Our results establish that ZIKV and DENV share epitopes that are targeted by neutralizing, protective human antibodies. The availability of potently neutralizing human monoclonal antibodies provides an immunotherapeutic approach to control life-threatening ZIKV infection and also points to the possibility of repurposing DENV vaccines to induce cross-protective immunity to ZIKV.Zika virus (ZIKV) is a mosquito-borne flavivirus responsible for thousands of cases of severe fetal malformations and neurological disease since its introduction to Brazil in 2013. Antibodies to flaviviruses can be protective, resulting in lifelong immunity to reinfection by homologous virus. However, cross-reactive antibodies can complicate flavivirus diagnostics and promote more severe disease, as noted after serial dengue virus (DENV) infections. The endemic circulation of DENV in South America and elsewhere raises concerns that preexisting flavivirus immunity may modulate ZIKV disease and transmission potential. Here, we report on the ability of human monoclonal antibodies and immune sera derived from dengue patients to neutralize contemporary epidemic ZIKV strains. We demonstrate that a class of human monoclonal antibodies isolated from DENV patients neutralizes ZIKV in cell culture and is protective in a lethal murine model. We also tested a large panel of convalescent-phase immune sera from humans exposed to primary and repeat DENV infection. Although ZIKV is most closely related to DENV compared to other human-pathogenic flaviviruses, most DENV immune sera (73%) failed to neutralize ZIKV, while others had low  that potently neutralizes ZIKV in addition to all four DENV serotypes points to a potential immunotherapeutic to combat ZIKV. This is especially salient given the precedent of antibody therapy to treat pregnant women infected with other viruses associated with microcephaly, such as cytomegalovirus and rubella virus. Furthermore, the identification of a functionally conserved epitope between ZIKV and DENV raises the possibility that a vaccine may be able to elicit neutralizing antibodies against both viruses. Flaviviridae family, which includes important human pathogens such as Japanese encephalitis virus (JEV), West Nile virus (WNV), yellow fever virus (YFV), and dengue viruses 1 to 4 (DENV-1 to -4) (6–Zika virus (ZIKV) is an arbovirus in the 1 to -4) , 3. Howe1 to -4) . Since t1 to -4) , 4, 6–8.to -4) 6–, 10. Theto -4) 6–.The emergence of ZIKV overlaps geographically with regions in which DENV is endemic, and ZIKV shares approximately 60% sequence identity with DENV . Moreove13–50s) of 8.9 × 10−4 and 3.4 × 10−4 µg/ml, respectively. Interestingly, EDE2 B7  and a strain circulating in the Americas in 2015 (PRVABC59). As expected, human and nonhuman primate type-specific MAbs that strongly neutralize DENV-1 (1F4), DENV-2 2D22), DENV-3 (5J7), and DENV-4 (5H2) did not neutralize ZIKV  17–20).–20.17–20–, DENV-3  EDE2 B7 , which s EDE2 B7 . Of note EDE2 B7 .in vitro may not necessarily be protective in vivo  of ZIKV H/PF/2013 or PBS in the footpad. The PBS-treated mice experienced 60% mortality following challenge (P < 0.05). The EDE1 C10-treated, infected mice largely exhibited no signs of illness, and their weight gain was more than that of mice that were infected with ZIKV and mock antibody but less than that of the mice that were not infected , DENV-2 (n = 4), DENV-3 (n = 5), and DENV-4 (n = 3) infections for cross-neutralization of ZIKV , and only one individual had strong ZIKV neutralization . Overall, secondary DENV immune sera poorly neutralized ZIKV compared to cross-neutralization phenotypes noted among other DENV serotypes, and only one individual (6%) strongly cross-neutralized ZIKV.A hallmark of secondary DENV infections is the induction of dengue virus serotype cross-neutralizing antibodies which reduce the risk of disease from subsequent DENV infections . We testThe fact that convalescent-phase DENV immune human sera displayed low cross-neutralization of ZIKV suggests that ZIKV is antigenically distantly related to DENV. To examine the antigenic relationships between ZIKV and DENV, we used antigenic cartography to calculate the Euclidean distances between sera, and metric multidimensional scaling was used to render the data in three dimensions  and, importantly, a strain circulating in the Americas in 2015 (PRVABC59). Indeed, the dose of EDE1 C10 administered to protect in vivo (two doses of 10 µg) is far less than the 500 µg required for the fusion loop-targeting mouse MAb 2A10G6  and the frequency of EDE antibodies was not broken down into EDE1 versus EDE2 , but there are several important caveats: the study had a small cohort  at 32°C. Vero-81 cells were grown in Dulbecco’s modified Eagle’s medium , and U937+DC-SIGN cells were maintained in RPMI 1640  at 37°C. All media were supplemented with 10% (Vero-81) or 5% (C6/36 and U937+DC-SIGN) fetal bovine serum , 0.1 mM nonessential amino acids , and 100 U/ml penicillin and 100 mg/ml streptomycin . U937+DC-SIGN medium was additionally supplemented with 2 mM GlutaMAX , 10 mM HEPES , and 2-mercaptoethanol . All cells were incubated in the presence of 5% CO2.All viruses were propagated in C6/36 ZIKV H/PF/2013 viral stocks were obtained from Michael S. Diamond . ZIKV PRVABC59 viral stocks were obtained from the Centers for Disease Control and Prevention . DENV strains used in the polyclonal neutralization panel  were obtained from natural isolates maintained in the laboratory of Aravinda M. de Silva. DENV strains used in the monoclonal antibody panel  were obtained from infectious clones in the laboratory of Ralph S. Baric .Deidentified human DENV immune sera and plasma were collected from individuals with naturally acquired DENV infections confirmed via serology. All donations were collected in compliance with the Institutional Review Board of the University of North Carolina at Chapel Hill (protocol 08-0895). Deidentified human immune sera previously collected from the Pediatric Dengue Vaccine Initiative were also used.Monoclonal antibodies were purified from hybridomas (1M7 and 1N5) or synthetically generated by Lake Pharma  from published sequences ; the latter are available upon request.2 and MgCl2. The virus-antibody mixtures were incubated for 45 min in a 96-well plate at 37°C. Following this incubation, 5 × 104 cells were added and the infection was allowed to proceed for 2 h at 37°C. The volume of medium in each well was increased to 200 µl, and the cells were returned to 37°C for a total of 24 h. After 24 h, the cells were fixed in paraformaldehyde, permeabilized, blocked with normal mouse serum in permeabilization buffer, and stained with Alexa Fluor 488-conjugated  4G2 antibody. Unbound antibody was washed off, and cells were resuspended in Hanks’ buffered salt solution  supplemented with 2% fetal bovine serum. Assays were performed twice and in duplicate. Samples were read on a Guava easyCyte 5HT flow cytometer (Millipore) as previously described by our group  fetal bovine serum and was supplemented with 2 mM CaClur group .2, at which time they were fixed with paraformaldehyde, permeabilized, blocked with normal goat serum  in permeabilization buffer, stained with 4G2 primary antibody followed by secondary horseradish peroxidase (HRP)-conjugated anti-mouse IgG , washed again, and developed with TrueBlue peroxidase substrate .Neutralization in Vero-81 cells was assessed by serially diluting the monoclonal antibodies 10-fold and mixing with approximately 150 focus-forming units of virus. Dilution medium contained reduced (2%) fetal bovine serum. The virus-antibody mixtures were incubated for 1 h in a 96-well plate at 37°C and then transferred to a monolayer of Vero-81 cells in a 96-well plate. Following a further 1-h incubation at 37°C, the monolayers were overlaid with Opti-MEM  containing 2% fetal bovine serum and 1% (wt/vol) carboxymethyl cellulose . Infected plates were incubated for 2 days at 37°C with 5% COp-nitrophenyl phosphate tablets , and signal was read at 405 nm.High-binding Microlon 600 96-well plates  were coated with 100 ng of 4G2 and 2H2 antibody in 0.1 M carbonate buffer, pH 9.6, overnight at 4°C. Unbound antibody was rinsed with wash buffer , and wells were coated with blocking buffer (TBS plus 0.05% Tween) for 1 h at 37°C. Virus was diluted in blocking buffer at a concentration sufficient to result in approximately equal reactivity with a human cross-reactive control serum and was added to the plate for 1 h at 37°C. Unbound virus was rinsed in wash buffer, and 1 µg of each primary antibody (or the control serum) diluted in blocking buffer was added to the plate for 1 h at 37°C. Unbound primary antibody was rinsed in wash buffer, and alkaline phosphatase-conjugated goat anti-human IgG antibody  at 1:2,500 in blocking buffer was added to the plate for 1 h at 37°C. Unbound secondary antibody was rinsed in wash buffer, the plate was developed with SigmaFast −/− and Ifngr−/−) on a C57BL/6 backbone were used in a single experiment. On days −1 and 9 postinfection, mice received either PBS (mock and virus only) or 10 µg EDE1 C10 antibody (virus with antibody) in a 100-µl dose delivered intraperitoneally. On day 0, mice received either PBS (mock) or 100 FFU of ZIKV H/PF/2013 (virus only and virus with antibody) in a 10-µl dose delivered subcutaneously in the hind left footpad  or two (mock) 5-week-old type I/II interferon receptor-knockout mice  equation of Prism 6 . Reported values were required to have: at least 5,000 recorded events per data point (in the case of the U937+DC-SIGN assay), an R-squared value of greater than 0.75, a Hill slope value of at least 0.7 for monoclonal antibodies and 0.5 for sera, and an EC50 within the range of the assay. Variation between groups was measured by one-way analysis of variance (ANOVA) with a Bonferroni post hoc test. P values of less than 0.05 were considered statistically significant.When analyzing neutralization assays, antibody and serum concentrations were logAbsorbance signals for each virus group in the binding assay were multiplied or divided such that the signal for that virus against the common control serum was set to 1. Each assay was run singly with technical duplicates. Means and standard deviations were calculated in Prism 6 .Survival rates in the animal experiment were analyzed using the log rank (Mantel-Cox) test in Prism 6 for Windows . The virus-with-antibody cohort was compared to the virus-only cohort.Figure S1 KJ776791.1 and KU501215.1, respectively). Contact residues are visualized as spheres, and disordered residues are visualized as sticks (EDE1 and EDE2 epitopes on ZIKV envelope. Location of the EDE1 (C8) (A), EDE1 C10 (B), and EDE2 B7 (C) epitopes on the ZIKV virion. Color coding is based on the infectious clones used in the text with ZIKV H/PF/2013 and ZIKV PRVABC59 , DENV-2 (B), DENV-3 (C), and DENV-4 (D) sera against ZIKV H/PF/2013 in a U937+DC-SIGN assay. Download Figure S2, TIF file, 0.1 MBFigure S3 Neutralization curves of DENV secondary sera with ZIKV H/PF/2013. Neutralization curves for all DENV secondary sera against ZIKV H/PF/2013 in a U937+DC-SIGN assay. Download Figure S3, TIF file, 0.1 MBMovie S1 Three-dimensional antigenic cartography of DENV primary and secondary immune sera. Antigenic cartography of all sera based upon the neutralization of DENV-1, -2, -3, and -4. Sera are colored according to either their DENV reactivity classification  (left) or their ability to neutralize ZIKV (right). Download Movie S1, MP4 file, 0.1 MBMovie S2 Three-dimensional antigenic cartography of DENV secondary immune sera. Antigenic cartography of all DENV secondary sera based upon the neutralization of DENV-1, -2, -3, and -4. Sera are colored according to their ability to neutralize ZIKV. Note that among the secondary sera, there is no distinct clustering of ZIKV-neutralizing versus ZIKV-nonneutralizing sera. Download Movie S2, MP4 file, 0.1 MBTable S1 KJ776791.1 and KU501215.1, respectively). Numbering is based on sequence of ZIKV envelope. Yes, identified contact residue; ?, contact status unknown (structure was too disordered to make a determination)  .Table S1, DOCX file, 0.1 MBTable S2 Characteristics of human plasma and sera used in neutralization assays. List of the human serum samples with the location of infection and, if known, the approximate date of the infection and sample collection.Table S2, DOCX file, 0.1 MB


Ascobolus immersus suggest that DNA methylation may suppress recombination rates and/or alter its distribution across the genome; however, these patterns appear complex, and more direct inquiries are needed. Unlike other organisms, Drosophila only have one known DNA methyltransferase, DNMT2, which is expressed in the ovaries and historically has been thought to be responsible for limited genomic DNA methylation. To test for a role of DNMT2 on the frequency and distribution of recombination, I compared recombination rates between Dnmt2 −/− and Dnmt2 +/− Drosophila melanogaster individuals in two euchromatic regions and one heterochromatic region across the genome. I failed to detect an altered pattern of recombination rate in the absence of DNMT2 in all regions surveyed, and conclude that other epigenetic effects are regulating recombination initiation in Drosophila.Epigenetics is known to be involved in recombination initiation, but the effects of specific epigenetic marks like DNA methylation on recombination are relatively unknown. Studies in Arabidopsis and the fungus  Transcripts of Dnmt2 were particularly enriched during early stages of embryonic development; expression in adult flies was limited to female ovaries, and there was no activity in male testes  D. melanogaster. I did not detect any change in recombination rate or distribution in the absence of Dnmt2 and conclude that other epigenetic factors are determining sites of recombination events in Drosophila.Therefore, to determine whether there is a detectable effect of DNA methylation and/or the DNA methyltransferase Dnmt2D. melanogaster  p-element excision line 99Dnmt2(D. melanogaster Zim29 to generate variability to score recombination events. (B) F1 females were crossed to a D. melanogaster chromosome 2L deficiency line over a balancer, Df(2L)BSC826/SM6a . F2 females were collected, and females carrying the SM6a balancer were identified by the curly wing phenotype and discarded.  The remaining F2 females  were crossed to wild-type males (Zim29) in single pair crosses, allowed to lay eggs, and subsequently genotyped after larvae appeared. Undesired genotypes (see section Scoring recombination for genotyping methods) were discarded, and F3 progeny were collected from remaining vials. Recombination was assayed in these individuals.For all crosses, virgin flies were collected, separated by sex, and aged for 7 d. The crossing scheme  consiste99Dnmt2/ Df(2L)BSC826 genotype were Dnmt2−/−, reverse-transcription polymerase chain reaction (PCR) was completed. For each line BSC826/SM6a, Zim29, 99Dnmt2/ Df(2L)BSC826), I prepped RNA from approximately 25 pooled flies of varying life stages using the QIAGEN RNeasy kit, QIAGEN QIAShredder kit, and QIAGEN DNase kit. For each line, I used genomic DNA, H2O, and a negative control reverse transcription product as controls. For the reverse transcription reaction, the following recipe was used: 2 µL of 10× PCR buffer (15 mM MgCl2), 1.5 µL of 50mM MgCl2, 0.8 µL of 25 mM dNTPs, 1 µL of 50 µM experimental R primer, 1 µL of 50 µM control intron R primer, 0.5 µL of 40U/µL RNase inhibitor, 0.1 µL of 200U/µL MMLV reverse transcriptase, 9.1 µL of diethylpyrocarbonate H2O, and 4 µL of RNA mix + H2O to get to 500 ng of RNA. The reverse transcription program consisted of: 15 min at 42° followed by 5 min at 99°. Then, a PCR was completed with the following recipe: 2.5 µL of 10× PCR buffer (15 mM MgCl2), 1.5 µL of 2mM dNTPs, 1.25 µL, 10 µM experimental F primer, 1.25 µL of 10µM experimental R primer, 0.3 µL of DNA polymerase, 16.2 µL of H2O, and 2 µL of reverse transcription reaction product. The PCR program consisted of an initial denaturing step of 95° (60 sec), three touch-down cycles of 94° (30 sec)−56° (30 sec)−72° (45 sec) each, three touch-down cycles of 94° (30 sec)−53° (30 sec)−72° (45 sec) each, followed by 33 main cycles of 94° (30 sec)−50° (30 sec)−72° (45 sec) each. I used the trade embargo (TREM) gene as a control with the following primers: forward: CAGTAAGTGTGAATCCTGCTTGGTTTGC; reverse: GCATGTCCATAATGTGCTGATGGGATC. The primers used for flanking the Dnmt2 intron were: forward: GGTCTTAGAACTATTTAGTGGCATTGGCG; reverse: TAATTGTGCGCATAAACCGCATTGGC.To ensure that individuals of Flies were collected in 96-well plates and frozen at −20°. DNA was extracted following the protocol of 2 females were genotyped at three loci on the X chromosome (Df(2L)BSC826/Zim29) across the region of interest, and at one locus on chromosome 2L (99Dnmt2/ Df(2L)BSC826  or Df(2L)BSC826/Zim29 . Vials from parents of the desired genotypes were kept, and the F3 progeny were collected; all other vials and their progeny were discarded.To identify crosses with desired genotypes, Fromosome  to ensurosome 2L  to ident3 progeny by genotyping at the same three markers on the X chromosome. The markers on the X chromosome delineate two regions, one of lower recombination (~1.8 cM/Mb) and one of higher recombination (~3.7 cM/Mb). A recombinant was called when an individual fly’s genotype changed from heterozygous to homozygous or vice versa for females, and when the fly’s genotype changed between the possible allele combinations for the males. In total, 1536 F3 control progeny and 1177 F3 experimental progeny were scored for the euchromatic regions.Recombination was scored in F2 females were selected based on exhibiting heterozygosity in this additional region of interest at two markers spanning the centromere of Chromosome 3 (BsoBI enzyme (New England BioLabs) was used with the following conditions: 10 μL of PCR product, 1.2 μL of CutSmart Enzyme buffer, 0.6 μL of BsoBI enzyme, and 8.2 μL of ddH2O, incubated at 37° for 60 min, 80° for 20 min, and 10° for 1 min. The digests were run on 1% agarose gels at 160V for 45 min. Recombination was scored in F3 progeny with the appropriate parental genotypes, a subset of the total F3 progeny described previously. This resulted in a more modest sample size of 200 F3 control progeny and 152 F3 experimental progeny scored for the heterochromatic region.An additional heterochromatic region was added later, and a subset of the same pool of Fmosome 3 . For thet-test . A power analysis was completed using the “pwr” package in R [Statistic:  and identified recombinants in two adjacent euchromatin regions on the X chromosome. Region 1 spans assembly positions 494,471 to 3,240,200 bp (Dnmt2.To identify an effect of the DNA methyltransferase gene Dnmt2+/− (control) individuals and 1177 Dnmt2−/− individuals were scored. In Region 1, I identified no significant difference between Dnmt2 −/− and control individuals  mother (Drosophila.Although DNA methylation influences the recombination landscape in fungus ) mother , but thi) mother . As suchDrosophila and other Dnmt2-only systems is a long-debated issue  (The presence of DNA methylation in osophila  may haveDnmt2 on meiotic recombination supports this recent data and points to other epigenetic mechanisms directing recombination in Drosophila. Indeed, there is some evidence that histone modifications in Drosophila may mimic the role of DNA methylation in transcriptional processes in other invertebrates (Drosophila (The finding that there is no effect of the gene osophila .Dnmt2 and Drosophila DNA methylation research has experienced a tumultuous 20 years, although new research appears to settle the question of the existence of DNA methylation in Drosophila (A. immersus and Arabidopsis is one question that remains to be elucidated, but I conclude based on the available results that DNMT2-dependent methylation has no detectable role in Drosophila recombination.Clearly,


Here, we report a facileone-step hydrothermal method for the controlled growth of single crystallineVO2(M/R) nanorods. Through tuning the hydrothermal temperature,duration of the hydrothermal time and W-doped level, single crystallineVO2(M/R) nanorods with controlled aspect ratio can be synthesized inlarge quantities, and the crucial parameter for the shape-controlled synthesis isthe W-doped content. The dopant greatly promotes the preferential growth of (110) toform pure phase VO2(R) nanorods with high aspect ratio for the W-dopedlevel = 2.0 at% sample. The shape-controlled process ofVO2(M/R) nanorods upon W-doping are systematically studied. Moreover,the phase transition temperature (Tc) of VO2 depending onoxygen nonstoichiometry is investigated in detail.Monoclinic VO For instance, Mott field-effect transistor, light modulator andoptical storage medium are potential products based on VO212 with a metal-insulator phase transition(MIT) is a key material for applying to thermochromic smart windows because it exhibitsa reversible structural transformation from an infrared-transparent monoclinic phase(VO2(M1)) at low temperature to an infrared-reflective rutilestate (VO2(R)) at higher temperature than the transition, while maintainingcertain visible transmittance4562 exhibitshysteresis in its phase transition properties and mechanical degradation on passingthrough the MIT because of stresses during the structural changec) of MIT(68 °C) is always too high for the practical application ofVO2-based materialsVanadium dioxide VO plays a 2c of undoped VO2 by synthesizingvarious sized nanostructures2(M1/R) possesses the probability of tuning hysteresis widthfor obtaining a sharper, more reproducible phase transition. Up to now, more than 20compounds of vanadium oxide  (the M1 phase is referred to as the M phase ofVO2 in this study) experiences a fully reversible MIT at the vicinity ofroom temperature (RT). Moreover, low temperature synthetic method has usually generatedVO2(B) nanobelts and subsequently can be transformed toVO2(M/R) by the post-heating treatment, but the nanostructure has been nearlydestroyed16172(M/R) with a shape controlled nanostructure.Nano-materials often exhibit extraordinary physical and chemical properties compared totheir bulk counterparts2 involves electron-correlation-driven (Mott transition)1921c by approx.20–26 °C/at% W for the bulk and by50–80 °C/at% W in nanostructures2425262(M/R) by controlling both the shape of nanostructures and theamount of W dopant could be a good strategy to narrow the hysteresis width whilereducing Tc for obtaining an excellent phase transition property ofVO2-based materials. Of note, systematically experimental investigationof nonstoichiometric effect in VO2 has been insufficient. The phasetransition behavior has been demonstrated to be also sensitive to vanadium or oxygenrelated vacancies, even a deviation in the oxygen stoichiometry by a few percent cancause the lattice structure change and result in several orders of magnitude differencein the resistivity transition or the phase transition temperature shift28c will contribute to the general understanding of the intrinsic MITmechanism in VO2.The ongoing debate associated to the fundamental origin of the phase transition behaviorin VO2(M/R) with desired morphology. It is inspiring to discover that the Wdopant promotes the generation of pure phase VO2(M/R) nanorods with highaspect ratio. Moreover, the effect of oxygen nonstoichiometry on the structural phasetransition and subsequently Tc of VO2 is discussed in detail.In this study, we successfully explored a one-pot hydrothermal method to prepareVOxV1−xO2 with x = 0,0.5, 1.0 and 2.0 at% respectively where the temperature being kept at280 °C but the different duration of the hydrothermal time beingapplied. For the undoped VO2, pure phase VO2(B) isobtained for the duration of the hydrothermal time for 6 h. Byincreasing the duration of the hydrothermal time from 12 to 72 h, thepeak of {011} for VO2(M) (M {011} at around 27.8°) appearsand becomes more significant. However, there always exists the secondary phaseVO2(B) in the final product. Serial SEM images in 2 upon increasing the duration of the hydrothermal time.Products of the metastable VO2(B) are the tangled nanobelts in themorphology for the 6 h-sample. By increasing the duration of thehydrothermal time from 12 to 72 h, VO2(B) nanobelts alwaysexist as partial morphology except the block or snowflake VO2(M). Inconclusion, we could not synthesize pure phase VO2(M) without Wdoping.2(B) nanobelts alwaysexist as partial morphology except the snowflake or rod-like VO2(M)for the duration of the hydrothermal time ≤48 h as shown in2(B) vanish and pure phase VO2(M)(Tc > RT) with uniformly rod-like morphologyis successfully synthesized for the 72 h-sample. Meanwhile, for thehydrothermal samples prepared at 280 °C with W-doped of 1.0 and2.0 at%, pure phase VO2(R) with uniformly rod-like morphology isobtained when the duration of the hydrothermal time ≥48 h and≥12 h respectively as shown in 2(M/R) is exactly free from the existing of the other V-Ocompounds and other VO2 phases, which was reported by our group inthe recently study2 issummarized in By increasing the W-doped level to 0.5 at%, VOxV1−xO2 with x = 4.0,6.0 and 10.0 at% were prepared at 280 °C for 72 h toinvestigate more W dopant on the crystalline phase metamorphic and morphologytransition behavior of the as-obtained products as shown in 2(R) is stillobtained for the 4.0 and 6.0 at% sample. When the W-doped level increases to10.0 at%, VO2(B) nanobelts are grown again besides the main phaseVO2(R). The results indicate a certain doping level of W couldpromote VO2(B) metamorphoses to pure phase VO2(M/R), whichagrees with the previous reports142(B) intoVO2(R) thoroughly. The intensity ratio between the XRD peaks of{110} and that of {101} of VO2(R) prepared at 280 °Cfor 72 h with different W-doped levels is listed in 2 nanowiresprepared at high temperatures by vapor transport31322(R) nanorods increasesfrom nearly 5 to 10 with the increased dopant. Whereas, if the W-doped levelincreases from 4.0 to 10.0 at%, the intensity ratio {110}/{101} decreases from2.5 to 1.1. Meanwhile, the aspect ratio of nanorods decreases with the increasedW dopant as shown in 2(R) is grown for the 10.0 at% sample. The results indicate acertain doping level of W can promote the preferential growth of R {110} and theincreased aspect ratio of VO2(R) nanorods, whereas the excess W wouldrestrain.W2 nanorods (synthesized at280 °C for 72 h) is about 2.5 μm with600 nm in diameter as shown in the low magnification TEM image in 2(R) nanorods is along [001]. The results demonstrate that thepreferential growth of nanorod' growth direction R (110) is responsiblefor the increased aspect ratio of VO2(R) nanorods. It is generallyknown that the greater the d-spacing, the atom arrange more closely on thecrystal plane. For the body-centered tetragonal VO2(R), (110) withthe largest d-spacing contributes to the lowest surface energy for thepreferential growth of VO2 grains. According to the SAED patternshown in the inset of 2(R). The three Bravais lattice points shown in the SAED of theinset of 2(R). Moreover, no fringespacing belongs to tungsten oxides or their derivatives are detected by HRTEM,which confirms the capture of W atoms into the crystal lattice of VO2as mother matrix and the formation of homogeneous solid-solution ofWxV1−xO2.The length of W-doped 4.0 at% VO1). The endothermic and exothermal transition temperature is62.3 and 49.3 °C during heating and cooling cycles respectively.Thus, the phase transition temperature /2) of the undoped micron-sized block andsnowflake-like sample is about 55.8 °C, which is much lower thanthe transition temperature of undoped bulk VO2 (about68 °C) reported by Morin2 (64 °C) reported by Whittakerc,h−Tc,c) ofthe undoped sample is about 13.0 °C.c for the HTh1 synthesizedundoped sample, we directly compare the DSC for this sample by the afterannealing  with that for thehydrothermal undoped one treated at 160 °C for 72 h andafter annealing   process, Tc,h andTc,c is about 59.7 and 47.2 °C respectively.Therefore, the Tc is about 53.5 °C, which is alsolower than the  fabricated undoped one(Tc being c.a 63.0 °C as shown in 2(B) vanish and all of the peaks can be indexed topure phase VO2(M) for the HTh1 synthesized undoped sampleby the after annealing, which is similar to the fabricated one. The inset close-upshows that M (011) peak shifts to low angles when comparing the  synthesized undoped sample with those by the synthesized one, whichindicates the lattice spacing of M (011) increases. Both micron-sized snowflakeand block-like morphologies are observed for the synthesized undoped sample asshown in 2 + Annealing) fabrication process. Thanks to the formationenergies of oxygen vacancies in rutile oxides are very high, the highhydrothermal temperature (280 °C) and reductive hydrothermalatmosphere for the  method may contribute to thegeneration of oxygen vacancies to form nonstoichiometricVO2-δ compared to the process (160 °C), and this would promote the lattice structuraltransition35To study the unusual low TTG) is about 10.4 %, 10.5 % and 9.6 % forthe HTh1 synthesized undoped VOx, undoped VOy and undoped VOzrespectively. The reaction equations for the oxidization of the samples can begiven as follows (1):To determine the oxygen stoichiometry, the thermogravimetric analysis of thesamples was conducted as shown in O and MVOx represent molar mass of oxygen andVOx respectively. When combining the above formulas (2) andexperimental results, we can work out x = 1.96,y = 1.95 and z = 2.00 respectively. The factdemonstrates that oxygen deficiency is formed in the HTh1 synthesizedundoped VO1.96 and  undopedVO1.95, and the precisely stoichiometric VO2.00 isformed in the  undoped sample. Sonand co-workers have synthesized monoclinic VO2 micro- andnanocrystals by optimizing the hydrothermal conditions2.00 microrods is around 68 °C. Usually, theTc of MIT for VO2 is affected by doping, nanoscaling,nonstoichiometry, strain and etc1230381 synthesized undoped micron-sized VO1.96, thereason for the unusual low Tc may be due to the oxygennonstoichiometry. The nano-size effect may be responsible for the relative lowerTc (63 °C) of the synthesized stoichiometricVO2.00 nanostructure.Where M1g), 191 (Ag), 223(Ag), 260 (Ag), 308 (Ag), 338(Ag), 388 (Ag), 437 (Ag), 442(Eg), 499 (Ag), 617 (A1g), and 826(B2g) cm−1 respectively, and theseRaman-active modes are the clear evidence of the existing of VO2(M)belonging to space group −1 (191/223) of the HTh1synthesized undoped VO1.96 is 1.6. When comparing the synthesized undopedVO2.00 with those by the synthesized undopedVO1.95, the intensity ratio decreases from 2.3 to 1.3. H. T. Kimand co-workers have studied Raman spectra for the MIT of the undopedVO2 in detail and deduced the conclusion that the Raman-activeAg modes at 191 and 223 cm−1 wereexplained by the pairing and the tilting of V cations respectively−1 peak suggests the depairing of Vcations and the occurring of the localized structural phase transition undoped VO1.95), and this might cause the transformation from theintrinsic structure of the matrix of VO2(M) to the localized rutilestructure. In addition, the local rutile structure is the structure-guideddomain, which will act as the initial nucleation site for the whole SPTc. However, this origin is still under the debate among theconcerned experts as cited in the literature by Y. Xie et al. for anexample, who pointed out that the atomic structure of isolated W dopant play arole in driving the nearby symmetric monoclinic VO2 lattice towardsrutile phase, resulting in the depression of Tc452(M/R) with controlled morphology weresuccessfully prepared via one-step hydrothermal method. The addition of a certainlevel of W (0.5–2.0 at%) is vital to synthesize the pure phaseVO2(M/R) nanorods. The assured level of W doping can promote thepreferential growth of {110} to form VO2(M/R) nanorods with high aspectratio. It must be emphasized that the unusual low Tc equals to 55.8 and53.5 °C is observed for the nonstoichiometric VO1.96 andVO1.95 in the bulk respectively, and the Tc is63.0 °C for the precisely stoichiometric VO2.00nanostructure. The present study demonstrates an improvement of the phase transitionbehavior and reduces the hindrances for the advanced applications ofVO2-based materials.In this study, pure phase VO2C2O4·2H2O, AR)and vanadium pentoxide  were used as sourcematerial to prepare the vanadium precursor solution. Deionized water(ρ = 18.2 MΩ.cm) was used to prepare allaqueous solutions. Ammonium tungstate hydrate((NH4)5H5[H2(WO4)6]·H2O,AR) was chosen as the W dopant. All of these reagents were used without furtherpurification.Oxalic acid  in molarratio) were directly added to 75 ml deionized water at RT. Then, acertain amount of W dopant was dispersed into the above solution with magneticstirring. After mixing for 1 h, the resulting precursor was transferredinto a 100 mL stainless steel autoclave with polyphenylene cup, thenbeing sealed and maintained at 280 °C for6–72 h. After the autoclave cooling to RT, a dark blueprecipitate was obtained. The product was washed with deionized water andacetone for several times, then centrifuged at 8000 rpm for8 min and dried in vacuum at 60 °C for 6 h.The detail of this part has been described in previous report4)5H5[H2(WO4)6]·H2Owas used as the W dopant, and the reported W-doped content here is based on thequantity of W atoms added in the feed. The sample synthesized by the duration ofthe hydrothermal time for 6 or 72 h is simplified to the 6 or72 h-sample.In this study, under the temperaturerange from 25 to 100 °C under the circulatory heating/coolingcycles. The thermogravimetric analysis (TG) of the samples was conducted on aNicolet 6700-Q50 thermal analyzer under dry air flow in the range of50–650 °C with a heating rate of 5 °Cmin−1.The phase purity of the products was examined by an X-ray diffractometer  in the 2θ range of5–80° with the step of 0.0083° using Cu-Kαradiation (λ = 1.54178 Å). The operatingvoltage and current were kept at 40 kV and 40 mA, respectively.The morphology and dimensions of the products were investigated using a fieldemission scanning electron microscope  under theoperating voltage of 2 kV. A JEOL-2100F instrument operated at200 kV was used to acquire high-resolutiontransmission-electron-microscopy (HRTEM) images and selected area electrondiffraction (SAED) patterns. Raman scattering spectra of the samples wererecorded on a LabRAM HR800 micro-Raman spectrometer using a 532 nmwavelength YAG laser. The phase transition properties depending on thesurrounding temperature of the as-prepared VOHow to cite this article: Chen, R. et al. Shape-controlled synthesisand influence of W doping and oxygen nonstoichiometry on the phase transition ofVO2. Sci. Rep.5, 14087; doi: 10.1038/srep14087 (2015).


EZH2), a histone 3 methyltransferase, is associated with aggressive behavior of many tumors and is a promising target of molecular therapy.Enhancer of zest homolog 2 , and it was significantly associated with HER2 positivity, high proliferative index, and high histologic grade . ER-positive breast carcinoma with low proliferative index (Ki67 < 14 %) showed the lowest expression and triple-negative breast carcinoma showed the highest overexpression of EZH2, 18.5 % (10/54) versus 90.9 % (50/55) . Intriguingly, 88 % (44/50) cases of grade 3 triple-negative breast carcinoma showed uniformly strong EZH2 expression with a multiplicative score of 9. The percentage of EZH2 overexpression in ER-positive breast carcinoma with a high proliferative index or HER2-positive cases were 61.2 and 74 %, respectively. Furthermore, EZH2 expression was significantly elevated in high-grade DCIS compared to benign lesions . However, there is no association between EZH2 expression and the status of histone 3 lysine 27 trimethylation or other clinicopathologic features.Of these cases, 138 (61.1 %) were defined as EZH2-overexpressing with a multiplicative score > 3. EZH2 expression was inversely related to the status of ER and PR (Chi-square, In summary, triple-negative breast carcinoma showed the highest overexpression of EZH2. EZH2 overexpression is associated aggressive pathologic features including high nuclear grade, high proliferative index, and positivity of HER2 of breast carcinoma. Breast carcinoma comprises an extraordinarily diverse group of diseases in terms of histology and clinical behavior and can be classified according to immunophenotypic features. ER-positive breast carcinoma is defined as ≥1 % of tumor cells being positive for ER, and usually these are also positive for PR . Triple-th AJCC TNM staging system. The histologic grade of invasive breast carcinoma was graded according to the WHO criteria of breast carcinoma classification. 20 benign breast lesions comprising of adenosis and usual ductal hyperplasia (UDH), 20 DCIS adjacent to high grade triple-negative carcinoma and 20 DCIS adjacent to low to moderate grade non-triple-negative carcinoma were also assessed for EZH2. This study was approved by the institution review board of Xi Jing Hospital.Two hundred twenty-six invasive breast carcinomas diagnosed between January 1998 and December 2014 were chosen from the archive at department of pathology in Xi Jing Hospital. The cases were reviewed by breast pathologists (S.P.G. and X.L.) and classified based on the World Health Organization criteria for breast carcinoma classification. The clinicopathologic information including patient age, menstrual status, tumor size, tumor stage, and lymph node stages were collected. The TNM staging was determined by the 7The tissue was fixed in 10 % buffered formalin and processed as usual for paraffin embedding. Representative tissue blocks of invasive breast carcinoma were selected by examination of the corresponding hematoxylin & eosin (HE) stained slides and 5 μm thick sections were stained with rabbit anti-human EZH2 antibody. Immunohistochemical staining was performed using the Bond-Max automated immunostainer , with a polymer-based detection system. EZH2 staining results was interpreted independently by two breast pathologists (S.P.G. and X.L.). Expression was assessed using a multiplicative score of intensity and percentage of cells stained for EZH2, where intensity  and percentage  were graded according to the method described previously , 9. A muThirty cases expressing a HER2 protein score of 2+ by IHC were assessed for the presence of HER2 amplification on 4 μm FFPE sections using a standard probe . Cases were regarded as HER2 amplified if the HER2/CEP17 ratio was ≥2.0 or the average HER2 copy number ≥ 6.0 signals/cell [p < 0.05. Data were analyzed using the Statistical Package for the Social Science Version 17.0 (SPSS 17.0).The associations between EZH2 expression and clinicopathologic features of the patients with breast carcinoma were analyzed using Pearson’s chi-square test. Statistical significance was defined as All 226 patients were female with a median age of 47 (range 30–87) years who presented with breast masses. Mastectomy, or breast conserving surgery with negative margins and axillary lymph nodes excision were performed in all patients. Out of 226 cases, 26 were well differentiated invasive carcinoma of nuclear grade 1, 86 were nuclear grade 2, and the remaining 114 were poorly differentiated carcinoma of nuclear grade 3. All cases were invasive carcinoma, not otherwise specific, except ten tubular carcinomas, five mucinous carcinomas which were included in the group of nuclear grade 1. Immunostaining data for ER, PR, HER2 were collected from the archive. 121 cases were ER positive, and 105 cases were negative. 110 cases were PR positive, and 116 cases were PR negative. Out of thirty cases expressing a HER2 protein score of 2+ by IHC, only nine cases showed the presence of HER2 gene amplification. 50 cases were positive for HER2 protein or showed HER2 gene amplification, 176 cases were negative for HER2 according to the results of IHC and/or FISH in total. Furthermore, the tumor were classified into three groups according to tumor proliferation index Ki67 , and the case number was 74, 73 and 79 in each group, respectively. The clinicopathologic information of the cases was summarized in Table p < 0.001) in ER- and PR-negative cases; the frequency of EZH2 overexpression in ER- and PR-positive cases was 42.1 % (51/121) and 43.6 % (48/110), versus 82.9 % (87/105) and 77.6 % (90/116) in ER- and PR-negative cases respectively. Overexpression of EZH2 was significantly higher in HER2-positive cases than HER2-negative, 76 % (38/50) versus 56.8 % (100/176) . EZH2 overexpression was significantly associated with the proliferative index, with 25.7 % (19/74), 68.5 % (50/73), and 87.3 % (69/79) of breast carcinomas having overexpression with a low (Ki67 < 14 %), moderate (Ki67 14 ~ 30 %) or high (Ki67 ≥ 30 %) proliferative index, respectively . The correlation between EZH2 overexpression and nuclear grade was statistically significant, with 38.5 % (10/26), 57 % (49/86), and 69.3 % (79/114) of grade 1, 2, and 3 breast carcinomas having overexpression of EZH2, respectively . Intriguingly, none (0/15) of the specific-type low-grade carcinomas  had EZH2 overexpression. No statistically significant difference was observed for other clinicopathologic features including age, menstrual status, tumor stage (pT), lymph node stage (pN) and expression of H3K27me3. The association of EZH2 expression and clinicopathologic characteristics of breast carcinoma is shown in Table Of 226 cases of breast carcinoma, 209 cases of breast carcinoma showed nuclear immunostaining of EZH2 with variable intensity and quantity. Low grade tubular carcinoma with well formed tubules and mild nuclear atypia Fig.  showed aHER2 gene amplification by FISH. The remaining fifty-five cases did not express either ER or PR nor showed overexpression of the HER2 protein or HER2 gene amplification; these were classified as triple-negative. ER-positive breast carcinoma with a low proliferative index showed the lowest percentage of EZH2 overexpression, whereas triple-negative breast carcinoma showed the highest percentage of EZH2 overexpression, 18.5 % (10/54) versus 90.9 % (50/55), . The percentage of EZH2 overexpression in ER-positive breast carcinomas with a moderate to high proliferative index and HER2 positive groups were 61.2 % (41/67) and 74 % (37/50) respectively. Intriguingly, EZH2 was uniformly strongly overexpressed in 88 % (44/50) of triple-negative breast carcinomas with the maximum multiplicative score of 9. Representative case of triple-negative breast carcinoma showed typical histologic features such as high grade tumor cells arranged in cords and nests with prominent necrosis and pushing border was shown in HE stained section , ER-positive with a moderate to high proliferative index (Ki67 ≥ 14 %), HER2 positive, and triple-negative. Of the 226 cases, 121 were classified as ER positive with at least 1 % positive tumor nuclei in the section. Of these, 54 cases were ER-positive with a low Ki67 index and 67 cases were ER-positive with a moderate to high Ki67 index. Fifty cases were regarded as HER2-positive either by demonstrating overexpression of the HER2 protein using IHC or ion Fig. . The tumion Fig. , c, d, aion Fig. . Strong ion Fig. .Fig. 2TrThe sensitivity and specificity of EZH2 overexpression for detecting triple-negative was 51 and 86 % respectively. EZH2 expression in ER-positive cases with moderate to high proliferative index or HER2 positive breast carcinoma was variable in both intensity and quantity, and 51.2 % (21/41) and 51.4 % 19/37) showed strong EZH2 overexpression with a maximum score of 9 in these two cohorts respectively. No case of ER-positive breast carcinoma with a low proliferative index showed strong overexpression of EZH2 with a score of 9 (Table 7 showed p < 0.001)  was also assessed. Compared with normal or benign hyperplastic lobules and ducts, EZH2 expression was elevated in high-grade DCIS adjacent to triple-negative breast carcinoma  Table . Breast 1) Table , b only 1) Table , e, wher1) Table , f, assoBreast carcinomas are extraordinarily heterogeneous with different clinicopathologic features and outcomes. The statuses of ER, PR and HER2 are important for treatment and prognosis. ER positive and HER2 positive breast carcinoma can benefit from anti-endocrine and anti-HER2 therapy, respectively. However, triple-negative carcinoma shows aggressive behavior but do not benefit from current available anti-endocrine or anti-HER2 targeted molecular therapy –5. TriplIn the present study, we found that EZH2 was significantly overexpressed in triple- negative breast carcinoma and was associated with high tumor cell proliferation, whereas low grade ER-positive breast carcinoma such as tubular carcinoma and mucinous carcinoma showed low EZH2 expression levels. A significant association between EZH2 overexpression with features of aggressive breast carcinoma including high nuclear grade and HER2 positivity was also found.EZH2 expression was previously evaluated in breast carcinomas and was reported to be significantly associated with increased tumor cell proliferation and as a marker of aggressive behavior , 22. Thep < 0.05. The significant association between EZH2 overexpression and HER2 also supports a role of EZH2 in regulation of tumor cell proliferation.We found that EZH2 expression in breast carcinoma was significantly associated with tumor cell proliferation: 87.3 % 69/79) of breast carcinoma with high Ki67 index (>30 %) showed high EZH2 levels, whereas only 25.7 % (19/74) invasive breast carcinoma with a low Ki67 index (<14 %) showed EZH2 overexpression. As EZH2 also regulates cell cycle in addition to its histone H3 methyltransferase function , and EZH/79 of brBCL11A is a novel triple-negative breast carcinoma oncogene, which was expressed in 66.7 % (16/24) of triple-negative breast carcinomas [in situ components [Recently, it was reported that rcinomas . We did rcinomas , 27, we 6.7 % 16/ of triplrcinomas , 27, andmponents , 27. OurThe reason that epithelial cells lose ER and PR without HER2 amplification during the process of malignant transformation in triple-negative breast carcinoma, however, remains unclear. It is reported that EZH2 forms a complex with NF-κB and activates transcription activity of NF-κB in ER-negative MDA-MB-231 cells , 28. As In summary, our study demonstrates that EZH2 overexpression is significantly associated with high grade triple-negative breast carcinoma as well as corresponding DCIS, plus many high-grade HER2-positive breast carcinomas. Because there was a strongly high association between the Ki67 proliferation index and EZH2 expression levels, we argue that EZH2 overexpression likely confers a proliferative advantage to EZH2-high cells which allows these tumor cells to escape cell cycle regulation. Thus, inhibiting EZH2 function may be relevant for treatment of triple-negative breast carcinoma, which currently has no targeted, effective therapy. Perhaps the next step is to assess the utility of EZH2 inhibitors in EZH2-overexpressing triple-negative breast carcinomas. EZH2 is a promising target for prognostication and treatment in the aggressive and otherwise unresponsive triple-negative breast carcinomas.


Bluetongue virus core protein VP6 is an ATP hydrolysis dependent RNA helicase. However, despite much study, the precise role of VP6 within the viral capsid and its structure remain unclear. To investigate the requirement of VP6 in BTV replication, we initiated a structural and biological study. Multinuclear nuclear magnetic resonance spectra were assigned on his-tagged full-length VP6 (329 amino acid residues) as well as several truncated VP6 variants. The analysis revealed a large structured domain with two large loop regions that exhibit significant conformational exchange. One of the loops (amino acid position 34–130) could be removed without affecting the overall fold of the protein. Moreover, using a BTV reverse genetics system, it was possible to demonstrate that the VP6-truncated BTV was viable in BHK cells in the absence of any helper VP6 protein, suggesting that a large portion of this loop region is not absolutely required for BTV replication. BTV particles have three consecutive proteins layers that are organized into two capsids; an outer capsid comprising VP2 and VP5 and an inner icosahedral capsid (core) composed of two major proteins, VP7 and VP3, which encloses the three minor proteins, VP1, VP4 and VP6, in addition to the viral genome. The viral genome consists of 10 linear dsRNA molecules, segment 1 to segment 10 (S1-S10). In addition to 7 structural proteins, BTV genome also encodes 3 or 4 nonstructural proteins  in infected host cells Bluetongue virus (BTV), the etiological agent of Bluetongue disease of livestock, is a member of the in vitro studies in vitroThe catalytic activities of both VP1 and VP4 have been confirmed by a range of Recently, we constructed VP6-deficient BTV strains using a reverse genetics (RG) system and a complementary BSR-VP6 cell line, demonstrating that VP6-deficient mutant viruses were replication-deficient in non-complementary cells  In this study, to further understand the importance of VP6, we assigned Multinuclear nuclear magnetic resonance (NMR) spectra of VP6 to provide insight into the VP6 structure. Based on NMR-derived secondary structure, it was possible to design a series of truncated proteins removing dynamic loop regions. Most importantly, in spite of the essential role of VP6 in the primary replication cycle of BTV, we show that the region between aa 34 and aa 92 of this large loop is not required for viral replication. However the second half of this loop (aa 93–130) plays an essential role in virus replication.22.1BSR cells (BHK-21 subclone) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma) supplemented with 4% (vol/vol) fetal bovine serum (FBS) (Life Technologies). The stable cell line, BSR-VP6 Preparation of dsRNA was as described previously 2.2A bacterial expression plasmid for the fusion of BTV-10 VP6 protein incorporating N-terminal hexahistidine tag (his-tag) was produced by inserting the coding region of S9 into the modified bacteria expression vector, pRSETA-imperial. A region, which includes transcript stabilizing sequence from gene 10 of phage T7, the Xpress™ epitope, and the enterokinase cleavage recognition sequence, was replaced with the thrombin cleavage recognition sequence.Mammalian expression plasmids for the RG system, pCAG-PBTV1VP1, pCAG-PBTV1VP3, pCAG-PBTV1VP4, pCAG-PBTV10VP6, pCAG-PBTV1VP7 and pCAG-PBTV1NS2, were as described previously Modification of VP6 and S9 was generated by site-directed mutagenesis, using the method as described previously Synthesis of uncapped BTV transcripts was performed as described previously 2.3E. coli strain, BL21, was transformed with each VP6 expression plasmid. The transformed cells were incubated in 10 ml of LB broth containing 100 μg/ml of ampicillin (ABPC) at 37 °C for 16 h. After incubation, 1 ml of culture was inoculated to 10 ml of the fresh LB broth containing 100 μg/ml of ABPC and was incubated at 37 °C for 1 h. The expression of his-tagged VP6 was then induced for 5 h with the presence of 0.5 mM of IPTG.An 600 ∼ 0.3–0.5. The cell pellets were transferred from the culture to 1 L of minimal media containing 15NH4Cl and 13C6-glucose in D2O for efficient 15N-13C-labelling and deuteration of VP6. Expression of the his-tagged protein was induced after 5 h by adding 0.5 mM IPTG, and then harvested after an overnight induction period at 28 °C. Note that for the expression of mutated VP6 proteins, an optimized expression protocol based on the double-colony selection method described previously was used For the production of labelled NMR, sample BL21 (DE3) was used. After the transformation, a clone for each protein was selected and amplified in 1 L of LB broth upon reaching ODl-arginine, 50 mM l-glutamic acid, 10 mM DTT and 10% D2O.The cells were collected by centrifugation and lysed by sonication. The his-tagged protein was purified by bench top chromatography using a nickel–nitrilotriacetic acid (Ni–NTA) resin. A second step of size exclusion chromatography was carried out on the previous protein enriched fractions. Protein samples were concentrated to 0.75 mM in 50 mM sodium phosphate pH7.5, 50 mM NaCl, 50 mM 2.41HN, 15N, 13Cα, 13Cβ and 13CO cross peaks were assigned using the double- and triple-resonance heteronuclear three-dimensional NMR spectra and in-house assignment algorithms NMR spectra were collected at 298 K on Bruker DRX600 and DRX800 spectrometers equipped with Z-shielded gradient triple resonance cryoprobes. The chemical shifts of 2.52 for 3 days to allow cytopathic effects (CPE) to appear. Note that as the recovery efficiency should be low due to uncapped transcription in the second transfection, the plaque-purification procedure was not performed after transfection in this study.Confluent monolayers of BSR-VP6 cells were transfected, first with six mammalian expression plasmids (100 ng each), coding VP1, VP3, VP4, VP6, VP7 and NS2, followed by the second transfection with ten BTV T7 transcripts (50 ng each) using Lipofectamine 2000 Reagent (Life technologies) as described previously 2.6To observe CPE, 10 μl of VP6-truncated BTV recovered from transfected BSR-VP6 cells was inoculated to normal BSR cells and the supernatant, containing viruses, was replaced with 1.5% low-melting agar in D-MEM supplemented with 5% FBS. At 2 days post-inoculation, cells were stained with 0.5% of crystal violet.To determine the virus replications, each 100 μl of VP6-truncated BTV recovered from transfected BSR-VP6 cells were once amplified in BSR-VP6 cells (P0), followed by the second (P1) and the third (P2) amplification in normal BSR cells. The virus titer of each collected supernatant (P0 ∼ P2) was determined by plaque assay using BSR-VP6 cells.33.1E. coli. The proteins were expressed well in soluble form (data not shown). The NMR analysis was first performed on the full-length VP6 (329 residues). Sequence specific assignment of NMR spectra of 15N, 13C-labelled VP6 could be completed for over half the protein sequence  only or the complete loop region  of VP6. The third truncated VP6, d3, had a deletion of the second loop region (aa 183–aa 220) and the last mutant VP6, d2.3, lacked both loop regions (aa 34–aa 130 and aa 183–aa 220). To reduce any detrimental effects on the stability of the core VP6 domain, we substituted the first loop in variants d1, d2 and d2.3 with 5 amino acid residues, Gly-Ala-Gly-Ala-Gly (GAGAG). All truncated VP6 proteins were expressed in in, BL21 B. Truncal-arginine, 50 mM l-glutamic acid, 10 mM DTT and 10% D2O. By simple comparison of the sequence specific assignment of WTVP6 on the 1H-15N-HSQC spectrum shown in Protein samples were concentrated to 0.75 mM in 50 mM sodium phosphate pH7.5, 50 mM NaCl, 50 mM 15N, 13C-double labeled sample of d2VP6 was expressed and purified as described above. The sequence assignment of 15N-13C-d2VP6 on the 1H-15N-HSQC spectrum was very similar to that of WTVP6, suggesting that loss of the loop did not affect the overall folding of the protein . To determine whether these truncated VP6 proteins were still functional in BTV replication, the two VP6-truncated viruses, which contained the same deletions in S9 RNA segment as those in his-tagged VP6, were generated . The twoEach virus was recovered in the BSR-VP6 complementing cell line and the profile of the genomic dsRNAs of each mutant virus was analyzed by native-PAGE B. The prTo assess the replication capability of the VP6 mutant viruses in normal BSR cells, 10 μl of VP6-truncated BTV recovered from transfected BSR-VP6 cells was inoculated into normal BSR cells. At 2 days post-inoculation, the presence of CPE was observed A. SurpriTo further confirm viability of d1BTV in normal BSR, the infectious virus titer  and the profile of genomic dsRNAs extracted from infected cells in each virus passage (P0 ∼ P2) were determined B and C. 4Orbivirus genus, is unique among the Reoviridae family in encoding VP6, a protein with nucleoside triphosphatase, RNA binding, and helicase activity in vitroBTV, a member of the Despite decades of attempts to generate an atomic structure of VP6, it has not been possible to crystallize the protein. VP6 protein is highly soluble and can only be precipitated in the presence of up to 90% (wt/wt) of the ammonium sulfate in vitro studies showed that this region of d2VP6 contains ATP-binding/ATPase motif http://www.imtech.res.in/raghava/atpint/), suggests that the 10 residues in d2VP6, which included the 5 linker residues inserted into the truncated loop, may interact with ATP. Therefore, it is conceivable that the insertion of the linker may have created an unexpected ATPase motif that allows d2VP6 to complement for VP6’s ATPase function during BTV replication.One of the truncations, the complete truncation of the first loop (d2), lost the function of VP6 in BTV replication whereas the half truncation of that (d1) still retained the active function. The lack of 38 residues between amino acid position 93 and 130 in VP6 is likely to be critical in BTV replication. Previous Recently, the fourth nonstructural protein of BTV, NS4, was identified The deletion of VP6 was believed to be replication-defective as VP6 protein is essential for the primary replication


Cardiac diffusion-weighted MRI is a non-contrast technique that has the potential to identify changes in tissue microstructure in acute myocardial infarction (MI) in humans and rats ,2. The t3, 3 slices, diffusion TEprep = 105 ms, b = 400 s/mm2, Gdiff = 43 mT/m, three orthogonal DW directions). Diffusion preparation was applied in the most quiescent phase identified by CINE imaging (35 phases). Contrast LGE GRE imaging was performed roughly 15 min post contrast injection . The lateral wall of the most basal slice (furthest from the LAD occlusion) was designated as the remote region. The region of infarction was determined by elevated trADC and signal intensity in LGE (both required μinfarct >μremote + 5σremote). For each pig, the mean trADC's calculated for the infarct and remote regions. The infarct area determined by elevated trADC was compared to the LGE infarct area. Differences in means were tested for significance with a two tailed paired t-test. Cardiac phase mismatch between trADC maps and LGE images was corrected using a nonlinear B-spline registration, whose transform was derived from two phases of CINE closest to the trADC map and LGE.Four chronic MI  mini pigs were scanned on a 3T MR system (Siemens Verio). Before contrast was administered, M1M2 motion compensated [-3 mm2/s and mean remote trADC values of 1.4 ± 0.4 × 10-3 mm2/s (p < 0.05). The location of elevated trADC infarct region agreed well with the LGE infarct area. The mean trADC and mean LGE infarct area was 0.9 ± 0.1 cm2 and 0.8 ± 0.1 cm2, respectively (p = 1). In the infarct regions, wall motion was akinetic.M1M2 diffusion-prepared TSE scans resulted in mean infarct trADC values of 2.4 ± 0.2 × 10In this preliminary study, infarct regions delineated by elevated trADC correlated well with infarct regions defined by protocol matched LGE in area size and location. The roughly 50% increase in trADC values found in the infarct region relative to the remote region was statistically significant. Further histological studies and larger sample size are needed to confirm the origin of the increase in trADC and its relationship with infarcted tissue.NIH/NHLBI RO1 HL38698.


Objective. To investigate changes in the glycated hemoglobin A1c (A1c) level and those in β cell function and insulin resistance in newly diagnosed and drug naive type 2 diabetes patients and to evaluate the relationship between them. Design and Methods. A total of 818 newly diagnosed diabetic individuals who were ≥40 years of age were recruited. The subjects were grouped by A1c values . The homeostasis model assessment (HOMA) was used to evaluate pancreatic β cell function (HOMA-β) and insulin resistance (HOMA-IR). ANOVA, t-tests, and binary logistic regression analysis were used for data analysis. Results. Compared with subjects with A1c values <6.5%, individuals with an A1c of 6.5–7% exhibited an increased HOMA-β index. However, the HOMA-β index was significantly decreased at A1c values ≥7% and further decreased by 9.3% and by 23.7%, respectively, at A1c values of 7-8% and 8-9%. As A1c increased to ≥9%, a 62% reduction in β cell function was observed, independently of age, gender, body mass index (BMI), blood pressure (BP), blood lipids, and hepatic enzyme levels. Meanwhile, insulin resistance was significantly increased with an increase in A1c values. Conclusions. Elevated A1c values (≥7%) were associated with substantial reductions in β cell function.  The hemoglobin A1c (A1c) value is an integrated measure of mean glycemia over the preceding 8–12 weeks and is considered the “gold standard” for monitoring metabolic control in subjects with diabetes [β cell function. However, little is known about the relationship between the A1c level and β cell function, especially in newly diagnosed and drug naive type 2 diabetic patients. This study was performed to investigate the changes in A1c along with β cell function and insulin resistance in newly diagnosed and drug naive type 2 diabetic patients and to evaluate the relationship between them.The main pathophysiological defects responsible for type 2 diabetes mellitus (T2DM) include sitivity . In the ioration . Accumuldiabetes . It has The present work consists of one part of the baseline survey from the Risk Evaluation of cAncers in Chinese diabeTic Individuals: a lONgitudinal (REACTION) study, which was conducted among 259,657 adults, aged 40 years and older, in 25 communities across mainland China from 2011 to 2012 . This stA total of 10,028 subjects were recruited (40 years of age and older) in Shandong province from January to April 2012. Based on previous medical histories and OGTT, we selected 818 newly diagnosed and drug naive type 2 diabetes patients. The exclusion criteria consisted of (1) previously diagnosed hepatic disease, including fatty liver, liver cirrhosis, and autoimmune hepatitis; (2) previously diagnosed diabetes; and (3) any malignant disease. A total of 818 subjects (508 women) were eligible for the analysis.β index was calculated as follows: 20∗fasting insulin concentration (mIU/L)/(FBG concentration (mmol/L) − 3.5) [The demographic characteristics, lifestyle, and previous medical histories were obtained by trained investigators through a standard questionnaire. All subjects underwent a baseline evaluation including body mass index (BMI), waist circumference (WC), and blood pressure (BP). Laboratory evaluations of fasting blood glucose (FBG), fasting insulin, cholesterol, triglyceride, ALT, and AST levels were also performed. OGTTs were conducted in all patients, using a glucose load containing the equivalent of 75 g of anhydrous glucose dissolved in water. The A1c level was measured by high-performance liquid chromatography . The homeostasis model assessment of insulin resistance (HOMA-IR) index was calculated as follows: fasting insulin concentration (mIU/L) × FBG concentration (mmol/L)/22.5. The HOMA-) − 3.5) .β cell function, we divided the subjects into the following five groups according to the A1c values: <6.5%, 6.5–7%, 7-8%, 8-9%, and ≥9%.Diabetic patients who were diagnosed based on the 1999 World Health Organization (WHO) criteria (FBG ≥ 126 mg/dL (7.0 mmol/L) and/or 2hPG ≥ 200 mg/dL (11.1 mmol/L))  were ideβ cell function/insulin resistance in three models. The following three models were constructed: Model 1 = not adjusted; Model 2 = adjusted for age, gender, BMI, and WC; Model 3 = Model 2 plus systolic BP, diastolic BP, cholesterol, triglycerides, ALT, and AST values. A value of P < 0.05 was considered statistically significant. The data were analyzed using the SPSS 16.0 software .Continuous variables with a normal distribution are expressed as the means ± standard deviation (SD), and variables with a nonnormal distribution are presented as medians (interquartile range). Categorical variables are presented as numbers (%). Between-group differences were evaluated with ANOVA. Binary logistic regression analysis was used to estimate the association between A1c levels and We recruited a total of 818 newly diagnosed and drug naive diabetic subjects, including 508 females and 310 males with an average age of 60.4 ± 9.6 years. The subjects were divided into five groups according to their A1c levels. As shown in β index to assess β cell function. As shown in β cell function. By contrast, the HOMA-β index was significantly decreased in individuals with an A1c ≥ 7%. As A1c increased to ≥9%, a 62% reduction in β cell function was observed. We further compared β cell function at different A1c levels in male and female subjects. Impaired β cell function was observed in subjects with an A1c ≥ 8% in both male and female patients; the values were decreased by 26% and 48%, respectively. Furthermore, the HOMA-β index values in female patients were significantly higher than those in male patients with an A1c ≥ 9%.We used the HOMA-We further used the HOMA-IR index to assess insulin resistance in different A1c groups . Insulinβ cell function using three models. We found that patients with A1c values of 8%-9% and ≥9% had a significantly decreased β cell function (odds ratio (OR) = 2.45 and 15.36, resp.). After adjusting for age, gender, BMI, and WC, these two groups still presented increased ORs . After further adjusting for systolic BP, diastolic BP, cholesterol, triglycerides, ALT, and AST, the patients with A1c levels of 8%-9% and ≥9% also showed an increased risk of impaired β cell function .As shown in P = 0.014).Similarly, we analyzed the association between increased A1c levels and insulin resistance using the three models . As expeβ cell dysfunction and decreased insulin sensitivity. Pancreatic β cell dysfunction plays a major role in determining dysglycemia from the onset of diabetes [β cell function of up to 50% at the time of diagnosis, and this value gradually increases with the progression of diabetes [β cell function protection should be a priority starting at the onset of diabetes.The prevalence of diabetes has increased significantly in recent decades and is now reaching epidemic proportions in China. The most recent national survey in 2013 reported that the prevalence of diabetes and prediabetes in Chinese adults was 11.6% and 50.1%, respectively [diabetes , 13. Studiabetes . Increasβ cell function. However, whether this trend is present in newly diagnosed and drug naive diabetes patients remains unclear. Moreover, β cell function changes at different A1c levels, and these changes in newly diagnosed T2DM patients have not been characterized. In this study, we divided the subjects into five groups based on their A1c levels and compared β cell function and insulin resistance at different A1c levels. We found that individuals with an A1c of 6.5–7% exhibited an increased HOMA-β index compared with subjects who had an A1c < 6.5%, indicating that a slight increase in the A1c level may induce increases in insulin secretion, which occur to compensate for rising insulin resistance. However, the HOMA-β index was significantly decreased in patients with A1c levels ≥ 7%; it was decreased by 9.3% in patients with an A1c of 7-8% and by 23.7% in patients with an A1c 8-9%. As the A1c level increased to ≥9%, a 62% reduction in β cell function was observed, which suggests that poor glycemic control may contribute to the decrease in β cell function. These results are consistent with a previous study [β cell function in newly diagnosed and drug naive type 2 diabetes patients, our present finding could have potentially important clinical implications. Attention should be focused on the A1c value to protect β cell function in diabetic patients. The underlying mechanisms of this process may be found in diabetic rodent studies. Sustained hyperglycaemia damages β cell function via several mechanisms such as an increase in oxidative stress, activation of the MAPK pathway, and reduction of the pancreatic and duodenal homeobox factor-1 (PDX-1) function [The A1c is an integrated measure of mean glycemia over the preceding 8–12 weeks, and the ADA has recommended the A1c value as a diabetes diagnosis standard . Previouus study . Becausefunction , 18.β cell function include age, obesity, hypertension, ALT, AST, and dyslipidemia. We adjusted for age, gender, BMI, systolic BP, diastolic BP, ALT, and AST, as well as WC, cholesterol, and triglyceride levels to ensure that our results were more reliable. After adjusting for the above risk factors, the A1c value was still significantly associated with impaired β cell function in patients with A1c levels ≥ 7%; no changes were observed in individuals with an A1c < 7%. To date, no study has detailed insulin sensitivity in Chinese subjects with newly presented type 2 diabetes mellitus stratified by A1c levels. Our study also demonstrated that insulin resistance increased with increasing A1c levels, compared to individuals with an A1c < 6.5%.In addition to hyperglycemia, the traditional risk factors of impaired β cell function and A1c levels during the development of diabetes.Our study also has some limitations. First, our study included only middle-aged and elderly Chinese subjects; therefore, the results might not be applicable to subjects of different ages or ethnicities. Second, we used the HOMA-IR index to evaluate insulin resistance instead of the “gold standard” (euglycemic-hyperinsulinemic clamp techniques). The HOMA-IR index is a mathematical model of the fasting state, and it represents hepatic insulin resistance only; therefore, it cannot reflect insulin resistance accurately. Third, this is only a cross-sectional study. Long-term prospective studies are needed to clarify the association between the changes in β cell function. The A1c value could be used as simple and practical index to evaluate β cell function and direct clinical treatment in newly diagnosed and drug naive type 2 diabetes patients.Elevated A1c levels (≥7%) were associated with substantial reductions in


At 3 days after extraction, the experimental group showed higher collagen density and lower numbers of polymorphonuclear leukocytes in the upper part of socket, as compared to the control group (p < 0.05). Gene expression of interleukin-1β, tumor necrosis factor-α and nuclear factor-κB were also lower in the experimental group than in the control group (p < 0.05). At 8 days after tooth extraction, there were no significant differences in collagen density, number of polymorphonuclear leukocytes and bone fill between the groups. Our results suggest that topical application of rCoQ10 promotes wound healing in the soft tissue of the alveolar socket, but that rCoQ10 has a limited effect on bone remodeling in rats.Accelerating wound healing after tooth extraction is beneficial in dental treatment. Application of antioxidants, such as reduced coenzyme Q10 (rCoQ10), may promote wound healing after tooth extraction. In this study, we examined the effects of topical application of rCoQ10 on wound healing after tooth extraction in rats. After maxillary first molars were extracted, male Fischer 344 rats (8 weeks old) ( Tooth extraction is a common dental treatment. However, it can lead to adverse effects such as bacteremia and excess inflammation. Furthermore, prolonged wound healing is disadvantageous for subsequent prosthodontic treatment after tooth extraction. Therefore, acceleration of wound healing after tooth extraction has potential benefits for dentists. In the wound healing process, production of reactive oxygen species (ROS) is necessary as a defence against bacterial pathogens ,2. HowevCoenzyme Q10 (CoQ10) is a vitamin-like, oil-soluble molecule. Its reduced form (rCoQ10) is an effective fat-soluble antioxidant ,6 and anIn the present study, we hypothesized that rCoQ10 improves inflammation, collagen density and bone fill after tooth extraction. Such conditions may also stimulate tissue regeneration, which is assessed based on expression of fibroblast growth factor-2 (FGF-2) ,12. The Forty male Fischer 344 rats  were housed in an air-conditioned room (23–25 °C) with a 12-h light-dark cycle. They had free access to powdered food  and drinking water. Experimental protocols were approved by the Animal Research Control Committee of Okayama University (OKU-2013410).n = 3), 0.1% experimental group (n = 3), 1.0% experimental group (n = 3) and 5.0% experimental group (n = 4). Under general anesthesia (2%–4% isoflurane delivered in O2 gas), ointment containing rCoQ10 was topically applied to periodontal tissue in rats in the experimental groups   [Rats were randomly divided into four groups; control group  . Controln = 6), 8-day control group (n = 7), 3-day experimental group (n = 7), and 8-day experimental group (n = 7). Rats were randomly divided into four groups; 3-day control group (2 gas) with a dental explorer and extracting forceps . After hemostasis using a cotton ball, rats received topical application of ointment containing 5% rCoQ10  or control ointment without rCoQ10 on the outside of extraction sockets. Ointment with or without rCoQ10 was applied with a cotton ball once a day for 3 or 8 days. Ointment included bee wax (33 g per 100 g ointment) and soybean oil (67 g per 100 g ointment). Applied ointment was wiped off using a cotton ball after 10 min. After 3 or 8 days, animals were sacrificed under general anesthesia with diethyl ether.Left and right maxillary first molars were extracted in all rats under general anesthesia  for 1 day. Animals with remaining palatal roots were excluded. After fixation with paraformaldehyde, maxillary samples were decalcified with 10% tetrasodium-EDTA aqueous solution (pH 7.4) for 2 weeks at 4 °C. Formalin-fixed tissue samples were embedded in paraffin following dehydration with ethanol  and immersion in xylene. Paraffin-embedded longitudinal sections (6 μm) from palatal roots were then stained with hematoxylin and eosin, Mallory’s aniline blue or other stains described below. For histological analysis, the right maxillary molar regions were resected Immunohistochemical staining of FGF-2 and 8-hydroxydeoxyguanosine (8-OHdG) (an indicator of oxidative stress) were performed. A commercial kit  was used to determine the levels of FGF-2 expression. Polyclonal antibodies against FGF-2  and 8-OHdG  were diluted 1:200 in phosphate-buffered saline ,15. ColoA blinded examiner performed the following histological analysis with a light microscope. Three sections stained with hematoxylin and eosin from each rat were selected for the analyses. Number of polymorphonuclear leukocytes was measured at 400× magnification at three sites within 100 μm of the bone surface in the socket as follows : upper pCollagen density was determined in tissue sections stained with Mallory’s aniline blue. Three histological sections from each rat were analyzed under a standard microscope equipped with a digital camera at 20× magnification. Images generated by the camera were transferred to a microcomputer and analyzed using mathematical morphology software  . To analGingival biopsy samples of the left extraction wound were homogenized using a frozen cell crusher . Total RNA was isolated from the gingival biopsy samples using Trizol reagent , in accordance with the manufacturer’s instructions. Isolated RNA was quantified by measuring the absorbance at 260 nm, and purity was determined by the 260/280 nm absorbance ratio. Only samples with a ratio of >1.8 were used . Total RAmplification conditions were as follows: 45 cycles at 95 °C (30 s), 58 °C (30 s), 72 °C (30 s) for MMP-3; 45 cycles at 95 °C (30 s), 58 °C (30 s), 72 °C (30 s) for TIMP-1; 45 cycles at 95 °C (15 s), 54 °C (20 s), 72 °C (20 s) for IL-1β and NF-κB; 50 cycles at 95 °C (30 s), 55 °C (30 s), 72 °C (10 s) for TNF-α; 50 cycles at 95 °C (30 s), 63 °C (60 s), 72 °C (60 s) for HO-1; and 45 cycles at 95 °C (10 s), 54 °C (20 s), 72 °C (10 s) for β-actin. The mRNA levels were calculated in terms of relative copy number ratio of each mRNA to β-actin for each sample.Left maxillae from 8-day rats were scanned . Computed tomography was performed with the following settings: voltage, 90 kV; electrical current, 20 μA; slice thickness, 8.598 μm. After scanning, three-dimensional (3D) models of the maxillae were prepared using a 3D image-analysis system  .t-test was used for statistical comparisons between control and experimental groups. α-levels of 0.05 were considered to be statistically significant.Results are presented as mean values ± standard deviation (SD). Student’s n = 3–4, mean ± SD, μg/g) following rCoQ10 application for 7 days were 3.3 ± 1.2 in the control group, 4.6 ± 1.6 in the 0.1% experimental group, 4.0 ± 0.6 in the 1.0% experimental group, and 5.1 ± 0.4 in the 5.0% experimental group. Total CoQ10 concentrations in the periodontal tissue increased in the 5.0% experimental group. There was a significant difference in total CoQ10 concentration between the control and the 5.0% experimental groups (p < 0.05).Periodontal concentrations of total CoQ10 (p < 0.05) and lower numbers of polymorphonuclear leukocytes (p < 0.05) in the upper part of the sockets, as compared to the control group. In the bottom and the middle parts of the sockets, there were no differences in collagen density or number of polymorphonuclear leukocytes between the experimental and the control groups at 3 days. At 8 days after tooth extraction, osteoid and woven bone were observed in both groups. No significant differences in collagen density and number of polymorphonuclear leukocytes in all parts of the socket between the control and the experimental groups were observed at 8 days.The results for collagen density and number of polymorphonuclear leukocytes in the alveolar socket are summarized in The experimental group also showed greater expression of FGF-2 in the upper part of the socket than in the control group at 3 days . HoweverAfter removing the rats having remaining roots after tooth extraction, the number of rats decreased to 6 per group. There were no differences in bone mineral density, trabecular thickness, trabecular separation and trabecular number between the two groups at 8 days .p < 0.05)  . The expThis is the first study to examine the effects of rCoQ10 on wound healing after tooth extraction. At 3 days after tooth extraction, the clot was mainly replaced with granulation tissue in both groups, and histological findings at 3 days were consistent with the granulation stage of wound healing . The expGene expression of HO-1, IL-1β, TNF-α and NF-κB, which are involved in oxidative stress and inflammation ,21,22, wThe experimental groups exhibited high expression of FGF-2 in the upper part of the socket at 3 days. Previous studies demonstrate that FGF-2 induces wound healing and tissue regeneration . The incOn the other hand, at 8 days after tooth extraction, osteoid and woven bone were observed in the socket. Thus, the histological findings at 8 days were indicative of the callus stage of wound healing. Therefore, healing of bone tissue is more important than that of soft tissue at 8 days. However, there were no bone morphogenetic differences between the experimental and control groups at 8 days. This indicates that topical application of rCoQ10 had little effect on bone remodeling in the socket. A previous study reported that CoQ10 suppresses osteoclast differentiation and enhances bone-forming osteoblast differentiation . HoweverCoQ10 is a safe material with very low toxicity. Previous studies have shown that oral application of CoQ10 is effective at improving periodontitis . AnotherIn this study, because rCoQ10 is fat-soluble, we used ointment containing soy bean oil. Previous studies have been shown that there is an inter-relationship between periodontal tissue and fat consumption ,31,32,33Our study has some limitations. First, the study was only performed for 8 days. In rats, it takes about 1 month for the socket to heal completely. Therefore, extraction wound healing in rats is generally studied for more than 8 days ,35,36. HTopical application of rCoQ10 promotes wound healing of the soft tissue in the upper part of the alveolar socket, but the effects of rCoQ10 on bone remodeling were low in rats.


Photoautotrophs (εlipid/water between −149 and −264‰) and chemoautotrophs (εlipid/water between −217 and −275‰) produce fatty acids depleted in D. Fatty acids become, in general, enriched by between 4 and 46‰ with growth phase which is minor compared to the influence of metabolisms. Therefore, the D/H ratio of fatty acids is a promising tool to investigate community metabolisms in nature.Microorganisms are involved in all elemental cycles and therefore it is important to study their metabolism in the natural environment. A recent technique to investigate this is the hydrogen isotopic composition of microbial fatty acids, i.e., heterotrophic microorganisms produce fatty acids enriched in deuterium (D) while photoautotrophic and chemoautotrophic microorganisms produce fatty acids depleted in D compared to the water in the culture medium (growth water). However, the impact of factors other than metabolism have not been investigated. Here, we evaluate the impact of growth phase compared to metabolism on the hydrogen isotopic composition of fatty acids of different environmentally relevant microorganisms with heterotrophic, photoautotrophic and chemoautotrophic metabolisms. Fatty acids produced by heterotrophs are enriched in D compared to growth water with ε In orde13C, 15N, 18O) for the label to be incorporated by active microorganisms into biomarkers like DNA, RNA and lipids. The labeled biomarkers can be then purified and identified  can be used for identification of methanotrophs due to the fact that they produce lipids depleted in 13C compared to other microorganisms  can be used to identify specific microorganisms which utilize particular substrates  of fatty acids reflect the central metabolism of microorganisms  and of growth phase  on the δD-values of fatty acids of different microorganisms which are mainly derived from aquatic environments with salinities ranging from almost freshwater or open marine. Thiocapsa roseopersicina (DSM-217) and Halochromatium glycolicum (DSM-11080) were grown on a modified Pfenning's medium containing 0.34 g NH4Cl, 0.34 g KH2PO4, 0.5 g MgSO4 × 7 H2O, 0.34 g KCl, 0.25 g CaCl2 × 2 H2O, 1.5 g NaHCO3, 0.4 g Na2S × 9 H2O, 0.02 g vitamin B12, and 1 mL trace element solution SL-12 . The cultures were incubated in air tight bottles at 25°C and a light intensity of ~1300 lux of a halogen lamp .The photoautotrophic purple sulfur bacteria Pfennig,  per liteThiobacillus denitrificans (DSM-12475) was grown on a medium containing 2 g KH2PO4, 2 g KNO3, 1 g NH4Cl, 0.8 g MgSO4 × 7 H2O, 2 mL trace element solution SL-4, 5 g Na2S2O3 × 7 H2O, 1 g NaHCO3, 2 mg FeSO4 × 7 H2O, 1 mL 0.1 N H2SO4 per liter of distilled water (pH 7.0). The trace element solution SL-4 contained 0.5 g EDTA, 0.2 g FeSO4 × 7 H2O, 0.01 g ZnSO4 × 7 H2O, 3 mg MnCl2 × 4 H2O, 0.03 g H3BO3, 0.02 g CoCl2 × 6 H2O, 1 mg CuCl2 × 2 H2O, 2 mg NiCl2 × 6 H2O, 3 mg Na2MoO4 × 2 H2O per liter of distilled water. T. denitrificans cultures were incubated at 25°C.The chemolithoautotrophic sulfide oxidizer Isochrysis galbana (CCMP 1323) was grown at a salinity of 35.5 practical salinity units (psu) as previously described .The photoautotrophic eukaryote Pseudomonas str. LFY10 obtained from Prof. Matt Sattley  was grown on an ammonium–glucose medium and an ammonium–acetate medium. The ammonium–glucose medium contained: 5 g glucose, 0.2 g MgSO4 × 7 H2O, 5 g NaCl, 1.3 g (NH4)2HPO4, 1 g KH2PO4, 2 mL trace element solution SL-4 per liter of distilled water (pH 7.1). The ammonium–acetate medium contained 5 g Na–acetate, 0.2 g MgSO4 × 7 H2O, 5 g NaCl, 1.3 g (NH4)2HPO4, 1 g KH2PO4, 2 mL trace element solution SL-4 per liter of distilled water. The pH was adjusted to 7.1. The cultures were incubated at 25°C.A recently isolated heterotrophic After inoculation cell densities, and thereby growth phase, were monitored regularly by flow cytometry  or by measuring the optical density (OD) at 600 nm with a spectrometer . Culture samples, including water samples for hydrogen isotope analysis, were taken during exponential, stationary and death phase. The water was stored with no headspace in 12 mL exertainers (Labco) in the dark at ~5°C until analysis. Biomass was collected by filtration over a 0.7 μm GF/F filter  or by centrifugation.2O (1/1), twice with MeOH, and three times with dichloromethane (DCM) (two volumes each). The extracts were combined and bidistilled H2O (six volumes) was added. The combined solutions were mixed and allowed to separate in a MeOH/H2O and DCM phase, the DCM phase was removed and collected. The MeOH/H2O layer was re-extracted twice with 3 mL DCM. The combined DCM layers were dried over a Na2SO4 column and the DCM was evaporated under a stream of nitrogen. The dried extracts were stored at 4°C before further workup.Bacterial biomass and filters were freeze dried and hydrolyzed directly by base hydrolysis with four volumes of 1 N KOH in methanol (MeOH) solution under reflux for 1 h at 190°C. Afterwards the pH was adjusted to 4 with 2 N HCl/MeOH (1/1) and the liquid was transferred into a separatory funnel. The residues were further extracted once with MeOH/H3–MeOH) for 5 min at 60°C. Then H2O and DCM were added (1 mL each). The aqueous layer was washed three times with 1 mL DCM, and the combined DCM fractions were cleaned over a Na2SO4 column and dried under a stream of nitrogen. In order to obtain a fatty acid fraction, the methylated extract was separated over an aluminum oxide (AlOx) column, eluting the methylated fatty acids with DCM.Fatty acids were methylated with a boron trifluoride–methanol solution (BF2/ether (60 mg/mL) were added to the fatty acids and incubated at 40°C overnight. After adding hexane, the iodine was deactivated by addition of a 5% aqueous solution of Na2S2O3. The aqueous phase was washed twice with hexane. The combined hexane layers were cleaned over a Na2SO4 column and dried under a stream of nitrogen. The dried extracts were stored at 4°C before analysis.In order to identify the position of double bonds in unsaturated fatty acids, the methylated fatty acids were derivatized with dimethyldisulfide (DMDS)  using a Agilent 7890A GC instrument and Agilent 5975C VL mass selective detector (MSD).The fatty acid fractions were analyzed by gas chromatography (GC) using an Agilent 6890 gas chromatograph with a flame ionization detector (FID) using a fused silica capillary column (25 m × 320 μm) coated with CP Sil-5 (film thickness 0.12 μm) with helium as carrier gas. The temperature program was the following: initial temperature 70°C, increase of temperature to 130°C with 20°C min−1). The GC temperature program was 70 to 145°C at 20°C min−1, then to 200°C at 4°C min−1 320°C for 15 min. Eluting compounds were converted to H2 at 1420°C in a ceramic tube before introduction to the mass spectrometer. An internal standard, squalane (δ D = −170‰), was co-injected with each fatty acid sample in order to monitor the precision of the measurements. The average δD of the internal standard was −1.7 ± 3‰. The δD of the individual fatty acids was measured in duplicates and corrected for the added methyl group.Hydrogen isotope analysis of the fatty acids was performed by GC thermal conversion isotope ratio monitoring MS (GC/TC/irMS) using an Agilent 7890 GC connected via Thermo GC Isolink and Conflo IV interfaces to a Thermo Delta V MS according to Chivall et al. . Sampleslipid/water following:The hydrogen isotopic composition of fatty acids compared to water was expressed as ε+ XL mass spectrometer following the procedure described by M'boule et al.  using a Thermo Finnigan TC/EA interfaced via a Thermo Finnigan ConFlo III to a Thermo Finnigan Deltae et al.  with NorThe δD-value of dry sodium acetate was also determined by EA/TC/irMS. NBS 22 mineral oil (δD = −120.0‰) and polyethylene IAEA-CH-7 (δD = −100.3‰) were used as standards. The δD-value of acetate was measured in triplicate.2O was added and the water layer was washed three times with hexane. The hexane was evaporated and the acetylated glucose was dissolved in ethyl acetate and analyzed by GC, GC–MS and GC/TC/irMS similar to the fatty acids. The δD-value of the non-exchangeable hydrogen of glucose was calculated by correcting for the added acetyl groups. The acetylated glucose was measured five times.The hydrogen isotopic composition of the non-exchangeable hydrogen of the glucose substrate was determined by analyzing the acetylated derivative of glucose. For this, glucose was acetylated using 0.5 mL acetic anhydride with a pre-determined δD-value and 0.5 mL pyridine for 3 h at 75°C. Afterwards, 1 mL distilled HStatistical analysis was done via One-Way ANOVA test with SigmaPlot Version 12.0 .Thiocapsa roseopersicina was grown photoautotrophically under anoxic conditions with CO2as sole carbon source and hydrogen sulfide as electron donor. Fatty acids that were present in all growth phases are C16:1ω 7, C16:0, and C18:1ω 7. In addition, during the death phase traces of C12:0, C14:0, and C17:1ω 7 were also detected. The C18:1 fatty acid (30-49%) was the most abundant fatty acid in all growth phases followed by C16:1 (21-32%) and C16:0 (~21%). Minor fatty acids were C12:0, C14:0, and C17:1 fatty acids with abundances between 7 and 14%  and C16:1 the least abundant (7-9%)  Table . In addi%) Table  and εlip0‰ Table , Table 1Isochrysis galbana was grown photoautotrophically under oxic conditions with CO2as sole carbon source  of all photoautotrophs, Thiocapsa roseopersicina, Halochromatium glycolicum, and Isochrysis galbana, is relatively similar and ranged from −187 to −233‰. The oxygenic photoautotroph I. galbana reduce NADP+ to NADPH using H2O as electron donor , the D/H ratio of water will not be substantially affected. The similar fractionation of anoxygenic and oxygenic photoautotrophs suggest that the steps leading to the production of NADPH under both conditions have similar fractionation factors. The same has been observed for various other algae and an anoxygenic, photoautotrophic bacterium  cycle, the pentose phosphate pathway  when grown on succinate. Like acetate, succinate is metabolized via the TCA cycle which may thus lead to production of NADPH enriched in D. Thus, for heterotrophs, growth on substrates that are directly involved in the TCA cycle apparently leads to a depletion in D of all fatty acids, including C16:0, when shifting from exponential to stationary phase. In contrast, growth on substrates that are involved in the pentose phosphate pathway, like glucose, apparently leads to enrichment in D of fatty acids in stationary phase compared to exponential phase.Zhang et al.  already lipid/water-values of the different fatty acids with the same initial biosynthetic pathways fall in a similar range and differences observed are due to the addition and/or removal of hydrogen atoms related with chain length and degree of unsaturation.Although growth phase changes result in changes in D/H ratios of fatty acids these are relatively minor compared to the impact of metabolism on fatty acid D/H ratios. Therefore, changes in growth phase in microbial communities in the environment can be considered to have relatively minor impact on the overall isotopic signal of the fatty acid pool of the whole community. Additionally, lipid identity does not play a major role on the hydrogen isotopic composition of fatty acids compared to metabolism. The εC16:0/water-values of different microorganisms grown as either photoautotroph, chemoautotroph or heterotroph . Chemoautotrophically grown microorganisms produce fatty acids with a mean εC16:0/water-value of −298‰ and ranging between −264 and −345‰  which is depleted by ca. 100‰ relative to fatty acids produced by photoautotrophs. Heterotrophically grown microorganisms typically show an enrichment in D of the lipids relative to the water . The differences between the different metabolisms are significant (P < 0.001) and should allow for the characterization of the dominant metabolism of microbial communities in the environment by analyzing the isotopic difference between C16:0 fatty acid and water. However, several issues should be kept in mind. Relatively few microorganisms have been analyzed for their fatty acid hydrogen isotopic composition, and although most of them fit the general pattern it is possible that exceptions to this pattern arise once other microorganisms are analyzed in the future. In the environment all microorganisms producing the C16:0 fatty acid will contribute to the fatty acid pool in varying amounts depending on the amount produced in the cell. Therefore, the εC16:0/water-value in an environmental sample will not so much represent the average εlipid/water-value of the whole microbial community but will also be affected by the relative abundance of the C16:0 fatty acid in the various contributing microbes. For instance, the pink streamer (PS) communities in Yellowstone National Park are dominated by chemoautotrophic Aquificales which mainly produce C20:1, C21:cyc with C18:0 and C16:0 fatty acids occurring only in minor amounts, while C16:0 fatty acid is abundant in members of the co-occurring genus Thermus which are heterotrophic. Thus, the D/H ratio of the C16:0 fatty acid reflects heterotrophy rather than chemoautotrophy in these PS communities despite the dominance of chemoautotrophs  εC16:0/water-value is a promising approach, possibly combined with the εlipid/water-value of other fatty acids and potentially biomarker lipids with a more restricted origin.Nevertheless, in order to obtain an idea of the dominating metabolism of a microbial community in the present, as well as in the past, the εThe hydrogen isotopic composition of fatty acids produced by a range of different microorganisms depends on the general metabolism expressed during growth. Both photoautotrophs and chemoautotrophs produce fatty acids strongly depleted in D, while heterotrophs produce fatty acids enriched in D compared to the growth medium. Fatty acids produced during different growth phases become somewhat enriched in D with increasing age of the culture in most of the experiments described here. Thus, growth phase likely plays a minor role in controlling the D/H ratio of fatty acids relative to metabolism in the natural environment. Our results suggest that an overall characterization of community metabolism via the D/H ratio of fatty acids is potentially feasible.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.