We have observed a decrease in MΦ number paralleled by an increase in the proportion of M2 MΦ. To understand this phenomenon, we then evaluated, in vitro, the effects of X-rays on the MΦ phenotypes and survival. We have found that X-ray radiation failed to modify the phenotype of the different MΦ. However, M1 MΦ were more sensitive to ionizing radiation than M2 MΦ, both in normoxia and in hypoxia, which could explain the in vivo observations. To conclude, M2 MΦ are more radioresistant than M0 and M1 MΦ and the present study allows us to propose that X-ray radiotherapy could contribute, along with other phenomena, to the increased density in the protumor M2 MΦ in GB.In some highly inflammatory tumors, such as glioblastoma (GB), macrophages (MΦ) represent the most abundant population of reactive cells. MΦ, initially denoted as M0 MΦ, can be polarized into two further phenotypes: the antitumor M1 MΦ, and the protumor M2 MΦ. The three phenotypes can reside simultaneously in the tumor mass and various external factors may influence MΦ polarization. Radiotherapy is a common modality of cancer treatment aiming to target tumor cells. However, the specific effects of X-ray radiation on the inflammatory cells are, so far, controversial and not fully understood. In the present investigation, we have first analyzed,  Glioblastoma (GB) are the most frequent and aggressive form of the primary malignant brain tumors in adults . ConventGB are highly heterogeneous tumors in which various cell types coexist, such as tumor cells, endothelial cells, fibroblasts and different cell types of the immune system . Of all Once differentiated, M0 MΦ exhibit a considerable degree of plasticity and can be polarized into two well established functional phenotypes, termed M1 and M2 MΦ . M1 MΦ, Ionizing radiations (IR), such as X- and γ-radiations, can also influence the tropism of MΦ in the tumor by an increased production of chemokines at the origin of MΦ migration. In support of the above, studies have demonstrated that irradiation promotes the recruitment of MΦ in brain tumors approximately 20 days post-radiation  by increin vivo, in various tumor types , was cultured in Roswell Park Memorial Institute media (Sigma-Aldrich) supplemented with 10% fetal calf serum (Eurobio), 1μg/ml penicillin/streptomycin  and 2mM of glutamine  at 37°C in a humidified atmosphere and in mycoplasma free conditions.Bone marrow-derived MΦ were obtained from mice  and were isolated from femora and tibiae by flushing the bones with 1ml of Iscove's Modified Dulbecco's Media  containing 60% Fetal Clone II  and 1μg/ml P/S. The marrow was passed through a 70μm strainer and MΦ (M0) were selected and cultured in IMDM enriched with 15% FCII, 1μg/ml P/S, 10 ng/ml recombinant mouse macrophage colony-stimulating factor  and 10 ng/ml recombinant mouse Fms-related tyrosine kinase 3-ligand  at 37°C in a humid atmosphere. M1 MΦ were obtained by culturing cells in 1g/l glucose Dulbecco's Modified Eagle Medium  supplied with 15% FCII, 1μg/ml P/S, 2mM Gln (Sigma-Aldrich), 100 ng/ml LPS (Sigma-Aldrich) and 10U/ml recombinant mouse interferon-gamma . M2 MΦ were obtained by culturing cells with 1g/l glucose DMEM supplemented with 15% FCII, 1% P/S, 2mM Gln (Sigma-Aldrich) and 50 ng/ml recombinant mouse interleukine 4 .2/30% O2) and GL261 cells were injected [1.105 cells in 3μl in 2mM Gln/phosphate buffer saline (PBS)] in the right caudate-putamen.Tumor models consist of an orthotopic injection of GL261 cells in C57/Bl6 mice . The animal investigations were performed under the current European directive (2010/63/EU). The license to investigate was given to SV (14-55) in authorized housing and laboratories (B14118001) and with the permission of the regional committee on animal ethics (N/04-01-13/04/01-16). Mice were operated under anaesthesia (induction in 5% and maintenance in 2% of isoflurane in 70% NO2) cells were cultured in a humidified 5% CO2/air atmosphere. Moderate  [2, the O2 level found around necrotic areas of GB) hypoxic cells were cultured in a humidified 5% CO2/balance N2 gas mixture in a hypoxic chamber  at 37°C. For radiation treatment, M0 MΦ were cultured either in normoxia or in hypoxia 6h before radiation. M1 and M2 MΦ were activated for 24h and then cultured in normoxia or hypoxia 6h before radiation with their respective conditioning media. The hypoxic culture medium was equilibrated for 30 min with the gas mixture contained in the hypoxia chamber before add it to cell cultures.Normoxia (20% Od in GB)  and seveAll radiation experiments were performed on the XRad225Cx .in vivo experiments, the radiation of ipsilateral hemisphere was realized seven days after the implantation of GB cells (tumor volume around 10mm3). Mice were anaesthetized as described above and irradiated thrice with 4Gy dose every two days at a dose rate of 3.3Gy/min. The animals were sacrificed 15 days after implantation of tumor cells for the non-irradiated animals and 7 days and 20 days after the first irradiation for the irradiated animals  at a dose rate of 2Gy/min. After radiation, cells were maintained under normoxic or hypoxic conditions until the end of the experiment.For At the end of the protocol, mice were deeply anaesthetized and were transcardially perfused with a 0.2M phosphate buffer (PB)/4% paraformaldehyde . Brains were removed and placed in 30% sucrose for 48h and 30μm thick freezing microtome sections were realized. Slices were blocked with PBS, 0.5% Triton, 3% bovine serum albumin  for 2h and then incubated overnight with anti-CD68  and anti-CD206  antibodies in PBS, 0.5% Triton, 1% BSA at 4°C. Sections were then incubated with an Alexa-555-conjugated anti-rat  or an Alexa-488-conjugated anti-rabbit  as secondary antibodies in PBS, 0.5% Triton, 1% BSA containing Hoechst 33342 .MΦ were fixed with a 0.2M PB/4% PFA solution. Cells were blocked with PBS, 0.1% Tween, 3% BSA for 30min and the cells were firstly incubated overnight at 4°C with the primary antibody in PBS 0.1% Tween, 1% BSA at 4°C. The following primary antibodies were used: phosphorylated histone H2AX (ser139)  and cleaved-caspase-3 . γH2AX and cleaved-caspase-3 are sensitive markers of DNA double-strand breaks  and apopThe effect of radiation on cell survival was measured by manually counting the cell number 2h, 24h and 72h after radiation. MΦ were fixed as mentioned above and cell nuclei were stained with Hoechst 33342 (10μg/ml).http://imagej.nih.gov/ij/). For immunohistochemistry, MΦ density was determined as the number of positive area for CD68 divided by the total tumor area using an automatic thresholding. The percentage of M2 MΦ was determined by a manual counting of the number of cells that expressed both CD206 (M2 MΦ) and CD68 (MΦ). For immunocytochemistry, the number of cells was manually counted. For γH2AX immunostaining, cells with at least 10 foci in nucleus were considered as positive. The percentage of γH2AX+ cells was determined by the number of γH2AX compared to the total number of cells (Hoechst 33342). The percentage of cleaved-caspase 3+ cells was determined by the number of cleaved-caspase 3+ cells compared to the total number of cells (Hoechst 33342). The presence of micronuclei  and then analyzed by ImageJ software (astrophe ) was assTM flow cytometer  with 20 000 events per determination. The analysis and determination of the cell distribution in each phase of the cell cycle was achieved based on the Kaluza® Flow Analysis software .The cell cycle of MΦ was studied by flow cytometry with the DNA-prep reagents kit according to manufacturer's instructions . Propidium iodide staining was analyzed by the GalliosTM flow cytometer and at least 20 000 events were collected per sample. Data was analyzed using Kaluza® Flow Analysis software.This experiment was performed using Annexin V-FITC Kit (Beckman Coulter), following the manufacturer's protocol. Briefly, cell samples  were washed with cold PBS and cell pellet was resuspended in 1X binding buffer and immediately kept on ice. Annexin V-FITC solution and Propidium Iodide (IP) were added and incubated for 15 minutes on ice in the dark. Subsequently, cells were analyzed by flow cytometry, using GalliosNO measurement in the supernatant of MΦ cultures was performed by the Griess reaction . Each saArg1 activity was determined by a standard colorimetric method in cell lysates as published . Each saSupplementary Materials about proliferation assay and cell debris analyses are available in the online version of the paper.Data are represented by the mean±standard deviation (SD), the circles representing results of individual experiment. Statistical analyses were performed with the JMP® program  and, unless otherwise stated, significances were calculated by the Tukey's HSD test after significant ANOVAs.


SLC2A5 and histone acetylation in the transcribed region are induced by differentiation associated-signals such as glucocorticoids and p44/42 mitogen-activated protein kinase (MAPK) inhibition in small intestinal Caco-2 cells.Expression of the fructose transporter gene Brd4+/− mice during weaning period were analyzed by qRT-PCR.We co-treated with glucocorticoid receptor agonist dexamethasone (Dex) and p44/42 MAPK inhibitor PD98059 (PD) in Caco-2 cells with or without Brd4 small hairpin (sh) RNA expression vector, and the cells were analyzed by qRT-PCR and chromatin immunoprecipitation assays. The small intestine of wild-type mice and SLC2A5. Brd4-protein depletion by shRNA revealed that the association of these proteins on the transcribed region of SLC2A5 promoted gene expression in a Brd4-dependent manner. Expression of small-intestine Slc2a5, but not another intestinal gene sucrase-isomaltase, during weaning period, was significantly lower in Brd4+/− mice compared with wild-type mice.Co-treatment with Dex and PD increased binding of the bromodomain-containing protein-4 (Brd4)–positive transcriptional elongation factor-b (P-TEFb)–RNA polymerase II complex to acetylated histones in the transcribed region of SLC2A5 gene in intestinal Caco-2 cells and in the small intestine of mice during weaning period.Brd4-P-TEFb plays a crucial role in differentiation-associated transcription of SLC2A5 expression in the small intestine.Histone acetylation and the transcription elongation factor Brd4 are important for  •SLC2A5 in human intestinal Caco-2 cells.Brd4 regulates expression of •SLC2A5.Brd4–P-TEFb and acetylated histones bind transcribed region of •Slc2a5 expression during weaning period of mice.Brd4 regulates jejunal  Indeed, previous studies, including our own, have shown that the expression of the hexose transporter, SLC2A5, particularly in the transcribed region of the gene SLC2A5 expression is regulated not only by activation of glucocorticoid receptors (GRs), but also by enhancing the histone acetylation on SLC2A5.Previous studies showed that hormone-induced gene expression, which frequently occurs in differentiating cells, is mediated by epigenetic memories which are acquired modifications on the chromatin such as modifications of the histone tail, including acetylation, methylation and phosphorylation, and the DNA methylation SLC2A5 was enhanced by co-treatment with PD and Dex in Caco-2 cells SLC2A5, are regulated by Brd4-P–TEFb in differentiated cells has not yet been elucidated.Acetylation of histones H3 and H4 in the promoter region of genes facilitates binding of the transcriptional machinery, including transcription factors, co-activators, the SWItch/Sucrose NonFermentable (SWI/SNF) complex, and RNA polymerase II SLC2A5 expression in Caco-2 cells. We also investigated the role of Brd4 in SLC2A5 induction in the small intestine during the suckling–weaning transition using Brd4 heterogeneous gene targeting in mice. Our results in current study suggest that epigenetic regulation via histone acetylation and the Brd4 play vital roles in induction of SLC2A5 expression during the intestinal differentiation.In this study, we examined the possible function of Brd4 downstream of glucocorticoids and p44/42 MAPK inhibition in regulating 22.14 cells/cm2 in 10-cm culture collagen plates  in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal calf serum (FCS), 1% non-essential amino acids , 20 mM HEPES (pH 7.4), 1× antibiotic–antimycotic mixed stock solution , and 2 mM l-glutamate (Invitrogen) at 37 °C in a humidified atmosphere of 5% CO2.Caco-2 cells  were seeded at a density of 0.6×10g) for 2 h. Transfected cells were selected with puromycin  for 7 days and cultured in the same conditions described above. After 2 days, cells had reached approximately 70% confluence and normal Caco-2 and control/Brd4-shRNA-expressing cells were continuously cultured in DMEM containing 10% FCS stripped of glucocorticoids by treatment with charcoal/dextran, with 1 µM Dex (glucocorticoid receptor agonist) and 50 µM PD (p44/42 MAPK kinase inhibitor), or vehicle (DMSO) alone for 48 h.Control short hairpin (sh) RNA- or Brd4-shRNA-expressing Caco-2 cells were constructed by inserting control or Brd4 shRNA into the pSUPERRNAi vector  2.2Brd4 heterozygous allele (Brd4+/−) mice were generated by our group in an NIH animal facility. The mice were derived from C57BL/6J mice and bred with wild-type mice purchased from Japan Oriental Yeast Co. Ltd. . Brd4+/− mice were divided into four groups analyzed at the following times after birth: 10 days , 14 days , 21 days , and 26 days . Animals were genotyped by polymerase chain reaction (PCR) using the following primers: WT forward: 5′-GGACTAGAAACCTCCCAAATGTCTACAA-3′; neo forward: 5′-TGAAGAGCTTGGCGGCGAATGGG-3′; both reverse: 5′-CCTGTGTGCACTTGCTCCCGAGGAGAGA-3′. The PCR conditions for WT were 94 °C for 1 min, and 34 cycles of 94 °C for 20 s, 60 °C for 20 s, and 72 °C for 2 min, and 72 °C for 7 min. The real-time reverse transcription (RT)-PCR conditions for neo were 95 °C for 5 min, and 30 cycles of 95 °C for 10 s, 60 °C for 10 s, and 72 °C for 1 min 30 s, and 95 °C for 10 s, 65 °C for 15 s, and 50 °C for 10 s. Mice were maintained in an air-conditioned room at 23±2 °C and a humidity of 55±5% with a 12-h light–dark cycle (7:00–19:00) at the Japan Oriental Yeast Co. Ltd. Mice were killed by decapitation between 12:00 and 15:00. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the University of Shizuoka.2.3SLC2A5; mice: Slc2a5, Si, and Brd4) and housekeeping genes  were amplified by real-time RT-PCR using a Light Cycler System  and SYBR Green I . The cycle threshold (CT) values detected by real-time RT-PCR were converted into signal intensities using the delta-delta method Total RNA was subjected to RT using Superscript III reverse transcriptase (Invitrogen). Test genes . Lysates were centrifuged at 10,000g for 10 min at 4 °C. The protein concentration of the soluble supernatants was determined by the Lowry method, and samples were stored at −20 °C.Total cell proteins were extracted in RIPA buffer /10 ml, and phosphatase inhibitors . The primer sequences used in ChIP assays are listed in Supplemental Cells were incubated in fixation solution  in 10% FCS/DMEM for 30 min at 37 °C. Reactions were terminated by adding glycine to a final concentration of 150 mM. After washing in fluorescence-activated cell sorting solution . The significance of differences among groups was determined by Tukey's multiple range test based on one-way analysis of variance (ANOVA) , two-wayt-test . A P val33.1SLC2A5 expression was significantly increased by co-treatment with Dex and PD for 8, 24, and 48 h, and SLC2A5 protein levels were significantly elevated by co-treatment with Dex and PD for 48 h. Brd4, Cyclin T1, Cdk9, and TFIIB were expressed in Caco-2 cells treated with DMSO, Dex, PD, or Dex and PD. However, Brd4 protein levels were decreased by PD or co-treatment with Dex plus PD  were slightly enhanced by co-treatment with Dex and PD for 48 h, and signals from 2000 bp of SLC2A5 were significantly higher. Co-treatment also notably increased the ChIP signals associated with Brd4 on the promoter/enhancer region and the transcribed region close to the initiation site of SLC2A5 from −2000 bp to 1000 bp. Co-treatment with Dex and PD for 48 h strongly increased the ChIP signals associated with Cdk9 on the transcribed region close to the initiation site of SLC2A5 at 1000 bp and 22,000 bp. ChIP signals were also significantly increased in association with Pol II on the promoter/enhancer region and the transcribed region close to the initiation site of SLC2A5 at −2000 bp, 1 bp, and 1000 bp  were markedly enhanced by co-treatment with Dex and PD for 48 h, particularly the transcribed regions of  1000 bp B.3.2SLC2A5 in Caco-2 cells co-treated with Dex and PD. Brd4 protein levels were reduced in these cells compared with controls. Levels of Cyclin T1, Cdk9, and TFIIB proteins were similar in cells subjected to different treatments in both control- and Brd4-shRNA-expressing cells. In contrast, co-treatment with Dex and PD for 48 h increased SLC2A5 levels in control cells, but not in Brd4-shRNA-expressing cells  increased after co-treatment with Dex and PD for 48 h in both control- and Brd4-shRNA-expressing cells  also increased after co-treatment with Dex and PD in both cell lines. We obtained similar results in terms of the associations of Brd4, Cdk9, and Pol II with SLC2A5 in control cells and in WT cells in Acetylation of histone H3 K9/14 on the promoter/enhancer and transcribed regions of ng cells B. Acetyl3.3Slc2a5 in the mouse small intestine by real-time RT-PCR using total RNA extracted from WT or Brd4+/− mice at 10, 14, 21, and 26 days after birth. The genetic background of Brd4 heterozygous (Brd4+/−) mice is shown in Brd4+/− mice compared with WT mice at each day after birth. We confirmed that Brd4 expression was significantly lower in Brd4+/− mice compared with WT mice. Slc2a5, but not Si expression was also significantly lower in Brd4+/− mice compared with WT mice at 21 and 26 days after birth , which regulates mRNA transcription elongation by regulating Pol II activity SLC2A5 may enhance gene expression by recruiting P-TEFb and Pol II to the acetylated histones in the transcribed region, thus enhancing mRNA transcription elongation.In this study, we demonstrated that co-treatment with Dex and PD induced SLC2A5 is induced in the small intestine of rodents during the suckling–weaning transition period when serum glucocorticoid levels increase and p44/42 MAPK is inactivated. Interestingly, we found that small-intestine expression of Slc2a5 was significantly lower in Brd4+/− mice compared with WT mice at 21 and 26 days after birth, suggesting that Brd4 regulates the induction of small-intestine Slc2a5 expression during the suckling–weaning transition in mice. It should be noted that body weight was lower in Brd4+/− mice compared with WT mice, and the lower Slc2a5 mRNA levels in Brd4+/− mice may thus be associated with delayed growth. However, mRNA levels of another intestinal gene, Si, were similar in Brd4+/− and WT mice, implying that the decreased Slc2a5 mRNA levels in Brd4+/− mice were likely related to reduction of Brd4 as a result of gene targeting, rather than delayed growth. Further studies are needed to determine if acetylated histone-Brd4–P-TEFb is recruited to the transcribed region of rat jejunal Slc2a5 during the suckling–weaning transition.SLC2A5 were enhanced by co-treatment with Dex and PD in Caco-2 cells Si gene during the suckling–weaning transition in mice may not be affected by Brd4 reduction via gene targeting. It is possible that the rates of expression of several genes are controlled by Brd4-associated transcription elongation and/or transcription initiation, while other genes may be controlled by Brd4. This issue should be examined in future studies.We previously demonstrated that both GR translocation from the cytosol to the nucleus, and GR binding to the promoter/enhancer region of SLC2A5. Because the enhancement of histone acetylation around SLC2A5 by co-treatment with Dex and PD was closely associated with Brd4–P-TEFb binding, co-treatment may enhance histone acetylation, which may in turn promote Brd4–P-TEFb recruitment. However, the specific histone acetyltransferase (HAT) that regulates histone acetylation around SLC2A5 has not yet been identified. GCN5 is a HAT that controls amino acid synthesis and is recruited around the transcription initiation site of intestinal genes such as Si and Slc5a1 in response to a high-carbohydrate diet in rats SLC2A5. A high-carbohydrate diet has also been shown to promote the association of another HAT, CREB-binding protein (CBP), with the Mgam gene, leading to increased Mgam expression in mice SLC2A5 expression. However, although many HATs have been identified It is still unclear which cascade promotes Brd4–P-TEFb recruitment to the transcribed region of SLC2A5 in human intestinal Caco-2 cells after co-treatment with a glucocorticoid hormone agonist and a MAPK inhibitor. This induction is mediated by recruiting P-TEFb to the transcribed region and enhancing mRNA polymerase II transcription elongation. We further demonstrated that Brd4 regulated the induction of Slc2a5 in the small intestine during the suckling–weaning transition in mice. These results suggest that Brd4–P-TEFb plays a crucial role in differentiation-associated transcription of SLC2A5 gene in intestinal Caco-2 cells and in the small intestine of mice during weaning period.In conclusion, we demonstrated that Brd4 enhances the induction of Conceived and designed the experiments: YI TG KM.Performed the experiments and analyzed the data: YI KM.Generated the Brd4 heterogeneous mice: AD AN KO.Wrote the paper: YI TG TK KO KM.Grant-in-Aid for JSPS Researcher Fellows for Young Scientists (23-9920), Grants-in-Aid for Young Scientists (22680054), a Grant-in-Aid for Scientific Research (26282023) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), and a Grant-in-Aid, and for Development of Core Technologies for Innovative Drug Development based upon IT from the Japan Agency for Medical Research and Development (AMED), and the Takeda Science Foundation.This work was supported by a The authors declare that they have no conflicts of interest.


Puccinia graminis f. sp. tritici Eriks. & E. Henn, can incur yield losses in susceptible cultivars of durum wheat, Triticum turgidum ssp. durum (Desf.) Husnot. Although several durum cultivars possess the stem rust resistance gene Sr13, additional genes in durum wheat effective against emerging virulent races have not been described. Durum line 8155-B1 confers resistance against the P. graminis f. sp. tritici race TTKST, the variant race of the Ug99 race group with additional virulence to wheat stem rust resistance gene Sr24. However, 8155-B1 does not confer resistance to the first-described race in the Ug99 race group: TTKSK. We mapped a single gene conferring resistance in 8155-B1 against race TTKST, Sr8155B1, to chromosome arm 6AS by utilizing Rusty/8155-B1 and Rusty*2/8155-B1 populations and the 90K Infinium iSelect Custom bead chip supplemented by KASP assays. One marker, KASP_6AS_IWB10558, cosegregated with Sr8155B1 in both populations and correctly predicted Sr8155B1 presence or absence in 11 durum cultivars tested. We confirmed the presence of Sr8155B1 in cultivar Mountrail by mapping in the population Choteau/Mountrail. The marker developed in this study could be used to predict the presence of resistance to race TTKST in uncharacterized durum breeding lines, and also to combine Sr8155B1 with resistance genes effective to Ug99 such as Sr13. The map location of Sr8155B1 cannot rule out the possibility that this gene is an allele at the Sr8 locus. However, race specificity indicates that Sr8155B1 is different from the known alleles Sr8a and Sr8b.Wheat stem rust, caused by  Puccinia graminis Pers.:Pers. f. sp. tritici Eriks. & E. Henn. (Pgt) is a devastating fungal disease of both common  and durum wheat (Triticum turgidum ssp. durum (Desf.) Husnot, 2n = 4x = 28, AABB), and can culminate in significant yield losses worldwide  that was first discovered in Kenya in 2006. Race TTKST is virulent to both T. turgidum ssp.) have contributed stem rust resistance genes such as Sr2, Sr9d, Sr9e, Sr9g, Sr11, Sr12, Sr13, Sr14, and Sr17  with the pedigree Marruecos 9623//Marruecos 9623/CItr 8155 that was characterized and selected as monogenic for stem rust resistance derived from CItr 8155  were used to map the TMLKC resistance utilizing the 90K Infinium iSelect Custom bead chip SNP genotyping platform. KASP assay-based markers were developed and evaluated on all the 143 BC1F2 families and on a set of 11 durum cultivars from the US. Eleven durum cultivars and five bread wheat genetic stock lines were evaluated with Pgt at the seedling stage and KASP assays to validate mapping results . Previously, durum cultivar Mountrail  using a method described by 1F2 plants from each of the 143 families were evaluated against Pgt race TTKST (isolate 06KEN19v3) along with Rusty and 8155-B1. Choteau, Mountrail, and the 4× and 6× Choteau/Mountrail populations were assessed in two biological replicates each for response to races TTKST and TTKSK . Inoculation of seedlings was performed according to previously described methods . The top 16 SNP markers (P < 0.03) significantly correlated with resistance to race TTKST were converted into KASP assays. Eight of these KASP assays were polymorphic and were evaluated on the 143 BC1F2 lines. These data were used to generate a linkage map using JoinMap version 4.0 , followed by 36 cycles of regular PCR: 94° for 20 sec, 57° for 60 sec, followed by fluorescence reading at 20°. A total of 3–9 additional cycles of PCR were added to obtain a good separation of clusters, as needed. Both thermal cycling and fluorescence reading were performed on an ABI StepOnePlus Real-Time PCR system. At least two replicates of each KASP assay were performed. If inconsistent results were observed between the two replicates, a third replicate was performed.Table S1 describes the wheat lines used in this study. Table S2 describes the Pgt isolates used in this study. Table S3 describes the number of Rusty/8155-B1 F2 progeny with specific infection types observed in response to Pgt race TMLKC. Table S4 lists 90K SNP markers identified as correlated with response to Pgt race TTKST in a selection of the BC1F2 population. Table S5 contains the alleles of markers linked to Sr8155B1 in Rusty*2/8155-B1 families. Table S6 contains the alleles of markers linked to Sr8155B1 in Choteau/Mountrail RILs that displayed recombination events near Sr8155B1. Table S7 contains alleles of markers mapped to chromosome 6A in the Rusty/8155-B1 F2 population. Table S8 contains seedling infection types observed on F2 plants from the Rusty/8155-B1 population. Table S9 contains seedling infection types observed on BC1F2 families of Rusty*2/8155-B1. Table S10 contains seedling infection types observed on Choteau/Mountrail RILs in response to races TTKST and TTKSK. Figure S1 displays a range of seedling infection types observed on Rusty/8155-B1 F2 progeny in response to Pgt race TMLKC. Figure S2 displays the genetic linkage map derived from Rusty/8155-B1 F2 progeny. Figure S3 displays the seedling infection types observed on Choteau and Mountrail in response to Pgt race TTKST.All data that we used to draw conclusions in this article are represented either within the article, or in the Supplemental Material. Figure S1). The F2 progeny segregated for resistance to race TMLKC, with 112 plants displaying infection types characteristic of 8155-B1-type resistance  and 361 plants not exhibiting this type of resistance  (Table S3). The presence of hypersensitive reactions “;” and “1” were considered indicative of the presence of resistance derived from 8155-B1. Segregation for resistance among the F2 plants did not deviate from the expected ratio for a single recessive gene . Out of the 75 F2 plants with infection type “0;” to “;”, 72 were selected for genotyping. Out of the 84 F2 plants with infection type “4,” 80 with adequate DNA extractions were used for genotyping.The durum line 8155-B1 exhibited an infection type of “0;” to “0;1,” whereas Rusty exhibited an infection type of “3+” to race TTKST  at both 1F2 families exhibited infection types (“23−” to “4”) to race TTKST when homozygous, and segregated for resistant infection types  and susceptible infection types (“23−” to “4”) when heterozygous. The segregation of resistance within BC1F2 families classified as heterozygous  did not deviate from the expected ratio for a single recessive gene . The ratio of homozygous vs. heterozygous families deviated from the expected 1:1 ratio with χ2 = 16.79 (P = 4.2 × 10−5). The linked KASP assay-based SNP markers also deviated from the expected 1:1 ratio , eight polymorphic KASP assay-based markers were evaluated on 143 BC1F2 families (Sr8155B1 (Table S5). Markers KASP_6AS_IWB61585 and KASP_6AS_IWB1550 flanked Sr8155B1 at 1.3 cM distal and 1.1 cM proximal, respectively. From the 152 F2 plants, we generated a linkage map of 116 cM . The linkage map clearly positioned Sr8155B1 in the short arm of chromosome 6A, and was tightly linked to three markers that also were linked to Sr8155B1 in the BC1F2 population: IWB64918, IWB43809, and IWB10558. Sr8155B1 was flanked by the SNP markers IWB55188 and IWB35219, being 7.3 cM proximal and 0.7 cM distal to Sr8155B1, respectively. The corresponding positions of these markers in the durum consensus map , KASP_6AS_IWB72958, and KASP_6AS_IWB61585 . The response of Mountrail to races TTKSK and TTKST is typical of cultivars postulated to possess both Sr13 and Sr8155B1 , likely Sr13. The response of the RILs to race TTKST included infection types ranging from “0;” to “3+”. Segregation of resistance to race TTKST fit a two-gene model , likely Sr13 and Sr8155B1. We classified RILs with resistant TTKST infection types that displayed lower infection types compared to the response to race TTKSK as possessing Sr8155B1 (Table S10). RILs with TTKST infection types that displayed similar infection types to race TTKSK were classified as lacking Sr8155B1 (Table S10). We postulated that 91 RILs possessed Sr8155B1 and 108 lacked the gene, which fit a single gene ratio . We were not able to confidently postulate presence or absence of Sr8155B1 in 20 of the 219 RILs (Table S10). We did observe a small quantitative difference between the effect of Sr8155B1 in hexaploid and tetraploid RILs of the Choteau/Mountrail population. The four most common infection types of hexaploid RILs (SXD1 through SXD135) postulated to possess Sr8155B1 were “;1−”, “;1”, “11+”, and “;13−”, but the most common infection types for tetraploid RILs (SXD136 to SXD232) with Sr8155B1 were “0;”, “;”, “;1−”, and “;1” (Table S10).Choteau displayed susceptible infection type “3+” in response to races TTKSK and TTKST. Mountrail displayed infection type “2−” in response to race TTKSK, and infection types “0;” to “;” in response to race TTKST , demonstrating their tight linkage and the tractability of KASP_6AS_IWB10558 in another population. These mapping results confirm the presence of both Sr8155B1 and Sr13 in the cultivar Mountrail.Both osome 6A . Sr8155Bet al. 2007, 2008). Monogenic line 8155-B1 line is unique because it is resistant to race TTKST, a variant of TTKSK with increased virulence, but susceptible to race TTKSK. 8155-B1 is also resistant to other variants in the Ug99 race group including TTTSK, TTKSF+, and TTKTT. In addition, 8155-B1 is resistant to TMLKC and TRTTF. Avirulence to Sr8155B1 is found in isolates of the Ug99 race group that vary in their avirulence to resistance genes Sr9h, Sr24, Sr31, Sr36, and SrTmp, and only one isolate has been characterized as virulent to Sr8155B1 . Although 04KEN156/04 is the oldest isolate in the Ug99 race group that we tested, we expect that ancestral Ug99 race group isolates are likely avirulent to Sr8155B1 based on the geographic and genetic variability of isolates that we confirmed as avirulent to Sr8155B1  and IWB6327 (BS00011010_51), which were linked to Sr8155B1 in our study, were also reported by Sr_TRTTF, which was predicted to be Sr8a. IWB64918, closely linked to Sr8155B1, mapped 3.3 cM from Sr_TRTTF, and IWB6327 mapped 30.2 cM away from Sr_TRTTF  resistance inherited from Choteau . Going forward, we recommend the combination of both Sr8155B1 and Sr13, such as in durum varieties Alkabo, Grenora, Mountrail, Munich, Renville, and Rugby in order to provide the maximum immediate protection of the durum crop in the Northern Great Plains of North America.The susceptibility of 8155-B1 to race TTKSK limits the value of www.g3journal.org/lookup/suppl/doi:10.1534/g3.117.300209/-/DC1.Supplemental material is available online at Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.


Trichoderma asperellum is one of the species which can be isolated from contaminated Pleurotus ostreatus cultivation substrate with green mold disease. This study focused on the relationship between high temperature and infectivity of T. asperellum to P. ostreatus. Antagonism experiments between T. asperellum and P. ostreatus mycelia revealed that high temperature-treated P. ostreatus mycelia were more easily infected by T. asperellum and covered by conidia. Microscopic observation also showed that P. ostreatus mycelia treated with high temperature could adsorb more T. asperellum conidia. Furthermore, conidia obtained from T. asperellum mycelia grown at 36°C featured higher germination rate compared with that incubated at 28°C. High temperature-treated T. asperellum mycelia can produce conidia in shorter periods, and T. asperellum mycelia were less sensitive to high temperature than P. ostreatus. Deactivated P. ostreatus mycelia can induce T. asperellum cell wall-degrading enzymes (CWDEs) and P. ostreatus mycelia subjected to high temperature showed induced CWDEs more effective than those incubated at 28°C. Moreover, T. asperellum showed higher CWDEs activity at high temperature. In dual cultures, hydrogen peroxide (H2O2) increased after 36°C, and high concentration of H2O2 could significantly inhibit the growth of P. ostreatus mycelia. In summary, our findings indicated for the first time that high temperature can induce a series of mechanisms to enhance infection abilities of T. asperellum to P. ostreatus mycelia and to cause Pleurotus green mold disease. Pleurotus ostreatus is one of the most important commercial edible mushroom crop in China. Its production is the third largest among all edible mushrooms in China. Pleurotus ostreatus is mainly cultivated using traditional agricultural cultivation methods in which production may be affected due to limited temperature control system. Green mold diseases caused by Trichoderma often occur during P. ostreatus cultivation [Trichoderma spp. mainly include T. pleurotum, T. pleuroticola, and T. harzianum [P. ostreatus farms, green mold diseases have resulted in severe crop losses and huge economic losses [P. ostreatus mycelia have suffered from high temperature in summer during spawn running period. However, little is known about the causes why high temperature-treated P. ostreatus mycelia are more vulnerable to green molds. Therefore, it is of positive significance to find out the causes of green mold disease.tivation . These Tarzianum ,3. As anc losses . InteresTrichoderma are characterized by thick green sporulation on substrate surface [Trichoderma conidia form quickly and profusely, and can quickly spread in the air due to their low weight. These floating conidia can be adsorbed onto fungal cell wall and recognized by lectin [Trichoderma growth and reproduction. Trichoderma conidia produced at high temperature exhibit maximum germination and growth [Green mold diseases caused by  surface . Trichody lectin . It was y lectin . Conidiad growth ,8.Trichoderma spp. have been widely used to control plant pathogenic fungi and improve soil environment [Trichoderma spp. are common pathogenic fungi for edible mushrooms [Trichoderma spp. can grow on the mushroom beds and finally cover the entire substrate. Trichoderma features a variety of mechanisms that inhibit or degrade other fungal mycelia [Trichoderma species should first recognize the host cell wall, coil around its mycelia, develop appressoria, and then secrete cell wall degrading enzymes (CWDEs) [Trichoderma species can produce a wide variety of CWDEs, such as chitinases, β-1,3-glucanases and proteases to hydrolyze the main components of host cell wall during antagonism [Trichoderma could be further induced [ironment . Such biironment . Howeverushrooms . Trichod mycelia . To infe (CWDEs) . Trichodtagonism , which itagonism . In the  induced .Pleurotus green mold diseases [Trichoderma asperellum has been reported as one of the species in Pleurotus substrates with green mold disease [P. ostreatus substrates subjected to high temperature were more easily infected by Trichoderma during P. ostreatus cultivation. The aim of this study is to investigate the effects of high temperature on T. asperellum infecting P. ostreatus mycelium and find out the relationship between high temperature and rampant breakout of T. asperellum in P. ostreatus spawn running stage.Previous studies have focused on isolation and identification of pathogenic fungi causing diseases ,18. Tric disease . StudiesPleurotus ostreatus P89 (CCMSSC 00389) was provided by the China Center for Mushroom Spawn Standards and Control.Trichoderma asperellum T11 (ACCC 32725) was deposited at the Agricultural Culture Collection of China. The internal transcribed spacer region (ITS) sequence was amplified by the primers ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’), and translation elongation factor 1α (tef1) sequence was amplified by primers EF1-728F (5’-CATCGAGAAGTTCGAGAAGG-3’) and TEF-LLErev (5’-AACTTGCAGGCAATGTGG-3’) [tef1 sequences of T. asperellum T11 were deposited in NCBI GenBank (KY368169 and MF049065)T11 ACCC 2725 was T. asperellum can cause Pleurotus green mold disease, Koch's Postulates were applied . One liter of MSM contained 5 g glucose, 0.2 g MgSOed water . For othFifteen pieces (a diameter of 5 mm punch) of P89 mycelial discs from solid medium were inoculated into 300 mL of PDB and incubated in a shaking incubator at 150 rpm and 28°C for 5 days. Then, the Erlenmeyer flasks were divided equally into four parts and incubated at 28°C, 32°C, 36°C and 40°C respectively for 2 days. Finally, mycelia were collected with filter paper , washed thrice with distilled water, dried under vacuum and grounded into powder .P89 mycelial discs (5 mm diameter) were placed at one side of PDA plates and incubated at 28°C for 3 days. Then, the plates were respectively placed in incubators with different constant temperatures of 28°C, 32°C, 36°C and 40°C for 2 days. Then, T11 mycelial discs were inoculated at the other side of plates and incubated at 28°C for 7 days ,22. The T. asperellum conidia and P. ostreatus mycelia, P89 was grown on a thin layer of PDA on microscope slides at 28°C for 3 days. Then, the plates were transferred to incubators with different temperature at 28°C and 36°C for 2 days. Conidial suspension (1.86×107 CFU/mL) in phosphate buffered saline (PBS) was used to observe agglutination reaction. Attachment of T11 conidia to P89 mycelia was observed in dual culture. Conidial suspension was poured over microscope slides with P89 mycelia. Reaction was carried at 28°C for 30 min. Then, the suspension was gently washed thrice with distilled water. Finally, attachment of T11 conidia to P89 mycelia was observed under light microscopes. An optical microscope  was used for observations. The magnification was 400 times.To examine agglutination between T11 disc was inoculated on PDA and incubated at 28°C for 10 days. Then, T11 plates with numerous of conidia were transferred to incubator at 36°C for various periods of time . Conidia were harvested separately with SDW. Conidial numbers in suspension were determined using a hemocytometer and adjusted to appropriate concentration. Then, 100 μL of diluted conidium suspension was spread onto 2% water agar plates . All plaT11 discs were inoculated on the center of plates and incubated at 28°C for 3 days. Plates were incubated at 36°C for different times  and then transferred to 28°C to observe formation of conidia. Conidium formation time was denoted as the time when the whole plate was covered with green conidia.Both P89 and T11mycelial discs were inoculated on the center of PDA plates and incubated at 28°C. When colony diameter reached 1 cm, the plates were transferred into different incubators at 28°C, 32°C, 36°C, and 40°C, respectively. Then, colony diameter in each plate was recorded daily .P. ostreatus mycelia. To detect the effect of temperature on enzyme activity, T11 conidia (1.80×107 CFU/mL) were inoculated in MSM and MSMP (using deactivated P. ostreatus mycelia cultivated at 28°C), and cultures were incubated for 3 days at 28°C with shaking at 150 rpm. Then, flasks were transferred to rotary shakers  with different temperature  for 2 days. Liquid culture was collected by centrifuging  at 6,000×g for 10 min and supernatants were used as crude enzyme solutions or stored at -80°C until assayed. To detect the effect of different substrates on enzyme activity, T11 conidia (1.58×107 CFU/mL) were inoculated in MSMP, which used deactivated P. ostreatus mycelia collected from cultures growing at different temperatures. Cultures were incubated for 3 days at 28°C with shaking at 150 rpm. Then, crude enzyme solutions were collected as above.For enzymatic assays, T11 conidia were inoculated into 100 mL MSM and MSMP. The formula of MSMP was exactly the same as MSM except that 5 g glucose was replaced by 3 g glucose and 2 g deactivated Chitinase activity was determined by the method of dinitrosalicylic acid (DNS) . The reaLaminarin (Sigma) (0.75% w/v) in 50 mM sodium acetate buffer (pH 5.0) was used to determine β-1,3-glucanase activity . The reaProtease activities were determined using casein as substrate and assayed according to the method of Satake et al.  with sli2O2 accumulation in antagonism between T. asperellum and P. ostreatus, T11 and P89 were evaluated in dual culture tests. P89 mycelia were inoculated into 250 mL Erlenmeyer flasks containing 100 mL PDB and incubated for 3 days at 28°C with shaking at 150 rpm. Then, 1 mL of T11 conidial suspension (1.69×107 CFU/mL) was inoculated into flasks containing P89 mycelia and incubated at 28°C for 1 day. Then, flasks were transferred to rotary shakers  at 28°C and 36°C, respectively for different time . PDB flasks inoculated with either T11 only or P89 only were used as controls at 28°C for 3 days. Extracellular fluid was collected by centrifugation for 5 min at 8,000 rpm. The H2O2 content was determined using a Hydrogen Peroxide Assay Kit , following the manufacturer’s instructions. Absorbance was measured at 405 nm.To detect H2O2 can inhibit the growth of mycelium. In order to assess sensitivity of T. asperellum and P. ostreatus mycelia to H2O2, P89 mycelial discs and T11 mycelial discs were inoculated on the center of PDA plates with different concentrations of H2O2 and incubated at 28°C. When the majority of plates were occupied by mycelia, diameter and morphology of colonies were observed.High concentration of HP ≤ 0.05) were applied to calculate and analyze data. Data were analyzed using SPSS version 20.0 software . Error bars represent the standard deviation of three replicates.Analysis of variance (ANOVA) and Duncan’s multiple range tests  higher germination rates than those at 28°C. Heat treatment time at 36°C can also affect conidia germination rates. Germination rates increased sharply upon heat treatment and reached the highest level at 6 h, then declined gradually and remained stable at high levels until 48 h . Growth trends of T11 and P89 mycelia in response to temperature were similar. As temperature elevated, colony diameter denoting growth rate of both strains declined significantly. T11 mycelia grew significantly (P ≤ 0.05) faster than P89 at 28°C, 32°C and 36°C  were exhibited at 32°C (P ≤ 0.05) higher than that at 28°C  induced enzyme activities of β-1,3-glucanases, chitinases and proteases, and their enzyme activities were significantly (P ≤ 0.05) higher than those cultivated with P. ostreatus mycelia collected from 28°C  and P89 mycelia only (0.38 mM) were significantly (P ≤ 0.05) less than that in dual cultures . Thus, effects of various concentration of H2O2 on T11 and P89 mycelial growth were detected. Results showed that H2O2 over 2.5 mM can significantly inhibit growth of P. ostreatus mycelium, and 10 mM of H2O2 almost completely suppressed the growth of P89 mycelium  or the ability of T. asperellum to infect P. ostreatus mycelium enhanced.Other studies have observed antagonism by er fungi ,36. In operature . In addiperellum . These pTrichoderma infection in other fungi. Recognition is a prerequisite in inhibiting and hydrolyzing other fungal cells. Trichoderma asperellum attaches to other fungal cell with cell wall carbohydrates that bind to fungal lectins [P. ostreatus mycelia treated at 36°C for 2 days can adsorb a large number of conidia  can produce conidia in shorter periods compared with that incubated at 28°C  and superoxide radicals (O2.-), during their direct confrontation by Trichoderma, and excessive ROS levels can cause cell death, damage cell membrane and increase membrane permeability, and cause electrolyte leakage [2O2 was detected in cultures with either T. asperellum only or P. ostreatus only; however, high concentration of H2O2 accumulated in dual cultures, and high temperature can further promote the accumulation of H2O2 which reached the highest at 36°C for 6 h. Furthermore, sensitivity of P. ostreatus mycelia to high concentration of H2O2 was significantly higher than that of T. asperellum mycelia. Therefore, it is shown that T. asperellum can produce H2O2 to inhibit and damage P. ostreatus mycelia in the process of mycoparasitism, and high temperature can further promote the accumulation of H2O2 to enhance the ability of inhibition.During antagonism, the fungal cell can induce synthesis of reactive oxygen species (ROS), such as hydrogen peroxide Click here for additional data file.S2 FigTrichoderma asperellum and P. ostreatus mycelial discs were inoculated in the center of plates with different concentrations of H2O2. T: mycelial growth rate of T. asperellum. P: mycelial growth rate of P. ostreatus. Data were analyzed by Duncan’s ANOVA test. Error bars represent the standard deviation of three replicates.(TIF)Click here for additional data file.


The IMS-localized Gpx3 contains an 18-amino acid N-terminally extended form encoded from a non-AUG codon. This acts as a mitochondrial targeting signal in a pathway independent of the hitherto known IMS-import pathways. Mitochondrial Gpx3 interacts with the Mia40 oxidoreductase in a redox-dependent manner and promotes efficient Mia40-dependent oxidative protein folding. We show that cells lacking Gpx3 have aberrant mitochondrial morphology, defective protein import capacity, and lower inner membrane potential, all of which can be rescued by expression of a mitochondrial-only form of Gpx3. Together, our data reveal a novel role for Gpx3 in mitochondrial redox regulation and protein homeostasis.Thiol peroxidases are conserved hydrogen peroxide scavenging and signaling molecules that contain redox-active cysteine residues. We show here that Gpx3, the major H  •A pool of yeast Gpx3 localizes to mitochondria via translation from a non-AUG codon•Loss of Gpx3 causes defects in mitochondrial architecture and membrane potential•Gpx3 interacts with the oxidative protein folding machinery in the IMS The redox sensor protein Gpx3 is imported into yeast mitochondria via a targeting sequence encoded from an upstream non-AUG codon. Kritsiligkou et al. show that mitochondrial Gpx3 acts in collaboration with the oxidative protein-folding machinery to ensure mitochondrial proteostasis and morphology. Disulfide bond formation is crucial for the native structure and stability of many proteins, while redox regulation through reversible cysteine oxidation is a common cellular strategy to adapt protein function to redox conditions. Conversely, oxidative stress may have detrimental effects on cell physiology through thiol oxidation, which is why cells have evolved several enzymatic mechanisms to cope with such conditions. These include the glutaredoxin and thioredoxin systems, which are the major cellular protein disulfide reduction systems .2O2) is a reactive oxygen species (ROS) that can lead to oxidative damage but can also act as a signaling molecule (2O2 are the processes of fatty acid oxidation in peroxisomes and disulfide bond formation in the endoplasmic reticulum (ER) and the mitochondrial intermembrane space (IMS). In yeast cells, the signaling role of H2O2 is primarily mediated by the Gpx3 thiol peroxidase . Under these conditions, we observed the co-localization of Gpx3 with mitochondria. Similarly, under respiratory conditions, Gpx3-GFP displayed predominantly cytoplasmic fluorescence, while co-localization with mitochondria was seen following H2O2 treatment   A and S3BGPX3 abrogates the mitochondrial membrane potential in mitochondria isolated from wild-type and gpx3 mutant strains by evaluating the incorporation of the fluorescent dye 3,3′-dipropylthiadicarbocyanine iodide (DiSC3(5)) in mitochondria with active membrane potential  than wild-type mitochondria isolated under physiological conditions  a genetic approach, where we deleted ry media C; and (2ry media D. The geexposure D. MitoSOexposure . Higher chondria D. The in3 mutant D could r3 mutant B and imp mutants C.gpx3 mutant cells transformed with a plasmid expressing wild-type Gpx3 (WT) or no Gpx3 (ev) showed import kinetics similar to those of WT and gpx3 strains  or the empty vector (ev) reflect the morphology of a WT and gpx3 strain, respectively  presequence that targets proteins to the IMS, upstream of Gpx3-myc, and is expressed under the control of the Mia40 promoter. Cyb2-Gpx3-myc is only found in the mitochondrial fraction, unlike the WT, which is dually localized in the cytosol and mitochondria A. The sucapacity C. We theo the WT D. In parT levels E.Given the role of Gpx3 in Yap1 oxidation, we asked whether Gpx3 has any similar redox-dependent interactions within the IMS. By drawing parallels with the ER, where PrxIV interacts with the PDI oxidoreductase , we focuWe used an additional approach to examine the potential interaction of oxidized Gpx3 with reduced Mia40, where radiolabeled Gpx3 was incubated with purified ΔN290Mia40His. To confirm the AMS labeling result, we compared the migration of the reduced and oxidized form of Gpx3 to that of a cysteine mutant of Gpx3 (Cys82Ala) E. OxidizA peptide scan array was used to independently confirm and map the putative interacting segments between Gpx3 and Mia40 . We usedMIA40 is an essential gene, and cells were grown on glucose-containing media to downregulate the expression of Mia40 prior to purification of these mitochondria. As a control, we examined the import of the MIA-dependent substrate Tim10, which was strongly reduced in the MIA40-depleted mutant , both Erv1 and Mia40 appear to be differentially oxidized   . We prop2O2 produced from the oxidative protein-folding machinery via Ero1 .A CellASIC microfluidic chamber was used to monitor live cells using a Delta Vision (Applied Precision) restoration microscope with a 100×/NA 1.40 Plan-Apo objective and fluorescein isothiocyanate (FITC) and Texas red band pass filters from the Sedat filter set (Chroma). The images were collected using a Coolsnap HQ (Photometrics) camera with a Z optical spacing of 0.25 μm. The raw images were deconvoluted using the Softworx software. All images were analyzed using ImageJ . They were then infiltrated with 1% NaIO4 in water for 1 hr and stained with 1% uranyl acetate in water for 1 hr. The pellet was cut into small pieces, which were infiltrated with 1.6 M sucrose and 20% polyvinylpyrrolidone overnight, put on aluminum stubs, and frozen in liquid nitrogen. Ultrathin sections were cut with a Leica Ultracut UC6 ultramicrotome with FC6 cryo-chamber at −120°C and retrieved with the mix of 2.1 M sucrose and 2% methylcellulose. Sections were thawed, washed with 0.1M phosphate buffer, fixed with 1% Glutaraldehyde, and after washing with distilled water embedded into 1.8% methylcellulose with 0.5% uranyl acetate. Cells observed with an FEI Tecnai 12 Biotwin microscope at 100 kV accelerating voltage. Images were taken with Gatan Orius SC1000 charge-coupled device (CCD) camera.3(5) (Molecular Probes) as described previously . The following reagents were successively added, and the change in fluorescence was recorded: DiSC3(5) ; 50 μg mitochondria  and, finally, valinomycin  to disrupt the potential. The difference in the fluorescence before and after the addition of valinomycin represents a relative assessment of the membrane potential. Each reaction was performed three times , and then averages were calculated and presented as percentage values.The membrane potential (Δψ) of isolated yeast mitochondria was assessed by measuring the fluorescence quenching of DiSCeviously . Measure5 cells collected by centrifugation. Cells were incubated in the dark with 2.5 μM MitoSOX (Life Technologies) for 30 min at 30°C. 30,000 cells from each experimental condition were analyzed using a flow cytometer .For measurements using flow cytometry, cells were grown to mid exponential phase in SCD media and 1035S-labeled precursor proteins were synthesized using the TNT SP6-coupled transcription/translation kit (Promega) and plasmid-vectors pSP64 containing the genes of interest. The radioactive material was then denatured in 8 M urea, 50 mM HEPES (pH 7.4), 5 mM EDTA, and 20 mM DTT for 40 min at 30°C. The precursor is imported in 50 μg WT yeast mitochondria in the presence of 2 mM ATP and 2.5 mM NADH for the indicated time points at 30°C. Mitochondria were resuspended in 1.2 M sorbitol and 20 mM HEPES (pH 7.4), followed by a treatment with 0.05 mg/mL trypsin to remove unimported material for 30 min on ice (inactivation with 0.5 mg/mL soybean trypsin inhibitor [SBTI] for 10 min on ice). Mitoplasts were produced by resuspending mitochondria in 1× import buffer . Finally, samples were resuspended in Laemmli sample buffer with β-mercaptoethanol as indicated, analyzed by SDS-PAGE, and visualized by digital autoradiography (Molecular Dynamics). The quantification of the imported material based on the 10% samples was performed using the TotalLab Quant program.t buffer  at 5 mg/Purified proteins were precipitated with ammonium sulfate and resuspended in 50 mM Tris (pH 8) with or without DTT (50 mM) to generate the oxidized or the reduced versions of these proteins. After 1 hr at room temperature (RT), 30 ng of each protein per reaction was incubated together for specific time points. Proteins were precipitated with 5% TCA, and AMS labeling was performed in buffer containing 50 mM Tris (pH 7.5), 3% w/v SDS, 3 mM EDTA, and 15 mM AMS for 30 min at 30°C and 30 min at 37°C (adapted from Conceptualization, P.K., A.C., C.M.G., and K.T.; Methodology, P.K., A.C., C.M.G., and K.T.; Investigation, P.K., A.C., G.C., A.M., C.M.G., and K.T.; Writing, P.K., A.C., G.C., A.M., C.M.G., and K.T.; Funding Acquisition, C.M.G., P.K., and K.T.; Supervision, C.M.G. and K.T.


The X-ray diffraction (XRD) experiments indicate that the obtained main phases of Li2.0MnTiO4 and the lithium-excess materials are monoclinic and cubic, respectively. The scanning electron microscope (SEM) images show that the as-prepared particles are well distributed and the primary particles have an average size of about 20–30 nm. The further electrochemical tests reveal that the charge-discharge performance of the material improves remarkably with the lithium content increasing. Particularly, the first discharging capacity at the current of 30 mA g−1 increases from 112.2 mAh g−1 of Li2.0MnTiO4 to 187.5 mAh g−1 of Li2.4Mn0.8TiO4. In addition, the ex situ XRD experiments indicate that the monoclinic Li2MnTiO4 tends to transform to an amorphous state with the extraction of lithium ions, while the cubic Li2MnTiO4 phase shows better structural reversibility and stability.Lithium-excess and nano-sized Li As the  LiMn2O4 ,4,5,6. A LiMn2O4 . Li2MTiOtructure .2MTiO4 material has a cubic cation-disordered rock salt structure with the Fm-3m space group, which means that all the metal atoms disorderly occupy the octahedral sites of the cubic close-packed array of anions. Particularly the involved strong Ti–O bond could stabilize the M3+/M2+and M4+/M3+ transition. The present Li2MTiO4 materials mainly include Li2NiTiO4, Li2FeTiO4, Li2MnTiO4, Li2x−VTiO4, and Li2CoTiO4 [2NiTiO4 shows a reversible capacity of 179 mAh g−1, which is 61.6% of the theoretical value for the exchange of 2 M lithium (290.6 mAh g−1). Li2FeTiO4 and Li2MnTiO4 show a comparable capacity of ~130 mAh g−1 if they are prepared within the carbon coating. In the case of Li2x−VTiO4, the maximum capacity is close to 350 mAh g−1 in a wide potential range between 1.0 V and 4.4 V. For Li2CoTiO4, the discharge capacity is 150.8 mAh g−1 if the particle size is much smaller. Although the achieved discharge capacities of the above materials were not bad, they were still far from their theoretical values. Thus, how to enhance the discharge capacity of these materials has become an urgent and important problem. In 2014, Ceder et al. pointed out that increasing the amount of lithium is an effective way to improve the electrochemical properties of cation-disordered materials, which had been achieved in a Li1.211Mo0.467Cr0.3O2 material [2MTiO4 materials is quite worthy of study.The Lii2CoTiO4 ,13,14,15material ,17. Inspx2+Mn1x/2−TiO4  cathode materials were synthesized and studied in this paper. The synthesis was done via a sol-gel method to obtain the nano-sized and well-distributed particles of the materials. The effects of different lithium amounts on the structure, morphology, and electrochemical properties were then investigated.Herein, the lithium-excess Lix2+Mn1x/2−TiO4 materials with different lithium content were prepared, namely Li2.0MnTiO4, Li2.2Mn0.9TiO4, and Li2.4Mn0.8TiO4. For brevity, they are respectively expressed as L2.0, L2.2, and L2.4 in the following discussions. Among them, L2.0 has a normal stoichiometric proportion while L2.2 and L2.4 contain excess lithium.Three representative Lix = 0, L2.0) and Li-excess proportions  are presented in The heat treatment process of the materials with different element proportions was investigated through the thermogravimetry and differential scanning calorimetry (TG-DSC) experiments. The typical curves of the precursors with normal proportion 2/3)) and 1/3)) cation layers alternate in the (1 1 1) cubic planes. This layer structure has a strong resemblance to monoclinic Li2TiO3 and possesses an ordered structure with lattice parameters of a = 5.048 Å, b = 8.833 Å, c = 9.629 Å; α = 90°, β = 99.939°, γ = 90°. A similar ordered phase of another member of the titanate family, Li2NiTiO4, was reported in a previous study [2MnTiO4 was consistent with the Fm-3m space group, as previously reported [a = b = c = 4.151 Å; α = β = γ = 90°, and a = b = c = 4.151 Å; α = β = γ = 90°. Additionally, the phase contents of cubic Li2MnTiO4 of the two materials are also much the same, both about 80%. The discrepancy in the structure between L2.0 and the lithium-excess materials indicates that the cation-disordered structure can be obtained more easily through increasing the content of the lithium, which can explain the difference of the TG-DSC curves between the materials.us study . For thereported ,9. The rx2+Mn1x/2−TiO4 materials was identified by field emission scanning electron microscope (FE-SEM) analysis, as shown in The morphology of the Li3+/Mn2+ and Mn4+/Mn3+ transition. Thus, the theoretical capacities of the L2.0, L2.2, and L2.4 can be calculated based on their Mn contents, and they are respectively 296.7 mA g−1, 273.2 mA g−1, and 248.6 mA g−1.Galvanostatic charge and discharge properties of the Lix2+Mn1x/2−TiO4 cathode were tested between 1.5 V and 4.8 V with constant currents of 30 mA g−1 and 60 mA g−1, as shown in −1 . For Li-excess electrodes, the voltage plateau of charging and discharging became more distinct, and the discharge capacity was also higher than that of L2.0. Specifically, the L2.4 electrode could deliver a capacity as high as 187.5 mAh g−1. When the current density increased to 60 mA g−1, the capacity gap between L2.0 and the lithium-excess materials became even clearer  can be calculated. It turns out that during the first charge process, the change of the lattice constant was only 0.45%, suggesting that cubic Li2MnTiO4 could be considered as a stable cathode material for lithium-ion batteries.To investigate the microstructure changes of the electrodes during the charge and discharge process, x2+Mn1x/2−TiO4  cathode materials was based on sol-gel reactions, followed by heat treatment under atmosphere protection. To form the sol, stoichiometric amounts of high-purity tetra-n-butyl titanate , lithium acetate dehydrate , and manganese acetate dehydrate  were dispersed in ethanol and then continuously stirred in oil bath at 80 °C for 24 h. The sol was evaporated at 80 °C overnight and further ball-milled sufficiently to get the xerogel powders. The powders were calcined at 600 °C for 10 h in argon atmosphere to obtain the LixMnx/22-TiO4 material.The synthesis of nanoscale LixMn2−x/2TiO4 materials. The precursors were heated at a rate of 10 °C min−1 under nitrogen atmosphere in a SDT Q600 instrument  to obtain the TG-DSC curves.Thermogravimetry (TG) and differential scanning calorimetry (DSC) experiments were carried out to investigate the heat treatment process of Lii.e., 5 kV).The phase composition and structure of the as-prepared materials were analyzed based on powder X-ray diffraction (XRD). The diffraction data were collected on an ARL™ X'TRA Powder Diffractometer using Cu Kα radiation in the two-theta range of 10–85°. Rietveld refinement was done to achieve the analyses. To characterize the morphology, scanning electron microscopy (SEM) images of the products were collected on a HITACHI  S-4800 field-emission SEM (FE-SEM) at a high potential  as binder. Aluminum foils were used as the current collectors. The cells were assembled in an argon-filled glove box. The electrolyte was 1 M LiPF6 dissolved in a mixture of ethylene carbonate (EC)-Ethyl Methyl Carbonate (EMC)-dimethyl carbonate (DMC) . The separators were made up of Celgard3501 (USA) microporous polypropylene membrane.Electrochemical tests were carried out using CR2032-type coin cells with Li metal as the anode. The LiThe charge-discharge performance of the batteries was investigated through a BT-2000 battery testing system . The tests were implemented at constant currents between voltage limits of 1.5–4.8 V at 30 °C.x2+Mn1x/2−TiO4  cathode materials were successfully synthesized via a sol-gel method. The TG-DSC experiments suggest that the excess lithium induces a significant impact on the synthesis process of the material. It is confirmed by the XRD detections that the obtained main phases of L2.0 and Li-excess materials are different, namely monoclinic Li2MnTiO4 and cubic Li2MnTiO4, respectively. The materials show similar morphology and the particles are all well distributed with an average size of about 20–30 nm. Then the electrochemical tests indicated that the charge-discharge performance of the material improves remarkably with the lithium content increasing. Particularly when the testing current density was 30 mA g−1, the first discharging capacity increased from 112.2 mAh g−1 of L2.0 to 187.5 mAh g−1 of L2.4. The performance improvement between L2.0 and the lithium-excess materials can be attributed to the phase transition induced by excess lithium. The improvement from L2.2 to L2.4 can be explained with the achievement of the percolating network of 0-TM channels to provide macroscopic diffusion. However, it should be noted that the Li content cannot be too high or it would lead to lower theoretical capacity and poor structural stability. Thus, there should be a limiting ratio corresponding to the optimal performance, and this is worthy of future studies. In addition, the ex situ XRD experiments revealed that the cubic Li2MnTiO4 phase shows better structural reversibility and stability compared with the monoclinic one. Additionally, a small volume change was confirmed as one advantage of the cation-disordered material, which means there would be fewer mechanical problems such as deformation or fracture.In this paper, lithium-excess and nano-sized Li


Recent studies have identified a critical role for lysophosphatidic acid (LPA) in the progression of ovarian cancer. Using a transcription factor activation reporter array, which analyzes 45 distinct transcription factors, it has been observed that LPA observed robustly activates the transcription factor hypoxia-induced factor-1α (HIF1α) in SKOV3.ip ovarian cancer cells. HIF1α showed 150-fold increase in its activation profile compared to the untreated control. Validation of the array analysis indicated that LPA stimulates a rapid increase in the levels of HIF1α in ovarian cancer cells, with an observed maximum level of HIF1α-induction by 4 hours. Our report demonstrates that LPA stimulates the increase in HIF1α levels via Gαi2. Consistent with the role of HIF1α in epithelial to mesenchymal transition (EMT) of cancer cells, LPA stimulates EMT and associated invasive cell migration along with an increase in the expression levels N-cadherin and Slug/Snail2. Using the expression of Slug/Snail2 as a marker for EMT, we demonstrate that the inhibition of Gαi2, HIF1α or Src attenuates this response. In line with the established role of EMT in promoting invasive cell migration, our data demonstrates that the inhibition of HIF1α with the clinically used HIF1α inhibitor, PX-478, drastically attenuates LPA-stimulates invasive migration of SKOV3.ip cells. Thus, our present study demonstrates that LPA utilizes a Gαi2-mediated signaling pathway via Src kinase to stimulate an increase in HIF1α levels and downstream EMT-specific factors such as Slug, leading to invasive migration of ovarian cancer cells. This isand Gα13  as well ene Gαi2 , 15. HowHIF1α has been shown to play a critical role in ovarian cancer malignancy, especially ovarian cancer cells found in the hypoxic conditions of the peritoneal cavity –18. WhilThe activation of HIF1α involves its dimerization with the constitutively expressed HIF1β . This isIn the present study, using a transcription array to identify transcription factors activated by LPA-mediated signaling, we demonstrate that LPA potently stimulates the activation of HIF1α via a pathway involving Gαi2 and Src. We further demonstrate that that the activation of LPA-Gαi2-Src-mediated signaling pathway induces EMT in ovarian cancer cells and subsequent invasive migration of ovarian cancer cells that can be inhibited by PX-478, a clinically tested inhibitor of HIF1α. Thus, our current study demonstrates that LPA stimulates a signaling nexus involving Gαi2, Src, and HIF1α to induce EMT and migration of ovarian cancer cells. Furthermore, we show that Gαi2 signaling is necessary and sufficient for hypoxia-mediated induction of HIF1α expression, which has not been shown, to our knowledge, by any previous studies to date.In order to identify possible mechanism utilized by LPA to drive the progression of ovarian cancer we employed a transcription factor array that can analyze the activation profile of fortyfive different transcription factors. SKOV3.ip cells were stimulated with LPA for 20 minutes along with the appropriate vehicle control and the lysates were subjected to the transcription array analysis. Our results indicated that LPA stimulation activated several transcription factors that have previously been shown to be stimulated by LPA including STAT3 , 31, 32 Next, we sought to identify the downstream G protein that mediates LPA- signaling in this process. Previous studies from us , 13, 37 It has been well established that the induction of HIF1α expression and its subsequent dimerization with HIF1β to function as a transcription factor in hypoxic conditions is involved in EMT and migration of many different cancer cell types , 27, 42.Our findings presented above Figures –3 indicaOvarian cancer cells are often found in the hypoxic environment of the peritoneal cavity and the core of the primary tumor . A previRecent reports from our lab , 15 haveNext, we investigated whether Src is involved in activating HIF1α and Slug. To test SKOV3.ip cells were treated with PP2, an inhibitor of the Src family of kinases, , 56, or Since EMT has been shown to promote invasive migration of cancer cells , 59, ourin vivo [Previously, we have shown the critical role of LPA in promoting cell proliferation and migration in ovarian cancer cells –9, 15 inin vivo . In the Previous studies have shown that the levels of HIF1α is regulated at multiple levels such as the inhibition of degradation of HIF1α, increased translation of HIF1α mRNA, and enhanced transcription of HIF1α gene . AlthougHIF1α signaling has been linked to EMT and cancer progression. There is voluminous reports that HIF1α and hypoxic conditions are linked to EMT . Indeed,Of critical importance, we show here that Gαi2 is also necessary for HIF1α activation independent of exogenous LPA signaling. Thus, there is a distinct possibility that LPA and/or other ligands that utilize Gαi2, such as CXCL12, could be responsible for activation HIF1α in hypoxic conditions via autocrine/paracrine signaling. However, this needs to be investigated further. Nonetheless, our report demonstrates that Gαi2 and Src are needed for HIF1α activation in hypoxic conditions, indicating that inhibition of this pathway can suppress hypoxia-induced resistance in ovarian cancer patients. This is also the first report to our knowledge that has shown the importance of LPA-signaling via Gαi2 in inducing EMT. Although a recent study reported the ability of LPA to induce EMT via Wnt/β-catenin signaling pathways , the undin vivo passaged variant of SKOV3 cells established by Yu et al., [2incubator. For serum-starvation, the media used was RPMI 1640 with 0.1% BSA , 50 U/mL penicillin and 50 mg/mL streptomycin (Mediatech). Lysophosphatidic acid (1-oleoyl-2-hydroxy-sn-glycero-3-phosphate) was obtained from Avanti Polar Lipids  and dissolved into 10 mM stock solutions in PBS with 0.1% BSA and stored at −80°C until use. Non-target control shRNA pLKO.1 vector construct was purchased from Sigma-Aldrich, St. Louis, MO (SHC002) whereas pLKO.1 vector constructs targeting Gαi2 (RHS3979-9596925) was purchased from Open Biosystems . The siGENOME Non-targeting siRNA (D-001206-13-05), siGENOME SMARTpool Gαi2 (M-003897-00-0005) and HIF1α (M-004018-05-0005) were purchased from Dharmacon . Peroxidase-conjugated anti-rabbit IgG was purchased from Promega , and peroxidase-conjugated anti-mouse was purchased from GE Healthcare . E-cadherin antibody was purchased from Santa Cruz Biotechnology . HIF1α antibody was purchased from BD Biosciences . Alexa 568 anti-mouse and Alexa 488 anti-rabbit antibodies were purchased from Invitrogen . DAPI was purchased from Life Technologies and used at a working concentration of 0.25 μg/mL.The ovarian cancer cell lines OVCAR2, OVCAR3, OVCAR5 and OVCA432 were kindly provided by Susan K. Murphy . SKOV3.ip1 cells (SKOV3.ip), an  et al.,  were kin6 cells per transfection cuvette were transfected with either non-targeting siRNA (100 nM), siRNA targeting Gαi2 (100 nM), Gαi2QL (2 μg) or pcDNA3 vector (2 μg) as indicated. After transfection, the cells were plated on 60 mm plates and allowed to adhere overnight. The following day the media was changed and the cells were allowed to grow until the end of the day. The cells were then were re-plated at a density of 5 × 105 cells per 100 mm plate and allowed to adhere overnight. For stable transfection, SKOV3.ip cells transfected as previously described with shGαi2 or control, nonsense shRNA and selected for the expression of shGαi2 or the nonsense vector with puromycin [All cells were transfected with a Nucleofector II system from Lonza  using the provided transfection protocol for SKOV3 cells as published previously , 15. SKOuromycin .2) were performed in INVIVO2 400 hypoxia workstation . Cells were incubated with 5% CO2 and 1% O2 at 37°C for the indicated lengths of time. After incubation, cells were collected and western blot analysis was carried out.Hypoxia treatments  was used to screen for different transcription factors upon LPA stimulation of SKOV3.ip ovarian cancer cells. Cells were seeded into wells  of the Cignal™ Finder 96-well plates  to transfect the reporters into cells via reverse transfection according to manufacturer's protocol. Briefly, reporter DNA constructs resident in each well of the plate were resuspended with 125 μl Opti-MEM and complexed with 25 μl of Lipofectamine 2000  transfection reagent. Each well is added with 5 × 104 cells suspended 25 μl of Opti-MEM media. Transfection was allowed to happen by incubating the plate for 24 h at 5% CO2 and 37°C. Following transfection, the cells were serum deprived for 16 h and treated with either vehicle (0.1% BSA in PBS) or LPA (20 μM) for 20 min. Differential activation of the transcription factors were determined by lysing the cells and measuring the luminescence intensity following the manufacturer's protocol.Cignal5 in 6-well plates with glass coverslips at the bottom. The cells were allowed to adhere overnight in a 37°C incubator with 5% CO2. The cells were washed 3× with sterile PBS and then serum-starved for 4 hours. After serum-starvation, the cells were treated with 10 μM of LPA for 4 hours. After LPA treatment, the cells were washed with ice-cold PBS one time and then treated with 4% paraformaldehyde for 15 minutes while rocking. The cells were then washed with PBS 1× and then stored at 4°C until they were stained. All treatment groups were lysed with 0.25% Triton X-100 for 10 minutes and then washed with PBS 3×. After washing, the coverslips were blocked with 1% BSA in PBS for 30 minutes at room temperature while rocking. After blocking, the coverslips were washed with PBS 1×. After washing, the primary antibody was applied in 1%-BSA in PBS and rocked for 10 minutes at room temperature. The coverslips were then transferred to 4°C and incubated overnight while rocking. The following day the primary antibody was removed and the coverslips were washed 3× for 5 minutes each. After washing, the coverslips were incubated with fluorescently tagged secondary antibody for 45 minutes at room temperature while rocking and covered with aluminum foil. After incubation with the secondary antibody, the coverslips were washed 1× with PBS and then stained with DAPI for 5 minutes. The coverslips were then washed 3× with PBS for 5 minutes each wash and then allowed to dry. Once dry, the coverslips were mounted with ProLong Gold antifade from Life Technologies  on glass slides. The coverslips were allowed to dry overnight at room temperature in the dark and then imaged the following day with a Nikon Eclipse Ni-U  at 600×.OVCA432 and SKOV3.ip cells were plated at density of 1 × 104 SKOV3.ip cells were suspended in 200 μL serum-free media were placed in the well of a 24-well companion plate. Each well contained 500 mL media containing serum-free media control or serum-free media containing 10 μM of LPA. The cells were incubated for 20 hours. Non-migrating cells on the proximal side of the inserts were removed with a cotton swab, and the migrated cells on the distal side of the inserts were fixed and stained with Hemacolor . Images were obtained at 100× in 5 random fields for each group. The experiments were repeated 3 times. SKOV3.ip cells were transfected with the indicated plasmid (shRNA) and plated into 6-well plates for a total of 48 hours. Twenty-four hours after transfection, the cells were serum starved for an additional 24 hours, trypsinized, counted, and placed into the transwell.The Collagen-1 migratory invasion assay was performed as previously published . CollageImmunoblot analysis with the indicated antibodies were carried out following previously published procedures , 74 and t-test with Welch's correction was performed to determine statistical significance.An unpaired two-tail


The experimental results have shown that the grain sizes of TiB2/7075 Al matrix composites firstly decreased, then increased, and finally decreased again with the increase of TiB2 content. The nucleation undercooling of TiB2/7075 Al matrix composites first increased, then decreased, and finally increased again with the increase of TiB2 content when the cooling rates was 5 and 10 °C/min respectively, but kept decreasing with the increase of TiB2 content at a cooling rate of 20 °C/min. The melting and solidification process showed no significant change with the decrease of cooling rate in 9.0% TiB2/7075 Al matrix composites. Most small particles can act as heterogeneous nucleus, which induced grain growth and were captured into the grain by the solid/liquid interface. At the same time, most of the larger particles and a minority of the small TiB2 particles are pushed into the grain boundary; locating in the grain boundary can hinder the Al atoms from diffusing during the solidification process and restrain α-Al phase growth. The influence of particles shifted from dominating by locating to dominating by nucleation as the quantity of TiB2 particles increased.The nucleation undercooling of TiB Finally, the refining mechanism of the composites is discussed.TiBaterials ,2,3,4,5.ensively ,10,11,12 a grain , but it  a grain . A free- a grain ,16,17. G refiner ,19,20. T refiner ,22,23. H2TiF6 (mass > 97%) and KBF4 (mass > 97%) mixture were put into molten 7075 Al alloy with the composition Al-5.52Zn-2.36Mg-1.51Cu-0.18Si-0.26Fe-0.15Mn-0.25Cr (in mass %) at 850 °C. After being stirred at the same temperature for 30 min and degassed by using C2Cl6, 3.0%  and 4.5%, 6.0% and 9.0% TiB2/7075 Al matrix composites were prepared. The molten TiB2/7075 Al composites at 720 °C were casted in a graphite mold 15 mm in diameter. The liquidus and solidus temperatures of composites were measured by NETZSCH Q100 differential scanning calorimetry (DSC) with weight of about 8–9 mg, and the heating and cooling rates were 1 and 5, 10 and 20 °C/min between 400 °C and 700 °C, respectively. Samples were held for 3 min under the protection of argon when the temperature reached 400 °C with the same heating rate, then all samples were heated to 700 °C with the different heating rate . Finally, the temperature was decreased to 400 °C with the different cooling rate  after holding for 3 min again. After sanding and polishing, the specimens were etched in a solution of mixed acids (1% HF + 1.5% HCl + 2.5% HNO3 + 95% H2O). The microstructures of composites were tested by ZEISS SUPRA55 scanning electron microscope (SEM) and CIKONG 4XCE optical microscope (OM). The grain size and area of the primary solid phase were analyzed statistically by a quantitative image analysis system (Image-Pro plus 6.0). The mean grain size was denoted by the average gain diameter S is the area of the primary phase.K2/7075 Al matrix composites after being poured into the graphite mold are pictured in 2/7075 Al matrix composites were 69 μm and 50 μm, respectively. However, larger dendrite grains could be found after adding 6.0% TiB2; the 9.0% TiB2/7075 Al matrix composite was also mainly composed of the dendrite grains smaller than that of 6.0% TiB2/7075 Al matrix composites.The microstructures of 3.0% and 4.5%, 6.0% and 9.0% TiB2/7075 Al matrix composites with cooling rate of 20 °C/min, 5 °C/min and 1 °C/min from 700 °C to 400 °C respectively. The grain sizes of TiB2/7075 Al matrix composites firstly decrease, then increase, and finally decrease with the increase of TiB2 content. The mean grain sizes of 3.0% and 4.5% TiB2/7075 Al matrix composites reached 107 μm and 66 μm, respectively, with a cooling rate of 20 °C/min in 2/7075 Al matrix composites increased with the decrease of cooling rate in 2/7075 Al matrix composites reached 236 μm and 165 μm, respectively, with cooling rate of 1 °C/min in 2/7075 Al matrix composites. Rosette grains in 9.0% TiB2/7075 Al matrix composites are also very strongly apparent in The microstructures of the samples after their solidification inside the DSC crucible have been compared. 2/7075 Al matrix composites decreased with the increase of heating rate; the liquidus temperatures of TiB2/7075 Al matrix composites were the opposite, but the solidus and liquidus temperatures of 9.0% TiB2/7075 Al matrix composites showed no remarkable change, as seen in L and Ts are the liquidus and solidus temperature, respectively; TI and TP are the initial solidification temperature and peak temperatures of solidification, respectively. The nucleation needs to satisfy the condition with 2/7075 Al matrix composites are on the decline, but the liquidus temperatures of TiB2/7075 Al matrix composites first decrease, then increase, and finally decrease again with the increase of TiB2 content. The liquidus and solidus temperatures of 7075 Al alloy were 637.7 °C and 475.0 °C, respectively, with heating rate of 10 °C/min [2/7075 Al matrix composites were 54.4 °C and 43.2 °C, respectively, which are only about a third and a quarter of 7075 Al alloy. Pysz et al. also found that the liquidus and solidus temperatures of 7075 Al alloy were 637.0 °C and 475.5 °C, respectively. The initial solidification temperature and peak temperatures of solidification were 632.8 °C and 625.1 °C [The solidus temperatures of TiBh ΔT>ΔTN ,(1)ΔTN=0 °C/min . The gap2/7075 Al matrix composites decrease with the increase of cooling rate, but 6.0% and 9.0% TiB2/7075 Al matrix composites are the opposite. The peak temperatures of solidification in TiB2/7075 composites all decreased with the increase of cooling rate. The initial solidification temperatures of TiB2/7075 Al matrix composites also firstly decrease, then increase, and finally decrease again with the increase of TiB2 content. The peak temperatures of solidification in TiB2/7075 Al matrix composites firstly increase, then decrease with the increase of TiB2 content at cooling rate of 10 and 20 °C/min, respectively, but keep decreasing with a cooling rate of 5 °C/min. The nucleation undercooling of TiB2/7075 Al matrix composites firstly increase, then decrease, and finally increase with the increase of TiB2 content at a cooling rate of 5 and 10 °C/min, respectively, but the nucleation undercooling of TiB2/7075 Al matrix composites keep decreasing at a cooling rate of 20 °C/min.The initial solidification temperatures of 3.0% and 4.5% TiB2 particles, and that the undercooling for free growth d are simply related by2/7075 composites firstly decrease, then increase, and finally decrease with the increase of TiB2 content from 2/7075 composites firstly increase, then decrease, and finally increase with the increase of TiB2 content at cooling rates 5 and 10 °C/min, respectively, from 2 content on the nucleation of 7075 Al alloy at large cooling rates such as 20 °C/min, so the nucleation undercooling of TiB2/7075 composites keep growing with the increase of TiB2 content.According to the free growth model ,16, two  Quested  also fou2/7075 composites at a cooling rate of 5 °C/min are 1.9 °C and 3.0 °C, 1.6 °C and 4.6 °C, respectively. The nucleation undercooling of 3.0% and 4.5%, 6.0% and 9.0% TiB2/7075 composites at a cooling rate of 10 °C/min are 5.5 °C and 7.3 °C, 3.3 °C and 4.6 °C, respectively. The gap of nucleation undercooling in TiB2/7075 composites at cooling rates of 5 °C/min and 10 °C/min are 3.6 °C and 4.3 °C, 1.7 °C and 0 °C, respectively, with the increase of TiB2 content. The change of the nucleation undercooling reflects the change of the critical nucleation radius of particles. The large change of nucleation undercooling with cooling rate means a wide range in particle diameter; the small change means a narrow range of particle diameter. That is to say, the nucleation undercooling of 9.0% TiB2/7075 composites has almost no change with changing cooling rate, which means the range of particle diameters is relatively narrow.The nucleation undercooling of 3.0% and 4.5%, 6.0% and 9.0% TiB2/7075 composites. Most of the TiB2 particles appear to be located on α-Al grain boundaries; however, small agglomerates of TiB2 particles could be found in the grains of 6.0% and 9.0% TiB2/7075 composites. TiB2 particles are distributed in the grain boundary area; most particles were about 600–1000 nm in 3.0% and 4.5% TiB2/7075 composites in 2 particles could also be found. The size of large TiB2 particles will decrease, the numbers of which will increase with increasing TiB2 particles, but the number of small TiB2 particles will also increase, because the heat of reaction of in-situ synthesis will also increase to refine the particles. More small TiB2 particles in 6.0% TiB2/7075 composites dispersed near large TiB2 particles in 2 particles are distributed in the grain boundary, and a large number of scattered small TiB2 particles with size of about 400 nm were distributed in the grain near the grain boundary in 2 particles will narrow the range of the size distribution; for example, the range of particle diameter changed from 1000–50 nm to 600–50 nm with the increase of TiB2 content, resulting in the change of nucleation undercooling.2 particles also varied from 570 nm to 430 nm, and then increased to 530 nm. Quested [2 particles, whereas the minimum grain size and the corresponding size of TiB2 particles were different in our research. TiB2 particles have the strongest nucleation abilities when the mean size of TiB2 particles is 250–500 nm, as shown in 2 particles smaller than 600 nm can be found in the grain near the grain boundary in 2 particles and parts of small TiB2 particles are distributed in the boundary area of grain in The research has shown that the grain size varied from 73.58 μm to 12.75 μm, and then increased to 23.11 μm with the increase of reaction holding time . At the  Quested  also fou2 from contact-angle measurements, 2/7075 Al matrix composites at a cooling rate of 5 °C/min is 188 nm and 123 nm, respectively, by Equation (2). From 2 particles are smaller than 200 nm (white dot in the picture) in the grain of 9.0% TiB2/7075 Al matrix composites, compared to 4.5% TiB2/7075 Al matrix composites. Comparing 2 particles will be captured into the grain by solid/liquid interface. At the same time, most particles larger than 600 nm and a minority of small TiB2 particles are pushed into the grain boundary by the solid/liquid interface; locating in the grain boundary can hinder the Al atoms from diffusing during the solidification of the 7075 Al alloy and restrain α-Al phase growth. The mean size of TiB2 particles is relatively larger in 3.0% TiB2/7075 composites, which particles value a major role locating in the grain boundary. With increasing of TiB2 particles, the number of TiB2 particles smaller than 600 nm will increase, resulting in the increase of the capability of heterogeneous nucleus, and nucleation is the main function of TiB2 particles at contents below 4.5%. That is to say, the parts of TiB2 particle valve a major role locating in the grain boundary, and others become the nucleus of α-Al phase. The influence of particles shifted from dominating by locating to dominating by nucleation as the quantity of TiB2 increased, which causes a change from dendrites to uniform rosette grain, and again to large dendrites. Meanwhile, small agglomerates of TiB2 particles could also be found in TiB2/7075 composites with the increase of TiB2 content, because the tiny particles have a larger surface energy, even in 4.5% TiB2/7075 composites.The nucleation at low cooling rates can best reflect the influence of particles on microstructures. We take the highest available value of 106J/Km3 , it can (1)2/7075 Al matrix composites firstly decreased, then increased, and finally decreased again with the increase of TiB2 content. The grain sizes of TiB2/7075 Al matrix composites increased with decreasing cooling rate, but the changes were not very noticeable from 20 °C/min to 5 °C/min. The mean grain size of 3.0% and 4.5% TiB2/7075 Al matrix composites could reach 236 μm and 165 μm, respectively, at a cooling rate of 1 °C/min.The grain sizes of TiB(2)2/7075 Al matrix composites firstly decreased, then increased, and finally decreased again with the increase of TiB2 content. The initial solidification temperatures and the nucleation undercooling of TiB2/7075 Al matrix composites firstly increased, then decreased, and finally increased again with the increase of TiB2 content. The melting and solidification process showed no significant change with the decrease of cooling rate in 9.0% TiB2/7075 Al matrix composites. A large change of the nucleation undercooling with cooling rate meant a wide range in particle diameter in TiB2/7075 composites, so a small change meant narrow range.The liquidus temperatures of TiB(3)2 particles were pushed into the grain boundary, locating in the grain boundary can hinder the Al atoms from diffusing during the solidification of the 7075 Al alloy and restrain α-Al phase growth. The influence of particles shifted from dominating by locating to dominating by nucleation as the quantity of TiB2 particles increased due to the decrease of the average diameter of TiB2 particles.Most small particles can act as heterogeneous nuclei, which induced the grain growth and were captured into the grain by solid/liquid interface. At the same time, most of larger particles and a minority of small TiB


Pleurotus ostreatus is a white rot fungus that grows on lignocellulosic biomass by metabolizing the main constituents. Extracellular enzymes play a key role in this process. During the hydrolysis of lignocellulose, potentially toxic molecules are released from lignin, and the molecules are derived from hemicellulose or cellulose that trigger various responses in fungus, thereby influencing mycelial growth. In order to characterize the mechanism underlying the response of P. ostreatus to lignin, we conducted a comparative proteomic analysis of P. ostreatus grown on different lignocellulose substrates. In this work, the mycelium proteome of P. ostreatus grown in liquid minimal medium with lignin, xylan, and carboxymethyl cellulose (CMC) was analyzed using the complementary two-dimensional gel electrophoresis (2-DE) approach; 115 proteins were identified, most of which were classified into five types according to their function. Proteins with an antioxidant function that play a role in the stress response were upregulated in response to lignin. Most proteins involving in carbohydrate and energy metabolism were less abundant in lignin. Xylan and CMC may enhanced the process of carbohydrate metabolism by regulating the level of expression of various carbohydrate metabolism-related proteins. The change of protein expression level was related to the adaptability of P. ostreatus to lignocellulose. These findings provide novel insights into the mechanisms underlying the response of white-rot fungus to lignocellulose. P. ostreatus is a white-rot fungus that can be easily cultivated on a variety of lignocellulosic substrates, owing to its ability to degrade cellulose, lignin, and hemicellulose through the action of complex oxidative and hydrolytic enzymatic systems  and laccase are the major oxidative enzymes secreted by P. ostreatus that are responsible for the oxidation of lignin and a wide range of lignin-analogous compounds -dependent proteins are a current research focus, as these enzymes play important roles in lignocellulose oxidation  was used in this study. The strain was maintained on potato dextrose agar (PDA) slants at 4°C and activated for 1 week on new PDA slant before use, then transferred into potato dextrose broth (PDB) medium for 7 days at 28°C as inoculum.−3 mol/L KH2PO4, 3 × 10−3 mol/L MgSO4.7H2O, 2 × 10−5 mol/L ammonium tartrate, 3 × 10−4 mol /L CaCl2.2H2O, 5 × 10−2 mol/L glucose, and 10 ml/L trace element contained: 7.8 × 10−3 mol/L amino acetic acid, 1.2 × 10−3 mol/L MgSO4.H2O, 2.9 × 10−3 mol/L MnSO4.H2O, 1.7 × 10−2 mol/L NaCl, 3.59 × 10−4 mol/L FeSO4.7H2O, 7.75 × 10−4 mol/L CoCl2, 9.0 × 10−4 mol/L CaCl2, 3.48 × 10−4 mol/L ZnSO4.7H2O, 4 × 10−5 mol/L CuSO4.5H2O, 2.1 × 10−5 mol/L AlK(SO4)2.12H2O, 1.6 × 10−4 mol/L H3BO3, 4.1 × 10−5 mol/L NaMoO4.2H2O.or 100 ml Kirk's liquid medium supplemented with 0.5 g lignin(Sigma), xylan(Sigma), or cellulose(Sigma). All experiments were accompanied by controls that lacked the lignocellulose amendment. The mycelia were collected after 7 days incubation in dark at 28°C with continuous stirring at 120 r/min, and the cultures were centrifuged for collection washed with sterilized MilliQ water for several times to separate from medium, then kept at −80°C for use.In order to exclude influence of other organics, the strain was inoculated into a 250 ml flask with 100 mL modified Kirk's liquid medium which just contain basal salt component as basic medium , and proteins were quantified by BCA method. Intracellular FAD concentration was measured using an FAD Colorimetric/Fluorometric Assay Kit (BioVision). Experimental methods refer to product description.P. ostreatus with ABTS as the substrate.Laccase activity was determined spectrophotometrically as previous study described by with 14 μmol of ABTS as the substrate , were considered as significant. The theoretical pIs were calculated using the ExPASy Compute pI/Mw tool (http://web.expasy.org/compute_pi/).Frozen mycelia were used to extract total myceliaproteins by the TCA-acetone precipitation method .Then, we performed MALDI-TOF/TOF to identify significantly changed spots in one or two cultures compared with that in the control. Spots from 2-DE gels were excised and digested with trypsin for 20 h. The resulting peptide mixtures were desalted using ZipTips C18 (Millipore), and eluted onto a 96-well MALDI target plate. Then, 2 mL samples on the plate were mixed with 1 mL supersaturated CHCA solution with 0.1% TFA and 50%ACN. Mass spectrometric analysis were measured on 5800 MALDI-TOF/TOF (AB SCIEX). Briefly, mass data acquisitions were piloted by 4000 Series Explorer Software v3.0 using batched-processing and automatic switching between MS and MS/MS modes The PMF data were collected and blasted in JGI database using MASCOT software  classification system  and that of 71 spots significantly decreased , the High-tech Research and Development Program of China (2012AA101805) and the National Natural Science Foundation of China (J1103514).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.