Cobetia marina alkaline phosphatase (CmAP) and Far Eastern holothurian Apostichopus japonicus mannan-binding C-type lectin (MBL-AJ) was performed. Expression of the fusion gene in E. coli cells resulted in yield of soluble recombinant chimeric protein CmAP/MBL-AJ with the high alkaline phosphatase activity and specificity of the lectin MBL-AJ. The bifunctional hybrid CmAP/MBL-AJ was produced as a dimer with the molecular mass of 200 kDa. The CmAP/MBL-AJ dimer model showed the two-subunit lectin part that is associated with two molecules of alkaline phosphatase functioning independently from each other. The highly active CmAP label genetically linked to MBL-AJ has advantaged the lectin-binding assay in its sensitivity and time. The double substitution A156N/F159K in the lectin domain of CmAP/MBL-AJ has enhanced its lectin activity by 25±5%. The bifunctional hybrid holothurian's lectin could be promising tool for developing non-invasive methods for biological markers assessment, particularly for improving the MBL-AJ-based method for early detection of a malignant condition in cervical specimens.A fusion between the genes encoding the marine bacterium  AJ, as tools for recognizing aberrant glycans or foreign microbial structures have been proposed in recent days AJ was successfully applied for differential diagnostics of benign and malignant neoplasms of uterine cervix by the analysis of contents of lectin-binding carcinoembryonic antigen (CEA) in vaginal secretion AJ has oligomeric forms with identical 17-kDa subunits and natural ligands among extracellular low-branched bacterial α-D-mannans composed of α-1,2- and α-1,6-linked D-mannose residues, participating in recognition of the altered structures in the invertebrate during embryogenesis, morphogenesis and the formation of the immune response AJ and human serum lectin MBL detected by the antibodies against MBL-AJ suggested the presence of common antigenic determinants AJ specificities resulted in the absence of the MBL-AJ interaction with the components of the healthy patient's serum have been found to facilitate the detection of the slight structural differences of glycans, excluding the wrong positive results in the assayed samples AJ has allowed identifying statistically reliable differences between the levels of the lectin-binding CEA between healthy women and patients with cervical cancer, and between patients with benign and malignant neoplasm AJ-based method gives a possibility to assess tumours noninvasively.The peculiar characteristic to recognize and bind specific carbohydrates made lectins of animal and plant origin useful tools for detecting changes in carbohydrate profiles and identifying aberrant glycans in neoplastic cells with the aim of more precise diagnostics and more accurate prognosis. The technique most common and widespread is the use of lectins in immunohistochemical assays AJ-binding assay for detection of aberrant patterns of glycosylation a novel bifunctional hybrid protein CmAP/MBL-AJ based on the Far Eastern holothurian lectin MBL-AJ gene fused with the gene of the marine bacterium alkaline phosphatase CmAP has been synthesized with E. coli cells. CmAP/MBL-AJ has the binding activity of lectin improved by mutations and the high enzymatic activity of alkaline phosphatase facilitating the identification of MBL-AJ ligands without the additional steps of the MBL-AJ-specific antibodies production and conjugation with an enzyme.To improve the MBL-CmAP/MBL-AJ has been constructed on the base of NcoI/SalI part of pET-40b(+) vector (Novagen) and the chimeric gene comprised the mature CmAP open reading frame (accession n. DQ435608) and cDNA fragment encoding mature lectin MBL-AJ (accession n. AAT42221) linked by the sequence encoding the peptide linker (G4S)3. For the CmAP gene amplification, marine bacterium Cobetia marina KMM 296 (Collection of Marine Microorganisms PIBOC FEB RAS) chromosomal DNA, Encyclo Taq Polymerase (Evrogen), and the gene-specific upstream primer, 5′– TATTCCATGGCAGAGATCAAGAATGTCATTCTGAT-3′, and downstream primer including the linker (G4S)3, 5′ – TTAAGAGCTCAGAACCACCACCACCAGAACCACCACCACCAGAACCACCACCACCCTTCGCTACCACTGTCTTCAGATACTGTCCT – 3′, were used. The recombinant plasmid pET40CmAP was obtained by ligation of CmAP gene into pET-40b(+) and used for the subsequent cloning of the lectin MBL-AJ gene.Expression plasmid encoding the fusion protein A. japonicus coelomocytes, the gene-specific upstream primer, Lect-X-dir: 5′– AGCTGAGCTCTGTCTGACGGCTTGTCCGGAGTTTTG– 3′, and downstream primer, Lect-X-rev: 5′– CAGTGTCGACCTCCAAATGATACTCGATACAGGGAAG-3′ (construction 1 –CmAP/MBL-AJ-Stop_codon), or X-Lect-pET40rev-TEV: 5′– ACTGGTCGACTTAGTGGTGGTGGTGGTGGTGGTGGTGACCCTGAAAATAAAGATTCTCCAAATGATACTCGATACAGGGAAG– 3′ (construction 2 – CmAP/MBL-AJ-TEV) were used. cDNA for MBL-AJ was produced as previously described CmAP/MBL-AJ was obtained by ligating the cDNA fragment encoding MBL-AJ into the recombinant vector pET40CmAP, linearized by SacI/SalI site-specific scission. Restriction endonucleases and T4 DNA ligase were purchased from Fermentas. The PCR product and linearized plasmid were purified from agarose gel with Gel Extraction kit (Qiagen). Correct recombinant plasmid sequences were verified by DNA sequencing using an automated PE/ABI 310 DNA sequencer and PE/ABI-ABI PRISM BigDye Terminator cycle sequencing Ready Reaction Kit (PE Applied Biosystems). Preparation of E.coli DH5α competent cells for plasmid propagation and heat shock transformation were carried out according to the standard method E. coli Rosetta (DE3) cells were used as a host for the expression of the recombinant hybrid CmAP/MBL-AJ.For the lectin gene amplification, cDNA of CmAP/MBL-AJ, the E.coli Rosetta(DE3) cells transformed with the recombinant plasmid pET40CmAP/MBL-AJ were grown on LB agar plate containing 25 mg/ml kanamycin overnight at 37°C. A single colony was picked and grown at 200 prm in 20 mL of LB medium with 25 mg/ml kanamycin at 37°C for 12 h, then transferred to 1 L of fresh LB with 25 mg/ml kanamycin. When the cell density reached an A600 of 0.6–0.8, 0.2 mM IPTG was added to induce the expression of the protein, and the incubation continued at 16°C up to 12 h at 200 rpm. E.coli Rosetta(DE3) cells were transformed with pET-40b (+) as a control.For the expression of E.coli cells were resuspended in 150 ml of the buffer A, containing 0.02 M Tris-HCl, pH 8.2, and 0.01% NaN3, and disintegrated by ultrasonic treatment, then centrifuged at 10000 g for 30 min. The supernatant was applied to a column (4×25 cm) of DEAE-52 cellulose (Whatman). Elution of the protein was performed with NaCl gradient (0.05 M–0.38 M) in the buffer A. The enzymatically active fractions were collected and applied to a column (1×2.5 cm) of Ni-agarose (Qiagen). Elution of the protein was carried out in buffer B . The active fractions were collected and desalted with DEAE-Toyopearl 650 M (Toyo Soda) column (0.7×2.5 cm), then incubated with TEV protease (New England Biolabs) at 21°C for 12 hours. Then the protein solution was applied to a gel filtration column (1.5×170 cm) of Superdex 200 (Sigma) equilibrated with buffer C . All purification steps were evaluated by SDS-PAGE according to Laemmli All purification steps were carried out at +6°C. After harvesting, the p-nitrophenylphosphate (pNPP)  in 0.1 M Tris-HCl buffer, pH 9.0, containing 0.2 M KCl, or in 1 M diethanolamine (DEA) buffer, pH 10.3, containing 15–20 mM pNPP. The release of p-nitrophenol (ε = 18.5 mM−1·cm−1) was monitored at 405 nm. One unit of AP activity was defined as the quantity of the enzyme required to release 1.0 µmol of p-nitrophenol from pNPP in 1 min. The specific activity was calculated as units per 1 mg of protein.The standard assay for alkaline phosphatase (AP) activity was carried out at 37°C using 2 mM CmAP/MBL-AJ with concentration 100 µg/mL were added to each well. TBS-T was used as negative control, and interaction of MBL-AJ with adsorbed antibodies under the same conditions served as a positive control. After that, the plate was kept for 1 h at room temperature and washed again. Then 150 µL of pNPP in the buffer for AP assay was added to each well. After 2–5 min of incubation, 100 µL of 2 M sodium hydroxide was added to stop the reaction. Absorbance of the resultant solution was measured at 400 nm with a plate spectrophotometer .The embryonic alpha-1-acid glycoprotein (0.1 µg/mL) was dissolved in 0.1 M carbonate buffer, pH 9.5, containing 0.15 M NaCl, and 150 µl was added to the first well and was two-fold serially diluted in the same buffer in each well of a polystyrene 96-well ELISA microtiter plate , and then incubated at 4°C overnight. After incubation, the plate was washed three times with the buffer containing 0.01 M Tris-HCl, pH 7.5, 0.15 M NaCl, 0.05% Triton X-100 (TBS-T) and then three times with water. After that, 160 µL of bovine serum albumin (1 µg/mL) in TBS-T was added to each well to block non-specific binding sites. The plate was then incubated for 1 hour at room temperature and washed as described above. 150-µL aliquots of the samples containing chimeric lectin CmAP/MBL-AJ was determined by gel filtration on the column (1.5×170 cm) of Superdex 200 (Sigma) in 20 mM Tris-HCl, pH 8.2, 100 mM NaCl at a flow of 0.2 ml/min at 6°C and calibrated using Bio-Rad standard molecular weight markers: bovine thyroglobulin (670 kDa), bovine γ-globulin (158 kDa), chicken ovalbumin (44 kDa), horse myoglobin (17 kDa), Vitamin B12 (1.35 kDa). The molecular mass of CmAP/MBL-AJ was determined by 12.5% SDS polyacrylamide gel electrophoresis (SDS-PAGE). An aliquot of the protein solution was mixed with Laemmli sample buffer and heated at 95°C for 5 min, then applied to SDS-PAGE and stained according to Laemmli CmAP/MBL-AJ was performed without SDS and heat treatment. The “native” gels were stained for AP by the casing over with the standard enzyme assay buffer containing 2 mM pNPP. Both PAGEs were performed at 25°C.The molecular size of the native hybrid CmAP and MBL-AJ were generated by comparative modeling approach for further modeling the putative spatial structure of the bifunctional hybrid CmAP/MBL-AJ. The target-template alignments and modeling of CmAP and MBL-AJ 3D-structures were carried out by the molecular modeling package Molecular Operating Environment version 2013.08 . The high-quality experimental structure of alkaline phosphatase from marine bacterium Vibrio sp. G-15 with 1.4 Å resolution  was used as a template for homology modeling of CmAP 3D-structure. The crystal structure of C-type lectin CEL-IV from holothurian Cucumaria echinata with 1.65 Å resolution (PDB code: 3ALU) was used as a template for homology modeling of MBL-AJ 3D-structure. The CmAP/MBL-AJ chimeric model was manually generated by adding (G4S)3 linker to the C-terminus of the CmAP model and by binding with N-terminus of MBL-AJ using program MOE. Structural optimization of CmAP, MBL-AJ and CmAP/MBL-AJ models were carried out by energy minimization method with force field Amber12:EHT using program MOE 2013.08. The structure of the model oligosaccharide α-D-Manp-(1→6)-[α-D-Manp-(1→2)-α-D-Manp-(1→2)]-α-D-Manp(1→6)-D-Manp was build and optimized in water with MOE. The single W100A, Q103H, A156N, F159K, double A156N/F159K MBL-AJ mutants were in silico generated with program MOE. The molecular docking of the MBL-AJ mutants with the model branched oligosaccharide α-D-Manp-(1→6)-[α-D-Manp-(1→2)-α-D-Manp-(1→2)]-α-D-Manp(1→6)-D-Manp was carried out for the calculation of binding energy and affinity values The structural models of CmAP/MBL-AJ were genetically engineered by site-directed mutagenesis. The single or double amino acid substitutions were introduced into the forward and reverse gene-specific primers to the expected gene regions. The resultant PCR products were purified from agarose gel and annealed both the DNA matrix and primers in the equivalent ratio for the next PCR. The resultant mutant gene was amplified with the primers Lect-X-dir and Lect-X-rev, and fused to the linearized vector pET40CmAP and expressed as described above.The single W100A, Q103H, A156N, F159K and double A156N/F159K mutants of CmAP/MBL-AJ colonies was carried out by the modified method described above. Mucin (0.1 µg/mL) was dissolved in 0.1 M carbonate buffer, pH 9.5, containing 0.15 M NaCl, and 150 µl was added to each well of a polystyrene 96 -well ELISA microtiter plate , and then incubated at 4°C overnight. After incubation, the plate was washed three times with the buffer, containing 0.01 M Tris-HCl, pH 7.5, 0.15 M NaCl, 0.05% Triton X-100 (TBS-T) and then three times with water. After that, bovine serum albumin (1 µg/mL) in TBS-T was added as described above. Samples containing chimeric lectin CmAP/MBL-AJ (100 µg/mL) were two-fold serially diluted in TBS-T and added in 150 µL aliquots to each well. TBS-T was used as a negative control. After that, the plate was incubated, washed and visualized as described above. All of the lectin activity assays were performed in 3 independent parallels for 3 to 5 times.The lectin-binding activity assay of the mutant All values presented in this article are representative of at least three independent experiments. Data were analyzed using the Student's t-test of the SigmaPlot 2000 version 6.0 program (SPSS Inc.). Differences from controls were considered significant at P≤0.05.AJ has been developed. The recombinant bifunctional hybrid CmAP/MBL-AJ expression and purification were monitored by the alkaline phosphatase CmAP activity facilitating the rapid and effective detection of the CmAP/MBL-AJ-containing fractions. Although the expression of all engineered the full-length mature CmAP and MBL-AJ genes fusions in E. coli cells resulted in a high-level production of AP activity, the construction (2) containing C-terminal 6XHis tags was more useful for the hybrid CmAP/MBL-AJ purification than the same construction (1) without C-terminal 6XHis tag. The additional His-tag drastically enhanced the binding of CmAP/MBL-AJ to Ni-agarose column. However, for increasing of the lectin-binding activity of CmAP/MBL-AJ, the step of the TEV protease cleavage of C-terminal 6XHis tag was preferable  cells culture carrying the expression recombinant vector pET40CmAP/MBL-AJ , MBL-AJ (17 kDa), peptide linker (G4S)3, the plasmid fusion protein DbsC (32.5 kDa) and N- and C-terminal 6 X His tags .The optimal conditions for the for 12 h . At the His tags , 3. The CmAP/MBL-AJ has an optimal AP activity at pH 9.0–10.3 in 1 M DEA or 0.1 M Tris-HCl buffer containing 0.2 M KCl or NaCl, and does not depend on the presence of the bivalent metal ions that accords with the properties of wild type of the enzyme . CmAP/MBL-AJ is stable at pH between 6.0–11.0, but CmAP is gradually inactivated below pH 6.0. CmAP was also gradually inhibited when the enzyme was exposed above pH 8.5 maintained by 0.1 M glycine . Inactivation of 50% of AP activity occurred in 45 min at 37°C. However, CmAP/MBL-AJ was stable up to 45°C with a half-life of 15 min at 47°C and 120 min at 45°C, when the enzyme was incubated with 10 mM MgCl2 (data not shown). pH 10.0 . CmAP/MBCmAP/MBL-AJ was estimated by its affinity to embryonic alpha-1-acid glycoprotein or mucin. The detailed carbohydrate-binding specificity of MBL-AJ determined by inhibition assay has been previously shown The level of lectin-binding activity of the bifunctional hybrid AJ has advantaged the lectin-binding assay in its sensitivity and time in comparison with the two-step method of the wild-type MBL-AJ conjugation with the lowly active horse peroxidase . The diluted of the highly purified CmAP/MBL-AJ preparation up to thousand fold was fully stable at −20°C during more than one-year storage in the dilution buffer .The lectin activity of the bifunctional hybrid CmAP showed that the crystal structure of the cold-active Vibrio sp. G15-21 alkaline phosphatase  obtained with 1.4 Å resolution Halomonas sp. 593 (HaALP) determined with 2.1 Å resolution recently Search for a template of CmAP, HaALLP and VAP have 75% and 69% of identity and 86% and 82% of similarity, respectively. The 3D-structure of cold-active alkaline phosphatase CmAP from marine bacterium C. marina (Uniprot Q1W622) was modeled using as a template the crystal structure of the cold-active Vibrio sp. G15-21 alkaline phosphatase  is 0.43 Å. The structure of the CmAP monomer is stabilized by 148 hydrogen bonds, 20 ionic bonds and 180 hydrophobic contacts. The disulfide bonds are absent in CmAP, HaALP and VAP structures. Superposition of the CmAP structural model and HaALP and VAP crystal structures showed that the value RMSD is 0.87 Å for 497 Cα atoms. The CmAP monomer structure consists of a catalytic domain and a “crown”-domain by analogy with VAP and HaALP. Catalytic domain displays a three-layer β-sandwich fold and contains β-sheet of nine β-strands surrounded by α-helices at both sides. The structure of the catalytic domain has a fold of (α-β-α)-type that is AP characteristic. The CmAP, HaALP and VAP “crown”-domains are the largest observed in known APs. The cold-active CmAP, HaALP and VAP are the AP variants with the highest known k(cat) value. Analysis of the CmAP 3D-structure models and crystal structures of VAP and HaALP showed that the structures of the active sites and metal binding sites of CmAP, HaALP and VAP are identical. The critical N-terminal region for the stability of dimeric ECAP from E. coli K12 is absent in CmAP as in VAP and HaALP. The VAP or HaALP crystal structures are the dimers but it was experimentally showed that the functional structure of the CmAP is a monomer Superposition of all Cα atoms of AJ was generated using as a template of the crystal structure of sea cucumber lectin CEL-IV (PDB ID 3ALU)   [24].AJ and the template CEL-IV revealed that they have 65% identity and 79% similarity. Spatial structure of the lectin MBL-AJ is characteristic of the packed C-type lectins and contains two α-helix and 12 β-strands, disordered loops and ion Ca2+ in carbohydrate-recognizing domain. 3D-superposition of Cα- atoms of the structural model and prototype showed the value of RMSD to be 0.4 Å. The lectin MBL-AJ has a longer loop near the carbohydrate-recognizing domain than CEL-IV lectin 3 and lectin MBL-AJ was generated using MOE 2013.08  for diagnosing of cervical cancer AJ and mammalian lectins, including human MBL, had common properties to recognize bacterial mannans, the differences in the carbohydrate-binding specificities were found to be significant AJ interacts with the promising cancer biomarkers in the following order of the lectin-binding affinity: CEA, embryonic α-1-acid glycoprotein, trophoblast-specific β1-glycoprotein and α-fetoprotein isolated from the abortive and retroplacental blood AJ is not inhibited by monosaccharide, which is in the content of the normal human glycoconjugate's carbohydrate chains, and does not interact with the human blood serum components. On the base of the unique carbohydrate-binding domain that defines the MBL-AJ ability to distinguish microheterogeneity of the malignant or normal cell glycoconjugates, a novel method of cervical cancer diagnosis has been developed AJ has allowed identifying statistically reliable differences between the levels of lectin-binding CEA between healthy women and patients with cervical cancer, and between patients with benign and malignant neoplasms AJ-based method give a possibility to assess tumours noninvasively. Loss of expression of the MBL-AJ ligands was found in cervical cancer. According to the results, the concentration of the MBL-AJ-binding CEA of the healthy patients and the patients with the benign neoplasm were determined to be 48.5±11.8 U/ml versus the concentration of 11.4±7.5 U/ml of the patients with the cervical cancer diagnosis. The total CEA, which can be synthesized by both malignant and normal cells, were excluded from analysis because the cervical specimens were collected from the local source of the cancer CEA biosynthesis. A high sensitivity and specificity of the MBL-AJ-CEA interaction allowed detecting the lectin-binding structures in the vaginal secretion in the concentration of CEA 3–50 ng/mL Currently, construction of the fusion or tagged proteins is a useful method to obtain chimeric molecules with an improved functionality or, moreover, dual mode of action such as enzymes with specific binding activities AJ derivation from the holothurian A. japonicus coelomic liquid has many restrictions, namely: low concentration of MBL-AJ in the native source, preservation of the wild life of the Far Eastern holothurian A. japonicus, the narrow habitat of the endemic species of the holothurian. A recombinant analogue of MBL-AJ was attempted to be produced in the E. coli Top10/pQE_80L expression system AJ by the level of interaction with the antibodies against MBL-AJ. The recombinant production of the bifunctional hybrid CmAP/MBL-AJ reached the full solubility and functionality of the lectin MBL-AJ. Moreover, the alkaline phosphatase CmAP fused with the lectin MBL-AJ by the long flexible linker facilitated the recombinant protein purification monitoring as well as the lectin binding assay, eliminating the additional steps, namely: production of the antibodies against MBL-AJ, labeling the MBL-AJ antibodies by horsh peroxidase or alkaline phosphatase and incubation of the labeled MBL-AJ antibodies with the ligand-binding lectin MBL-AJ.However, the wild-type lectin MBL-CmAP included in the hybrid CmAP/MBL-AJ has N-end and C-end fusion proteins, its activity is not limited. The N-ends of both lectin subunits in CmAP/MBL-AJ diverge angularly in the reverse direction from each other, permitting the AP modules of the dimeric hybrid lectin to function independently 3 connecting to the CmAP module, whereas they form else two disulfide bonds in the native homotetrameric MBL-AJ .Although the alkaline phosphatase endently . Howeverc MBL-AJ , and 9. CmAP/MBL-AJ in different buffers and high affinity and specificity of its lectin part ensure good efficacy and minimal side effects of reliable detection of different carbohydrate structures in the CmAP/MBL-AJ-captured glycoproteins in diagnostic applications.The applications for molecules that bind proteins keep expanding, and issues of affinity, specificity, robustness, and time for development are still in focus. The potential limitations of using chimeric fusion molecules in clinical medicine include induction of antibodies to novel biological agents, and this will need to be specifically investigated. However, high enzymatic activity and stability of


Arabidopsis thaliana and related species, leading to PT overgrowth and a failure to deliver the sperm cells. Here we show that ARTUMES (ARU) specifically regulates the recognition of interspecific PTs in A. thaliana. ARU, identified in a genome-wide association study (GWAS), exclusively influences interspecific—but not intraspecific—gametophyte interactions. ARU encodes the OST3/6 subunit of the oligosaccharyltransferase complex conferring protein N-glycosylation. Our results suggest that glycosylation patterns of cell surface proteins may represent an important mechanism of gametophyte recognition and thus speciation.Species-specific gamete recognition is a key premise to ensure reproductive success and the maintenance of species boundaries. During plant pollen tube (PT) reception, gametophyte interactions likely allow the species-specific recognition of signals from the PT  by the embryo sac , resulting in PT rupture, sperm release, and double fertilization. This process is impaired in interspecific crosses between  Species-specific gamete recognition is needed to maintain species boundaries. Here, Müller et al. show that ARTUMES regulates pollen tube recognition between different Arabidopsis species, representing the first gene known to exclusively influence inter- but not intraspecific gamete interaction in plants. Species evolve and are maintained by a variety of hybridization barriers that prevent interspecific gene flow and thus the formation of potentially unviable or sterile hybridsIn plants, successful fertilization starts with the deposition of intraspecific (same-species) pollen  onto the stigma of a gynoecium. The subsequent steps involve extensive communication between the male and female tissues, leading to pollen adherence, hydration, and the germination of a pollen tube (PT). Within its cytoplasm, the tip-growing PT transports the two sperm cells through the transmitting tract of the pistil to the embryo sac , which is deeply embedded in the ovule, the precursor of seed. During its journey, the PT is guided towards the embryo sacs by attractants secreted by female tissuesferonia/sirène (fer/srn), lorelei (lre), nortia (nta), turan (tun), evan (evn), and Zea mays embryo sac 4 (ZmES4) RNAi-lines13141516171819FER has been proposed to be involved in interspecific PT recognitionZmES4 is sufficient to trigger PT growth arrest and rupture in a species-preferential mannerHowever, if a pollen grain originating from a different species (interspecific pollination) is placed on a plant's stigma, all the communication processes described above have the potential to act as pre-zygotic post-pollination barriers. Several studies describe a species-preferential behaviour of molecular factors involved in pollen adherence to the stigma, PT growth, and PT guidance towards the ovules47891012A. thaliana accessions in interspecific PT reception, we identified ARTUMES (ARU) as an indispensable factor for the recognition of interspecific A. lyrata PTs by A. thaliana embryo sacs. In contrast, aru mutants do not affect gametophytic communication in intraspecific A. thaliana crosses. ARU encodes the OST3/6 subunit of the oligosaccharyltransferase complex, which is known to regulate site- and substrate-specific N-glycosylation of proteins in yeast22A. thaliana OST3/6  pollen germination is inhibited at the A. lyrata stigma, A. lyrata PTs are guided towards A. thaliana embryo sacs, but PT reception fails  but also between species that are only partially reproductively isolated and do interbreed (A. arenosa × A. lyrata).Self-fertilizing on fails . Such un. lyrata , betweenArabidopsis, we assessed PT overgrowth in 86 A. thaliana accessions that were pollinated with A. lyrata pollen . Examples of accessions with extreme phenotypes are Lz-0  and Kz-9  also with pollen of A. halleri and A. arenosa. Although OG/S in the accessions pollinated with A. lyrata or A. halleri pollen was highly comparable . Intraspecific PT reception was normal in all the tested accessions , we crossed them with cessions , indicatA. thaliana, we used publicly available single-nucleotide polymorphism (SNP) data from the 86 accessions to perform a genome-wide association study (GWAS)Arabidopsis have identified previously known candidate genes, with only a few studies identifying novel regulatory genes in the respective pathways28To identify loci causing the variation in interspecific PT reception in At1g61780, At1g61790 and At1g61810; as well as At1g61795, for which no expression data was available; At1g61790 (n=18 siliques) of ovules display A. lyrata PT overgrowth, significantly more than in the Col-0 wild-type control  after the Etruscan goddess of night, nature, and fertilityaru-1. A second mutant allele, aru-2, carrying an EMS-induced premature stop-codon after aa residue 129  than the wild type  in aru-1 mutants. These promoters are highly active in synergids13aru-1 plants expressing a functional copy of ARU-GFP in their synergids displayed wild-type-like PT reception in interspecific crosses  is highly expressed in wild-type synergids , although there was more line-to-line variability than when the pMYB98 promoter was used We assessed the correlation of amino acid differences in the  P<0.05, , suggestcessions , but theARU in different accessions, we transformed high OG/S accessions  with pMYB98::ARU-GFP to ensure strong expression in synergid cells and assessed interspecific PT reception in these lines. Of several independent transformants of all accessions, none showed a significant reduction of OG/S  from Ga-0 could not better complement the aru mutant—which is in the Col-0 background—than the Col-0 allele, as it would be expected if differences in ARU alone caused the phenotypic difference between Ga-0 and Col-0 , but do not fall into the 5% tail of the estimated genomic distribution of D in A. thalianaARU locus. In addition, we estimated Fay and Wu's H, another test statistic to detect positive selectionP<0.02), and fall into the very extreme tails of both an empirical and a simulated distribution of H calculated with 12 accessions, which represent a world-wide distribution and are a subset of the accessions used in this studyARU locus. The fact that D for the upstream region was very close to the 5% tail and the strongly negative values for H provide evidence for possible positive selection and indicate that ARU may have undergone a recent selective sweep. This is consistent with the lack of variation in ARU amino acid sequence and expression level but is difficult, although not impossible, to reconcile with the fact that we identified ARU in a GWAS for variation in the OG/S phenotype. As pointed out above, this may be due to epistatic interactions with additional factors involved in this complex process. Nevertheless, as in animal speciation genes, selective pressures appear to have contributed to shaping the genetic basis that underlies interspecific PT reception in A. thaliana.Genes involved in reproductive isolation and speciation are often subject to selective pressures driving rapid divergenceARU encodes the OST3/6 subunit of the hetero-oligomeric plant oligosaccharyltransferase complex (OST), which catalyses the co- or posttranslational transfer of pre-assembled carbohydrate oligomers (Glc3Man9GlcNAc2) to asparagine (N) residues of polypeptidesN-glycosylation affects the substrate protein's folding, targeting, and/or processing through the ER. Subsequently, the N-linked glycan can be modified in the Golgi apparatus in a cell-type and species-specific manner, accounting for the functionality and binding specificity of the glycoprotein22A. thaliana ost3/6 (aru) mutant only a subset of glycoproteins is misglycosylated and therefore non-functionalaru has previously been identified in an EMS-screen for cell wall mutantsFER and NTA, have been implicated in the perception of cell wall perturbations, pathogen resistance, and innate immunity1744A. lyrata, fer heterozygous mutants show higher OG/S (74.9%) than wild-type segregants  and LRE (a glycosylphosphatidylinositol-anchored protein) are likely to be glycosylated1316aru ovules. We included NTA, which itself does not contain any putative glycosylation sites but whose subcellular localization depends on FER signallingaru embryo sacs: FER-GFP and LRE-Citrine were observed at the micropylar end of the synergids, and NTA-GFP was re-localized there upon PT arrival receptors, bind putative ligands from intraspecific PTs both via specific interactions with carbohydrates on the receptor protein and via direct protein–protein interactions, a mechanism similar to the proposed ‘domain-specific model' in mammalian sperm-egg binding0 ovules . It is cific PTs , while leception .The crosstalk between gametophytes constitutes a specific form of cell–cell communication. Cellular interactions are often mediated by specific binding of an extracellular ligand to a receptor, triggering downstream signalling cascades in the recipient cell. Most extracellular ligands and receptors are heavily glycosylated49A. thaliana accessions were part of the Nordborg collection for GWAS27The The accessions were grouped into early- , mid-, and late flowering plants  according to the flowering timeA. lyrataA. halleri , and A. arenosa  plants were stratified for 10 days and grown in the same greenhouse chamber. Plants were vernalized to induce flowering (see above).At1g61790, aru-1), SALK_137883C (At1g61780), SALK_052207C and SALK_026074C (At1g61795), SALK_104077 (At1g61810). The EMS allele aru-2 (ref. SALK-lines were obtained from NASC: SALK_067271 (u-2 ref.  was a giFlowers were emasculated and the pistils were pollinated 2 days after emasculation (dae). Siliques were collected two days after pollination and fixed for aniline blue staining in 9:1 ethanol:acidic acid. Aniline blue staining was performed as described previouslyA. thaliana 250 K Affymetrix SNP genotyping data sethttps://cynin.gmi.oeaw.ac.at/home/resources/atpolydb. GWAS analyses were performed using a compressed mixed linear model, using population parameters previously determined51Association mapping was conducted using the mean values of the proportions of ovules with PT overgrowth per silique (OG/S) as phenotypes. An pMYB98/pFER::ARU-GFP the complete coding sequence of ARU without the stop-codon was amplified using gene-specific primers with attB-sites for Gateway cloning: 5′- GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCGCTCAAATCAAAACTCGTC -3′ and 5′- GGGGACCACTTTGTACAAGAAAGCTGGGTCACGCCAACTCGATGGCCAATACGGA -3′. We introduced the PCR-fragment into pDONR207, and subsequently into the destination vector . The destination vector was a modification of the plant Gateway vector pMDC83, which contains the 2x35S-promotor before and GFP after the Gateway cassetteFERMYB98 , and the PCR fragments were introduced into the dephosphorylated vector backbone by ligation. pDONR207 carrying the ARU coding sequence and the modified destination vector were combined in an LR reaction. The resulting vectors, pFER::ARU-GFP and pMYB98::ARU-GFP were transformed into Agrobacterium tumefaciens strain CV1310, and homozygous aru-1 plants were transformed by the floral dip methodaru-1 mutation. For experiments with pMYB98::ARU-GFP in accessions with high OG/S, the construct was transformed into Fei-0, Kz-1, and Kz-9, and OG/S was assessed in heterozygous plants (T1 generation).For C83 ref.  with HinARU under the control of its endogenous promoter, the ARU fragment  was amplified from Col-0 and Ga-0 genomic DNA using primers 5′- TTTTACTAGTAGGCAATTCCATCAGTTGTT -3′ and 5′- TTTTGGTACCGTTACTTCACTTTCTCGAGT -3′, introducing a SpeI and a KpnI restriction site, respectively. The fragments were cloned into pMDC99 (ref. aru mutants (Col-0 background).For the constructs with C99 ref.  using repARU::ARU-GFP was cloned by amplification of a part of ARU coupled to GFP-tNOS from pFER::ARU-GFP with primers 5′- GCGTTAACGCTTTACCTCA -3′  and 5′- TTTGGATCCAGTAACATAGATGACACCGCG -3′ (introducing a BamHI site after tNOS). This fragment was introduced by ligation into the pMDC99 vector carrying the genomic fragment of ARU (Col-0). By cutting this vector with HpaI and BamHI, part of the ARU coding sequence and the downstream sequence were removed and replaced with the respective fragment of ARU coupled to GFP-tNOS, resulting in pARU::ARU-GFP, which was transformed into Col-0.pLRE::LRE-Citrine was cloned with overlapping PCR fragments that were assembled using the Gibson cloning Master Mix from New England Biolabs according to the manufacturer's recommendations. The 779-bp long promoter sequence with the predicted signal peptide from LRECitrine was amplified from the transgenic line CS36962 , using gene-specific primers with overhang primers for the signal peptide of LRE and overhang primers for the GPI-anchor of LRE: 5′- GGCCGGCCTGGAGGTGGAGGTGGAGCTGTGAGCAAGGGCGAGGAGCT -3′ and 5′- GGCCCCAGCGGCCGCAGCAGCACCAGCAGGATCCTTGTACAGCTCGTCCA -3′. The GPI-anchor of LRE was amplified with overhang primers for pMDC99: 5′- TGCTGGTGCTGCTGCGGCCGCTGGGGCCTCGGGTATGTCTTTTTGTTGTC -3′ and 5′- AGCTCCACCGCGGTGGCGGCCGCTCTAGAAGTCTCGCTTCTTCTTTTGT -3′. pMDC99 was amplified with overhang primers for the LRE promoter and the GPI-anchor using primers: 5′- ACTTAATCGCCTTGCAGCAC -3′ and 5′- TCTAGAGCGGCCGCCACCGCGG -3′. All the constructs were verified by sequencing. pFER::FER-GFP and pNTA::NTA-GFP were described previously13pFER::ARU-GFP construct for microprojectile bombardment of onion epidermal cells and co-localized it with the ER-marker pER-rk (mCherry) obtained from ABRCWe used the For visualizing GFP expression in the synergids, flowers were emasculated and pistils were dissected 2 dae to ensure the development of mature, unfertilized embryo sacs. The tissue was mounted on slides in 1 M glycine, pH 9.6. Images were captured on Leica Confocal Microscopes SP2 and SP5 (Leica Microsystems).RNA from pistils , inflorescences, and ovules  was extracted using the Trizol reagent (Invitrogen) according to the manufacturer's recommendations. Pistil and inflorescence cDNA was reverse transcribed using Oligo-dT primers and Superscript II reverse transcriptase from Invitrogen. Ovule cDNA was amplified using the Ovation Pico SL WTA system V2 from Nugen.ARU was done using primers 5′- CAATGTGCTTGTTCGAGTG -3′ and 5′- ATCCAGTCTTCCAGTTATCCA -3′. For quantitative RT and digital droplet PCR of ARU in A. thaliana accessions, the primers 5′- GTTTGTTACCAATGTGCTTGTTCG -3′ and 5′- TCCATATCCAGTCTTCCAGTTATCC -3′ were used and expression levels were normalized against UBIQUITIN C . For digital droplet PCR on ovule cDNA, the UBC9 assay was performed as an EvaGreen assay, whereas ARU transcripts were detected using a gene-specific probe (5′-FAM- TACTGCACAAAGGTTG -MGB-3′). The samples were analysed with the QX200 system from Bio-Rad.Reverse transcriptase PCR (RT–PCR) of ARU gene structure and UTRs is based on annotations in the ARAMEMNON databaseARU and 1,000 bp up- and downstream flanking regions were downloaded from http://signal.salk.edu/atg1001/3.0/gebrowser.php. For accessions for which no sequences or only sequences with missing data were available, we amplified the whole region from genomic DNA using primers 5′- TTTGCTATAGGCACATGTGT -3′ and 5′- GACCCGAAATTGTCAAATGA -3′, and sequenced the resulting PCR products of Bay-0, Fab-2, Fab-4, Omo-2-3, Knox-10, Kz-1, LL-0, Lz-0, Mr-0, Mrk-0, Zdr-6. The upstream region was sequenced additionally from Got-7, Pu2-23, and Spr1-6 (primer 5′- TTTGCTATAGGCACATGTGT -3′ and 5′- CGGAGGTTAGGAATTTTGAGA -3′), and the downstream region from Got-7, Pu2-23, Kz-9, Mz-0, Pro-0, Van-0, and Var2-1 (primer 5′- CAATGTGCTTGTTCGAGTG -3′ and 5′- GACCCGAAATTGTCAAATGA -3′). Tajima's D and Fay and Wu's H were calculated separately for the 1,000 bp up- and downstream as well as the coding sequence with the set of 96 accessionsA. lyrata was used as outgroup. P values against the null model were obtained by running 10,000 coalescent simulations and for Tajima's D, the 5% quantile was calculated using previously published estimates for DDetermination of How to cite this article: Müller, L. M. et al. A subunit of the oligosaccharyltransferase complex is required for interspecific gametophyte recognition in Arabidopsis. Nat. Commun. 7:10826 doi: 10.1038/ncomms10826 (2016).Supplementary Figures 1-10, Supplementary Tables 1-2 and Supplementary ReferencesCorrelation of SNPs (1000 bp downstream region) with OG/S phenotype of accessions


However, use of more than two PET tracers in a single clinical trial is quite challenging. In this study we carried out a longitudinal investigation of + human breast cancer ZR-75-1 models were established in female mice that were then randomly assigned to a treatment  or vehicle group. Micro-PET/CT imaging with 18F-FES, 18F-FDG, and 18F-FMISO was performed on days 0, 3, 14, and 21 after treatment. The uptake value  for each probe in tumor (T) tissue and contralateral muscle (M) was measured for quantitative analysis and T/M calculation. Tumor volume was measured to record tumor growth at each time point. Tumor tissues were sampled for immunohistochemical staining of ER expression. Correlations for tumor volume and ERα levels with uptake data for the probe were tested.ER18F-FES in ZR-75-1 tumor tissues corresponded well with tumor response to endocrine therapy, but not for 18F-FDG and 18F-FMISO, according to longitudinal micro-PET/CT imaging and quantitative correlation analysis. There was a significant positive correlation between 18F-FES uptake and ER levels . Notably, 18F-FES uptake on day 3 was significantly correlated with the day 21/baseline tumor volume ratio .Uptake data for 18F-FES, 18F-FDG, and 18F-FMISO probes revealed that 18F-FES PET/CT molecular imaging can provide a precise early prediction of tumor response to endocrine therapy in ER+ breast cancer in a ZR-75-1 xenograft model. This molecular imaging strategy with 18F-FES PET/CT will be useful in evaluating the efficacy of endocrine therapies and in developing new endocrine drugs.Comparison of  Unfortunately, approximately half of patients with breast cancer cannot benefit from endocrine therapy because of intrinsic or acquired drug resistance [+ breast cancer.Breast cancer is one of the most common causes of cancer-related death, and approximately 75–80% of breast cancers are estrogen receptor (ER)-positive at the time of primary diagnosis . Endocrisistance –3. There18F-Fluorodeoxyglucose (18F-FDG) reflects glucose metabolism and is the most widely used PET probe for evaluating therapeutic efficiency [18F-FDG cannot be used for direct assessment of changes in ERα expression in breast cancer induced by endocrine therapy [18F-Fluoromisonidazole (18F-FMISO), an analogue of nitroimidazole, is a PET probe commonly used for hypoxic imaging as it reflects the degree of hypoxia in solid tumors in vivo [18F-FMISO PET/CT could predict primary letrozole resistance in ER+ breast cancer [18F-FMISO cannot target ER, this is also an indirect approach for predicting response to endocrine therapy. 18F-Fluoroestradiol (18F-FES) is a specific ER-targeted molecular probe for PET evaluation of ER expression in breast cancer [18F-FES PET can reliably detect ER+ breast cancer lesions and that 18F-FES uptake correlates well with ERα immunohistochemical (IHC) scoring [18F-FES PET could be used to predict the response of breast cancer to neoadjuvant chemotherapy and could help in individualizing treatment for breast cancer patients [Two methods are currently used to assess the efficacy of cancer therapy in clinical practice. The conventional method for evaluating treatment responses relies on changes in tumor size according to response evaluation criteria in solid tumors (RECIST) . However therapy . 18F-Flu in vivo –11. In at cancer . Howevert cancer . Clinica scoring –16. We ppatients –18.18F-FES, 18F-FDG, and 18F-FMISO PET probes for early prediction and monitoring of the response to endocrine therapy in a mouse xenograft model of ER+ breast cancer s in this preclinical study.For early prediction and monitoring of breast cancer response to endocrine therapy, it is important to assess the use of multiple PET imaging probes. However, it is quite challenging to apply more than two PET tracers in a single clinical trial. Thus, we carried out a longitudinal molecular imaging investigation using All animal studies were conducted under a protocol approved by the Institutional Animal Care and Use Committee of Fudan University (LASFDI-20140179A032). All invasive animal procedures were performed under anesthesia  and all efforts were made to minimize suffering.+ breast cancer cell line was purchased from Cell Bank, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. The cells were grown in RPMI 1640 medium with L-glutamine, penicillin 100 μg/mL, streptomycin 100 μg/mL and 10% fetal calf serum in a humidified 5% CO2 atmosphere at 37°C. Female BALB/c nude mice aged 4–6 weeks were purchased from the Department of Laboratory Animal Science, Fudan University, and housed in laminar flow cabinets under specific pathogen-free conditions and provided with food and water ad libitum. During the entire study period, the body weight and behaviors of mice were monitored by a balance and visual observation every two day. No significant body loss and abnormal behaviors were observed. The mice were sacrificed by overdose of anesthesia at each imaging time point, at the study end, or when the maximum tumor size in long diameter reached 20 mm.The ZR-75-1 human ER6 cells in 100 μL of medium mixed with 100 μL of Matrigel (BD Biosciences)) were injected into the mammary fat pad on the right thorax of mice. All invasive procedures were performed under anesthesia . Tumor growth was followed via caliper measurement of the perpendicular axes. Tumor volume was calculated as V = α × (b2)/2 [α is tumor length and b is tumor width.Estrogen pellets  were implanted into the body of mice 3 days before tumor cell inoculation and were left until the tumors reached 6.0–8.0 mm (~14 days after inoculation). ZR-75-1 (5×10× (b2)/2 , where αn = 10 per group), and the estrogen pellets were surgically removed before treatment initiation. The dose and protocol for each group were as follows: vehicle, 0.9% sodium chloride, 50 μl/mouse/week, s.c.; and fulvestrant, 5 mg/mouse/weekly, s.c., prepared according to standard methods. The treatment duration was 21 days.When tumors had grown to 6–8 mm in diameter, mice were randomly assigned to a treatment or vehicle group  in our center as previously reported [18F-FES, 18F-FDG, or 18F-FMISO into the tail vein. The animal numbers for each PET probe were 10, 9, 8, and 7 on days 0, 3, 14, and 21, respectively, because of tumor sampling for IHC. Considering 18F decay, microPET/CT imaging were performed in the order of 18F-FDG, 18F-FES and 18F-FMISO with 16-20h interval for a specific imaging time point [18F-FDG administration, mice were kept fasting for at least 6.0 h. 18F-FMISO was injected 90 min before the scan start [18F-FES and 18F-FDG 60 min before the scan start. Isoflurane was administered 10 minutes before the scan start, and mice were maintained under anesthesia during the scanning period. The images were reconstructed using a three-dimensional ordered-subset expectation maximization (OSEM3D)/maximum algorithm. For data analysis, the region of interest (ROI) was manually drawn to cover the whole tumor on fused images. A similar circular ROI was drawn on the muscle of the opposite foreleg of the mouse on fused images. The percentage injected dose per gram (%ID/g) in the tumor and muscle ROIs was recorded. The tumor-to-muscle ratio (T/M) was calculated by dividing %ID/g for tumor tissue by that for muscle. T/M before and after therapy were denoted as T/M day0 and T/M dayn, and %ID/g as %ID/g day0 and %ID/g dayn, respectively. Changes after therapy are denoted as ΔT/M = (T/M dayn − T/M day0)/ T/M day0 × 100% and Δ%ID/g = (%ID/g dayn − %ID/g day0)/ %ID/g day0 × 100%. [The reported –21. Micrme point . Before an start , and 18FIHC for ERα was performed using standard protocols according to the manufacturers’ instructions , where the tumor tissues were sampled from both treatment and control groups at each imaging time point. IHC analysis for quantitative ERα expression was based on standard procedures for breast cancer . The tot+ breast cancer groups were assessed using analysis of variance (ANOVA) models. An unpaired t-test was used to determine statistical significance between experimental and control groups. Pearson correlation coefficients were calculated to determine the association between treatment-induced changes in18F-FES uptake and ER expression according to IHC. P values < 0.05 were considered statistically significant. All statistical analyses were performed using SPSS version 19.0 (SPSS-IBM).Data are expressed as mean ± SD. Within-group comparisons (before and after treatment) and differences among ERP > 0.05), but a significant difference was observed between the groups on days 14 and 21 (P < 0.001).The changes in tumor volume are shown in 18F-FES by ZR-75-1 tumor tissues corresponded well to the efficacy of endocrine therapy according to longitudinal 18F-FES micro-PET/CT imaging (18F-FES %ID/gmax in the vehicle group showed no significant change compared with baseline at any time point (P > 0.05). By contrast, tumor uptake of 18F-FES in the treatment group decreased remarkably from day 3. 18F-FES %ID/gmax significantly decreased on day 3 , day 14 , and day 21  compared to baseline. Moreover, significant differences in 18F-FES tumor uptake between the groups were observed on days 3, 14, and 21 (P < 0.001). T/M ratios for 18F-FES exhibited similar results to those for %ID/gmax , day 14 , and day 21  compared to baseline (18F-FDG %ID/gmax fluctuated within a very narrow range and there was no significant difference compared to baseline at any time point (P > 0. 05)  . Compari%ID/gmax .18F-FMISO micro-PET/CT imaging , day 14 , and day 21 compared to baseline. In the treatment group, a similar trend was observed from day 0 to day 3 , day 14 , and day 21 . Comparison of the vehicle and therapy groups revealed no significant difference in 18F-FMISO uptake at any time point after endocrine therapy (P > 0.05). For18F-FMISO T/M ratios within and between the groups, similar results to those for %ID/gmax were observed . This result indicates that quantitative changes in 18F-FES uptake by tumor tissue could be an early predictor of responders to endocrine therapy in ER+ breast cancer. Most importantly, changes in 18F-FES uptake occurred before measurable changes in tumor size, which could be beneficial for evaluation of endocrine therapies for ER+ breast cancer patients in translational investigations in the near future.We demonstrated the feasibility of t cancer . As a revestrant . By cont18F-FES PET imaging can measure the pharmacodynamics of endocrine drugs in ER+ breast cancer over time. Tumors treated with fulvestrant showed the most significant decrease in 18F-FES %ID/gmax and T/M on day 3 after treatment. However, tumor 18F-FES uptake days 14 and 21 did not differ from that on day 3 (P > 0.05). By contrast, tumor 18F-FES uptake in the vehicle group gradually increased during the course of treatment. Therefore, 18F-FES PET/CT imaging offers a noninvasive way to determine quantitatively changes in the tumor oestrogen receptor during endocrine therapy.Apart from the between-group findings, longitudinal 18F-FES PET/CT has brought clinical benefits to breast cancer patients by making appropriate hormonal treatment based on the measurement of oestrogen receptor [18F-FES uptake appear complex and multiple aspects, such as concomitant therapies and menopausal status [18F-FES uptake decreased in fulvestrant-resistant tumor model after endocrine therapy [Currently, receptor –28. Howel status . For exa therapy . To addr therapy .18F-FES was superior to 18F-FDG and 18F-FMISO as a PET imaging probe in predicting response to endocrine therapy in ER+ breast cancer. According to 18F-FDG uptake by ER+ breast cancer between the treatment and vehicle groups until day 21, so 18F-FDG PET could not differentiate between responders and non-responders to endocrine therapy in the early phase. The reason might be that glucose metabolism is not directly affected by endocrine therapy in breast cancer and thus a longer time might be required for significant outcomes [max, T/M) for 18F-FMISO uptake in the treatment group generally exhibited a transient decrease on day 3 and a continuous increase thereafter. However, there was no significant difference in 18F-FMISO uptake by ER+ breast cancer between the vehicle and therapy groups. Thus, 18F-FDG and 18F-FMISO as PET probes are not the first choice for prediction of early response to endocrine therapy for ER+ breast cancer.Finally, our findings indicate that outcomes . Similar18F-FDG and 18F-FMISO was not performed because there was no correlation between their uptake and treatment response. Second, only a single cell line and one model of breast cancer were used. Third, the role of 18F-FDG and 18F-FMISO PET/CT in evaluating endocrine therapy response of breast cancer was not as good as that we expected, which indicated that more research efforts should be paid on multiple PET imaging probe strategy for diagnosis and therapy of breast cancer. Further studies are needed to investigate clinical translation of PET molecular imaging for response prediction and follow-up of endocrine therapies for different breast cancer mechanisms.Our study has several limitations. First, IHC of Glut-1 and HIF-1 in relation to 18F-FES, 18F-FDG, and 18F-FMISO probes showed that 18F-FES PET/CT imaging is suitable for precise and early prediction and monitoring of response to endocrine therapy in ER+ breast cancer. Among the three probes evaluated, 18F-FES will provide the greatest benefit in evaluating the efficacy evaluation of endocrine therapy.Comparison of S1 FileTable A and Table B in S1 File are the tumor volume changes in vehicle and fulvestrant groups at different times, respectively.(PDF)Click here for additional data file.S2 File18F-FES PET/CT images of vehicle and fulvestrant groups on days 0, 3, 14, and 21 after treatment, respectively. Table A and Table B in S2 File are the value of 18F-FES %ID/gmax in vehicle and fulvestrant groups, respectively. Table C and Table D in S2 File are the value of 18F-FES T/M in vehicle and fulvestrant groups, respectively.Figure A and Figure B in S2 File are (PDF)Click here for additional data file.S3 File18F-FDG PET/CT images of vehicle and fulvestrant groups on days 0, 3, 14, and 21 after treatment, respectively. Table A and Table B in S3 File are the value of 18F-FDG %ID/gmax in vehicle and fulvestrant groups, respectively. Table C and Table D in S3 File are the value of 18F-FDG T/M in vehicle and fulvestrant groups, respectively.Figure A and Figure B in S3 File are (PDF)Click here for additional data file.S4 File18F-FMISO PET/CT images of vehicle and fulvestrant groups on days 0, 3, 14, and 21 after treatment, respectively. Table A and Table B in S4 File are the value of 18F-FMISO %ID/gmax in vehicle and fulvestrant groups, respectively. Table C and Table D in S4 File are the value of 18F-FMISO T/M in vehicle and fulvestrant groups, respectively.Figure A and Figure B in S4 File are (PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


To explore the genetic and molecular events that control subclones exhibiting distinct invasive/migratory capacities derived from human epithelial ovarian cancer (EOC) cell line A2780 and SKOV3.Single-cell subclones were isolated and established that were derived from the SKOV3 and A2780 cell lines through limiting dilution methodology. Transwell insert assays and MTT assays were performed to screen and identify the subclones exhibiting the highest and the lowest invasive/migratory capacities, and the selected subclones were renamed as A-H (A2780 high), A-L (A2780 low), S-H (SKOV3 high), and S-L (SKOV3 low). Their biological characteristics were evaluated. RNA-Seq was conducted on the targeted subclones.P < 0.001 and = 0.001, respectively). A-H/S-H cells displayed a clear reduction in doubling time , and a significant increase in the percentage of cells in S phase . Additionally, the apoptotic rates of A-H/S-H cells were significantly lower than those of A-L/S-L cells . At both mRNA and protein levels, caspase-3 and caspase-7 expression were reduced but Bcl-2 expression was increased in A-H/S-H cells. The TrkB (anoikis-related) and Beclin1 (autophagy-related) levels were consistently high and low, respectively, in both A-H/S-H cells. Resistance to chemotherapy in vitro and higher capacities on tumor formation in vivo was presented in both A-H/S-H cells. PI3K/AKT/mTOR pathway components, PIK3CA, PIK3CD, AKT3, ECM1, GPCR, mTOR and PRKCB were increased but that the Nur77 and PTEN were decreased in A-H/S-H cells, identified by RNA-Seq and consistently confirmed by RT-PCR and Western blot analyses.Compared with their corresponding counterparts, A-H/S-H cells exhibited significantly higher invasive/migratory capacities (Heterogeneous cell subpopulations exhibiting distinct invasive and migratory capacities co-exist within the SKOV3 and A2780 cell lines. PI3K/AKT/mTOR pathway activation is associated with higher invasive and migratory capacities in subpopulations of human ovarian cancer cell lines. Inhibiting this pathway may be useful for the chemoprevention or treatment of EOC. Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy . IntratuHeterogeneity is a common phenomenon of parental tumor cell lines, and highly metastatic tumor cell variants pre-exist in parental tumor cell populations –9. SKOV32 incubator.The principal materials used in this study are shown in Limiting dilution methodology was performed as described by Chen et al. . A2780 a6) in the logarithmic growth phase were suspended in 200 μl of serum-free RPMI-1640 and seeded in the upper chambers of Matrigel-coated Transwell plates. RPMI-1640 containing 20% FBS (600 μl) was added to the lower chamber as a chemotactic factor. Each subclone was seeded in triplicate. All of the Transwell chambers were then incubated at 37°C for 24 h. Non-invading cells that remained on the upper surface of the filter were carefully removed with a swab. The Transwell chambers were then immersed in 500 μl of culture medium containing 0.5 mg/ml MTT and were incubated in the dark at 37°C for 4 h. Then, the liquid was discarded, 500 μl of dimethyl sulfoxide (DMSO) was added, and the cells were incubated at 37°C for 10 min to fully dissolve the formazan crystals. The absorbance at 490 nm was measured using a microplate reader. Based on their absorbance values, the subclones were classified into groups exhibiting high, moderate or low invasive/migratory capacity. Groups exhibiting high or low invasive/migratory capacity were retained and re-subjected to the above test. The subclones exhibiting the highest or the lowest invasive/migratory capacity were identified based on repeated comparisons and were renamed as A-H (A2780 high), A-L (A2780 low), S-H (SKOV3 high), and S-L (SKOV3 low). To investigate the stability of the invasive/migratory potential, populations of the A-H, A-L, S-H, and S-L cells were serially passaged to approximately the thirtieth generation, and the variation in the invasive/migratory capacities of the targeted subclones was evaluated.Transwell insert assays and 3--2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assays were performed to assess and compare the invasive/migratory capacities of the subclones. Polyvinylpyrrolidone-free polycarbonate (PVP-free) filters with a pore size of 8.0 μm, which were either uncoated (migration assay) or coated (invasion assay) with 50 μl of Matrigel diluted 1:3 in serum-free RPMI-1640, were placed in Transwell chambers. Cells  were seeded in each well. Each subclone was seeded in six wells of each 96-well plate . All of the 96-well plates were incubated at 37°C. One 96-well plate was removed from the incubator each day. Twenty microliters of MTT  was added to each well, and the plates were incubated in the dark at 37°C for 4 h. The liquid was then discarded, and 200 μl of DMSO was added to dissolve the formazan crystals. The optical density (OD) was measured at 490 nm using an enzyme-linked immunosorbent assay (ELISA) plate reader. The OD values for each subclone were recorded each day. Growth curves were plotted, and the doubling time was calculated.MTT assays were performed in 96-well plates. Cells , and then incubated at −20°C for at least 30 min. The cells were centrifuged, and the supernatant was discarded. After the cells were washed twice with PBS, they were resuspended and stained with 500 μl of PBS solution containing 50 μg/ml propidium iodide (PI), 100 μg/ml RNase A, and 0.2% Triton X-100. The samples were incubated in the dark at 4°C for 30 min and then analyzed via flow cytometry. Approximately 20,000 cells were examined per sample. This assay was performed in triplicate.The four subclones were collected in the logarithmic growth phase and counted. A minimum of (5–10) × 106/ml in 1× binding buffer. The cell suspension (100 μl) was placed in a Falcon tube and was sequentially incubated in annexin V-FITC (5 μl) followed by PI (5 μl) at 20°C–25°C in the dark for 15 min. Then, 1× binding buffer (400 μl) was added, and the samples were analyzed by flow cytometry within one hour.The apoptotic rates were measured using an Annexin V-FITC/PI Apoptosis Detection Kit. Cells were collected in the logarithmic growth phase, washed twice with cold PBS, and adjusted to a density of 1 × 10(−ΔΔCT) comparative method [The mRNA and protein levels of caspase-3, caspase-7, and Bcl-2 (apoptosis-related factors), TrkB (anoikis-related factor), and Beclin1 (autophagy-related factor) were detected. Total RNA was extracted using TRIzol reagent according to the manufacturer's instructions, and cDNA was then synthesized using a QuantScript Reverse Transcription Kit. All of the primers are listed in e method . Each sag for 10 min at 4°C, and the supernatants were collected. Approximately 50 μg of total protein was denatured for 10 min at 95°C, separated on a 10–15% SDS-polyacrylamide gel, and transferred to a nitrocellulose membrane. The membrane was blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 2 h at room temperature and then incubated in primary antibodies overnight at 4°C. The primary antibodies were detected by incubating the membranes in horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature, and the signals were visualized using a SuperEnhanced Chemiluminescence Detection Kit. Details of the primary and secondary antibodies are presented in Cells were lysed in cold radioimmunoprecipitation assay (RIPA) buffer containing freshly added 0.01% protease inhibitor for 30 min. After the lysed cells were incubated on ice for 10 min, they were centrifuged at 12000 × Cells were seeded at a density of 8,000 cells per well in 96-well plates. After the cells were incubated for 24 h, they were treated with different concentrations of cisplatin and taxol. MTT assays were performed after a 48-h incubation. Dose-response curves were then generated, and the 50% inhibitory concentration (IC50) values were calculated. Each assay was performed in triplicate.6 cells. The tumor volumes were estimated using the formula (width)2 × length/2. The mice were euthanized after 30 days of observation, and the tumors were harvested. All procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Peking Union Medical College Hospital.Female BALB/c nude mice (4–5 weeks-old) were maintained in micro-isolator cages. Forty mice were randomly divided into 4 groups of 10 mice each. Xenografts were established by subcutaneously injecting the mice with 1 × 10First, the library for Illumina sequencing was prepared. Briefly, 150 μg of total RNA was extracted from each sample using TRIzol reagent, according to the manufacturer's instructions. The quality of the extracted RNA was evaluated via 1.5% agarose gel electrophoresis and PI taining. The extracted RNA was then dissolved in RNase-free water and purified using an RNeasy Mini RNA Purification Kit. Illumina-compatible libraries were constructed using a TruSeq RNA library preparation kit. Briefly, mRNA was purified from the extracted RNA using oligo-dT magnetic beads and was fragmented. First-strand cDNA was synthesized using random hexamers and reverse transcriptase. Second-strand cDNA was synthesized using high quality deoxyribo nucleotide triphos phates (dNTPs), ribonuclease H (RNase H), and DNA polymerase. Double-stranded (ds) cDNA was synthesized via PCR amplification using sequencing adaptors. Fragments of 250–500 bp were selected, subjected to 2.5% agarose gel electrophoresis, excised from the gels, and purified using a Qiagen Gel Extraction Kit. The final library was sequenced on a HiSeq™ 2000 platform (Illumina).P-value < 0.05 and a-fold change in expression > 2.0 were considered to be significantly differentially expressed.Second, the obtained raw reads were analyzed by trimming, filtering, and the sequences aligning to the human genome (hg19) using TopHat (version 1.3.3) ; CufflinRT-PCR and Western blot analyses were performed to validate genes that exhibited significant differential expression in the RNA-Seq data for the targeted subclones. The primers and antibodies used are shown in ® Version 9.2 . All tests were 2-sided, and P < 0.05 was considered to be statistically significant. The invasive/migratory capacities, cell doubling time, and tumor volumes were compared using two- tailed independent- sample t tests. Chi-square tests or Fisher's exact tests were performed to compare the apoptotic rates and cell cycle distribution.All statistical analyses were performed using SASSingle adherent A2780/SKOV3 cells were observed as early as 4–5 h after isolation and inoculation Fig.  and 1B. For the A2780 cell line, single adherent cells were observed in 258 wells of the eight 96-well plates. Of these 258 single-cell clones, 166 died and disappeared, and 16 were eliminated due to bacterial contamination. Of the remaining 76 single-cell clones, 18 died and disappeared, and 13 were subsequently eliminated due to bacterial contamination during the transfer process from 96-well plates to 48-, 24-, 12-, and 6- well plates and, ultimately, to culture dishes. For the A2780 subclones, the contamination rate was 11.2%, and the mortality rate was 80.3%. Forty-five (17.4%) cell subclones were obtained and named A1–A45. For the SKOV3 cell line, single adherent cells were observed in 288 wells of the eight 96-well plates. Of these 288 single-cell clones, 148 died and disappeared, and 20 were eliminated due to bacterial contamination. Forty (19.2%) single-cell subclones were obtained and named S1–S40.P < 0.001; invasion: 2.340 ± 0.322 vs. 0.350 ± 0.082, P = 0.001; Fig. P = 0.001/0.002; A-L vs. parental: P = 0.044/0.001). Similarly, S15 (S-H) and S4 (S-L) exhibited most distinct invasive/migratory abilities  among all of the SKOV3 subclones. The invasive/migratory capacities of both S-H and S-L were significantly different from those of their parental cell line . The respective invasive/migratory capacities of A-H, A-L, S-H, and S-L were not significantly altered by serial passaging to approximately the thirtieth generation . These results indicated that the selected subclones had relatively stable biological traits. However, the invasive/migratory capacities of these subclones were slightly increased following passaging for thirty generations, with the exception of the migratory capacity of A-L, which decreased slightly, possibly due to experimental error. Consequently, subclones in similar generations were used, unnecessary passaging was avoided, and subclones in no greater than the 20th generation were used in the further comparative analyses.In the Matrigel invasive/migratory assay, subclone A6 (A-H) demonstrated the highest invasive/migratory activities, and subclone A41 (A-L) displayed the weakest ability to invade and migrate through the membrane  and a concomitant decrease in the percentage of cells in G0/G1 phase , suggesting that the enhanced proliferation of A-H/S-H cells might be due to the activation of DNA synthesis . Similarly, the respective taxol IC50 values of A-H and S-H cells were 0.095 ± 0.004 μg/ml and 0.074 ± 0.006 μg/ml, which were significantly higher than those of A-L cells  and S-L cells . The subclones exhibiting higher invasive/migratory capacities were more resistant to chemotherapyCell viability after cisplatin or taxol treatment was significantly inhibited in A-L/S-L cells compared with A-H/S-H cells, as demonstrated by drug cytotoxicity assays Fig. . The subth day vs. 19th day; 7th day vs. 20th day; Fig. P = 0.008). Additionally, the A-H/S-H tumors were significantly larger than the A-L/S-L tumors . The A-H/S-H cells exhibited higher capacities for in vivo tumor formation.After subcutaneous injection, A-H/S-H tumors were observed much earlier than A-L/S-L tumors  and PKC (PRKCB), are PI3K/AKT/mTOR pathway components. The PI3K pathway is involved in stimulating cell proliferation and migration and in inhibiting apoptosis; additionally, this pathway plays a pivotal role in many human malignancies –36. The The survival mechanism initiated via PI3K is primarily activated through AKT. Increased AKT activation has been noted observed in high-grade and late-stage serous ovarian cancer –49, in wBriefly, the 5 differentially expressed genes that were identified by RNA-Seq, as well as PIK (PIK3CA and PIK3CD subtypes), mTOR and PTEN, which function in the PI3K/AKT/mTOR pathway, were selected for further validation of the RNA-Seq results. RT-PCR and Western blot analyses consistently confirmed that PIK3CA, PIK3CD, AKT3, ECM1, GPCR, mTOR and PRKCB expression was increased but that Nur77 and PTEN expression was decreased in A-H/S-H cells compared with A-L/S-L cells. PI3K/AKT/mTOR pathway activation was associated with enhanced invasive and migratory capacities in human ovarian cancer cell lines.Heterogeneous cell subpopulations exhibiting distinct invasive and migratory capacities co-exist within human ovarian cancer cell lines. The subclones displaying higher invasive and migratory capacities exhibit significantly increased independent viability, which might play an important role in tumor metastasis and chemotherapeutic resistance. PI3K/AKT/mTOR pathway activation is associated with higher invasive and migratory capacities in subpopulations of human ovarian cancer cell lines. Inhibiting this pathway may be useful for the chemoprevention or treatment of EOC.


By contrast, the nucleotide excision repair activity is completely abrogated when the canonical cytosine base opposite the B[a]P-dG adducts is replaced by an abasic site in duplex DNA. However, base excision repair of the abasic site persists. In order to understand the structural origins of these striking phenomena, we used NMR and molecular spectroscopy techniques to evaluate the conformational features of 11mer DNA duplexes containing these B[a]P-dG lesions opposite abasic sites. Our results show that in these duplexes containing the clustered lesions, both B[a]P-dG adducts adopt base-displaced intercalated conformations, with the B[a]P aromatic rings intercalated into the DNA helix. To explain the persistence of base excision repair in the face of the opposed bulky B[a]P ring system, molecular modeling results suggest how the APE1 base excision repair endonuclease, that excises abasic lesions, can bind productively even with the trans-B[a]P-dG positioned opposite the abasic site. We hypothesize that the nucleotide excision repair resistance is fostered by local B[a]P residue—DNA base stacking interactions at the abasic sites, that are facilitated by the absence of the cytosine partner base in the complementary strand. More broadly, this study sets the stage for elucidating the interplay between base excision and nucleotide excision repair in processing different types of clustered DNA lesions that are substrates of nucleotide excision repair or base excision repair mechanisms.The nucleotide excision repair of certain bulky DNA lesions is abrogated in some specific non-canonical DNA base sequence contexts, while the removal of the same lesions by the nucleotide excision repair mechanism is efficient in duplexes in which all base pairs are complementary. Here we show that the nucleotide excision repair activity in human cell extracts is moderate-to-high in the case of two stereoisomeric DNA lesions derived from the pro-carcinogen benzo[ Eukaryotic nucleotide excision repair (NER) is an important mammalian defense system against DNA lesions derived from environmental genotoxic agents that include polycyclic aromatic hydrocarbons (PAH). The NER process involves ~ 30 proteins and entails the excision of a 24–30-mer oligonucleotide that contains the lesion, followed by repair synthesis of the resulting gap . A puzzla]pyrene have played an important role in elucidating the features of damaged duplexes that lead to their recognition by the NER recognition protein XPC-RAD23B. The major adduct derived from the metabolic activation of benzo[a]pyrene through the well-characterized diol epoxide pathway [S-(+)-trans-B[a]P-dG (trans-B[a]P-dG) adduct [trans-B[a]P-dG adduct completely abolishes the NER response in human HeLa cell extracts [R-(+)-cis-B[a]P-dG adduct (cis-B[a]P-dG)  adduct  shown in) adduct , is gene) adduct –11, but extracts . Howeverextracts . A simil[a]P-dG) ; this ade adduct , 19, 20.e adduct . The inta]P-dG adduct has been attributed to extensive stacking interactions between the intercalated polycyclic aromatic ring system and the adjacent base pairs, so that the insertion of the β-hairpin is hindered [trans-B[a]P-dG adduct opposite a tertrahydrofuran (THF) abasic site (AB); the AB site lacks only the cytosine base in the complementary strand opposite the modified guanine, but not its 2’-deoxyribose residue and phosphodiester group  of the 11/11-mer duplexes. The Tm values, defined as the temperature at which 50% of the duplexes are dissociated, were determined by monitoring the absorbance of DNA duplexes at 260 nm as a function of increasing temperature from ~ 15 to 65–70 0C under standard conditions in 20 mM sodium phosphate buffer solution, pH 7.0, 100 mM NaCl, 20 μM DNA duplex concentration.The single-stranded, site-specifically modified 11-mers, 5’-CCATCG*CTACC with a eviously –26. ThisThis duplex was used in the new NMR structural studies reported here, and the base numbering system is defined in a]P and nucleic acid protons in the trans-B[a]P-dG:AB abasic 11-mer duplex. To visualize the imino protons, 2D nuclear Overhauser effect spectroscopy (NOESY) data for the modified duplex in 10% H2O buffer  at 10 0C were recorded; a mixing time of 200 ms employing a jump-return pulse sequence for solvent suppression on a Bruker 500 Mhz NMR spectrometer equipped with a TXI probe was utilized. The corresponding NOESY spectra in D2O buffer at 10°C were recorded at a mixing time of 300 ms. Through-bond correlation spectroscopy (COSY) and total correlation spectroscopy (TOCSY) at a 300 ms mixing time were recorded using a Bruker 800 MHz NMR instrument equipped with a cryoprobe at the New York Structural Biology Center (NYSBC). Peak assignments were obtained using the SPARKY program [Through-space nuclear Overhauser effects and through-bond correlated 2D spectra were recorded and analyzed in order to assign the B[ program .trans-B[a]P-dG adduct opposite a THF AB site adopts a base-displaced intercalated conformation in the 11-mer duplex with the THF moiety opposite the lesion. We utilized an equilibrated initial model of this duplex  was employed for visualizing and model building. The figures and movies were prepared with Pymol .32P-endlabeled before ligation so that the 24–30-mer dual incision products could be visualized after separation of the dual incision fragments from the 135-mer duplexes by gel electrophoresis methods. The B[a]P-dG adduct opposite the abasic site was positioned at the 68th nucleotide counted from the 5’-side, in the middle of the 135-mer duplex. Briefly, the 11-mer oligonucleotide 5'-d(CCATCG*CTACC) was 5'-end labeled with [γ-32P]ATP  and incorporated into a 135-mer duplex using T4 ligase as described previously P-dG adduct in a full duplex, with all base pairs intact, assumes a minor groove conformation with a 5’-orientation relative to the modified guanine base [trans-B[a]P-dG adduct in the oligonucleotide 5’-C1-C2-A3-T4-C5-G6*-C7-T8-A9-C10-C11 at position G6*  and NOESY (300 ms mixing time with solutions of different concentrations) data sets, and then confirmed by water NOESY (250 ms mixing time) and COSY methods. These results are summarized in trans-B[a]P-dG:AB duplex in 10% H2O buffer, pH 6.8 at 10°C is plotted in a]P-dG adducts [trans-B[a]P-dG adduct, positioned inside the helix between G16 and G18. The absence of the dG6* imino proton resonance suggests that this guanine imino proton may be looped out of the helix and is exposed to solvent where it undergoes rapid exchange with water. The imino proton signals of the terminal base pairs are not detectable because of fraying effects that are also characterized by rapid exchange with solvent.The 1D exchangeable NMR spectrum of the  adducts , 35 and 2O buffer at pH 6.8 and at 10°C is shown in trans-B[a]P-dG:AB duplex. The A(H2) protons also show NOE interactions to their adjacent guanine imino protons  which suggest base pair stacking [2O buffer (trans-B[a]P-dG are weaker (An expanded region of the water NOESY contour plot (250ms mixing time) in 10% Hstacking  at thoseO buffer  confirm trans-B[a]P-dG adduct than the G16:C7 and A15:T8 base pairs on the 3’-side of G6*. These results are also consistent with the weaker NOEs between C5—G18(NH1) than those of the 5’-flanking C7—G16(NH1) base pair   .2O buffer, recorded at 10°C are shown in Expanded contour maps of a NOESY spectrum (300 ms mixing time) depicting interactions between the base and the sugar H1’ protons in Db and a, respectively). The NOESY walk for the complementary strand is from G22 to G18 on the 5’-side of the G6*-THF site of the duplex and from G16 to G13 on its 3’-side (a) is consistent with the absence of the G6* imino proton in the exchangeable proton spectrum  and the sugar protons H2’ (0.770 ppm) and H2”(1.46 ppm) of C5  of C5  Tables, m) of C5 .The base H8 (7.96 ppm), sugar H1’ (6.49 ppm), H2’ (2.64 ppm) and H2” (3.02 ppm) protons of the G6* are shifted downfield, which indicates that the G6* base is, at least in part, displaced from the interior of the double helix and is less well stacked with adjacent bases. The greater distortion of the C5:G18 base pair is also supported by its weakened imino-imino proton cross-peak as shown in a]P ring system were assigned by analysis of through-space NOESY connectivities and through-bond TOCSY resonances . For cor duplex . In all ormation , 22, 35, 8.5 ppm . This up 8.5 ppm . In the a]P residue protons and nearby exchangeable and non-exchangeable base and sugar protons were identified and assigned , B[a]P(H3), B[a]P(H4) and B[a]P(H5) protons with DNA protons of G18 and G16 on the THF-containing strand, as well as NOE connectivities between B[a]P (H12) and B[a]P (H1) with the base and sugar protons of C5. The B[a]P benzylic ring position on the major groove side of the DNA duplex is determined by the NOE between the B[a]P(H10) and C7(H5).A total of 30 NOEs between the non-exchangeable aromatic B[trans-B[a]P-dG adopts a base-displaced intercalated conformation, with the B[a]P aromatic rings inserted into the helix and the damage guanine displaced; accordingly, we created such an initial model of the trans-B[a]P-dG adduct opposite THF in a B-DNA duplex, and carried out restrained MD simulations in explicit solvent and with neutralizing sodium ions, as detailed in the Methods section. We then obtained five representative structures from the ensemble  the formation of intercalative duplexes is accompanied by a hypochromic effect and a final red-shifted absorption maximum at 352–354 nm due to the fully developed duplexes; (2) in the case of the minor groove B[a]P-dG adduct, a hyperchromic effect and a final blue-shifted absorption maximum at 346 nm is observed P-dG strands by cB and tB, and the abasic and fully complementary strands by a and f, respectively, the (cB or tB)/(a or f) ratios ranged from 0.1 to 1.0 in stepwise aliquots, and the absorption spectra were taken after each addition , the absorption maximum gradually blue-shifts from 350 nm to 346 nm  causes a red-shift in the absorption maximum from 352 to 355 nm in the duplex  of the unmodified dG:AB 11/11mer duplex without the B[a]P adduct is 26.2±0.5°C; this duplex is thus strongly destabilized relative to the full unmodified 11/11mer duplex with all 11 Watson-Crick base pairs intact —Tm(um) ~ +4°C, where um and mod refer to unmodified and B[a]P-modified duplexes, respectively. In the case of the stereoismeric (+)-cis-B[a]P-dG duplex we obtained a ΔTm(AB) = + 17°C value.The melting points of the modified 11-mer duplexes are summarized in 1±0.5°C, ). Thus, trans- or cis-B[a]P-dG opposite the abasic site in 135-mer duplexes , or opposite dC in analogous normal full duplexes , were internally 32P-labeled , known to be an excellent substrate of human NER [cis-Pt adduct showed a robust dual incision activity as expected for NER-active cell extracts [cis- and trans-B[a]P-dG adducts opposite dC in the partner strand in fully complementary duplexes exhibited about the same relative NER activities  [a]P-dG adducts, were reproducible (data not shown). None of the replicates showed any NER activity whatsoever in the case of duplexes with abasic sites opposite the same B[a]P-dG adducts that exhibited robust NER activity with a C opposite the same adducts.Results of typical NER assays in human HeLa cell extracts are depicted in -labeled , panel Auman NER  served auman NER , while aextracts . The cisf ~ 5:1) , 23. Howf ~ 5:1) , the dif32P 5’-end-labeled complementary strands, robust incisions at the abasic THF site are evident from the appearance of 67-mer oligonucleotides  trans- and cis-B[a]P-dG adducts are readily incised by APE1 in vitro. Since no incisions are observed in the control experiments indicating that the THF abasic sites are stable under our experimental conditions, the incision activities shown in In the case of the a]P-dG adducts incised by NER mechanisms , we suppressed the BER incisions at the THF abasic site by employing selective competition experiments. We used an excess (1 μM) of a 17-mer oligonucleotide strand with a single THF abasic site (5’-CCACCAACGCTACCACC) annealed with its fully complementary strand as a competitor for BER proteins in HeLa cell extract NER assays. The NER , while NER dual incision products accumulate more slowly as a function of time . We ther The NER  and BER  The NER  incision32P-labeled complementary strand (cis- or (+)-trans-B[a]P-dG adducts (lanes 9–12). However, in the presence of 1 μM 17-mer AB-competitor duplexes, the incision of AB sites in the same set of duplexes as in panel A is reduced by 80–85%, and is independent of the absence or presence of the B[a]P-dG adduct, or its stereochemical characteristics. The lack of NER signal in the case of G*:AB sites with 80–85% of the AB-containing complementary strands intact (cis- and (+)-trans-B[a]P-dG adducts are indeed resistant to NER when positioned opposite intact abasic sites in the complementary strand.By contrast, base excision repair of the y strand  is strontrans-B[a]P-dG adduct opposite a tetrahydrofuran THF abasic site, a stable analogue of a natural abasic site  enzyme that hydrolyzes AB sites by cleaving the phosphodiester bond on their 5’- and 3’-sides , 51. APEHF sites  that are adducts , 48.a]P-dG lesion does not, however, prevent the repair of the THF abasic site by the BER mechanism as shown in the present work and earlier [trans-B[a]P-dG and cis-B[a]P-dG lesions can be filled in by Pol β [The presence of the B[ earlier . The mamby Pol β .a]P-dG adducts [cis- than the trans-B[a]P-dG adduct opposite the THF abasic site  = Tm B[a]P-dG:AB—Tm dG:AB = + 4°C in the case of the (+)-trans-B[a]P-dG:AB duplex 11-mer. In the case of the stereoismeric cis-B[a]P-dG duplex that is also intercalated according to spectroscopic data, ΔTm(AB) = + 17°C. These results, indicate that the bulky polycyclic aromatic residues stabilize the THF AB duplexes by stacking interactions; this is analogous to the effects exerted by the same adducts on the stabilities of deletion duplexes that lack the entire nucleotide in the complementary strand opposite the B[a]P-dG adduct, and also adopt base-displaced intercalated conformations [m(Del) = Tm B[a]P-dG:Del—Tm dG:Del = +19°C in the case of the (+)-cis-B[a]P-dG [m(Del) = +6°C for the (+)-trans adduct [a]P-dG adducts in fully complementary 11-mer duplexes destabilize the double-stranded DNA by ΔTm (Full) = -11°C ((+)-cis), and = -10°C ((+)-trans) [a]P-dG adducts has a similar impact as removing the full nucleotide on their adduct conformations, thermodynamic stabilities of the DNA duplexes, and abrogation of NER activities as summarized in a]P DNA stacking properties due to stereoisomeric effects are likely responsible for the different impacts on the ΔTm values of the base-displaced intercalated cis-and trans-B[a]P-dG adducts [The structures and thermal melting data provide insights into the NER resistance for the s intact . Howeverrmations : ΔTm(DelB[a]P-dG  or ΔTm(Ds adduct . In shar)-trans) . We conc adducts trans- and cis-B[a]-dG lesions opposite an AB site stems in part from the local stabilization provided by the intercalated B[a]P ring system, which would impede insertion of the XPC BHD3 β hairpin. Furthermore, the absence of the partner base would weaken binding of the protein to the damaged DNA because the binding affinity of XPC depends on a competition between DNA opening and the time spent by the XPC protein at the lesion site [trans- and cis-B[a]P-dG lesions in the deletion duplexes P-dG lesions affects two important characteristics of the local adduct binding sites that impact the downstream NER dual incision events. The intercalation of the bulky aromatic B[a]P residues locally stabilizes the DNA duplex by stacking interactions with adjacent base pairs; we hypothesize that this local stabilization hinders the strand separation near the lesion that is required for the insertion of the β-hairpin and thus the binding of the XPC-RAD23B NER recognition factor to the damaged DNA site. In addition, the absence of one of the complementary strand bases opposite the DNA adduct, that would normally be flipped into the XPC protein, weakens the interactions of the damaged DNA site with the protein [The structural properties of DNA lesions that foster or inhibit NER remain of fundamental interest from a mechanistic perspective, and defining them has important implications for elucidating the characteristics that allow lesions to persist and cause harmful mutations. We have found that the missing partner base in the abasic site on the complementary strand opposite the  protein . Since tS1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 Fig(DOCX)Click here for additional data file.S4 Fig(DOCX)Click here for additional data file.S1 Methods(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.