv) high abundance: europium has crustal abundance of 10−6 wt, much higher than the noble metals  used in commercial OLEDs. Thus, we believe that 5d–4f transition materials, represented by Eu2+ complexes, will be the next unexplored but promising field in OLED emitters.The uniqueness of 52+ complexes are strongly limited by their air stability according to the standard potential φ(Eu3+/Eu2+) = −0.38 V. To the best of our knowledge, there was only one report of OLED device based on Eu2+ complexes, with unsatisfied performance in external quantum efficiency (EQE) of 0.01% and maximum luminance of 10 cd m−2 considering the high photoluminescence quantum yield (PLQY) of the complex to be 85%26. Thus, more efforts must be worked on the rational design of Eu2+ complexes and the deep understanding of the electroluminescent process to boost efficiency and luminance. We propose that the steric effect of cryptate ligands and coordinate interaction could improve the stability of Eu2+ complexes. The steric effect prevents Eu2+ from O2 by a more rigid structure. Improving the coordination interaction between the ligand and Eu2+ can largely enhance the thermodynamic stability. Thus, two ligands, 1,4,7,10-tetraazacyclododecane (N4) and 1,4,7,10,13,16,21,24-octaazabicyclo[8.8.8]hexacosane (N8) are chosen for the design of four Eu2+-containing azacryptates named as EuX2–Nn . Series of crystal analysis, spectral, stability, and theoretical studies were undertaken to reveal the photophysical nature of these Eu2+ complexes. Then EuX2–N8 complexes were chosen as emitters in OLEDs for their high efficiency and good thermal/air stability. As a breakthrough, the optimized device using EuI2–N8 exhibits excellent performance with a maximum EQE of 17.7% and a maximum luminance of 25470 cd m−2.Despite the advantages mentioned, Eu2+ complexes EuX2–Nn  were synthesized in glovebox by mixing EuX2 and corresponding ligands in methanol27. The purified products were identified by elemental analysis. Then, single-crystal X-ray diffraction (SCXRD) was performed to investigate the coordinate geometry of these Eu2+-containing azacryptates 2]2+ with different orientations in one cell, locating in the edge center and body center, while the eight iodide ions intersperse therein 2]2+ ions.The four Eutes Fig. . EuBr2–N2+ complexes, steady-state spectra and transient spectra were collected. Crystalline powder of EuX2–N4 shows orange-red emissions with maximum wavelength (λmax) of 605 nm (X = Br) and 613 nm (X = I), respectively  and 515 nm (X = I). The lifetimes for these complexes were found to be hundreds of nanoseconds  for these complexes in solid powder (40–45 nm) are relatively narrow comparing with luminescent materials featuring in charge-transfer (CT) mechanisms. The excitation bands of these complexes are broad and featureless, ranging from 230 to 500 nm (EuX2–N8) and 230 to 600 nm (EuX2-N4) as shown in Supplementary Fig. 2+ ion, where the ground state is 4f7 [8S7/2] and the excitation state is 4f6[7F0]5d1, specifically.To systematically study the photophysical properties of EuX2–N4 in common solvents, we only studied the photophysical properties of EuX2–N8 in methanol solution (1.5 mM) under N2 atmosphere. The EuX2–N8 solutions show bright yellow emission with λmax of 579 nm, and the emission spectra of the two complexes are almost identical. The emission is red-shifted by about 70 nm comparing with their solid samples, which is presumed to be caused by the differences in conformation of N8 ligand in solid and solution, outweighing the effect of different halogens. As shown in Fig. fz3 to 5dz2 and from 4fz3 to 5dxy, respectively30. The UV–visible spectra  and low-energy absorption peak at 404 nm  and 405 nm , respectively, which is consistent with their excitation bands. The large molar absorptivity is on par with the reported Eu2+ complexes due to the Laporte- and spin-allowed nature of f–d transition31. The time-dependent density functional theory (TD-DFT) calculation was conducted for EuX2–N8 and EuBr2–N4. The calculation prediction of EuX2–N8 is very close to the experimental data. For the N4 complex, EuBr2–N4 has two possible conformations (high symmetry: cubic geometry and low symmetry: square antiprism), and calculation result suggests that the different conformations exhibit distinct absorption bands.Due to the insolubility of Eu2–N8 exhibits mechanochromic property, showing a fluorescence color change from green to yellow under moderate mechanical grinding in Fig. 2+ center at a marginally different coordination environment. We tentatively attribute the mechanochromic property to the change in ligand conformation upon grinding, resulted from the relatively weak lattice energy of EuI2–N8. And the reversible process is essentially recrystallization in certain solvent atmosphere, like methanol34. Furthermore, the longer-wavelength emission can be pronouncedly enhanced by fast precipitation in antisolvent (tetrahydrofuran) to get amorphous powder of EuI2–N8. As shown in Fig. 2–N8  are around 270 °C, 265 °C, 393 °C, and 436 °C for EuBr2–N4, EuI2–N4, EuBr2–N8, and EuI2–N8 in Fig. X2–N4 are reasonably low (~3%) while the REs of EuX2–N8 are too high . The element analysis is employed to exclude the possibility of impurities in EuX2–N8. Thus, we believe these EuX2–N8 compounds undergo both decomposition and sublimation around Td, which will result in a large deviation in the final weight percentage.Thermal properties of these four compounds are studied by thermogravimetric analysis (TGA), which is of great significance for their further applications in OLEDs. The deposition temperature  at a small scale of 50 mg. It is notable that there will be obvious decomposition at large-scale sublimation, probably due to the uneven heating in the sublimation boat. A similar property is found for EuI2–N8 with a higher sublimation temperature at 350 °C.Then the sublimation properties of these compounds were tested under high vacuum of 108 complexes are potential candidates used in OLEDs as emitters. However, Eu2+ ion is traditionally known to be easily oxidized to Eu3+ by O2, hence the air stability is a critical parameter in terms of further applications. To shed light on their air stability at room temperature, the PLQYs of EuX2–N8 were measured as the function of time. As shown in Fig. 2–N8 does not change after exposure in air over 450 h and EuI2–N8 is metastable towards air. To explain the differences in stability for future design of Eu2+ complexes, the analysis of buried volume (%Vbur) was calculated to estimate the steric protection by N8 ligands as shown in Fig. 3c, d36. %Vbur is defined as the fraction of volume of ligand over the total volume of sphere centered on the metal. It determines the steric effect of a given ligand regard to the first coordination sphere . EuBr2–N8 has a slightly higher %Vbur than EuI2–Nn due to the closer distance between Eu2+ and Br-. Thus, the divergence in air stability between these two N8 complexes is related to the different lattice energy considering the similar %Vbur. The EuI2–N8 has smaller lattice energy than EuBr2–N8 because of weaker static interaction and the existing of solvent methanol in crystal, which is also applied to explain their different mechanochromic behaviors.Considering the high thermal stability and near-unity PLQY, the NX2–N8 complexes are better candidates used in OLEDs as emitters. Prior to device fabrication, the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) energy levels of the two complexes were deduced from their ultraviolet photoelectron spectroscopy  and electron transporting layer (ETL), adjusting the thickness of the emission layer in Supplementary sections 2–N8 device in Supplementary sections Based on the photophysical and stability studies, the Eu3 (2 nm)/N,N′-bis-N,N′-bis-phenyl--4,4′-diamine /cyclohexylidenebis /EuX2–N8:4,4′,4″-tris[phenyl(m-tolyl)amino]triphenylamine /diphenyl[4-(triphenylsilyl)phenyl]phosphine oxide /4,7-diphenyl-1, 10-phenanthroline /LiF (0.7 nm)/Al. The best EuBr2–N8 device gives pretty good performance with a turn-on voltage (Von) of 6.2 V, a maximum luminance (Lmax) of 10,200 cd m−2, a maximum current efficiency (CEmax) of 52.8 cd A−1 and a maximum EQE of 15.5%. While the champion device is obtained by using EuI2–N8 as the emitter, the Von, Lmax, CEmax, and EQEmax are 6.5 V, 25,470 cd m−2, 62.4 cd A−1, and 17.7%, respectively. These results shown in Fig. 2+-based OLEDs, with EQEmax of 0.01%, Lmax of 10 cd m−2 and Von of 20 V26. In addition, the electroluminescence lifetimes of the champion device are determined at 2.5 mA cm−2 and 10 mA cm−2, which are little shorter than tris(2-phenylpyridine)iridium (Ir(ppy)3) based control device, a well-studied phosphorescence emitter in the same spectral region . To understand this phenomenon, the photophysical properties of films fabricated by doping 10 wt% EuX2–N8 in m-MTDATA onto quartz substrates in a vacuum chamber at high vacuum (10−5 Pa) were studied. The pure films of EuX2–N8 were also fabricated as reference. The emission of doping films is mainly located as two bands, where the 400–470 nm band (τ~1.2 ns) is attributed as fluorescence from host materials and the 500–650 nm band (τ~102 ns) is from EuX2–N8 in Supplementary Fig. 2+ complexes, without complete energy transfer in the photoluminescence process. Intriguingly, the electroluminescence spectra only exhibit emission from Eu2+ complexes with doping concentrations (7 wt%) lower than those in the photoluminescence study. Thus, we tentatively propose that the carrier recombination dominantly occurs in the doping materials instead of host materials, where the excitation of ligand results in a high Von.It is notable that the Eu2+-containing azacryptates EuX2–Nn  were synthesized, showing promising photoluminescent properties: high PLQY (~100% for N8 complexes), short excited-state lifetime (102 ns) and easily tunable emission by ligand field. Intriguingly, EuI2–N8 exhibits reversible mechanochromic property under grinding, which is attributed to the potential flexibility of N8 ligand and recrystallization. Furthermore, the EuX2–N8 complexes were chosen as the emissive materials in OLEDs due to their good air-/thermal-stability. After optimization of design, the best device showed excellent performance with a maximum EQE of 17.7% and luminance of 25,470 cd m−2. Our work deepens the understanding of photoluminescence and electroluminescence properties in Eu2+ complexes and proves their promising applications in OLEDs.In summary, four Eu4 ligand was commercially available. 1H-NMR spectra were recorded on a Bruker-400 MHz NMR spectrometer. Tetramethylsilane (TMS) was used as an internal reference for the chemical shift correction, where δ(TMS) equals 0. Elemental analyses were performed on a VARIO EL analyzer . All the synthesis of Eu2+ complexes was conducted in glovebox. All spectral tests of solid Eu2+ complexes were carried out by paraffin encapsulation between two quartz plates and the solution was protected by capped cuvettes under N2 atmosphere. The commercially available paraffin was purified by oxidation using KMnO4 and column chromatography to remove fluorescent whitening agents.All chemical reagents used in the synthesis process were commercially available and were used as received unless otherwise mentioned. The N1,4,7,10,13,16,21,24-octaazabicyclo[8.8.8]hexacosane (N8ligand): The synthesis of N8 ligand is carried out by an improved version of a reported method38. Tris(2-aminoethyl)amine , NEt3 (12 mL), and 2-propanol (250 mL) were added to a 2-neck 1-L round-bottom flask equipped with mechanical stirring and a drip funnel containing a dilute solution of glyoxal (7.5 g). The flask was cooled to −78 °C and the glyoxal solution was added slowly (1 drop s−1). After the completion of addition, the yellow solution was stirred at room temperature overnight. Then the solvent was removed under vacuum at 40 °C, yielding a yellow solid which was dispersed in 300 mL CHCl3 and stirred for 2 h with the generation of lots of yellow translucent gels. The gels were removed by filtration and the resulting CHCl3 was removed under vacuum at 40 °C. The crude intermediate was dissolved in 300 mL MeOH, cooled with ice water. Excess NaBH4 (14 g) was gradually added to the solution to prevent an intensive reaction. The cloudy solution was stirred for 4 h and the solvent was removed under vacuum yielding white solid, which was extracted by CH2Cl2 (200 mL×3). The removal of CH2Cl2 gave the crude product N8. Further purification was conducted by thermal gradient sublimation (160−80 °C) at low pressure (~5 Pa). 1H-NMR : δ 2.79 , 2.75 , 2.58 .EuBr2-N4: EuBr2  was dissolved in 6 mL MeOH in a clean glass bottle. N4  was dissolved in 3.5 mL MeOH, which was slowly added to the EuBr2 solution without stirring. The colorless solution turned orange-red and red crystals suitable for SCXRD analysis formed as the evaporation of solvent (yield is 85% based on Eu). Elemental analysis for C16H40Br2EuN8, C, 29.28%, H, 6.14%, N, 17.07%. Found: C, 29.05%, H, 6.05%, N, 16.69%.EuI2-N4: EuI2  was dissolved in 6 mL MeOH in a clean glass bottle. N4  was dissolved in 3.5 mL MeOH, which was slowly added to the EuI2 solution without stirring. The light-yellow solution turned orange-red and red crystals suitable for SCXRD analysis formed soon after the completion of mixing (yield is 70% based on Eu). Elemental analysis for C16H40EuI2N8, C, 25.61%, H, 5.37%, N, 14.93%. Found: C, 25.84%, H, 5.54%, N, 14.42%.EuBr2-N8: EuBr2  was dissolved in 10 mL MeOH in a 50-mL round-bottom flask under magnetic stirring. N8  was dissolved in 10 mL MeOH, which was slowly added to the EuBr2 solution. The colorless solution turned orange. The solvent was removed under reduced pressure to get crude product. (Yield is 86% based on Eu). The complex was further purified by thermal gradient sublimation (320–250–60 °C) at low pressure (10−5 Pa). Green crystals suitable for SCXRD analysis were obtained by slow evaporation of MeOH as a solvent. Elemental analysis for C18H42Br2EuN8, C, 31.68%, H, 6.20%, N, 16.42%. Found: C, 31.75%, H, 6.18%, N, 16.39%.EuI2-N8: EuI2  was dissolved in 10 mL MeOH in a 50-mL round-bottom flask under magnetic stirring. N8  was dissolved in 10 mL MeOH, which was slowly added to the EuI2 solution. The light-yellow solution turned orange and green crystals suitable for SCXRD analysis formed as the evaporation of solvent without stirring (yield is 72% based on Eu). Elemental analysis for C18H42I2EuN8, C, 27.85%, H, 5.45%, N, 14.43%. Found: C, 28.04%, H, 5.41%, N, 14.28%.Supplementary InformationPeer Review File