Firstly, the enhanced green fluorescence protein gene (egfp) was used as the reporter protein to fusion the Pir1p gene and treS gene to construct the recombinant plasmids containing treS-egfg-Pir1p fusion gene, and electrotransformed into P. pastoris GS115 to analyze the surface display characteristics of fusion gene by Western blot, fluorescence microscopy and flow cytometry. The analysis shown that the treS-egfg-Pir1p fusion protein can be successfully displayed on the surface of yeast cell, and the expression level increased with the extension of fermentation time. These results implied that the Pir1p-treS fusion gene can be well displayed on the cell surface. Secondly, in order to obtain surface active cells with high enzyme activity, the enzymatic properties of TreS displayed on the cell surface was analyzed, and the fermentation process of recombinant P. patoris GS115 containing pPICZαA-Pir1p-treS and pGAPZαA-Pir1p-treS was studied respectively. The cell surface display TreS was stable over a broad range of temperatures (10–45°C) and pH (6.0–8.5). The activity of TreS displayed on cell surface respectively reached 1,108 Ug−1 under PAOX1 control for 150 h, and 1,109 Ug−1 under PGAP control for 75h in a 5 L fermenter, respectively. Lastly, the cell-surface displayed TreS was used to product trehalose using high maltose syrup as substrate at pH 8.0 and 15°C. The surface display TreS cells can be recycled for three times and the weight conversion rate of trehalose was more than 60%. This paper revealed that the TreS can display on the P. pastoris cell surface and still had a higher catalytic activity after recycled three times, which was suitable for industrial application, especially the preparation of pharmaceutical grade trehalose products.Yeast cell-surface display technologies have been widely applied in the fields of food, medicine, and feed enzyme production, including lipase, α-amylase, and endoglucanase. In this study, a  E. coli  was used to confirm the localization of TreS at the surface by Western blot analysis and flow cytometric analysis.Aimed at obtaining a yeast that displays TreS on its cell surface as an efficient and recyclable whole-cell biocatalyst, the cell-surface display system of TreS in Pseudomonas putida ATCC47054 containing the TreS gene was obtained from the American Type Culture Collection . P. pastoris GS115  was used as the host for cell-surface display. Saccharomy cescerevisiae CICC 32919, containing the Pir1p gene, was preserved in our laboratory. The pPICZαA expression vector, containing zeocin resistance, was purchased from Invitrogen  and zeocin was obtained from Cayla. Restriction enzymes, T4 DNA ligase, and an agarose gel DNA purification kit were all purchased from TaKaRa . Primers shown in Table Pseudomonas putida ATCC47054 was prepared with a bacterial genomic DNA extraction kit (TaKaRa). Primers F1 and F2 were used to amplify the TreS gene (GenBank accession number 26986745). The Pir1p gene from the genomic DNA of Saccharomy cerevisiae CICC 32919 was amplified by PCR using the primers F3 and F4, which were designed based on NCBI GenBank accession number D13740. Primers F5 and F6 were used to clone the egfp gene from the plasmid pEGFP-N1  and thePir1p gene and egfp gene were fused with primers (F4 and F5) by overlap extension PCR. The targeted PCR products were gel-purified, inserted into pZero-BluntT vector, and transformed into E. coli DH5α.Genomic DNA containing the TreS gene from EcoRI and NotI and inserted into the EcoRI and NotI sites of pPICZαA, transformed into E.coli DH5α, and screened. The pZero-BluntT-EGFP-Pir1p vector containing the egfp-Pir1p fusion gene fragments and the pZero-BluntT-Pir1p vector containing thePir1p gene fragments were digested with SpeI and XbaI, and inserted into the SpeI and XbaI sites of pPICZαA-TreS. The new recombinant plasmids named pPICZαA-TreS-EGFP-Pir1p and pPICZαA-TreS-Pir1pwere linearized using PmeI and then transformed into P. pastoris GS115 competent cells, respectively. Integration of the linearized pPICZαA-TreS-EGFP-Pir1p and pPICZαA-TreS-Pir1p plasmids into the P. pichia GS115 genome were confirmed by PCR using the 5′-AOX1 and 3′-AOX1 primers. Transformants were selected on YPD plates  containing 100 μg zeocin ml−1 as the selective marker. For expression of the TreS gene under the PGAP promoter, the primers G1 and G2 were used for PCR amplification of the TreS -Pir1p fragment from plasmid pPICZαA-TreS-Pir1p. The PGAP promoter was cloned, using the G3 and G4 primers, from P. pastoris CICC 32919. The pPICZαA-TreS-Pir1p gene, without an AOX1 gene, was ligated with the PGAP promoter by overlap PCR and the newly generated expression vector was named pGAPZαA-TreS-Pir1p.The PCR product containing the TreS gene from pZero-BluntT-TreS was digested with 600) of 3 to 5. the culture were centrifuged at 4,000 × g for 5 min and resuspended in BMMY medium to an OD600of cells were induced for the fusion protein expression at 25°C with 100% methanol every 24 h to a final concentration of 0.5%. P. pastoris GS115/pPICZαA-TreS-Pir1p was used as negative control. After 96 h of induction, growth medium was collected and centrifuged at 6,000 × g for 5 min. The cells were then harvested and disrupted in buffer  by Bead beater  at 4°C. After centrifugation at 300 × g at 4°C for 5 min to remove the unlysed cells, the supernatant was collected and the cell wall fractions were isolated  to induce the expression of fusion protein. Cells were harvested at different time points  and diluted with ice-cold phosphate-buffered saline  to a final OD600 of around 0.2. Flow cytometry analysis was performed on a FACSCalibur flow cytometer  using cell Questsoftware. For each detection, a total of 100,000 cells were analyzed. After induction 96 h, the recombinant P. pastorisGS115/pPICZαA-TreS-EGFP-Pir1p cells were collected and washed twice with phosphate buffered saline (PBS). Cells were observed using the fluorescence microscope system (NIKON Eclipse Ci-L). The P. pastoris GS115/pPICZαA-TreS-Pir1p induced for 96 h in BMGY was used as control.The recombinant P. pastoris GS115/pPICZαA-TreS-Pir1p and P. pastoris GS115/pPICZαA(GAP)-TreS-Pir1p inocula were prepared by cultivating cells for 16–18 h at 30°C in three 500 mL shake flasks containing 100 mL YPD medium. Then, inoculum was transformed to 5 L fermenters containing 2 L modified medium, which contained (per liter): KH2PO4, 42.9 g; (NH4)2SO4, 5 g; CaSO4, 0.6 g; K2SO4, 14.3 g; citric acid anhydrous, 1.92 g; MgSO4·7H2O, 11.7 g; glycerol, 40 g; and 2.0 mL PTM4 trace salt solution. Glycerol and MgSO4·7H2O were sterilized separately. The PTM4 solution contained (per liter): CuSO4·5H2O, 2.0 g; NaI, 0.08 g; MnSO4·H2O, 3.0 g; Na2MoO4·2H2O, 0.2 g; H3BO3, 0.02 g; CaSO4·2H2O, 0.5 g; CoCl2, 0.5 g; ZnCl2, 7 g; FeSO4·7H2O, 22 g; biotin, 0.2 g; and 1 mL concentrated H2SO4. The pH was controlled by adding 20% ammonium hydroxide and dissolved oxygen (DO) was controlled by the stirring speed (200–800 rpm) and aeration rate (0.1–2.0vvm). The fermentation process of recombinant P. pastoris GS115/pPICZαA-TreS-Pir1p comprised three phases. In the first phase (the batch phase), the culture temperature and pH were controlled at 30°C and pH 6.0 until the glycerol in the medium was used up, with a sudden increase in the DO level indicated the end of this phase. In the second phase, the glycerol-fed batch phase, the culture density was increased to an OD600 of approximately 150 by feeding medium containing 500 g L−1 glycerol and 2 ml L−1 PTM4 solution. Then, glycerol feeding was terminated for 30 min to exhaust the glycerol. Meanwhile, the pH was adjusted to 7.0 and the temperature was reduced to 25°C. In the third phase, the induction phase, pure methanol containing 2 ml L−1 PTM4 solution was fed to start the induction and the methanol concentration was adjusted to a final concentration of 0.5% once every 2 h. The DO level was maintained between 20 and 30% by adjusting the agitation and aeration rates. The fermentation process of recombinant P. pastoris GS115/pGAPZαA-TreS-Pir1p comprised the first two stages above but using 40 g L−1 glucose as the carbon source in the modified medium described above. Samples were taken at regular intervals and analyzed for biomass and TreS activity after fermentation. Cell density was expressed by grams of dry cell weight (DCW) per liter, which was obtained by centrifuging 10 mL samples in centrifuge tubes at 8,000 × g for 10 min, washing twice with deionized water, and drying the pellets to a constant weight at 105°C for about 150 min. The dry weight of the pellets was measured every half hour.Fermentation studies were conducted in a 5 L fermenter . P. pastoris GS115 and cell surface display Tres expressed by pGAPZαA-TreS-Pir1p) were mixed with 10 mL pure maltose solution (600 g L−1) and then diluted to 20 mL with fresh phosphate buffer. The maltose final concentration in the reaction system was adjusted to 300 g/L. The reaction mixture was incubated at different temperatures  and pH  for 1 h and then heated to 100°C for 10 min. Then, the weight content  of trehalose in the samples were identified by comparing with the trehalose standard curves of various concentrations determined by high-pressure liquid chromatography . The apparatus was equipped with an Inertsil-NH2 column  at 40°C. Separation was achieved by pumping acetonitrile: water (75:25 v:v) at a flow rate of 1.0 mL min−1 for 20 min. One unit (U) of TreS was defined as the amount of enzyme required to produce 1 μmol trehalose per hour. Optimum temperature and pH were determined by individually changing the conditions of the surface-displayed TreS activity assay (temperature 10–80°C and pH 3.0–10.0).The harvested yeast cells obtained through enzymolysis by laminarinase -TreS-Pir1p were constructed as described in the Methods. The construction process for the recombinant vectors is shown in Figure e Figure .P. pastoris GS115 cell surface. The recombinant P. pastoris GS115/pPICZαA-TreS-EGFP-Pir1p and P. pastoris GS115 as control were induced in BMMY medium for 96 h, the cells were collected to extract the cell wall proteins. The extracts from the purified cell walls, and concentrated samples of cell contents and growth medium from recombinant P. pastoris GS115/pPICZαA-TreS-EGFP-Pir1p and P. pastoris GS115 were analyzed by Western blot using an EGFP antibody. The fusion proteins in the extracts from the cell of P. pastorisGS115/pPICZαA-TreS-EGFP-Pir1p and P. pastoris GS115 were detected in Figure P. pastoris GS115/pPICZαA-TreS-EGFP-Pir1p and P. pastoris GS115  were observed by fluorescence microscopic, respectively. Green fluorescence was clearly observed on the cell surface P. pastoris GS115/pPICZαA-TreS-EGFP-Pir1p and no green fluorescence was observed on the P. pastoris GS115. This result showed that the fusion protein TreS-EGFP was successfully anchored and displayed on the cell surface of P. pastoris GS115  and temperature (10–50°C) with the optimum pH 8.0 and temperature at 25°C. When the temperature is 25°C and pH is 8.0, the enzyme activity of TreS was defined as maximum activity to produce 1 μmol trehalose per hour using pure maltose (300 g/L) as substrate. The displayed TreS kept more than 95% of its maximum activity at 15°C and pH7.0, but the relative activity of displayed TreS was lower than 40% when the temperature went over 60°C or the pH was less than 6.0.In order to keep the enzyme activity better during the fermentation and the transformation process. The characterization of surface display TreS in relation to temperature and pH was investigated in this study, as shown in Figures P. pastoris GS115/pPICZαA-TreS-Pir1p. TreS expression under the PAOX1 promoter was controlled using methanol as an inducer. Following an increase in DO, due to a carbon source limitation after approximately 20 h of fermentation, the glycerol fed-batch process was conducted for another 4 h until the DCW biomass reached about 35 g L−1. After glycerol depletion, the culture respiratory activity decreased with a fast DO spike. The methanol was feed every 24 h. After methanol-initiated induction, the culture respiratory activity recovered and the DO concentration decreased. During the first 4 h of the induction phase, methanol accumulated in the fermenter and DO levels were erratic while the culture adapted to the methanol. After methanol adaptation, a constant feed rate was maintained throughout the remainder of the fermentation. We also found that the methanol concentration should be kept constant to maintain limited growth after adaptation and then increased gently 60 h after induction, signaling that fermentation had stopped. At the process of the fermentation, the culture DCW reached 102 g L−1at 130 h and the enzyme activity reached 1,108 U g−1at 150 h  at 15°C and 0.01 M sterilized phosphate buffer (pH 8.0) in a 10 L bioconversion and stir at 60 rpm for conversion was used as substrate at a final maltose concentration of 300 g LonFigure . The firP. pastoris GS115/Pir1p-TreS also proved this point.Pir1p is reported to improve the performance of the catalyst by displaying an enzyme anchored by the C-terminus without hindering the catalytic site, which has been applied to display the reporter protein GFP and human α-1-antitrypsin  had stable activity at a temperature of 55°C, a pH of 7.4, and catalyze 59% maltose into trehalose with a substrate concentration of 30%. The higher temperature can effectively avoid contamination of miscellaneous bacteria, but it also caused certain enzyme activity loss. On the other hand, the maximum trehalose yield (approximately 81%) catalyzed by the TreS from Thermus thermophilus HB-8 (ATCC 27634), expressed in E. coli BL21 (DE3), was obtained at an optimum temperature of 65°C and a pH of 7.0 and was independent of substrate concentration , in a 50 L fermentor was reached with 6% glucose generated as byproduct using maltose syrup (30 wt%) as substrate in the bioreactor system via whole-cell biocatalysis . When the temperature was over 60°C or the pH was less than 6.0, the relative activity of displayed TreS activity was lower than 40%. Our results were inconsistent with the previous study (Wei et al., Heterologous expression did not change the properties of the enzyme; however, the characteristics of the biocatalyst can be affected by immobilization (Moura et al., PAOX1 is a strongly regulated promoter from alcohol oxidase I and has been chosen for production of a variety of recombinant proteins in P. pastoris. PGAP, a strong constitutive promoter, has also been used for constitutive expression of some heterologous proteins (Zhang et al., PGAP is more efficient than PAOX1 for the expression of eukaryotic heterologous proteins (Döring et al., PAOX1 as promoter resulted in higher production than using the PGAP promoter (Boer et al., PAOX1 expression system, methanol is applied to induce protein expression. However, methanol is a toxic substance that may not be appropriate for the production of food products and easily causes a fire hazard, especially in large-scale fermentations (Wang et al., PGAP expression system, cell growth and heterologous protein production occur simultaneously in the process of fed-batch fermentation. Also, the hazard and cost associated with the storage and delivery of a large volume of methanol are eliminated. In this study, we successfully expressed the treS gene from Pseudomonas putida ATCC47054 under the control of PAOX1 and PGAP on the cell surface of P. pastoris. The highest activity of recombinant TreS under the control of PAOX1 reached above 1,100 U g−1 in a 5 L fermenter after 140 h of culture, and the recombinant TreS produced in the PGAP promoter expression system exhibited a maximum activity of above 1,109U g−1 using glucose as the carbon source in a 5 L fermenter after 75 h of culture. Our results further demonstrated that the PGAP expression system can be an alternative to the PAOX1 system for heterologous expression of TreS in P. pastoris, without obvious differences in expression level.P. pastoris GS115/Pir1p-EGFP-TreS to prove the fusion protein displayed on the P. pastoris GS115 cell surface using Pir1p as anchor protein by Western blot localizaton, fluorescence microscopy observation and flow cytometry analysis. The recombinant P. pastoris GS115/Pir1p-TreS can successfully display TreS and used to produce trehalose. The expression levels of TreS obtained in the 5 L fermenters indicate that the PGAP system is a suitable alternative to the PAOX1 system for large-scale production of recombinant TreS in P. pastoris GS115 by a high cell density culture, and the enzyme activity reached above 1,100 U g−1. It can be concluded that constitutive TreS expression in a continuous culture of P. pastoris may be easy to scale up and has good prospects for decreasing production costs for large-scale industrial fermentations.We successfully constructed the recombinant TW and RW designed the project. SY, XL, and XW performed experiments. SY, XL, XW, and JW discussed and interpreted results. SY, XL, and TW wrote the manuscript. All authors agreed to be accountable for accuracy, integrity and appropriateness of the manuscript. Authors initials in order: SY, XL, XW, JW, RW, TW.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Metastasis is the most dangerous risk faced by patients with hereditary non-polyposis colon cancer (HNPCC). The expression of matrix metalloproteinases (MMPs) has been observed in several types of human cancers and regulates the efficacy of many therapies. Here, we show that treatment with various concentrations of prostaglandin E2  promotes the migration ability of the human LoVo colon cancer cell line. As demonstrated by mRNA and protein expression analyses, EP2 and EP4 are the major PGE2 receptors expressed on the LoVo cell membrane. The Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt cell survival pathway was upregulated by EP2 and EP4 activation. Following the activation of the PI3K/Akt pathway, β-catenin translocated into the nucleus and triggered COX2 transcription via LEF-1 and TCF-4 and its subsequent translation. COX2 expression correlated with the elevation in the migration ability of LoVo cells. The experimental evidence shows a possible mechanism by which PGE2 induces cancer cell migration and further suggests PGE2 to be a potential therapeutic target in colon cancer metastasis. On inhibition of PGE2, in order to determine the downstream pathway, the levels of PI3K/Akt pathway were suppressed and the β-catenin expression was also modulated. Inhibition of EP2 and EP4 shows that PGE2 induces protein expression of COX-2 through EP2 and EP4 receptors in LoVo colon cancer cells. Colorectal carcinoma (CRC) is one of the most prevalent cancers worldwide ,2 and isIn observational studies, PGE2 induced COX2 expression, and we used receptor antagonists ,19,20 toThe expression of COX2 in LoVo cells was evaluated by immunoblot assay and was found to increase following treatment with PGE2 at various concentrations  A. AnalysEP2 and EP4 are also major proteins upregulated by the PI3K/Akt pathway. As the results show, PI3K and Akt were activated within 1 h of PGE2 treatment. The downstream proteins p-GSK3β and β-catenin were also activated 6 to 12 h after treatment. Finally, PI3K/Akt pathway activation increased COX2 expression .In order to verify this pathway LoVo cells were pretreated with A EP2 antImmunofluorescence assays were performed in LoVo cells pretreated with the EP2 antagonist AH6809 (40 μM), the EP4 antagonist AH23848 (75 μM), EP2-specific siRNA (40 nM) or EP4-specific siRNA (40 nM) and treated with PGE2 (5 μM) using an anti-β-catenin antibody  followed by 4′,6-diamidino-2-phenylindole DAPI nuclear counterstaining (blue). The confocal microscopy images show that PGE2 treatment caused β-catenin to translocate into the nucleus in LoVo cells. In addition, the translocation of β-catenin was controlled by PGE2 antagonist treatment A,B. The The migration of the PGE2-treated LoVo cells was determined by a migration assay.As the results show, PGE2 treatment  efficiently promoted the migration abilities of LoVo cells A. AdditiInflammation is emerging as a new hallmark of cancer metastasis and invasion and cyclooxygenases (COXs) and lipoxygenase (LOX) play major roles in arachidonic acid-related inflammatory cascades ,24. PrevHereditary colon cancers include familial adenomatous polyposis (FAP) and hereditary non-polyposis colon cancer (HNPCC) that accounts for more than 5 percent of all cases of colorectal cancer ,32,33. SPGE2 levels are generally at higher concentrations in tumor tissues when compared to the normal tissues . PGE2 meTo prove that the receptors influence downstream proteins, we used receptor antagonists to block the interaction of PGE2 with EP2 and EP4 and thereby reduce the activation of the downstream proteins. The PGE2 receptor antagonists significantly suppressed the expression of the downstream proteins. Further, when the receptors were inhibited the downstream pathway was also suppressed. It is unclear what role the EP receptor plays in supporting smooth muscle cells and pericytes in the tumor vascular system. Targeting EP receptors can decrease the potential for angiogenesis in one cell type, but may have an adverse effect on other cell types and the entire organ system ,36. We uBoth immunofluorescence assays and analysis of the nuclear and cytosolic protein fractions were used to determine the localization of β-catenin. The translocation of β-catenin from the cytosol to the nucleus also plays a role in the transcription and translation of COX2. Our results indicated that a decrease in the expression of COX2 results in a corresponding reduction in cell migration. LoVo cells were pretreated with PGE2 and harvested and subsequently their migration ability was determined by migration assay. We used receptor antagonists and siRNA-mediated knockdown of the receptors to determine if COX2 inhibition affected cell migration. In this study, we showed that PGE2 treatment can activate the expression of COX2, β-catenin and the PI3K/Akt pathway. Through the receptor antagonists and specific siRNAs, we demonstrated here that the PGE2 receptors EP2 and EP4 play an important role in these pathways. PGE2 suppresses apoptotic effect by mediating the IGF-II/IGF-I receptor signaling-induced PI3k/Akt activation and by increasing the ability of Ras and PI3K association which is dependent on the activation of EP4 receptor . PGE2 inIn canonical Wnt signaling, phosphorylation of β-catenin at specific locations by glycogen synthase kinase-3β (GSK-3β) leads to ubiquitination and degradation of β-catenin ,52. COX-The human colon cancer cell line LoVo was obtained from the American Type Culture Collection (ATCC) . LoVo cells were established from a metastatic nodule resected from a 56-year-old colon adenocarcinoma patient.We utilized antibodies against the following proteins: phospho-PI3K, phospho-Akt, COX2, GSK3β, β-catenin, LEF-1 and HADAC-1 ; EP-2 and EP-4 ; and TCF-4 . Antibodies to α-tubulin or β-actin  were used as loading controls. The goat anti-mouse IgG, goat anti-rabbit IgG and rabbit anti-goat IgG antibodies, all conjugated to horseradish peroxidase, were purchased from Santa Cruz Biotechnology, Inc. in California, USA.2 culture dishes in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM glutamine, 1 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, and 10% Clontech fetal bovine serum in humidified air (5% CO2) at 37 °C. LoVo cell passpage were re-plated at a density of 1 × 105 cells in 6 cm2 culture dishes.LoVo colon cancer cells from the American Type Culture Collection (ATCC)  were cultured in 10 cmg, dried under vacuum for 10 min, dissolved in 50 μL of diethylpyrocarbonate-treated water, and incubated for 15 min at 60 °C. The RNA was quantified and checked for purity by spectrophotometry at a wavelength of 260 nm/280 nm. An aliquot of total RNA (0.5 μg) was reverse transcribed using 0.5 μM oligo d(T) primers in a reaction solution (50 μL) containing 75 mM KCl, 50 mM Tris–HCl (pH 8.3), 3 mM MgCl2, 10 mM dithiothreitol (DTT), RNase inhibitor , 0.8 mM total deoxynucleotide (dNTPs), and 200 U of Moloney murine leukemia virus reverse transcriptase (Promega). The sample was incubated at 42 °C for 1 h and at 95 °C for 5 min before being chilled on ice for 10 min.Total RNA was extracted using the Ultraspec RNA Isolation System  according to the manufacturer’s directions. LoVo cells were thoroughly homogenized (1 mL Ultraspec reagent) with a homogenizer in a polypropylene tube, and then total RNA was isolated using a standard method. The RNA precipitate was washed twice by gentle vortexing with 70% ethanol, collected by centrifugation at 12,000× Primers were as follows: human EP-1 forward primer CTGGGCGGCTGCATGGTCTTCTTC, reverse primer AGTGGCCGCTGCAGGGAGGTAG; human EP-2 forward primer CGAGACGCGACAGTGGCTTCC, reverse primer CGAGACGCGGCGCTGGTAGA; human EP-3 forward primer CGGGGCTACGGAGGGGATGC, reverse primer ATGGCGCTGGCGATGAACAACGAG; human EP-4 forward primer TCGCGCAAGGAGCAGAAGGAGAC, reverse primer GACGGTGGCGAGAATGAGGAAGGA.RT-PCR was carried out with the Ex Taq polymerase using Ex Taq Master Mix kit . One microliter of reverse transcription reaction was used for quantitative PCR in a total volume of 25 µL. RT-PCR was performed using 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s, 53 °C for 15 s and 72 °C for 10 s.g for 15 min at 4 °C, and the protein concentration was determined by the Bradford method. Samples containing equal amounts of protein (60 μg) were analyzed by immunoblot. Briefly, proteins were separated by 12% SDS-PAGE and transferred onto PVDF membranes . The membranes were blocked with blocking buffer  for at least 1 h at room temperature. Membranes were incubated in 1:1000 primary antibodies  in the above solution on an orbital shaker at 4 °C overnight. Following the primary antibody incubations, membranes were incubated with horseradish peroxidase-linked secondary antibodies  and soaked and rocked in 1:1000 secondary antibody (Santa Cruz Biotechnology) solutions for 1 h at room temperature. The signal detection was performed using enhanced chemiluminescence (ECL) reagent and a digital imaging system.To isolate total proteins, cultured LoVo cells were washed with cold PBS and resuspended in lysis buffer , pH 7.5, 10% glycerol, 1 mM β-mercaptoethanol, 1% IGEPAL-630 and a proteinase inhibitor cocktail . After a 30-min incubation on ice, the supernatant was collected by centrifugation at 12,000× 2, 1% Triton X-100, 50 mM HEPES, 1 mM EDTA, 150 mM NaCl, 10% glycerol, 1 mM NaVO3, 10 mM NaF, 10 mM β-glycerophosphate, and 5 mg/mL protease inhibitor). The cells were then scraped and centrifuged for 10 min at 12,000× g at 4 °C. For each group, 100 μg of the total protein sample was added into each microcentrifuge tube with enough lysis buffer, without protease inhibitor added, to bring the final volume to 500 μL. A total of 7 μL of Gα-s antibody (K-20)  was then added, followed by vortex at 4 °C for 1 h with a vortex mixer. The samples were then centrifuged at 4 °C and 1300× g for 30 s. The supernatant was then removed. For each group, 2.5 μL of a Gα-s antibody was added to each of the microcentrifuge tubes. The microcentrifuge tubes were vortexed at 4 °C overnight with a vortex mixer. An aliquot of 20 μL of Protein G PLUS Agarose was added, followed by vortexing at 4˚C for 2 h with the vortex mixer and then centrifuged at 4 °C and 1300× g for 30 s. After the supernatant was removed, 1 mL of lysis buffer without protease inhibitors was added to wash the pellet, followed by centrifugation at 4 °C and 1300× g for 30 s. Western blot analysis was performed to detect the Gα-s, EP2 and EP4 proteins.LoVo colon cancer cells were treated with PGE2. Cells were lysed for 30 min on the culture plates in 200 μL Co-IP cell lysis buffer . The siRNAs specific to EP2 and EP4  and the negative control Non-Targeting Pool (NT)  were mixed with serum-free medium. The detailed procedure has been described in our previous study.LoVo colon cancer cells were seeded into 15 cm4 LoVo cells/well cultured in a 48-well Boyden chamber plate  and polycarbonate membrane filters with a pore size of 8 µm. The lower compartment was filled with DMEM containing 20% FBS. LoVo cells were placed in the upper part of the Boyden chamber with serum-free medium and incubated for 48 h. After incubation, the cells on the membrane filter were fixed with methanol and stained with 0.05% Giemsa for 1 h. The cells on the upper surface of the filter were removed with a cotton swab. The filters were then rinsed in double-distilled water until any additional stain was leached. The cells were then air-dried for 20 min. The migratory phenotypes were determined by counting the cells that had migrated to the lower side of the filter with microscopy at 200× and 400× magnification. Ten fields were blindly selected and counted as the mean cell number in each filter, and each sample was assayed in triplicate.The migration assay was performed using a density of 5 × 102, 10 mM KCl, 1 mM DDT, and proteinase inhibitor) and nuclear lysis buffer (20 mM HEPES (pH 8.0), 1.5 mM MgCl2, 10 mM NaCl, 1 mM DDT, 0.2 mM EDTA, 0.25 M glycerol, and proteinase inhibitor). In brief, following the treatments, cells were resuspended in PBS and membrane lysis buffer was added. After a 10-min incubation on ice, the cells were centrifuged at 12,000 rpm for 2 min to pellet the nuclei. The supernatant was stored for use as the cytosolic fraction, and the nuclei pellet was lysed with nuclear lysis buffer to obtain the nuclear fraction.Cytosolic and nuclear proteins were isolated using an extraction reagent-containing membrane lysis buffer (10 mM HEPES (pH 8.0), 1.5 mM MgClLoVo cells were treated with increasing concentrations of PGE2 dosage  for 6 h. The cells were fixed by 4% paraformaldehyde and the immunofluorescence assay was performed in LoVo cells using an antibody against β-catenin  followed by DAPI nuclear counterstaining (blue), and the results were analyzed by confocal microscopy. Merged images of β-catenin (green) with DAPI (blue) were obtained using confocal microscope .p < 0.05 or 0.01 levels.Each experiment was repeated in triplicates. The results were presented as the mean ± SD, and statistical comparisons were made using one way ANOVA. Significance was defined at the EP2 and EP4 mediate the PGE2-induced COX2 expression and cell migration in LoVo colon cancer cells correlated with COX2 expression. PGE2 interaction with the EP2 and EP4 receptors results in upregulation of the PI3K/Akt pathway, and this survival pathway can control the p-GSK3β/β-catenin pathway. In addition, β-catenin also translocates into the nucleus and interacts with LEF-1 and TCF-4 at the COX2 promoter to activate the transcription of COX2.


The objective of this study was to evaluate the effect of platinum-based drugs on nuclear-factor erythroid2 like 2 (NRF2) signaling in non-small cell lung cancer cell lines with or without Kelch-like ECH-associated protein 1 (KEAP1) mutations and to determine the role of NRF2 and KEAP1 on platinum-based drug treatment.We used real-time PCR to assess relative mRNA expression and used western blotting and immunofluorescence assays to assess protein expression. Small interfering RNA and shuttle plasmids were used to modulate the expression of NRF2, wild-type KEAP1, and mutant KEAP1. Drug sensitivity to platinum-based drugs was evaluated with Cell Count Kit-8.We found that platinum-based therapies modified the NRF2 signaling pathway differently in KEAP1-mutated non-small cell lung cancer (NSCLC) cell lines compared with wild-type KEAP1 cell lines. The reactive degree of NRF2 signaling also varies between nedaplatin and cisplatin. The modification of NRF2 or KEAP1 expression in NSCLC cell lines disrupted downstream gene expression and cell sensitivity to platinum-based drugs. Finally, gene expression data retrieved from The Cancer Genome Atlas (TCGA) consortium indicated that KEAP1 mutation significantly affects NRF2 signaling activity in patients with NSCLC.Our findings suggest that NRF2 signaling plays an indispensable role in NSCLC cell sensitivity to platinum-based treatments and provides a rationale for using NRF2 as a specific biomarker for predicting which patients will be most likely to benefit from platinum-based treatment. Non-small cell lung cancer (NSCLC) is associated with considerably high mortality worldwide . SurgeryNuclear-factor (NF)-E2-related factor 2 (NRF2) is a core transcription factor involved in the cellular response to oxidative stress . Under bIn urethane-induced lung carcinogenesis model, NRF2 activation suppresses tumorigenesis, but somatic activation of NRF2 in cancer cells enhanced growth of tumors . StudiesUnless otherwise stated, all chemicals were purchased from Sigma-Aldrich . Antibodies against NRF2 (ab62352) were purchased from Abcam; antibodies against HO1 (#5853) and NQO1 (#3187) were purchased from Cell Signaling Technology; and antibodies against KEAP1 (10503-2-AP), GCLM  (14241-1-AP), and β-actin were purchased from Proteintech.The A549 (human lung adenocarcinoma), H292 (human mucoepidermoid carcinoma), H460 (human large cell lung cancer), and SKMES-1 (human squamous lung cancer) cell lines were obtained from the Cell Resource Center, Peking Union Medical College. The cells were cultured in RPMI-1640, with the exception of SKMES-1 cells, which were cultured in MEM. All media were supplemented with 10 % fetal bovine serum . No antibiotics were added to the media.G333C-forward, TGATCTACACCGCGGGCTGCTACTTCCGACAGTCG333C-reverse, AGCCCGCGGTGTAGATCAGGCGGCCCACCD236H-forward, AGCCCGCGGTGTAGATCAGGCGGCCCACCD236H-reverse, GGTCCCGGCTGATGAGGGTCACCAGTTGGThe small interfering RNAs used to silence NRF2 were:Sense, GGUUGAGACUACCAUGGUUTTAntisense, AACCAUGGUAGUCUCAACCTTThe wild-type KEAP1 coding sequence (CDS) was cloned into the pEnter shuttle plasmid. The Fast Mutagenesis system  was used to introduce the G333C and D236H mutations. The primers used were:Transfection was conducted with Lipofectamine 2000 (Invitrogen) according to a previously published method .2O. Cycling conditions were as follows: 95 °C for 10 min and 40 cycles of 95 °C for 15 s and 60 °C for 60 s. Cycle thresholds were calculated as the expression fold-changes according to the 2−△△CT algorithm. The primers used for real-time PCR are:NRF2, forward, TCCAGTCAGAAACCAGTGGATReverse, GAATGTCTGCGCCAAAAGCTGHO1, forward, AAGATTGCCCAGAAAGCCCTGGACReverse, AACTGTCGCCACCAGAAAGCTGAGNQO1, forward, GAAGAGCACTGATCGTACTGGCReverse, GGATACTGAAAGTTCGCAGGGGCLM, forward, TGTCTTGGAATGCACTGTATCTCReverse, CCCAGTAAGGCTGTAAATGCTCTotal messenger RNA (mRNA) was extracted from cells using TRIzol according to the manufacturer’s protocol . Seven hundred fifty-nanogram total mRNA was used to synthesize first strand complementary DNA (cDNA) using the cDNA synthesis kit (ThermoFisher Scientific). Quantification reactions were performed with the ABI 7900 HT platform. Each reaction consisted of 5 μl SYBR Green/ROX qPCR Master Mix (ThermoFisher Scientific), 1 μl cDNA, 1 μl 10 μM forward and reverse primer mix, and 3 μl ddHFragment 1, forward AGAGGTGGTGGTGTTGCTTATReverse TGGAGATGGAGGCCGTGTAFragment 2, forward CAGGTCAAGTACCAGGATGReverse GATGAGGGTCACCAGTTGFragment 3, forward ATCGGCATCGCCAACTTCReverse AGGTAGCTGAGCGACTGTFragment 4, forward CAGAAGTGCGAGATCCTGReverse GCTCTGGCTCATACCTCTFragment 5, forward GCCCTGGACTGTTACAACReverse GTCTCTGTTTCCACATCGTAFragment 6, forward GCTGTCCTCAATCGTCTCReverse AGTTCTGCTGGTCAATCTGNRF2 exon2, forward TCGTGATGGACTTGGAGCTGReverse AGCATCTGATTTGGGAATGTGTotal RNA was extracted and used to synthesize the first chain cDNA according to the procedure described in the real-time PCR analysis description. cDNA was used as a template to synthesize six segments of the KEAP1 gene using the following cycling conditions: initial denaturing step at 94 °C for 5 min followed by 30 cycles at 94 °C for 40 s, 60 °C for 40 s, and 72 °C for 55 s. Products were separated on 1 % agarose gels, and bands were visualized with ethidium bromide. The products were sequenced using the ABI3730 XL DNA Analyzer  and analyzed with Chromas 2.4.3 software. Primers used to amplify the KEAP1 gene fragments were:Sections were spliced together after manual inspection and compared with the reference sequence with BLAST to identify potential mutations.Cytoplasm and nuclear protein were extracted by using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (ThermoFisher Scientific). For total protein extraction, we used standard protocols for protein isolation and antibody detection, as previously described , 25. CelImmunofluorescence was performed as previously reported with some modifications . Cells wv/v) CCK-8 was added to each well. Absorbance at 490 nm was measured after a 2-h incubation at 37 °C. Cell survival was calculated as the ratio between the treated group and control groups. Ratios were graphed in line charts with Graphpad Prism 6 software.Cellular proliferation was evaluated using a Cell Count Kit-8 (CCK-8) with some modifications . Lung cahttp://www.compgenome.org/TCGA-Assembler/) in an R program. KEAP1 mutation status was defined as previously described [Expression data from The Cancer Genome Atlas (TCGA) squamous lung cancer patients was retrieved using the package TCGA-Assembler  Fig.  and thatp < 0.01, **p < 0.05) and H460 , nedaplatin induced a weak but significant increase in three downstream gene expression. Cisplatin treatment, however, significantly enhanced HO1, NQO1, and GCLM expression in both A549 and H460 cell lines after 24 h of treatment.To profile the expression of NRF2 signaling pathway components in response to platinum treatment, the four NSCLC cell lines  were treated with 30 μM nedaplatin or 30 μM cisplatin for 24 h. Total RNA from each group was extracted, and HO1, NQO1, and GCLM gene expression levels were assessed with real-time PCR. In the wild-type KEAP1 cell lines H292 Fig.  and SKMETo evaluate protein levels, the four cell lines were treated with 30 μM nedaplatin or 30 μM cisplatin for 3 or 24 h. Total proteins were extracted, and HO1, NQO1, GCLM protein expression was evaluated by using western blot analysis. In the wild-type KEAP1 H292 and SKMES-1 cell lines Fig. , a mild The results of immunofluorescence analysis of HO1 protein expression were consistent with the results of the western blot analysis. Over a 24-h treatment period, 30 μM nedaplatin or cisplatin induced a mild increase in HO1 protein expression in H292 and SKMES-1 cells Fig. . In the p < 0.001, **p < 0.05) and H460 , constitutive expression of wild-type KEAP1 over a 72-h period reduced the expression of the NRF2 downstream target genes HO1, NQO1, and GCLM at the mRNA and protein levels. Overexpression of wild-type KEAP1 sensitized A549 cells  and H460 cells  to nedaplatin and cisplatin over a 24-h treatment period.To investigate the impact of KEAP1 mutations status on NRF2 signaling, the KEAP1 CDS was cloned into the pEnter vector and transfected into the NSCLC cell lines. In the mutant KEAP1 cell lines A549  and SKMES-1 cells  transfected with the mutant KEAP1 construct. An identical trend was observed at the protein level  and SKMES-1  treated with 60 μM cisplatin or nedaplatin.Next, we introduced the G333C and D236H mutations into the KEAP1 pEnter construct. The mRNA expression levels of the NRF2-associated genes HO1, NQO1, and GCLM were significantly elevated in H292 cells  and SKMES-1 , siRNA transfection over a 72-h period reduced the expression of NRF2 and its downstream targets HO1, NQO1, and GCLM at the mRNA and protein levels. Pretreatment with siRNA targeting NRF2 increased sensitivity to nedaplatin and cisplatin in H292 cells  and SKMES-1  over a 24-h treatment period.To assess the effects of NRF2 expression on NSCLC cells exposed to platinum-based drugs, we designed a small interfering RNA (siRNA) construct targeting NRF2 mRNA to inhibit NRF2 activity. In the wild-type KEAP1 cell lines H292  and SKMES-1 , administration of the NRF2 activator over a 24-h period significantly increased the expression of the NRF2 downstream genes HO1, NQO1, and GCLM at the mRNA and protein levels. Pretreatment with 60 μm t-BHQ conferred chemo-resistance to nedaplatin and cisplatin in H292 cells  and in SKMES-1 cells  over a 24-h treatment period.In addition, NSCLC cell lines were treated with t-BHQ, a specific activator of NRF2. In the wild-type KEAP1 NSCLC cell lines H292  and KEAP1  were observed between the mutated patients with mutant KEAP1 cases and with wild-type KEAP1. However, expression levels of the NRF2 downstream target genes HO1 , NQO1 , and GCLM  were significantly enhanced in patients with mutant KEAP1 cases compared with those with wild-type KEAP1.A total of 100 individuals diagnosed with NSCLC were selected from the TCGA group for analysis. KEAP1 mutation status was defined as previously described . We founA large genomic study has demonstrated that multiple oncogenic and tumor suppressor pathways are involved in the initiation and progression of lung cancer . In addiFunctional KEAP1 represses NRF2 activity by recruiting the NRF2 protein to the actin cytoskeleton . SequencAfter exposure to cisplatin, the KEAP1 mutant cell lines A549 and H460 exhibited considerably increased levels of NRF2 signaling, whereas when the KEAP1 wild-type cell lines H292 and SKMES-1 exhibited no changes or only mild changes. Cisplatin is a weak inducer of ARE in the MCF-7 human breast cancer cell line (without KEAP1 mutation ) and indHowever, nedaplatin induces only weak activation or no activation of NRF2 signaling. The antineoplastic activity of nedaplatin, a cisplatin derivate developed in 1983 , might bAn early study by Devling et al.  has revet test p value 0.0116). This effect was also observed in Fig. t test p value 0.0218). We speculated that under siRNA intervention or activator treatment, some other pathways such as NF-kappa B and BACH1 are compensatorily involved in the process [NRF2 activity is involved in chemosensitivity in breast cancer  and ovar process . Our worTo confirm the significance of KEAP1 mutation in vivo, we evaluated public gene expression data from the publically available TCGA consortium. As expected, KEAP1 mutation did not significantly change alter the mRNA expression of NRF2 and KEAP1. However, mRNA levels of NRF2 downstream target genes were significantly higher in patients with KEAP1 mutations compared with patients with wild-type KEAP1, suggesting that KEAP1 and NRF2 interact primarily at the protein level and that KEAP1 mutations strongly affect NRF2 signaling Fig. . The assIn summary, the mRNA and protein expression profile of NRF2 and components of the NRF2-associated genes in four NSCLC cell lines were characterized and their responses to platinum-based therapies were analyzed. A causative effect of KEAP1-NRF2 signaling on platinum-based treatment was defined by engineered expressing either wild-type or mutant KEAP1 and knocking-down or activating NRF2. It is reasonable to hypothesize that KEAP1/NRF2 plays a key role in the cellular response to platinum chemotherapy in NSCLC and that KEAP1 could be explored as a specific biomarker for predicting a patient’s response in personalized therapy.


It has been shown previously that BALB/c strain embryos tend to contribute poorly to mouse aggregation chimaeras. In the present study we showed that BALB/c cells were not preferentially allocated to any extraembryonic lineages of mouse aggregation chimaeras, but their contribution decreased during the early postimplantation period and they were significantly depleted by E8.5. The development of BALB/c strain preimplantation embryos lagged behind embryos from some other strains and the contribution that BALB/c and other embryos made to chimaeras correlated with their developmental stage at E2.5. This relationship suggests that the poor contribution of BALB/c embryos to aggregation chimaeras is at least partly a consequence of generalised selection related to slow or delayed preimplantation development. The suitability of BALB/c embryos for maximising the ES cell contribution to mouse ES cell chimaeras is also discussed. Summary: BALB/c strain embryos contributed poorly to mouse aggregation chimaeras by E8.5. Selection appears linked to slow BALB/c development and might also explain the good ES cell contribution in BALB/c↔ES-cell chimaeras. Fig. S1 and Mouse aggregation chimaeras provide powerful research tools for a wide range of investigations , but theFig. S1A-C; Fig. S1A-C; These experiments showed that BALB/c cells tended to contribute poorly to the foetus, amnion and yolk sac mesoderm , and also relatively poorly to the yolk sac endoderm and placenta , pTE (overlying the ICM) and mTE (surrounding the blastocyst cavity) in chimaeric blastocysts, carrying the ransgene . These wransgene  and 2. Transgene  and imagransgene . The perContributions of tauGFP-positive (BF1×TP6.3) cells did not differ significantly among mTE, pTE and ICM regions of the chimaeric blastocysts in the balanced AAF2↔(BF1×TP6.3) strain combination 4B A or the a/aGpi1↔b/bGpi1 chimaeric conceptuses between E4.5 and E8.5 by GPI1 electrophoresis  plus three BF1 (b/bGpi1) oocytes with that in mixtures of three BALB/c (a/aGpi1) plus three BF1 oocytes . There were trends for more E6.5-E8.5 conceptuses in the unbalanced series 3U to be non-chimaeric and for more of these to be 0% GPI1A, rather than 100% GPI1A, than in the balanced series 3B (Table S1). The mean percentage of GPI1A was significantly less in the unbalanced series of BALB/c↔(BF1×TGB) chimaeras than in the balanced series of AAF2↔(BF1×TGB) chimaeras by E8.5, if the non-chimaeras were excluded, or by E7.5, if the non-chimaeras were included  series 3U than in the balanced AAF2↔(BF1×TGB) series 3B at this stage  used to produce the two chimaera series in the corresponding chimaera experiment. For example, embryo development experiment 1 compared the embryonic development of the three strains used to produce E12.5 chimaeras series 1U and 1B in chimaera experiment 1. Embryos were collected at E2.5 and cultured for 24 h, equivalent to the culture period used to make chimaeras. The overall developmental status of each embryo strain was summarised as the mean developmental score at E2.5 and E3.5, as explained in the Materials and Methods, and the percentage of blastocysts at E3.5. The developmental scores for the a/aGpi1 and b/bGpi1 strains used in a series of chimaeras, were used to calculate a relative developmental index for the a/aGpi1 strain in that chimaera series. The relative developmental index was calculated as the developmental score of the a/aGpi1 strain, expressed as a percentage of the sum of the scores for the a/aGpi1 and b/bGpi1 strains that were combined in the chimaeras .We compared the development of embryos from the strains used to produce the E12.5 chimaeras, shown in chimaera experiments 1-3 in Table S2) and, for experiments 1 and 2, they remained lagging behind after 24 h in culture (Table S3). The E2.5 BALB/c embryos in embryo development experiment 3 also lagged behind those in experiment 1, because embryos were collected about 2 h earlier . When embryos were collected at E2.5, in embryo development experiments 1-3, BALB/c embryos and  embryos were lagging behind the others (Tables S2 and S3) are consistent with a maternal effect contributing both to the slower or delayed development of BALB/c embryos and their poorer contribution to chimaeras.Overall, these results imply that the contribution that an embryo makes to the foetus of an E12.5 aggregation chimaera correlates with its stage of development at E2.5. Furthermore, the differences between embryos from the reciprocal crosses between BALB/c and AF1 mice (P<0.0001 by Mann-Witney U-test). The mean numbers of cell doublings completed were estimated as 2.69 for BALB/c and 3.30 for (BF1×TGB) embryos, from the log2 of the mean cell number, suggesting that BALB/c embryos lagged behind by about 0.61 cell doublings.Finally, to quantify the developmental lag of BALB/c embryos in terms of cell doublings, we compared cell numbers in 139 BALB/c and 127 (BF1×TGB) embryos at E2.5. As expected, the BALB/c embryos had significantly fewer cells than the (BF1×TGB) embryos. Median cell numbers were 6 and 9, respectively, and mean±95% confidence intervals were 6.47±0.34 cells for BALB/c and 9.87±0.47 cells for (BF1×TGB) embryos  and BALB/c↔(BF1×TP6.3) chimaeras, if hybrid cells outgrow BALB/c cells. However, this does not explain the differences in compositions of (BALB/c×AF1)↔BF2 and (AF1×BALB/c)↔BF2 chimaeras , or the Ped (preimplantation embryo development) gene complex  embryos lag behind reciprocal  embryos by E2.5, is consistent with their poorer contribution to E12.5 chimaeras  and sugg2(7.6/4.5)] and 0.59 doublings at 89 h [log2(33.1/22.0)]. This is comparable to our estimate that BALB/c embryos lagged behind (BF1×TGB) embryos by approximately 0.61 cell doublings at E2.5. Overall these data suggest that BALB/c embryos lagged behind C57BL/10 and (BF1×TGB) embryos by 0.59 to 0.76 cell doublings at these stages. If the cleavage cell cycle time is about 11-12 h, this is equivalent to a lag time of approximately 6-9 h, but this will be imprecise because cell divisions retain some synchrony.3H-thymidine label on cell numbers, results for giant chimaeric blastocysts, produced by aggregating four embryos, indicated that the more advanced embryo(s) contributed more to the ICM then expected by chance in some, but not all, chimaera combinations (Some evidence suggests that delayed or slow preimplantation development of BALB/c embryos might be predicted to cause a poorer contribution of BALB/c cells to the ICM than the trophectoderm (TE) in aggregation chimaeras. Studies of intact preimplantation mouse embryos , or embrinations . In addiinations . Howeverinations .a/aGpi1↔b/bGpi1 strain combination, showed that younger 4-cell embryos and half embryos at the 8-cell stage both contributed less well than whole 8-cell stage embryos to the foetus, amnion, yolk sac mesoderm, yolk sac endoderm and placenta  cells are also eliminated from mixed cultures of ES cells by a competitive process, which occurs when pluripotent ES cells differentiate. This depends on the presence of wild-type cells and involves establishing lower levels of c-Myc in cells that are later eliminated by apoptosis  used are summarised in , the partially congenic C57BL-albino strain C57BL/Ola.AKR-a,TyrcGpi1/Ws (abbreviated to ‘BC’), (BC×BALB/c)F1 hybrids (abbreviated to AF1), (BALB/c×A/J)F1 hybrids (abbreviated to ‘AAF1’) and stocks ‘TGB’ and ‘TP6.3’ were bred and maintained under conventional conditions at the University of Edinburgh with a light cycle of 14 h light (05:00 h-19:00 h) and 10 h dark or 12 h light (06:00 h-18:00 h) and 12 h dark.All animal work was performed in accordance with institutional guidelines and UK Home Office regulations . The strains of mice (TgN(Hbb-b1)83Clo transgene (abbreviated to Tg)  and c/cGpi1 as described previously (Stock TGB was homozygous for the reiterated d to Tg)  and was d to Tg) . Stock Transgene . Hemizygeviously .Female mice, 5-7 weeks old, were induced to ovulate by intraperitoneal injections of 5 i.u. PMSG  at 12:00 h, followed by 5 i.u. hCG , 48 h later. For collection of unfertilised oocytes, superovulated females were culled at approximately 10:00 h on the morning after the hCG injection, oocytes were collected from the oviduct and cumulus cells were dispersed in a solution of 100 units hyaluronidase  per ml of phosphate-buffered saline (PBS). For collection of embryos, females were housed individually with males overnight, and the following morning (designated E0.5) the female was checked to determine if a vaginal plug, indicative of mating, was present. Plugged females were killed by cervical dislocation on the appropriate day, depending on the age of embryos required, and preimplantation embryos were flushed from the reproductive tract, washed and kept in M2  or KSOM-c/cGpi1, CF1 hybrid strain, pseudopregnant recipients as described elsewhere  for 24 h, then transferred to fresh pre-equilibrated drops of culture medium under mineral oil in a WillCo HBSt-3522, thin glass-bottomed dish . This was placed in an environmental chamber on top of the pre-heated stage  of a Leica DMIRB/E inverted confocal microscope, and the atmosphere within the chamber was maintained at 37°C, 5% CO2 in air, as described elsewhere  would be calculated as 100A/(A+B) and expressed as a percentage. In some experiments, cells of E2.5 embryos were counted directly with a dissecting microscope.Females were induced to ovulate (see above) and paired overnight with males. Preimplantation embryos were flushed from the reproductive tract of pregnant females at E2.5 and cultured singly in drops of M16 culture medium  under paThree embryo development experiments were undertaken to compare the preimplantation development of the embryos with genotypes used in E12.5 chimaera experiments 1-3, shown in P<0.05), mean percentage GPI1A differences that were smaller than those previously published for E12.5 chimaeras of the same strain combination . An online statistical calculator (http://vassarstats.net/index.html) was used for Fisher's exact tests. Where data are plotted as box and whisker plots, they show the median , upper and lower quartiles (top and bottom of boxes) and the minimum and maximum of all the data (ends of whiskers).Minimum group sizes were guided by previous experience and power calculations. For the compositions of E6.5-E8.5 chimaeras, chimaera group sizes were chosen to ensure sufficient power to detect, as significant (


Bos taurus papillomaviruses infect cattle, which has both animal health and economic consequences. This is the first report and sequence of Bos taurus papillomavirus type 1 isolated from warts in cattle in Morocco. The double-stranded DNA (dsDNA) genome was 7,945 bp in size, and eight open reading frames were identified. Bos taurus (formerly bovine) papillomaviruses (BPV) belong to the family Papillomaviridae, and to date, 21 distinct BPV types have been identified (Deltapapillomavirus (Papillomavirus Episteme site (pave.niaid.nih.gov) (omavirus . The firnih.gov) . The gennih.gov) . In addinih.gov) , 6.Toward the end of 2015, sporadic cases of epithelial lesions affecting cattle on farms around Ain Harrouda, Morocco, were reported. Five to 10 cattle used for dairy and meat were affected. In January 2016, a lesion (50 mm in diameter) was completely excised with local treatment from a diagnosed adult crossbred cow  and stored (−80°C).Total DNA was extracted from the lesion using a DNeasy blood and tissue kit  and prepared per the manufacturer’s instructions. Two overlapping 4.1-kb sections of the genome were amplified by PCR using Phusion high-fidelity DNA polymerase  and the primer sets 5′-GGTTGGACTGTCTGTGGTG-3′ and 5′-AGGTCATAGGCACTGGCAC-3′ and 5′-CCCTGCTCAGATTTTATATGG-3′ and 5′-GAAAGTCTTTGACCATGCAC-3′, based on the sequence with GenBank accession number AB626705. The thermocycling conditions were initial denaturation at 98°C for 30 s, 30 cycles of 10 s at 98°C, 30 s at 55°C, and 150 s at 72°C, and final elongation at 72°C for 5 min. The amplified DNA was cloned using the CloneJET PCR cloning kit  per the manufacturer’s instructions, and the sequence of the DNA was determined using primer walking . The sequences were assembled using DNAman version 5.2.9 (Lynnon BioSoft).The full-length genome was compared to the reference BPV-1 genome (GenBank accession number X02346). The sequence was identical in size to the reference genome at 7,945 bp, and eight open reading frames were identified, containing the early genes E1, E2, E4, E5, E6, and E7 and the late genes L1 and L2. The sequence differed from the reference genome by a total of 36 nucleotides, with nonsynonymous mutations affecting the E1, E2, L1, and L2 genes. One insertion affected both the E2 and E4 genes, and there were two deletions and one insertion within the NCR. Our sequence shared greater than 99.4% identity with the reference sequence and 99% identity with other complete BPV-1 genome sequences in GenBank.This is the first report of a full-length genome of any BPV from Morocco. The report indicates the highly conserved nature of BPV-1 in comparison to the reference sequence described decades earlier and on another continent.Bos taurus papillomavirus type 1 was deposited in GenBank under the accession number KY746722.The complete genome sequence for


Amphibalanus amphitrite. Glucosamine isocyanides with an ether functionality at the anomeric position exhibited potent antifouling activity, with EC50 values below 1 μg/mL, without detectable toxicity even at a high concentration of 10 μg/mL. Two isocyanides had EC50 values of 0.23 and 0.25 μg/mL, comparable to that of CuSO4, which is used as a fouling inhibitor (EC50 = 0.27 μg/mL).Biofouling, an undesirable accumulation of organisms on sea-immersed structures such as ship hulls and fishing nets, is a serious economic issue whose effects include oil wastage and clogged nets. Organotin compounds were utilized since the 1960s as an antifouling material; however, the use of such compounds was later banned by the International Maritime Organization (IMO) due to their high toxicity toward marine organisms, resulting in masculinization and imposex. Since the ban, there have been extensive efforts to develop environmentally benign antifoulants. Natural antifouling products obtained from marine creatures have been the subject of considerable attention due to their potent antifouling activity and low toxicity. These antifouling compounds often contain isocyano groups, which are well known to have natural antifouling properties. On the basis of our previous total synthesis of natural isocyanoterpenoids, we envisaged the installation of an isocyano functional group onto glucosamine to produce an environmentally friendly antifouling material. This paper describes an effective synthetic method for various glucosamine-based isocyanides and evaluation of their antifouling activity and toxicity against cypris larvae of the barnacle  Many essential human activities, such as transportation and fishing, are carried out in the ocean, and submarine structures—seawater intakes pipes for power plants, breeding nets, and so on—are also widely used. Ocean-based activities such as offshore energy generation will continue to increase in the future . Any str1), obtained from nudibranchs of the family Phyllidiidae by Okino, shows strong antifouling activity (50% effective concentration (EC50) = 0.14 μg/mL) against cypris larvae of the barnacle Amphibalanus amphitrite, comparable to CuSO4 (EC50 = 0.27 μg/mL), along with low toxicity  >10 μg/mL) [1 and examined the biological activity of a synthetic sample against the same cypris larvae, obtaining similar activities  compared to those of the natural compound [2 and the nitrile 3, synthetic intermediates in the total synthesis of 1, were found to have less potency than 1. Other isocyanide compounds such as 3-isocyanotheonellin (4) also exhibited potent antifouling profiles [5), obtained from crustaceans such as crabs and shrimp as a cheap and abundant biomass platform, through organic synthesis, because 5 possesses an amino group, which is essential for introduction of the isocyano group. In addition, the four hydroxy groups in 5 enable easy access to a wide variety of synthetic isocyanides. Furthermore, the compound, which is derived from a sugar-based structure, was expected to have low toxicity. In this study, glucosamine-based isocyanides (6) were designed and synthesized in short reaction sequences. The antifouling activities and toxicities of the synthetic compounds 6 against the cypris larvae of Amphibalanus amphitrite were evaluated to prove the feasibility of these compounds as alternative environmentally friendly antifouling agents, including through investigation of substituent effects at the C-1 and C-3, 4, and 6 positions.In current research on green antifouling agents , natural0 μg/mL) , as showcompound ,27,28. Aprofiles ,31,32,336a and the tetrapalmitate 6b were prepared from d-glucosamine hydrochloride (5) by a sequence of formamide formation with HCO2Me and NaOMe [2O or palmitoyl chloride , respectively, and the dehydration of formamides 7a and 7b with POCl3 and Et3N [6a and 6b were evaluated against the cypris larvae of Amphibalanus amphitrite after a 48 h incubation period, showing moderate activity  and very low toxicity .Initially, the tetraacetate nd NaOMe , the acyand Et3N  , benzyl (CH2Ph), allyl (CH2CH = CH2), isopropyl (CH(CH3)2, iPr), and 2,2,2-trifluoroethyl (CH2CF3) groups were chosen as the substituent R of the C-1 alkoxy group. 6c–g. For the synthesis of 6c–g, the commercially available d-glucosamine derivative 8 was employed as a starting compound because glycosidation reactions between formamide 7a and alcohols such as isopropyl alcohol under various conditions did not afford the desired glycosidation products. In glycosidation reactions of 8 promoted by BF3·OEt2 (Step A), two solvent systems  were employed for optimization of chemical yield. Phenyl glycoside 9c and trifluoroethyl glycoside 9g were obtained in higher yields by allowing 8 to react with phenol and 2,2,2-trifluoroethanol at room temperature in CH2Cl2. However, treatment of 8 with benzyl alcohol, allyl alcohol, and isopropyl alcohol at 80 °C in MeCN produced benzyl glycoside 9d, allyl glycoside 9e, and isopropyl glycoside 9f in better yields. During each of the glycosidation reactions, the β-anomer was formed as the sole glycosidation product. The β-configurations at the anomeric position (C-1) of 9c–g were determined based on coupling constant analysis. Thus, a large vicinal coupling constant, J12 = 9 Hz , was observed in all the 1H NMR spectra. The conversion of 9c–g into formamides 7c–g was accomplished in the following three steps (Step B): (i) removal of the phthaloyl and acetyl groups with ethylenediamine (EDA); (ii) a formamide formation reaction with HCO2Me and NaOMe; and (iii) acetylation of the three resulting hydroxyl groups with Ac2O and pyridine. Isocyanides 6c–g were derived from 7c–g using the same dehydration protocol with POCl3 and Et3N (Step C) that was used in the preparation of 7a and 7b. The biological activities of synthetic 6c–g against the cypris larvae of Amphibalanus amphitrite were then evaluated to reveal enhanced potency for 6c–g (EC50 = 0.23–0.71 μg/mL) relative to 6a and 6b . Furthermore, it was noteworthy that mortality among the cypris larvae was not observed during the assay, even at a high concentration of 10 μg/mL.Next, the effect of replacement of the C-1 ester groups of 6f with other substituents. As illustrated in 9f followed by formamide formation, octanoyl (COnC7H15), palmitoyl (COnC15H31), benzoyl (COPh), and TBS (SiMe2tBu) groups were installed to afford isopropyl glycosides 7h–k, respectively. Each of 7h–k was transformed to the corresponding isocyanide (6h–k) under dehydration conditions that were the same as those used for the preparation of 6a–g. Isocyanides 6h–k were tested against Amphibalanus amphitrite and exhibited similar antifouling activities (EC50 = 0.25–0.81 μg/mL) to 6f (EC50 = 0.23 μg/mL). Again, none of 6h–k showed detectable toxicity (LC50 > 10 μg/mL).Further modifications were made by replacing the acetyl groups at C-3, 4, and 6 of the isopropyl glycoside 1. Although the chemical yields should be improved by further optimization, the synthetic scheme seems to be a reliable one for the synthesis of various isocyanides with latent antifouling activity. All of the samples obtained in the current study showed not only antifouling profiles but also extremely low toxicity against the cypris larvae of Amphibalanus amphitrite. Compounds 6a and 6b with the ester group at the C-1 position had EC50 values above 5 μg/mL, but 6c–g with the ether group at the C-1 position were more active, with EC50 values below 1 μg/mL. Compounds 6f and 6k exhibited EC50 values of 0.23 and 0.25 μg/mL, comparable to that of CuSO4, which is used as a fouling inhibitor (EC50 = 0.27 μg/mL) [50 values of 6a and 6b compared to 6c–g, suggesting that more detailed investigations of ether substituents at this position may enable the discovery of more potent compounds, and that substituents at the C-1 position affect the antifouling mechanism through steric or electronic effects [These procedures are short syntheses  allowing access to various glucosamine-based isocyanides using inexpensive reagents. In contrast, 27 steps were required for the total synthesis of  effects ,37  spectrometer , methanol (MeOH), and acetonitrile (MeCN) were purchased from Kanto Chemical Co. Inc. . Dichloromethane (CH2Cl2) was distilled from CaH2. All commercially available reagents were employed as received. Analytical TLC was carried out using pre-coated silica gel plates (TLC silica gel 60 F254). The silica gel used for column chromatography was Wakogel 60N 63–212 μm.Optical rotations were obtained using a Horiba SEPA-300 instrument . IR spectra were recorded on a JASCO FT/IR 4100 spectrometer using an NaCl cell. eter see . N,N-dimd-glucosamine hydrochloride (5) (1.0 equiv) in MeOH (0.40 M) and HCO2Me (1.0 M), NaOMe (1.4 equiv) and Et3N (1.0 equiv) was added at room temperature under an Ar atmosphere. The mixture was stirred at room temperature overnight and then concentrated in vacuo. To a solution of the crude formamide in pyridine (15 equiv), Ac2O (6.0 equiv) for 7a or palmitoyl chloride (6.0 equiv) for 7b was added at room temperature under an Ar atmosphere. After stirring at room temperature overnight, MeOH (6.0 equiv) was added at 0 °C. The mixture was extracted with EtOAc, washed successively with 1.0 M NaOH, 3.0 M HCl, and brine, dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography (EtOAc/hexane = 40:60) to yield 7a or 7b, respectively.To a solution of 7a–k (1.0 equiv) in CH2Cl2 (0.20 M), Et3N (9.0 equiv) and POCl3 (3.0 equiv) were added dropwise at 0 °C under an Ar atmosphere. After stirring at 0 °C for 10 min, the mixture was warmed to room temperature, stirred at room temperature for 1 h, quenched with saturated NaHCO3, and extracted with EtOAc. The combined organic extracts were washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography (EtOAc/hexane = 10:90) to yield isocyanides 6a–k, respectively.To a solution of each of the series of formamides 8 (1.0 equiv) in MeCN (0.25 M), benzyl alcohol, allyl alcohol, or isopropyl alcohol (1.0 equiv) and BF3·OEt2 (3.0 equiv) were added at room temperature under an Ar atmosphere. The mixture was stirred at 80 °C overnight, quenched with saturated aqueous NaHCO3, and extracted with EtOAc. The combined organic extracts were washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo. The residue was then purified by silica gel column chromatography (EtOAc/hexane = 20:80) to yield 9d–f, respectively.To a solution of 8 (1.0 equiv) in CH2Cl2 (0.20 M), phenol or 2,2,2-trifluoroethanol (2.0 equiv) and BF3·OEt2 (2.0 equiv) were added at room temperature under an Ar atmosphere. The mixture was stirred at room temperature overnight, quenched with saturated aqueous NaHCO3, and extracted with AcOEt. The combined extracts were washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo. The residue was then purified by silica gel column chromatography (EtOAc/hexane = 30:70) to yield 9c or 9g, respectively.To a solution of 9c–g (1.0 equiv) in MeCN (0.20 M), EDA (4.7 equiv) was added at room temperature under an Ar atmosphere. The mixture was stirred at 80 °C overnight and concentrated in vacuo. To a solution of the crude amine in MeOH (0.40 M) and HCO2Me (0.40 M), NaOMe (1.2 equiv) was added at room temperature under an Ar atmosphere. After stirring at room temperature overnight, DOWEX was added for neutralization. The mixture was filtered through a celite pad and concentrated in vacuo. To a solution of the crude formamide in pyridine (30 equiv), Ac2O (25 equiv) was added at room temperature under an Ar atmosphere. After stirring at room temperature overnight, MeOH (25 equiv) was added at 0 °C. The mixture was extracted with EtOAc, washed successively with 1.0 M NaOH (25 equiv), 3.0 M HCl, and brine, dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography (EtOAc/hexane = 40:60) to yield 7c–g, respectively.To a solution of each of 9f in two steps, octanoyl chloride (6.0 equiv) was added at room temperature under an Ar atmosphere. The mixture was stirred for 24 h, quenched with 3.0 M HCl (60 equiv), diluted with AcOEt, washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (EtOAc:Hexane = 5:95) to yield 7h (24% over three steps) as a white solid.To a solution of crude formamide in pyridine (50 equiv) and DMAP (0.50 equiv), obtained from 9f in two steps, DMF (0.050 M), palmitic acid (5.0 equiv), DMAP (0.50 equiv), and EDCI (5.0 equiv) were added at room temperature under an Ar atmosphere. The mixture was stirred for 20 h, supplemented with EtOH (2.0 equiv) and EDCI (2.0 equiv), quenched with saturated aqueous NH4Cl, diluted with AcOEt, washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography (EtOAc:hexane = 10:90) to yield 7i (16% over three steps) as a white solid.To a solution of crude formamide obtained from 9f in two steps, benzoyl chloride (5.0 equiv) was added at 60 °C under an Ar atmosphere. The mixture was stirred for 24 h, quenched with 3.0 M HCl (60 equiv), diluted with AcOEt, washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (EtOAc:hexane = 30:70) to yield 7j (46% over three steps) as a white solid.To the solution of crude formamide in pyridine (15 equiv) and DMAP (0.50 equiv), obtained from 9f in two steps, in DMF (0.050 M) and 2,6-Lutidine (15 equiv), TBSOTf (20 equiv) was added at 120 °C under an Ar atmosphere. The mixture was stirred for 1.5 h, quenched with saturated aqueous NaHCO3, diluted with AcOEt, washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography (EtOAc:hexane = 10:90) to yield 7k (43% over three steps) as a white solid.To the solution of crude formamide, obtained from Amphibalanus amphitrite) procured from oyster farms at Lake Hamana and a pier at Shimizu bay, Shizuoka, were kept in an aquarium at 20 °C and fed on Artemia salina nauplii. Broods were released as I–II stage nauplii upon immersion in seawater after drying overnight. The nauplii thus obtained were cultured in filtered natural seawater  containing penicillin G (20 μg/mL) and streptomycin sulfate (30 μg/mL) and were fed on the diatom Chaetoceros gracilis at concentrations of 40 × 104 cells/mL. Larvae reached the cyprid stage in 5 days. The cyprids were collected and stored at 4 °C until use (0 days old).Adult barnacles  and six 2-day-old cyprids were added. The plates were kept in the dark at 25 °C for 48 h. The numbers of cyprids that attached, metamorphosed, died, and did not settle were counted under a microscope. Three or four trials were done for each concentration. Probit analysis was used to calculate the EC


The small concentration of intercalated Li ions has a strong influence through steric hindrance of transverse fluoride ion vibrations, which directly controls the thermal expansion. Redox intercalation of guest ions is thus likely to be a general and effective method for controlling thermal expansion in the many known framework materials with phonon-driven negative thermal expansion.Thermal expansion properties of solids are of fundamental interest and control of thermal expansion is important for practical applications but can be difficult to achieve. Many framework-type materials show negative thermal expansion when internal cages are empty but positive thermal expansion when additional atoms or molecules fill internal voids present. Here we show that redox intercalation offers an effective method to control thermal expansion from positive to zero to negative by insertion of Li ions into the simple negative thermal expansion framework material ScF 3 framework offers effective control of the thermal expansion for this simple material.The positive thermal expansion exhibited by most materials at increased temperatures is a severe issue for many high precision applications. Here, Xing and co-workers show that redox intercalation of Li ions into a ScF Sc3+ is not easily reducible, and direct reactions using n-butyllithium failed to form LixScF3 products, so partial substitution of Sc by a reducible metal is needed. The solid solution (Sc0.9Fe0.1)F3 (SFF) was thus synthesized and then lithiated, as described in Methods. The ionic radii of Sc3+, Fe3+ and Fe2+ are, respectively, 0.745, 0.645 and 0.780 Å, so reduction of Fe3+ to Fe2+ in this framework is favoured by lowering of lattice microstrain. Characterization results for SFF and the lithiated product Lix(Sc0.9Fe0.1)F3 are shown in Insertion of Li ions into ScFx(Sc0.9Fe0.1)F3, respectively (3+ to Fe2+. The structure and composition of the lithiated Lix(Sc0.9Fe0.1)F3 product have been determined by joint studies of structure refinement based on neutron powder diffraction (NPD) data, spherical aberration-corrected scanning transmission electron microscopy (STEM) and X-ray absorption near-edge structure (XANES). A neutron scattering Fourier difference map clearly demonstrates that the negative peak at the  is the position of Li ions (0.06(Sc0.9Fe0.1)F3  electron micrographs of LSFF-1 and SFF directly reveal the Li sites within the structural model  to 3.99927(4) Å for SFF and Liectively , in keep Li ions , since L Li ions . The cheal model , and hen3+ to Fe2+ after the lithiation reaction F3 found by NPD refinement.XANES spectra were collected from samples SFF and LSFF-1. There is no change in the Sc K-edge XANES , which sreaction . The prereaction , correspFe ratio . For SFF3 and SFF have NTE behaviour with average linear thermal expansions (in the range 150–425 K) of αl=−7.47 and −5.01 × 10−6 K−1, respectively. However, introduction of a small amount of Li at the A-sites results in a change to PTE with αl=1.03 × 10−6 K−1 for LSFF-1 as shown in 2 atmosphere resulting in loss of Li as LiF and Fe as Fe3O4, and α-Fe2O3 (x(Scy1-Fey)F3 products (samples LSFF-2 to 4) were generated by this route  with αl=−0.75 × 10−6 K−1 is achieved in Li0.04(Sc0.94Fe0.06)F3 (LSFF-2) annealed at 475 K, while Li0.02(Sc0.97Fe0.03)F3 (LSFF-3) has moderate NTE of αl=−2.59 × 10−6 K−1. Annealing at 575 K gives sample LSFF-4 with αl=−7.40 × 10−6 K−1 the same as for ScF3 3, we have performed vibrational analysis of the fluorine atoms using first-principles calculations. The results reveal that the vibrations of fluorine ions are strongly perturbed by the inserted Li ions, and the directions of all transverse modes are inclined as shown for the representative mode with the lowest frequency in −1) vibrational mode for undoped ScF3 where the vibrational motion of fluorine ions is perpendicular to the Sc–F–Sc linkages leading to NTE in ScF3. Interestingly, intercalation of Li ions into the ScF3 cages strongly perturbs this vibrational mode (x∼0.04) is sufficient to suppress the overall NTE behaviour, owing to the dual effects on the amplitude and direction of transverse vibrations of fluorine ions.A previous inelastic neutron scattering study revealed that the NTE behaviour of ScFnal mode . The vecU33) of F ions is weakened, while the longitudinal one (U11) is enhanced. As shown in U33/U11. Larger U33/U11 corresponds to stronger transverse thermal vibration of fluorine ions, and a more negative expansion coefficient. The change of thermal expansion from PTE to NTE is accompanied by an increase in the ratio of U33/U11.The effects of Li intercalation on thermal expansion are further supported by the anisotropic atomic amplitude of fluorine ions calculated in the structure refinements from NPD data. 0.9Fe0.1)F3 switches the thermal expansion behaviour from NTE to PTE, consistent with the Li guest ions in Li0.06(Sc0.9Fe0.1)F3 providing steric hindrance to the transverse vibrations of fluorine ions. Thus, it is likely that NTE can be controlled in many frameworks by adjusting the concentration of guest ions, as previously found for several frameworks by varying the concentration of guest water molecules34353637The above results demonstrate that introducing a small concentration of Li ions into (Sc0.9Fe0.1)F3 and thermal decomposition to delithiate leading to impurity formation, standard electrochemical methods used in batteries or sensors could be used to introduce or remove cations reversibly. A similar Li insertion and extraction has been reported to occur reversibly and rapidly at room temperature for ReO3-type FeF3 via an electrochemical methodThe present study may provide an effective method to achieve thermal expansion control engineering by means of the redox intercalation of guest ions or molecules into the pores of a NTE framework. Although we have used chemical intercalation to introduce lithium into F3 (SFF) and ScF3 samples were prepared via the solid-state reaction with the precursors of high purity (99.99%) Sc2O3, Fe2O3 and NH4F. These precursors in stoichiometric proportions were pressed into a small pellet (radius and height 5 × 5 mm) and covered with NH4F powder and pressed into a larger pellet (10 × 10 mm). The pellet was loaded into Pt crucible and transferred to a furnace with heating at 600 °C for 5 h, and slow cooling to room temperature. The SFF sample was found to be phase pure by X-ray diffraction.The (Sc0.06(Sc0.9Fe0.1)F3 sample was obtained by the chemical intercalation of the SFF powder with n-butyllithium  at room temperature for 24 h in a glove box with high purity argon atmosphere. The as-prepared sample powder was washed with hexane several times, and then dried under N2 flow at 80 °C for 10 h. The product did not decompose on heating up to 425 K giving composition Li0.06(Sc0.9Fe0.1)F3 (sample LSFF-1). Samples Li0.04(Sc0.94Fe0.06)F3 (LSFF-2), Li0.02(Sc0.97Fe0.03)F3 (LSFF-3) and ScF3 (LSFF-4) were obtained by partial thermal decomposition in an inert N2 atmosphere, respectively, at 475, 525 and 575 K.The Li3-type phases.A small amount (0.8 wt%) of impurity phase LiF was observed in LSFF-1. Further impurity phases of iron oxides in LSFF-2 to 4 were generated by high-temperature decomposition as shown in 0.06(Sc0.9Fe0.1)F3 sample was collected from 298 to 573 K at the NIST Center for Neutron Research on the BT-1 high-resolution neutron powder diffractometer. The wavelength of the neutron beam was 1.5398 Å. The chemical compositions and anisotropic atomic displacement parameters of fluorine ions of the LSFF samples were determined by structure refinement using NPD data. All structure calculations were performed using FULLPROF softwareTemperature dependent X-ray diffraction data were collected from 150 to 650 K by using a PANalytical, PW 3040-X-PertPro X-ray diffractometer. The lattice constant was refined using a cubic structural model (space group: 0.06(Sc0.9Fe0.1)F3 powder with boron nitride powder. The EXAFS spectra were collected in transmission mode in the energy range 4.3–5.7 keV for Sc K-edge EXAFS, 6.9–8.3 keV for Fe K-edge EXAFS, with an energy step varying from 0.1 eV in the near-edge region to about 4 eV at the highest energies, to obtain a uniform wave vector step Δk ≅ 0.03 Å−1. The X-ray beam was monochromatized by a Si(111) double-crystal monochromator. The sample was mounted in a high-temperature furnace and the temperature was stabilized and monitored through an electric heater controlled by a feedback loop, ensuring a thermal stability within ±1 K.Sc and Fe K-edge XANES and extended X-ray absorption fine structure (EXAFS) spectroscopy measurements were performed from room temperature to about 700 K at the XAFS beamline of ELETTRA synchrotron radiation facility in Trieste . The samples for EXAFS were prepared by mixing and pelletizing the SFF and LiSTEM was performed using a JEM-ARM 200F  that operated at 200 kV and was equipped with double aberration-correctors for probe-forming. Imaging lenses were used to perform high-angle annular-dark field (HAADF) and ABF imaging. The attainable spatial resolution of the microscope is 78 pm at the incident semi-angle of 25 mrad. To observe Li ions directly using ABF collection geometry, the acceptance semi-angle in this study was fixed between 12 and 25 mrad.s1, Sc 3d14s2, Fe 3d64s2 and F 2s22p5) and atom cores were modelled by optimized norm-conversing pseudopotentials−1 in the Brillouin zone were chosen. To consider the effect of the intercalation of Li ions on the vibrational property, a 3 × 3 × 3 super cell was built in which one Sc atom was replaced by Fe atom and one Li atom was inserted in the neighbouring A-site. Before vibrational property calculation, the crystal structure was geometrically optimized to find the energy minimum. The Broyden–Fletcher–Goldfarb–Shanno (BFGS) minimization scheme−5 eV per atom, 0.1 eV Å−1, 0.2 GPa and 5.0 × 10−3 GPa for energy, maximum force, maximum stress and maximum displacement, respectively. On the basis of crystal configuration in the minimal energy, the frequencies of the phonon modes were calculated by linear response formalismThe first-principles vibrational analysis was performed by CASTEP44The data relevant to the findings of this study are available from the corresponding authors on reasonable request.How to cite this article: Chen, J. et al. Tunable thermal expansion in framework materials through redox intercalation. Nat. Commun.8, 14441 doi: 10.1038/ncomms14441 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Tables, Supplementary Notes and Supplementary References


CYP11B1 mutations, and pathogenic CYP11B2/CYP11B1 chimeras contribute to a minority of cases. Heterozygote cases (chimeras combined with missense mutation) are very rare, and genetic analysis of these cases is difficult.11β-Hydroxylase deficiency (11OHD) is a common form of congenital adrenal hyperplasia that has been shown to result from inactivating CYP11B2/CYP11B1 with a novel breakage point located at g.9559–9742 of CYP11B2. Interestingly, the other allele exhibited a new mutation, p.L340P, in CYP11B1. Bioinformatics and molecular dynamics simulation indicated that p.L340P decreased the stability and changed the surface configuration of 11β-hydroxylase, indicating a disease-causing mutation. Further pedigree study, PCR and next-generation sequencing indicated that the proband carried both the chimera and p.L340P, and coexistence of the two increased the severity of 11OHD in this family. After treatment with combined medications, blood pressure and clinical parameters improved.We describe an 11OHD patient presenting with precocious pseudopuberty and hypokalemia hypertension who harbored a chimeric CYP11B1 mutation screening should be simultaneously conducted, and pedigree study is necessary.Our results suggest that chimera screening and The online version of this article (10.1186/s12902-018-0249-z) contains supplementary material, which is available to authorized users. CYP11B1 inactivating mutations clustered in exons 2, 6, 7 and 8 [CYP11B1 mutations may also occur as a result of aberrant incorporation/chimerism of the gene with the highly homologous aldosterone synthase (CYP11B2) gene sequence. These CYP11B2/CYP11B1 chimeric genes are relatively rare, with breakpoints dispersively distributed, and have been speculated to be pathogenic because of a loss of function in the zona fasciculate/reticularis, despite maintained function in the zona glomerulosa [Congenital adrenal hyperplasia (CAH) is one of the most common inheritable metabolic disorders and is characterized by virilization, precocious pseudopuberty and accelerated skeletal maturation, progressing in some cases to severe dehydration, shock, and even death , 2. An a 7 and 8 , and appmerulosa –9. In a CYP11B2/CYP11B1 on one allele with a breakpoint range (g.9559–9742) in the CYP11B2 gene in the junctional zone of exon 6 and intron 6. Interestingly, the other allele held a novel mutation, p.L340P, and bioinformatics and molecular dynamics simulation indicated that this was a disease-causing mutation. We then performed a pedigree analysis, which revealed that both the missense mutation and chimera were synergistically pathogenic in the proband.In the present paper, we report an 11OHD case who harbored a chimeric A 14-year-old boy  was admitted in September 2015 because of precocious puberty and recurrent episodes of periodic paralysis without special intervention. His sexual development, such as pubic hair, laryngeal prominence and spermatorrhea, occurred earlier than that in peers. He was born after an uneventful full-term pregnancy to a non-consanguineous healthy couple of Chinese origin with no family history of congenital adrenal hyperplasia. He was taller than boys of the same age until he was 12 years old. In addition, axillary hair and laryngeal prominence had appeared at 7 years old, and spermatorrhea and pubic hair growth had occurred at age 9. Physical examination showed hypertension (143/106 mmHg), slightly black skin, and external genitalia maturation , which confirmed precocious puberty Fig. . LaboratCYP11B1 gene and identified the novel mutation p.L340P (CYP11B1mut)  and confirmed a chimeric CYP11B2/CYP11B1 gene. Interestingly, extended familial analysis revealed that the chimera was also present in his mother, uncle and grandfather  of rs6391 in this family with 3 single nucleotide polymorphisms (SNPs)  Fig. . Bioinfoher Fig. , and mulPs) Fig. . Our resSince the blood pressure of the patient was poorly controlled by hydrocortisone, the proband was treated with a combination of dexamethasone (0.75 mg/everyday) and levamlodipine (2.5 mg/everyday) for 20 months. After treatment, clinical parameters, such as blood pressure, 17OHP and ACTH decreased, and the volume of the adrenal glands was reduced  to cortisol and corticosterone, respectively, and excess androgens due to DOC accumulation . AlthougCYP11B2/CYP11B1 gene with a novel breakpoint (g.9559–9742) on one allele, which differs from previous reports. Interestingly, the other allele contained a new disease-causing mutation, p.L340P, which altered the free energy and stability of 11β-hydroxylase. The chimeric mutation leads to deletion of CYP11B1 exons 1 to 6 on one allele, and unfortunately, a pathogenic missense mutation appeared on exon 6 of CYP11B1 in the other allele, which resulted in a seemingly homozygous missense mutation that was predicted by bioinformatics and molecular dynamics simulation to be a disease-causing mutation. At present, only one case of a pathogenic p.G314R mutation combined with a nonfunctional chimeric CYP11B2/CYP11B1 has been reported [CYP11B1 mutation screening should be simultaneously conducted and that pedigree study is necessary.From our investigation of the genetic contribution to pathogenesis, this patient was found to harbor a chimeric Additional file 1:Table S1. The laboratory and endocrinological evaluation of the proband pre- and post-treatment. (PDF 200 kb)Additional file 2:Table S2. Bioinformatics analysis of the free energy alteration in CYP11B1mut (PDF 82 kb)Additional file 3:Figure S1. Graphs of sequence copy number around the mutation and multiple genes resulting in CAH of the proband and his mother by qPCR and targeted next-generation sequencing. (TIF 11910 kb)Additional file 4:Figure S2. The sequencing chromatogram near the mutation in the proband’s relatives. The box indicates the mutation location. (TIF 28380 kb)Additional file 5:Figure S3. Radiation imaging of adrenal gland after treatment. A and B present abdominal CT images after treatment for 6 months showing that the sizes of the left and right adrenal gland are 75.2 mm × 22.1 mm and 67.3 mm × 38.7 mm, respectively. Similarly, C and D present abdominal CT images after treatment for 9 months showing that the sizes of the left and right adrenal gland are 65.2 mm × 24.4 mm and 63.7 mm × 35.7 mm, respectively. The arrow shows the bilateral adrenal lesions. (TIF 63145 kb)Additional file 6:Main methods used in the study. (DOC 112 kb)Additional file 7:Consent form from the mother of the proband. (PDF 95.7 kb)