Our novel direct synthesis of graphene/TiO2 heterostructure is achieved by C60 deposition on transition Ti metal surface using a molecular beam epitaxy approach and O2 intercalation method, which is compatible with wafer scale growth of heterostructures. As-grown heterostructures exhibit inherent photosensitivity in the visible light spectrum with high photo responsivity. The photo sensitivity is 25 times higher than that of reported graphene photo detectors. The improved responsivity is attributed to optical transitions between O 2p orbitals in the valence band of TiO2 and C 2p orbitals in the conduction band of graphene enabled by Coulomb interactions at the interface. In addition, this heterostructure provides a platform for realization of bottom gated graphene field effect devices with graphene and TiO2 playing the roles of channel and gate dielectric layers, respectively.Heterostructures consisting of two-dimensional materials have shown new physical phenomena, novel electronic and optical properties, and new device concepts not observed in bulk material systems or purely three dimensional heterostructures. These new effects originated mostly from the van der Waals interaction between the different layers. Here we report that a new optical and electronic device platform can be provided by heterostructures of 2D graphene with a metal oxide (TiO In graphene field effect transistors (GFET), graphene provides the active layer where the channel carriers are modulated by gate biasing through the dielectric layer. In graphene optical devices a dielectric layer is needed to induce a built-in potential within the structure so that photo-excited electrons can be extracted from the photosensitive materials into the graphene layer on top. Currently the photosensitive 2D materials such as MoS2 or WS2 are widely investigated to enhance photo-responsibility. Recent studies of graphene/photosensitive 2D heterostructures have revealed new physical phenomena such as Moire patterns582/Gr heterostructure resulted from Van Hove singularities2 has been considered since it is a well-known material that has been widely used for light harvesting2 a very attractive material for the realization of both electronic and photonic devices. The schematic diagram of our growth approach is shown in 2 dielectric layer. We demonstrate this dual functionality by depositing two top metal contacts on the Gr/TiO2 heterostructure to serve as source and drain electrodes. The gating voltage is applied through the underlying TiO2 on n-type SiC in a bottom gated FET configuration. With the same device configuration, the photosensitivity under visible light illumination is measured by the change of photoconductivity with zero gate voltage. We believe that the observed photo response at zero gate voltage is caused by the optical transitions between the valence band maximum, associated O 2p orbitals in TiO2, and the conduction band minima, formed by the C 2p orbitals of the graphene in the Gr/TiO2 heterostructure. The schematic diagrams of device and measurement configurations are shown in 2 heterostructure have been conducted in air.Graphene (Gr) is a very attractive material for diverse optoelectronic and electronic device applications due to its extraordinary transport properties, broad spectral-bandwidth, and fast response time12360 flux dependence were studied. The growth temperature was kept at 1400 °C during these experiments. First, the C60 flux was held at 1.6 × 10−7 Torr while the growth time varied. As growth time increased, the 2D peak position shifted linearly to lower wavenumber. After 30 min. growth, the 2D peak was located at ~2712 ± 5 cm−1. With longer growth time (90min.), it shifted to 2705 ± 1.5 cm−1. The FWHM of the 2D peak showed similar behavior to peak position. The value of the FHWM decreased from 71 ± 9 cm−1 (30 min. growth) to 56.5 ± 2 cm−1 (90 min. growth). The quality of the graphene was estimated from the ratio of the D and G peaks. The growth time dependence of the D/G ratio also exhibited similar behavior to position and FWHM of the 2D peak. Increased growth time resulted in a decrease of the D/G ratio from 0.32 ± 0.07 (30 min. growth) to 0.23 ± 0.01 (90 min. growth). The C60 flux dependence with two different fluxes was also evaluated. The growth was done at 1400 °C for 2 hr. With increasing flux from 0.8 × 10−7 to 1.6 × 10−7 Torr, we observed improvement of graphene quality with a decrease of the D/G ratio and 2D FWHM by 40% and 20%, respectively. The peak position also shifts to lower wavenumber from 2706 cm−1 to 2703 cm−1. Next, the effect of O2 intercalation at 500 °C at various times was studied with Raman analysis. The 2D peak position shifted slightly to lower wavenumber. A 4hr. intercalation led a 2D peak shift of only 5 cm−1, to lower wavenumber. The 2D FWHM showed entirely different behavior. Initially it broadened compared to that of as-grown graphene  and then started to narrow . The graphene quality variation was estimated by comparing the D/G ratio before and after intercalation. We did not observed any variation of the ratio for intercalation up to 60 min. but a significant increase was observed beyond 60 min.To investigate the graphene growth on Ti metal surfaces, the growth time and C2 surfaces. 60 deposition on both TiO2 (red color curve) and Ti (blue color curve). As seen in 3/2 and Ti 2p1/2 peaks for TiO2 are located at 459.1 eV and 464.8 eV respectively. However, our results  shows no O 1s peak corresponding to TiO2. Therefore, we conclude that C60 deposition results in graphene and TiC formation on both Ti and TiO2. The formation of TiC upon C60 deposition could be explained by a carbon thermal reduction reaction2 reacts with carbon to form TiC and releases CO gas above 1300 °C. The high substrate temperature (1400 °C) during C60 deposition could initiate carbon thermal reduction reactions between molecular C60 and the TiO2 surface.Graphene growth was attempted on both Ti and TiO results  show the2 has been obtained from oxidation of TiC in the temperature range from 623 K to 1073 K in air2 was accomplished with an O2 intercalation procedure. Intercalation was conducted under controlled O2 pressure (10−4 Torr) in the XPS chamber (details in Method). 2 intercalation progressed. Besides peaks for as-grown graphene at 284.7eV and TiC at 281.5 eV, three peaks associated with C-O (286.1 eV), C = O (288.1 eV), and the π → π* (291.5 eV) transition were observed in the C 1s spectra (not shown here). Since oxygen-carbon bonding was observed in the as-grown sample as well, the existence of C and O bonding might be due to air exposure during transfer from the growth chamber to the XPS chamber. In as-grown graphene/Ti structure, graphene peak is located at 284.7 eV with ~1.2 eV FWHM. However, the shift of peak position to 284.2 eV and the FHWM narrowing from 1.2 eV to 1 eV were observed as intercalation proceeds. We attribute the shift in binding energy to substrate interactions between the Ti and TiO2. The conversion of TiC into TiO2 during intercalation is shown in 4+ cation doublet located at 458.8 eV and 464.6 eV and the metallic type bonding (titanium carbide) Ti0 peaks located at 454.7 eV and 460.9 eV were observed. These peaks are associated with Ti 2p3/2 and Ti 2p1/2 transitions, respectively. The evolution of chemical composition at different temperatures and intercalation times is also shown in 4+ and Ti3+ along with TiC peaks  and its peak intensity increases with further intercalation. We calculated the activation energy for TiO2 conversion by O2 intercalation and graphene etching by the O2 (detail in Method). The calculated activation energy for TiO2 conversion is about 0.29 eV while activation for graphene etching is 0.41 eV, which indicate that TiO2 conversion is the dominant reaction in our intercalation experiments.Intercalation between epitaxial graphene and its substrate has been widely studied as a method of decoupling the strong interaction from the substrate14151660 deposition on Ti metal and O2 intercalation were also investigated with Raman measurements. 2 exposure. In the as-grown sample, Raman measurements show the typical Raman fingerprint of graphene with the presence of the 2D, G, and D peaks. Importantly, a fit to the 2D peak gave a single Lorentizan at ~2700 cm−1. Typically, C60 deposition on SiC resulted in Bernal stacked few layer graphene−1. This suggests that graphene on Ti metal is not Bernal stacked but is very similar to turbostractic stacking of graphene2 intercalation (2 layer (Eg = 3.2 eV) after O2 intercalation enhances the transmittance of visible incident Raman laser into SiC substrate. It results in Raman activation of SiC substrate and convolution of SiC and graphene Raman spectrum. The formation of TiO2 after intercalation was confirmed by observation of TiO2 vibration modes , an A1g and B1g mode doublet at 516 cm−1, and a B1g mode at 399 cm−12, A1, and E1 modes (black colored annotations), while two distinct TiO2 related Raman peaks (orange color annotations) were observed after O2 intercalation. The peaks at ~143 cm−1 and ~640 cm−1 are attributed to the Eg mode (O-Ti-O bending vibration) of anatase TiO2.The Con modes . TiO2 ha2/n-SiC heterostructures we fabricated bottom gated FET devices. Ti/Au contacts were deposited for source and drain contacts on top of the 1 cm × 1 cm samples.  The channel length was about 6 mm. The schematic diagram of GFET structure is shown in 2. The gate response was measured for a gate voltage range of ±20 V and is shown in g ≈ +3 V, which indicates that the intercalated graphene exhibits hole doping. d ≈ 2.9 mA at Vg = 15 V and Vd = 0.5 V). These results might be due a high capacitance due to the TiO2 dielectric layer.To explore the electrical properties of our Gr/TiO2 has been widely used as photosensitive material. However, its large bandgap (3.2 eV) has restricted its use to ultraviolet applications since the photo response is only activated under ultraviolet radiation. Several approaches have been proposed to extend its photo response to a broader range such as dye sensitization, doping, and coupling with semiconductors23242/n-SiC heterostructures at room temperature (RT). The schematic diagram of measurement is shown in −1 conductance. Upon illumination, the device showed an increased current flow through the source-drain contacts with increased current flow at high bias voltage. The conductance of the graphene improved to 0.06 Ω−1, indicating a visible light photoresponse in our heterostructure. The calculated photo-responsivity of the heterostructure is about 278 mAW−1, which is much higher than that reported for modified graphene photo-detectors such as graphene metal junctions (6.1 mAW−1)−1)−1)g = −20 V, the graphene photoconductivity resulted in a conductance of 0.052 Ω−1 while it increased to 0.06 Ω−1 at 20 V. The difference between negative and positive gate voltage might be related to variations of net current flow through the channel. The response to turning the light on and off was measured under 0.2 V bias between source-drain as shown in −1, which is lower than that of our photo detector. Since the large bandgap (3.2 eV) of TiO2 does not allow for a photo-response to visible light, the photo-response observed here might originate from the Gr/TiO2 heterostructure. A possible mechanism for the enhanced visible light photoresponse seen in our Gr/TiO2 heterostructures may be provided by Geng, Liu and Yao2/graphene composites they calculated the electronic properties of atomic scale TiO2 clusters on graphene films and predicted anchoring of the clusters by charge rearrangement at the interface due to an interaction between the Ti 3d orbitals of the TiO2 and the p orbital of the graphene sheet. This results in extra states in the bandgap of TiO2 which enable absorption of visible light by the composite and the transfer of photoexcited electrons from the TiO2 valence band to the graphene conduction band. The subsequent separation of electrons and holes in graphene and TiO2 leads to long carrier lifetimes. In addition, since the interface between TiO2 and graphene plays a critical role, they also suggested that increasing the interface contact area could improve the photo response. We speculate that a similar process may be taking place in our comparatively large area heterostructures. The absorption of light takes place primarily in the TiO2 closest to the interface where their density of states is modified by the presence of the graphene layer. Electrons then transfer to the graphene layer and increase the electron density. The separation of electrons and holes reduces recombination and the holes in the TiO2 can screen the gating effect under positive bias while inducing more holes in the graphene layer under negative bias, leading to a reduced photoconductivity at negative bias. This model explains our experimental results shown in the gate response characteristics  with a metal carbide interface layer (TiC). After O2 intercalation the byproduct TiC layer was completely converted into TiO2, which resulted in a graphene/oxide heterostructure. We showed that this heterostructure exhibits dual functional properties, showing both electronic field effect phenomena and an enhanced visible light photosensitity. The field effect phenomena and enhanced photosensitivity were demonstrated in bottom gated GFET devices.We demonstrated that C60 molecules were deposited on the Ti layers in a high temperature molecular beam epitaxy system. The growth temperature and carbon flux were 1400 °C and 1.6 × 10−7 Torr. The growth time was 30 min. For the study of graphene growth on TiO2 surfaces, the as-deposited Ti/n-SiC sample was annealed in an O2 environment at 800 °C for 1 hr. The oxidation of the metallic Ti layer was confirmed by XPS analysis. The graphene growth was performed with the same growth conditions as those for Ti/n-SiC. After graphene growth, the sample was exposed to air while being transferred to the XPS chamber for O2 intercalation experiments.Metallic titanium layers (20 nm) were grown on an n-type SiC wafers by e-beam evaporation. CFor the field effect transistor device tests, Au/Ti metal contacts (source and drain) were deposited on the graphene surface. The photo response experiment was performed by illumination with a white LED at room temperature.2 was estimated by high resolution XPS spectra. The rate constant (k) for TiO2 formation due to intercalation was obtained by analyzing the temporal change in the ratio of TiC to TiO2 atomic concentrations at each intercalation temperature. By fitting an Arrhenius plot (Log (k) vs. 1/T), the activation energy (Eg) for oxidation was calculated. The graphene etching effect during O2 intercalation was monitored by the reduction of the graphene C 1s peak (284.7 eV) intensity. The etching activation energy was also calculated by estimating rate constant (k) and fitting the Arrhenius plot (Log (k) vs. 1/T). The temporal variation of C 1s ratio between as-grown and intercalated samples was used to calculate the rate constant (k) at each intercalation temperature.The temporal change in atomic concentration of TiC and TiO−8 Torr the sample was transferred to the analysis chamber. After initial XPS scans were acquired the sample was transferred back into the load-lock chamber for O2 intercalation. For the intercalation experiments research grade O2 was introduced into the chamber with a leak valve. The background pressure in the chamber was maintained at 1.0 × 10−4 Torr. Once the desired pressure was reached the sample was heated from the backside of the sample holder with a 10.6 μm CO2 IR laser. The laser output power was adjusted until the target temperature was reached. The temperature of the sample was measured with a pyrometer. After the intercalation the chamber was pumped down until a background pressure 3.0 × 10−8 Torr was reached. The sample was then transferred back into the analysis chamber for XPS data acquisiton.After graphene growth, the sample was transferred to the load-lock of the XPS system. When the background pressure of the load lock chamber reached 3.0 × 107/2 (84.0 eV) peak with an overall resolution of ~1 eV. Each spectrum was fit with a Shirley background followed by fitting convoluted Gaussian and Lorentzian peak shapes for each chemical state in the region of interest. For the C 1s graphene and Ti 2p metal peaks an additional asymetry parameter was added to the Gaussian-Lorentzian convolution. The fitting procedure consisted of a Levenberg-Marquardt routine that minimizes χ2.The XPS data was acquired with an Scienta Omicron standard Mg Kα (1253.6 eV) X-ray source and Argus hemispherical analyzer. High resolution scans of the C 1s, O 1s, and Ti 2p regions were acquired at 20 eV pass energy with 0.1 eV steps. The binding energy scale was calibrated against the Au 4fHow to cite this article: Park, J. et al. Approach to multifunctional device platform with epitaxial graphene on transition metal oxide. Sci. Rep.5, 14374; doi: 10.1038/srep14374 (2015).


This study focused on a collection of strains representing Ophiostoma piliferum, Ophiostoma pluriannulatum and related species that cause blue-stain; these fungi colonize the sapwood of trees and impart a dark stain. This reduces the value of the lumber. The goal was to examine the mtDNA rns intron landscape for these important blue stain fungi in order to facilitate future annotation of mitochondrial genomes (mtDNA) and to potentially identify mtDNA introns that can encode homing endonucleases which may have applications in biotechnology.Based on previous studies, it was suspected that the mitochondrial Comparative sequence analysis identified five intron insertion sites among the ophiostomatoid fungi examined. Positions mS379 and mS952 harbor group II introns, the mS379 intron encodes a reverse transcriptase, and the mS952 intron encodes a potential homing endonuclease. Positions mS569, mS1224, and mS1247 have group I introns inserted and these encode intact or eroded homing endonuclease open reading frames (ORF). Phylogenetic analysis of the intron ORFs showed that they can be found in the same insertion site in closely and distantly related species.rns introns), strains representing O. pilifera, O. pluriannulatum and Ophiostoma novae-zelandiae could not be resolved. Phylogenetic studies suggest that introns are gained and lost and that horizontal transfer could explain the presence of related intron in distantly related fungi. With regard to the mS379 group II intron, this study shows that mitochondrial group II introns and their reverse transcriptases may also follow the life cycle previously proposed for group I introns and their homing endonucleases. This consists of intron invasion, decay of intron ORF, loss of intron, and possible reinvasion.Based on the molecular markers examined  contains supplementary material, which is available to authorized users. Syd.] and some are plant pathogens , there is evidence that an rns GII intron could be associated with inducing hypovirulence . An Erlenmeyer flask containing fifty ml of PYG was inoculated with agar blocks (~1 × 1 mm) derived from an agar plate culture and the liquid cultures were incubated in the dark at 20 °C for 4–6 days. The protocol used for DNA extraction was previously described by Hausner et al. (2EDTA·2H2O (pH 8.0), 1.5 M NaCl, 2 % (w/v) cetyltrimethylammonium bromide (CTAB)] was added along with 3 g of glass beads and 660 μl of 20 % (w/v) sodium dodecyl sulfate (SDS). This mixture was vortexed for about 2 min. Thereafter, the lysate was incubated at 55 °C for 2 h. Cell debris and contaminants were removed by chloroform extraction and centrifuging for 20 min at 2000 rpm. The top aqueous layer was recovered and 2.5 volume of ice cold 95 % ethanol was added to precipitate the nucleic acids. Finally, the nucleic acids were recovered by centrifuging for 30 min at 3000 rpm. The DNA pellets were washed with 1 ml of 70 % ethanol and resuspended in 300 µl TE buffer [10 mM Tris–HCl (pH 7.6) and 1 mM Na2EDTA·2H2O (pH 8.0)].Fungal strains examined in this study are listed in Table r et al. . Brieflyrns sequences were previously described by Hausner and Wang . Automated fluorescent DNA sequence analysis was performed by the Manitoba Institute of Cell Biology DNA Sequencing Facility .Primers and conditions for the polymerase chain reaction (PCR) for obtaining the internal transcribed spacer (ITS) regions and mtDNA and Wang  and Hafeand Wang , respectand Wang . Primers Hausner  and Hafe Hausner ; additio Hausner . All PCRhttp://doua.prabi.fr/software/cap3). The resulting contigs were used as templates for designing new primers. Once completed, the rns sequences were applied to the basic local alignment search tool  to identify similar sequences. Sequences were compiled and aligned by the online multiple sequence alignment program, MAFFT . The literature was also consulted with regard to comparing introns identified in this study with introns and their putative folds, characterized in other studies  were also consulted. Intron sequences  were applied to RNAweasel  alignments were analyzed with three tree building methods  and the bootstrap option was implemented (2000 replicates) in order to assess the level of support for the tree topologies generated by the respective methods. With regard to NJ analysis, the maximum composite likelihood method and its defaults were selected along with the complete deletion of gaps option. For the PARS method, the complete deletion option was also selected. In the ML method, the best model was first determined with the “best model” function as implemented in MEGA. Therefore, for ML analysis, the Whelan and Goldman plus Freq. model was selected and the complete deletion option was selected to remove segments of the alignments that contained gaps.rns gene among the strains of Ophiostoma examined in this study. Based on previous reports (Hafez and Hausner rns genes that do not have insertions and rns derived amplicons greater than 1.2 kb have insertions (introns). Among the 23 strains surveyed, only the following strains, based on the size of rns-derived PCR amplicons, appear to have insertions within the rns gene: Ophiostoma californicum WIN(M)505 , Ophiostoma pluriannulatum WIN(M)539 [(Hedgc.) Syd. & P. Syd.], Ophiostoma carpenteri WIN(M)855 (J. Reid & Georg Hausner), Ophiostoma perfectum WIN(M)823 [(R.W. Davidson) de Hoog], and O. piliferum WIN(M)972. The sizes of the amplicons were as follows: O. perfectum WIN(M)823 yielded a 7 kb PCR amplicon while O. pluriannulatum WIN(M)539, O. piliferum WIN(M)972, O. californicum WIN(M)505, and O. carpenteri WIN(M)855 yielded rns-derived PCR amplicons of about 5 kb 823 contains three introns at the following positions 972 and O. californicum WIN(M)505 have introns inserted at positions mS952 and mS1224. O. carpenteri WIN(M)855 has one intron at position mS952. O. pluriannulatum WIN(M)539 also has an intron at mS952 in addition to an intron inserted at mS569.As summarized in Fig. Ophiostoma species . Sequences for strains representing Leptographium and Grosmannia were included as outgroups. All Ophiostoma species were clearly separated from the outgroup species. The taxonomy of O. piliferum is a complex issue 823 revealed to be the most complex with regard to intron arrangements  Georg Hausner, J. Reid & Klassen] with strong bootstrap support and both of these sequences are related to the mS379 ORFs from the Ophiostoma minus [(Hedgc.) Syd. & P. Syd.] rns genes. The phylogenetic tree also shows that mS379 RT ORFs are related to an RT ORF that exists in the cox1 gene of Podospora anserina. Although, at lower node support values, basal to the above grouping, are additional RT ORFs mostly encoded within cox1 introns with the exception of the Nitella hyalina rns intron-encoded RT ORF. A node that received high bootstrap support (94 %) suggests monophyly for RT ORFs encoded within bacterial genomes such as Pseudomonas syringae, Pseudomonas putida, Pseudomonas aeruginosa, Ktedonobacter racemifer, and the cox1 and cox3 intron-encoded RT ORFs from various fungi, brown algae  and liverwort (Marchantia polymorpha) along with the fungal mS379 ORFs.The ORF Finder program identified an intact GIIA RT ORF 510 aa) in the 0 aa in tO. perfectum WIN(M)823 and O. pluriannulatum WIN(M)539, showed that the ORF is degraded in O. perfectum due to some frame shift mutations but otherwise nearly identical to the intact sequence found in O. pluriannulatum. A data set was assembled by extracting related sequences from GenBank depending on the O. perfectum WIN(M)823 sequence data as a query. The alignment was analyzed with three programs contained within MEGA6 . Peltigera malacea was used as an outgroup  and O. pluriannulatum WIN(M)539 (286 aa) and for O. californicum WIN(M)505 (323 aa) but for O. pluriannulatum WIN(972) (173 aa) the ORF only has one LAGLIDADG domain. The latter single domain ORF may be an indication that this ORF has eroded due to mutations that generated premature stop codons. The phylogenetic tree shows that the O. carpenteri GII intron-encoded LHE ORF (representing the other mS952 ORFs uncovered during this study) is found in a variety of fungi including several members of the Ophiostomatales: Ophiostoma stenoceras [(Robak) Melin & Nannf.], Ophiostoma brevicolle [(R.W. Davidson) de Hoog & R.J. Scheff.], O. torulosum, Leptographium species and O. minus. A LHE sequence from the nad5 i2 ORF from Rhizoctonia solani was used as the outgroup 505 (319aa) and O. piliferum WIN(M)972 (441aa) suggests that these ORFs are closely related the ms1224 intron ORFs that exists in Ophiocordyceps sobolifera [AB027350], Agrocybe aegerita [AAB50391] and Leptosphaeria maculans [FP929115]. The clade is supported with high node support values. The rns i4 intron (=mS1210) ORF from C. parasitica joins the above clade at a node that received only moderate support (72 %). The remaining nodes representing deeper branching patterns within this tree received only poor support values and thus, distant relationships cannot be extracted from this analysis.The mS952 GII intron LHE ORF from rpenteri 22 aa andO. perfectum (rns i3 ORF) as a query in blastp. The Amoebidium parasiticum rnl-i2 ORF was used as the outgroup in this analysis  due to frameshift mutations but 418 aa could be assembled. The O. perfectum mS1247 ORF grouped with mS1247 ORFs from O. sobolifera [AB027350], L. maculans [FP929115] and Chaetomium thermophilum [JX139037]. However, the node support values are moderate for the clade which included all four mS1247 ORF sequences; but the node linking the O. sobolifera with O. perfectum received strong support (99 %). With regard to deeper nodes, the phylogenetic tree received poor node support values so no further conclusions could be reached as to possible relationships of the mS1247 with other LAGLIDADG type ORFs.Another data set was constructed by using the mS1247 intron ORF sequence from Ophiostoma species sampled in this study, many lacked introns within the rns gene, but a few members did contain introns . This is suggestive of either rapid gain or rapid loss of introns. Gain of introns could be achieved by horizontal transfer or by vertical transmission of these introns, random loss of introns could be achieved by the original intron containing allele being replaced by a cDNA version of the gene  due to the presence/absence of introns and associated ORFs. Also, introns appear to be inserted at the same positions among related and distantly related fungi, which is a reflection of the homing mechanism promoted by the intron-encoded homing endonucleases. Homing endonucleases recognize long target sites and therefore, require conserved regions for their long term survival and for moving laterally from one species to another 832 rns gene is 7 kb in length and has three introns inserted in highly conserved regions within the rns gene. The mS379 intron is a GII intron, type AI, that was previously reported in three O. minus strains by Hafez and Hausner (O. perfectum WIN(M)823 intron at mS379 encodes a complete RT ORF that is inserted into domain II of the GII intron. The latter is unusual as organellar GII intron ORFs are typically located in domain IV , a life cycle of invasion and degeneration for HEGs and their hosting GI introns. Our study suggests that RT ORFs in GII introns can follow a similar life cycle; i.e. invasion into an empty spot, followed by slow degeneration initially of the ORF, and eventually complete loss of the ORF, and presumably, complete loss of the intron, regenerating a potential site available for reinvasion.The analysis of sequence data has shown that the  Hausner . In thesand Burt  predicteOphiostoma mS379 RT ORF within a grouping that includes sequences from brown algae, bacteria, plants and fungi. Thus, it appears this intron ORF has a complex evolutionary history that could include horizontal transfers. It has been shown that GII intron ORFs probably co-evolve with their host introns but these composite elements can evolve independently from the host genomes that encode them due to lateral transfers 823 is the mS569 GI intron, type C2, with a LHE ORF. This intron is also found in O. pluriannulatum WIN(M)539. Hafez et al. 855, O. pluriannulatum WIN(M)927, O. subannulatum WIN(M)539, and O. californicum WIN(M)505. This GII intron was previously found in other Ophiostoma species by Mullineux et al. 972 and O. californicum WIN(M)505 have a GI intron, type C2, at position S1224 and a LHE as an IEP. This intron and ORF are also found in rather distantly related fungi, L. maculans, O. sobolifera, A. aegerita and C. parasitica 823 is located at position mS1247. The mS1247 intron is a GI, type C2, encoding a LHE ORF. So far within Ophiostoma, the mS1247 intron has only been observed in O. perfectum. Hafez et al.  and GII introns . Among intron-encoded ORFs, the LAGLIDADG family appears to have invaded four of these introns, with the mS379 GII intron maintaining an RT ORF. Intron landscapes provide information for those that annotate mtDNAs or for those searching for sources of polymorphisms. In addition, ribozymes and intron-encoded ORFs (HEases and RTs) have applications in biotechnology, genome editing, or as functional genomics tools (Stoddard With regard to the Stoddard . Intron Stoddard .Phylogenetic investigations suggest that the intron ORFs evolve rapidly and thus, most trees had poorly supported topologies. In general, among members of the Ophiostomatales, introns appear to be gained and lost frequently. The intron ORFs tree topologies do not follow the expected host species/genome trees, which hints at the possibilities of horizontal transfers of these elements among distantly related lines. Movement of introns along with their IEPs is facilitated by their encoded proteins that target specific sites in cognate intronless alleles (Colleaux et al.


Fungal secretome consists of various functional groups of proteins, many of which participate in nutrient acquisition, self-protection, or manipulation of the environment and neighboring organisms. The least characterized component of the secretome is small secreted proteins (SSPs). Some SSPs have been reported to function as effectors, but most remain to be characterized. The composition of major secretome components, such as carbohydrate-active enzymes, proteases, lipases, and oxidoreductases, appear to reflect the lifestyle and ecological niche of individual species. We hypothesize that many SSPs participate in manipulating plants as effectors. Obligate biotrophs likely encode more and diverse effector-like SSPs to suppress host defense compared to necrotrophs, which generally use cell wall degrading enzymes and phytotoxins to kill hosts. Because different secretome prediction workflows have been used in different studies, available secretome data are difficult to integrate for comprehensive comparative studies to test this hypothesis. In this study, SSPs encoded by 136 fungal species were identified from data archived in Fungal Secretome Database (FSD) via a refined secretome workflow. Subsequently, compositions of SSPs and other secretome components were compared in light of taxa and lifestyles. Those species that are intimately associated with host cells, such as biotrophs and symbionts, usually have higher proportion of species-specific SSPs (SSSPs) than hemibiotrophs and necrotrophs, but the latter groups displayed higher proportions of secreted enzymes. Results from our study established a foundation for functional studies on SSPs and will also help understand genomic changes potentially underpinning different fungal lifestyles. R)-gene mediated defense as avirulence factors  rich in cysteine residues; and (e) presence of a conserved motif within effector candidates like in oomycete and fungal effectors  with individual refined secretomes as queries against BLAST databases of these enzyme sets. Fungal oxidoreductases were downloaded from Fungal Peroxidase Database  and conserved SSPs (CSSPs) were annotated using pre-computed InterPro terms, which were retrieved from Comparative Fungal Genomics Platform 2.0  clustering. Resulting data were clustered via MCL with the inflation option 1.4 for high granularity  to 1940  and the highest being 11.0% . Both P. jirovecii and M. oryzae belong to the phylum Ascomycota, illustrating high degrees of variance within individual phyla  and one Ascomycota species (P. jirovecii) displayed large proportions  and a saprotroph (Kluyveromyces lactis), did not have any SSSPs. The remaining SSPs were encoded by at least two species and were termed conserved SSPs (CSSPs), which may correspond to general fungal proteins or elicitors.Effector-like SSPs were divided into two classes. Proteins of 300 aa or shorter that appeared to be encoded only by one species were considered species-specific SSPs (SSSPs); a total of 8275 SSSPs were found from 133 species. The fasta formatted SSSP sequences available in Supplementary Data Sheet InterPro domain analysis was performed to predict potential functions of both SSSPs and CSSPs. Most SSPs, 7960 out of 8275 (96.2%), displayed no defined InterPro terms with only 315 SSPs being annotated with 97 different terms , one biotroph (Mixia osmundae), and one saprotroph  had higher AT-contents at 52, 72, 44, and 60%, respectively  and B. graminis (Ascomycota) have the highest numbers. In contrast, two species in Ustilagomycotina (Ustilago maydis and Sporisorium reilianum) and three Ascomycota species  encode small numbers of SSSPs, similar to those in saprotrophs. In addition, wood-decaying necrotrophs and hemibiotrophs have similar numbers of SSSPs.The number of SSSPs within species ranged from 0 to 466. Although not all plant-associated fungi have higher numbers of SSSPs than saprotrophs and animal pathogens Figure , in geneU. maydis and S. reilianum) contained greater numbers of SSSPs compared to other members. In Ascomycota, the range was widest in Pezizomycotina with many outliers being present at both sides of the mean. Three hemibiotrophs, including M. oryzae, Leptosphaeria maculans, and Mycosphaerella graminicola, one nectrotroph Stagonospora nodorum, one biotroph B. graminis, and one saprotroph Pyronema confluens encode much larger SSSPs than the mean. Saccharomycotina and Taphrinomycotina encode noticeably smaller SSSPs than Pezizomycotina.The range of SSSPs numbers was also analyzed with regard to taxonomic positions Figure . The meaT. deformans, the only biotroph in Taphrinomycotina, also shows a high number of SSSPs. However, A. gossypii, the only biotroph in Saccharomycotina, has a lower number and proportion of SSSPs compared to other members  were conserved in 91 species, all belonging to Pezizomycotina or Agricomycotina (Figure Botrytis cinerea and Fusarium spp. have undergone duplication events. The wide distribution of the genes encoding cerato-platanin domain proteins in species with pathogenic lifestyles suggested that their products play an important role in pathogenesis. However, numbers of this group were also found in 4 symbionts, 2 nematophagous fungi, and 19 saprotrophs, suggesting other roles associated with them. The gene family identified using the B. graminis effector candidate BEC1040 has gone through 16 duplications and 12 losses, and its numbers were present in 50 species in Ascomycota and Punctularia strigosozonata in Basidiomycota (Supplementary Figure We predicted 13 families of CSSPs containing PHI-base effectors Table . Among ta Figure . In totaRapid progresses in sequencing fungal genomes, in combination with various “omics” tools, have facilitated large-scale comparative genomic analyses to uncover the genetic and evolutionary basis of various traits or functions of fundamental and practical significance. In this study, we developed a pipeline for mining SSPs as effector candidates from fungi with different lifestyles and taxonomic positions in order to conduct their kingdom-wide comparative analysis. It has been commonly hypothesized that biotrophs and symbionts secrete more effectors than necrotrophs, as biotrophic associations require the modulation of the host defense system to keep host cells alive for nutrient acquisition while preventing the launch of strong defense responses. Since necrotrophs utilize CAZymes and toxins to kill host cells to obtain nutrient, such manipulations of host defense likely play less critical roles. Because only a small number of fungal proteins in selected plant pathogens have been identified as effectors, we tested this hypothesis by comparing SSSPs as effector candidates.F. graminearum (Brown et al., S. reilianum and U. maydis are similar to those reported by Schirawski et al. (F. graminearum was compared to the data by Brown et al. (We refined secretomes to identify SSSPs as previously reported in analyzing the secretome of i et al. . The corn et al. , their 5n et al.  for kingM. oryzae is a zinc metalloprotease that acts as an avirulence factor in its host rice (Zhang and Xu, F. graminearum suppresses callose formation in wheat and is required for host infection (Blümke et al., The roles of fungal effector proteins regarding lifestyles are previously discussed by Lowe and Howlett  and Lo PL. bicolor (Martin et al., Tuber melanosporum (Martin et al., Neurospora crassa, a saprotrophic fungus that has been reported to be associated with pine trees in harsh conditions and even pathogenic (Kuo et al., In general, phytopathogenic fungi tend to have larger secretomes than non-pathogens. Although, Lowe and Howlett  suggesteC. fulvum and P. confluens. However, only the proteins secreted with canonical pathways were considered in this analysis, which means the minimum size of protein is bound to the length of signal peptide that is 15 to 40 aa (Choo et al., Another concern is that the number of small proteins could be affected by the minimum protein size used to annotate each genome. However, we strictly used the annotated data associated with published genomes, thus we can assume that the numbers of annotated proteins were comparable to each other. In addition, the majority of genome data were also from JGI and Broad Institute, which followed the conventional JGI annotation process and Broad Gene Finding Methods, respectively. If the minimum cutoff length for gene prediction was stated, only short peptides without EST support were eliminated. Since, the majority of fungal genome studies did not show the cutoff size for protein-coding gene prediction, we analyzed and compared the length distribution of the annotated proteomes for validation (the last column of Supplementary Table C. fulvum effector Ecp2 (Stergiopoulos et al., B. graminis, e.g., BEC1040, BEC1019, and BEC1005, were found in fungi having different lifestyles other than biotroph. These observations were further supported by the facts that these proteins are involved in fungal development, and resemble metalloprotease and glucanase, respectively (Pliego et al., Many genes coding candidate effectors in plant pathogens and virulence-associated genes of animal pathogens have been reported to reside in gene-sparse, AT-rich, and telomere-proximal genomic regions (Schmidt and Panstruga, In conclusion, different secretome components reflect lifestyle-associated genomic adaptations in fungi. Results from this comparative study provide new insights into the genetic basis and molecular evolution of fungal lifestyles and also establish a solid foundation for future discovery and functional validation of effectors.KK and YL designed this project. KK, JJ, HS and GC performed computational analyses. JC and KC provided the secretome and the genome data for analyses. KK, JJ, SK, and YL wrote the manuscript. SK and YL supervised the research. All authors read and approved the manuscript.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.


Together, our data suggest that CDH23-C is a CAMSAP3/Marshalin-binding protein that can modify MT networks indirectly through its interaction with CAMSAP3/Marshalin.Cadherin-related 23 (CDH23) is an adhesive protein important for hearing and vision, while CAMSAP3/Marshalin is a microtubule (MT) minus-end binding protein that regulates MT networks. Although both CDH23 and CAMSAP3/Marshalin are expressed in the organ of Corti, and carry several protein-protein interaction domains, no functional connection between these two proteins has been proposed. In this report, we demonstrate that the C isoform of CDH23 (CDH23-C) directly binds to CAMSAP3/Marshalin and modifies its function by inhibiting CAMSAP3/Marshalin-induced bundle formation, a process that requires a tubulin-binding domain called CKK. We further identified a conserved N-terminal region of CDH23-C that binds to the CKK domain. This CKK binding motif (CBM) is adjacent to the domain that interacts with harmonin, a binding partner of CDH23 implicated in deafness. Because the human Usher Syndrome 1D-associated mutation, CDH23 R3175H, maps to the CBM, we created a matched mutation in mouse CDH23-C at R55H. Both  CDH23 is widely expressed in various tissues including brain, heart, lung, kidney, nose, eye, and ear1CDH23 is known to cause various degrees of hearing impairment and blindness, including Usher Syndrome 1D6CDH23, an atypical cadherin protein, belongs to the cadherin superfamily that plays a major role in cell adhesion. CDH23 isoforms  and minus end tracking proteins (−TIPs), each of which targets the corresponding end of the MT and assists in maintaining the dynamic nature of the cytoskeletal network. In contrast to +TIPs, for which many have been identified, only a few −TIPs have been reported. Previously, we identified Marshalin (KIAA1543), a MT minus-end binding protein, as a potential CDH23-associated protein through a membrane-based yeast two hybridization screeningin vitro pull-down assays and in vivo CAMSAP3/MT bundle formation methodologies. We demonstrate that the CKK domain of CAMSAP3/Marshalin binds to the N-terminus of CDH23-C, which we named the CKK binding motif (CBM). Interestingly, a missense mutation has been found within the CBM of CDH23 (R3175H) in Usher Syndrome 1D patientsIn this study we investigated the relationship between CAMSAP3/Marshalin and CDH23-C proteins, as the two proteins are co-expressed in many cells including cochlear hair cells. Since both CDH23 and CAMSAP3/Marshalin carry several protein-protein interaction domains, we systemically characterized the authenticity of interactions between CAMSAP3/Marshalin and CDH23-C using both Previously, we showed that overexpression of CAMSAP3/Marshalin-Ld  resulted in dramatic reorganization of MT networks into bundles composed of both CAMSAP3/Marshalin and tubulinp = 0.0013) and CDH23-C2-GFP (p = 0.0037): by 21.3% for CDH23-C1 and by 20% for CDH23-C2, respectively. Thus, type I cells dominated when OK cells co-expressed CAMSAP3/Marshalin-Ld and CDH23-C isoforms. In contrast, a majority of cells co-expressing CAMSAP3/Marshalin-Ld and Ceacam16 showed type III MT patterns similar to the expression patterns of cells expressing CAMSAP3/Marshalin-Ld and GFP . Thus, CDH23-C1-GFP co-expression interferes with CKK’s ability to induce MT bundles, similar to that seen for full-length CAMSAP3/Marshalin-Ld.To understand the molecular mechanisms underlying the inhibitory role of CDH23-C isoforms on CAMSAP3/Marshalin-induced MT bundle formation, we first determined the functional domains that are responsible for the CDH23-C and CAMSAP3/Marshalin interaction. CAMSAP3/Marshalin-Ld was identified as a potential CDH23-associated protein using the membrane-based yeast two-hybridization (Y2H) approachin vitro pull-down assays. GST-tagged CDH23-C1 and CDH23-C2 fusion proteins were expressed in bacteria and immobilized on glutathione-agarose. The glutathione resins with GST-alone or GST-CDH23-C isoforms were then incubated with bacterial lysates containing the CKK domain, and bound proteins were eluted and detected on SDS-PAGE. The presence of GST, and GST-CDH23-C isoforms were detected by Coomassie Blue staining ), exon 68 (Cdh23 (Δ108)), and NBM (Cdh23 (Δ143) (see Methods for details), did not abolish binding between CKK and CDH23 (Cytoplasmic regions of CDH23 have been reported to interact with other proteins including harmonin through the PDZ domain-binding motif (PBM), exon 68, or the N-terminus domain binding motif (NBM) as shown in Fig. 1C1718nd CDH23 . This rend CDH23  were mixnd CDH23 , top, whnd CDH23 , bottom.Our data demonstrated that CDH23-C binds to CAMSAP3/Marshalin’s CKK domain but the minimum binding region did not include one of the harmonin binding sites (NBM). To learn more about the relationship among CAMSAP3/Marshalin, harmonin, and CDH23-C isoforms, we tested whether CDH23-C isoforms could still bind to CAMSAP3/Marshalin in the presence of harmonin. GST-tagged CDH23-C1 and CDH23-C2 proteins were first immobilized on glutathione-agarose along with GST as a negative control. GST-agarose and CDH23-C-GST-agarose were then simultaneously incubated with lysates from bacteria expressing CKK and lysates from bacteria expressing harmonin. In paired comparisons, glutathione-agarose with GST or GST-CDH23-C isoforms were incubated with CKK lysates and uninduced harmonin bacterial lysates, which were derived from the same bacterial transformants without IPTG treatment. The latter serves as the negative control as it contains similar proteins except for harmonin. As shown in CDH23 cause Usher Syndrome type 1D. One disease-associated missense mutation occurs within the intracellular region that changes arginine to histidine at the amino acid position 3175 of CDH23-A1 (R3175H)Cdh23-C1, which is also conserved among all species. We suspected that mutation of this highly conserved, positively charged amino acid might affect CDH23-C and CAMSAP3/Marshalin-Ld binding. Therefore, we introduced the same mutation within our GST-CDH23-C1 construct at position 55 (CDH23-C1_R55H), which corresponds to the R3175H mutation found in Usher Syndrome type 1D patients. This point mutation altered migration of the CDH23-C1 protein compared to the wildtype in SDS-PAGE (p < 0.05) as compared to GFP controls, albeit less effectively than wildtype CDH23-C1. In other words, more cells showed the type III phenotype in OK cells co-expressing CAMSAP3/Marshalin-Ld and CDH23-C1_R55H-GFP than OK cells co-expressing CAMSAP3/Marshalin-Ld and wildtype CDH23-C1-GFP (p < 0.05). Since the R55H mutation is within the CBM of CDH23-C isoform, we also introduced R55H into the CDH23-C1_1–67 construct, and performed the GST pull-down assay. The resulting GST-CDH23-C1_1–67_R55H fragment also exhibited a slight increase in the apparent molecular weight on the gel compared to the WT (p = 0.0141). These data suggest that the mutation within the CBM of CDH23-C isoforms alters the interaction between CDH23-C and CAMSAP3/Marshalin-Ld.Mutations in SDS-PAGE . The incSDS-PAGE , while tSDS-PAGE . The pero the WT , top. Coin vivo MT bundle formation assays within mammalian cells and in vitro GST pull-down assays. We have identified the binding sites between CDH23-C and CAMSAP3/Marshalin-Ld in the N-terminus of CDH23-C (CBM) and the CKK domain of CAMSAP3/Marshalin-Ld, respectively. We further showed that mutation in the CMB of CDH23, which was discovered in an Usher Syndrome 1D patient (R3175H), resulted in a marked decrease in CDH23-C’s ability to interact with CAMSAP3/Marshalin both in vivo and in vitro. These data suggest that CDH23-C plays an important role in regulating MT networks via its interaction with CAMSAP3/Marshalin, and that alteration of this interaction can lead to pathological conditions such as Usher Syndrome.CDH23 is an essential protein for auditory and visual signal transduction and is important for cell adhesion. Function of the cytoplasmic isoforms, however, is not fully understood even though CDH23-C is detected in various tissues and implicated in cochlear hair cell developmentAs an adhesive protein, CDH23 is generally thought to localize to the cell surface. Therefore, it was puzzling when CDH23 was also found in MT-rich areas such as centrosomes and basal bodies that are internal structures1013CDH23 is known to bind to the PDZ domain of harmonin through multiple domains including PBM, exon 68, and NBM18Our data suggest that CDH23-C can regulate MT networks indirectly through its interaction with CAMSAP3/Marshalin. As a new modifier of MT networks, additional studies are required to further define CDH23-C’s roles in various physiological contexts that may ultimately impact our understanding of the fundamental mechanisms associated with hearing and vision.CAMSAP3/Marshalin-Ld or the CKK domain  was inserted into a pcDNA6/V5His vector to introduce a V5-His tag to the C-terminusGFP-Cdh23-C1 and GFP-Cdh23-C2 constructs were kindly provided by Dr. FriedmanGFP-Cdh23-C1 using a QuickChange Site-Directed Mutagenesis Kit (Stratagene) to generate GFP-Cdh23-C1_R55H. For bacterial expression, CKK domains of mouse CAMSAP3/Marshalin-Ld  were inserted into the NheI/SalI site of the pET24(a) vector. Both constructs contained a His-tag at the C-terminus. We also made a construct encoding CKK1 cDNA without the His-tag. Glutathione S-Transferase (GST)-tagged mouse CDH23-A cytoplasmic tail constructs, CDH23, CDH23 (+68), CDH23 , CDH23 (Δ108), and CDH23 (Δ143), were generous gifts from Dr. BartlesCdh23-C1 and Cdh23-C2 fragments were inserted into the EcoRI/XhoI site of the pGEX-4T-2 vector with an N-terminal GST tag to generate the following constructs: Cdh23-C1, Cdh23-C2, Cdh23-C_1–67 (first 67 aa of CDH23-C), CDH23-C_1–67_R55H (first 67 aa of CDH23-C with the R55H mutation).For mammalian expression, the cDNA of mouse 2 Fragment , 1:1,000 for IF.Anti-MarshalinCdh23-C1 and Cdh23-C2 fragments, His-tagged CKK1/2 and harmonin were induced by 1 mM Isopropyl β-D-1-thiogalactopyranoside  in E. coli BL21 (DE3) cells (Invitrogen). Cells were then lysed in PBS containing 1X protease inhibitor (Sigma), 1 mM phenylmethanesulfonylfluoride , 1 mg/ml lysozyme (Sigma), and 370 unit/ml Benzonase (Sigma) by sonication. The bacterial lysates were incubated at 4 °C for 30 minutes, then centrifuged at 14000 rpm for 30 minutes at 4 °C to separate insoluble fragments from supernatant. GST-tagged Cdh23 tail proteins and various Cdh23-C1 and Cdh23-C2 fragments were then immobilized on glutathione-agarose (Thermo) at 4 °C on a rotator for 1.5 hours. Unbound proteins were washed away with chilled PBS. For Cdh23- CAMSAP3/Marshalin pull-downs, Cdh23 bound glutathione-agarose (Cdh23-GST-agarose) was incubated with CKK lysate at 4 °C on a rotator for 1.5 hours or overnight. In some experiments, CKK proteins were purified first using a MagneHis Protein Purification System (Promega) before adding 30–60 ng/ml of CKK protein to the Cdh23-GST-agarose. For pull-downs including harmonin, uninduced harmonin bacterial lysate (no harmonin expression) and induced harmonin bacterial lysate were combined with CKK lysate in a 1:1 ratio, and added the Cdh23-GST-agarose. Following incubation, the resins were washed with chilled PBS, and bound proteins were eluted with 100 mM L-Glutathione (Sigma), and then loaded onto 12% (w/v) polyacrylamide-SDS gels. In some experiments, CKK proteins and harmonin were purified first using a MagneHis Protein Purification System. The imidazole in elution buffer was removed and replaced with PBS using PD MiniTrap G25 . Protein concentrations were measured used a Pierce 600 nm Protein Assay Reagent (Thermo Scientific). Appropriate molar concentrations of CKK1 and harmonin were then added to the Cdh23-C1-GST-agarose. To visualize enriched GST-tagged proteins/fragments, Coomassie Blue (Thermo) was used to stain the gels, or 5% Ponceau S (Sigma) to stain the nitrocellulose membranes. To probe for CKK domains, membranes were incubated with anti-Marshalin followed by HRP-conjugated secondary antibodies and a SuperSignal West Pico Chemiluminescent Substrate (Thermo). Images were taken with a Kodak Imaging System and the intensities of the expected CDH23 and CKK bands measured using Molecular Imaging software from Kodak . For statistical analysis, the signal intensities of CKK were normalized to the intensities of Cdh23-GST bait, and means from 4 independent experiments were plotted. Significance was determined using one-way ANOVA with the Tukey’s multiple comparisons test in Prism 6 (GraphPad).Expression of GST-tagged CDH23 cytoplasmic tail proteins or various 2 in air at 37 °C. 24–45 hours post transfection, cells were treated with colchicine at a concentration of 250 ng/ml for 24 hrs or 1 μg/ml for 3 hrs. We also utilized LLC–PK1–CL4 cells (CL4 cells) cloned from LLC–PK1 cells provided by Dr. Bartles (Northwestern University). Detailed culture conditions for these cells were described in a previous publicationPlasmids encoding V5-His tagged CAMSAP3/Marsalin-Ld or simply its CKK domain were transiently co-transfected with GFP, CDH23-C1-GFP, and CDH23-C2-GFP into opossum kidney (OK) cells as previously describedGFP-Cdh23-C1 or GFP-Cdh23-C1_R55H were harvested 2 days after transfection and lysed in cold lysis buffer  supplemented with 1X protease inhibitor cocktail and 1 mM PMSF (Sigma). Insoluble material was removed by centrifugation at 15,000 g for 15 minutes at 4 °C. Proteins were resolved using 8% SDS-PAGE, followed by immunoblotting using anti-GFP (Rockland). Signals were detected using a SuperSignal West Pico Chemiluminescent Substrate (Thermo). A Kodak Imaging System was used to capture the images.OK cells transiently transfected with How to cite this article: Takahashi, S. et al. Cadherin 23-C Regulates Microtubule Networks by Modifying CAMSAP3’s Function. Sci. Rep.6, 28706; doi: 10.1038/srep28706 (2016).


Tumor PD-L1 expression is associated with increased tumor-infiltrating lymphocytes (TILs) in diverse solid tumors, including lung cancer. In addition, PD-L1 upregulation in tumor and/or stromal immune cells has been associated with increased clinical benefit to PD-1/PD-L1 axis blockers. The significance of PD-L1 expression in different immune cell subpopulations at the tumor microenvironment remains poorly understood. Here, we measured PD-L1 protein specifically in tumor-associated macrophages using objective methods and analyzed its clinical significance in human lung cancer.Using multiplexed quantitative immunofluorescence (QIF) we measured the levels of PD-L1 protein  in 554 stages I-IV FFPE lung carcinomas represented in two tissue-microarrays, one from Yale University  and one from Greece . PD-L1 was measured using multispectral QIF in CD68-positive cells  or in tumor cells  based on fluorescence co-localization. The levels of CD3 , CD8  and CD20  were also determined in the tumor samples. Associations between the macrophage and tumor PD-L1 levels, clinico-pathological variables and survival were analyzed.2]=0.311 in the Yale cohort and R2=0.43 in the Greek collection). Using the median score as cutpoint, elevated macrophage PD-L1 content was significantly associated with high CD8+ and CD20+ cells in both cohorts. In addition, increased macrophage PD-L1 signal was marginally associated with longer overall survival in the Yale set , but not in the Greek group .In lung cancer samples, PD-L1 was detected in both macrophages and tumor cells with a predominant membranous staining pattern. Overall, the levels of PD-L1 in the CD68+ population were lower than in tumor cells in both cohorts . In both collections, the macrophage and tumor PD-L1 signal showed a positive non-linear association (Linear regression coefficient [RPD-L1 is expressed in tumor-associated macrophages of human lung carcinomas. The macrophage PD-L1 signal is lower than in tumor epithelial cells and they show a positive association. Elevated macrophage PD-L1 is prominently associated with increased cytotoxic T lymphocytes and B cell infiltrates in lung tumors, but has limited prognostic value. Further studies will be required to evaluate the possible predictive role of PD-L1 expression in macrophages and other immune cell types.


Isolators, or optical diodes, are devices enabling unidirectional light propagation by using non-reciprocal optical materials, namely materials able to break Lorentz reciprocity. The realization of isolators at terahertz frequencies is a very important open challenge made difficult by the intrinsically lossy propagation of terahertz radiation in current non-reciprocal materials. Here we report the design, fabrication and measurement of a terahertz non-reciprocal isolator for circularly polarized waves based on magnetostatically biased monolayer graphene, operating in reflection. The device exploits the non-reciprocal optical conductivity of graphene and, in spite of its simple design, it exhibits almost 20 dB of isolation and only 7.5 dB of insertion loss at 2.9 THz. Operation with linearly polarized light can be achieved using quarter-wave plates as polarization converters. These results demonstrate the superiority of graphene with respect to currently used terahertz non-reciprocal materials and pave the way to a novel class of optimal non-reciprocal devices. Optical isolators, or optical diodes, allow electromagnetic radiation to travel in one direction but not the other. Here, the authors achieve unidirectional propagation of terahertz waves by taking advantage of the non-reciprocal nature of optical conductivity in magnetostatically biased graphene. The potential applications of graphene for terahertz passive and active devices have received considerable attention in recent scientific literature123456789101112131415161718192016819Apart from ferrite and graphene, alternative materials have also been proposed to achieve efficient terahertz non-reciprocity, and several works have been recently published presenting experimental characterization of the properties of these materials. One example is given by other free carrier based materials such as doped silicon27In the following we aim to exploit the non-reciprocity of magnetically biased monolayer graphene using a reflection configuration to achieve isolation for circularly polarized waves. The concept of isolator for circularly polarized light has been presented theoretically in a transmission configuration for graphene and other magneto-optical materials19N of graphene sheets are placed on a back-metallized thin silicon layer of 10 μm thickness (for our device N=3). The sheets are separated by thin poly methyl methacrylate (PMMA) layers (∼60 nm), while the thickness of the metallization (chromium and platinum) is 200 nm. The whole structure is bonded to a pyrex wafer, which has solely the function of mechanical support. A magnetostatic field B is applied orthogonally to graphene.A non-reciprocal isolator is a device possessing at least one electromagnetic mode showing unilateral propagation, that is, it allows the transmission of an electromagnetic wave in a given direction while strongly attenuating it in the other one The device operation is based on reflecting incident left hand circularly polarized (LHCP) plane waves as right hand circularly polarized (RHCP) ones, while absorbing RHCP incident waves. The device thus achieves non-reciprocal unidirectional propagation and isolation for circularly polarized waves1923η of free space , causing total absorption  rotating waves (incident RHCP or reflected LHCP) a total surface impedance equal to the impedance rization , taking σCW=σd+jσo and σCCW=σd−jσo for circularly polarized waves.which has two eigenvalues B=7 T the real part of σCW is very close to (3η)−1 and thus the total impedance of the three monolayers stack is very close to η. The role of the metallized silicon reflector is to cancel the imaginary part of σCW at the working frequency in order to enable full impedance matching in the CW case. Additional details can be found in the full design procedure provided in The yellow region in θ between the two polarizers. The magnetostatic field is normal to the sample surface, while the light k vector is close to normal. B=0 T. One can see that the normalized reflection shows strong absorption dips. These strong absorption features have a periodicity of 4.65 THz, corresponding to Fabry–Perot oscillation in a silicon layer of 9.43 μm in very good agreement with the nominal value. B=7 T. This is also shown better by the polarization diagrams in ϕ goes up to 90° and continues from −90° to 0°.The fabricated device has been measured using a Fourier transform infrared spectrometer connected to a split-coil superconducting magnet. A polarizer is used to create a linearly polarized incident light, while an analyser  is used in front of the detector see . The refThe isolation (ISO) and insertion loss (IL) of the device are defined as:Even though our measurement setup is equipped only with linear polarizers, both these quantities can be computed accurately from the measured polarization parameters . The sought isolation and insertion loss can then be retrieved as:Emax and Emin are the major and minor axis of the mapped elliptical polarization of the reflected electric field and Einc is the linearly polarized incident electric field. The full algorithm is actually more complex and it enables the compensation of polarizer imperfections  is a device figure of merit of the isolator, which depends on the isolation and on the insertion loss, and it defines the shape of the forbidden region. Notably, it forbids points having at the same time high isolation and low insertion loss, which represent the target for any isolator design.The right member For the fitted parameters, it can be shown that the forbidden region is frequency-independent in the band from 0 THz to 20  THz. The performance of the device is just 1 dB below the theoretical upper bound, which means that the device is near optimal.While the presented isolator operates with circularly polarized light, it is clear that most terahertz applications need components able to handle linearly polarized waves. It is however, quite simple to adapt our isolator to linear polarization operation using one or two quarter-wave plates as polarization converters, as shown in In this work we demonstrated the possibility of designing and implementing close to optimal terahertz isolators based on graphene. To achieve this goal, we proposed a reflection structure that exploits the Fabry–Perot resonances in a thin layer of silicon to obtain isolation using just three graphene monolayers. The operation of the device can be fully understood in the framework of Maxwell's equations using a 2D linear conductivity tensor to model carrier dynamics in magnetostatically biased graphene. One of the most significant aspects of this design is its ability to be very close to the optimal performances available with the used graphene45k vectors at the same time. Electric field effect in gated graphene could be used to fine tune the device to virtually infinite isolation, as demonstrated also at microwave frequenciesIn addition, it is worth mentioning some additional advantages related to this particular device geometry. First, due to the fact that the device is planar , it works for incident waves having different Because the device exploits Fabry–Perot resonances, it is relatively narrowband; however, the absolute bandwidth is in the order of 50 GHz, which is an excellent value for telecommunications and for continuous wave applications. This value can possibly be increased using more complex designs including patterned graphene, added metal patterns and more complex multilayer structuresFinally, the reflection configuration of the device eliminates completely the input impedance mismatch (return loss) issue, which is instead a concern for any device operating in a transmission configuration. For waves with a non-zero incidence angle  this fact is evident considering that the device is invariant to translation and hence it operates with a single diffraction order. This implies that the wave cannot possibly be reflected to the receiver. For normally incident waves, this is due to the fact that an incident left hand wave can only be reflected as right hand and vice versa, because the device is invariant to rotation.B biasing field is to use graphene with higher mobility2 V−1 s−1. With a mobility of 40,000 cm2 V−1 s−1 and a biasing field of 1 T (easily generated by rare earths permanent magnets) the insertion loss for perfect isolation would be as low as 0.3 dB according to the upper boundThe main drawbacks of our device are the need of high magnetic field (7 T) and an insertion loss of more than 7 dB. Both these issues cannot be solved by improving the design, since it is already quasi-optimum in this sense. Hence, the only way to lower the insertion loss and reduce the required 2 box layer of 4 μm. Platinum (200 nm) was evaporated on the top side of the silicon on insulator wafer and then it was bonded using parylene to a support pyrex wafer. The handle and box layers were then ground and etched leaving the device layer exposed. Three layers of graphene were subsequently transferred on the substrate keeping the PMMA support film between each layer. More details about fabrication and measurements are available in the The reflective substrate has been fabricated starting with an silicon on insulator wafer having a high resistivity silicon device layer with thickness 10 μm a handle layer of 500 μm separated by an SiOReflection coefficients and derived quantities (insertion loss and modulation depth) are defined in terms of electric fields, and consequently the corresponding decibel unit is defined as How to cite this article: Tamagnone, M. et al. Near optimal graphene terahertz non-reciprocal isolator. Nat. Commun. 7:11216 doi: 10.1038/ncomms11216 (2016).Supplementary Figures 1-5, Supplementary Notes 1-4 and Supplementary References


We show that electron charge is transferred across the interface and magnetization is induced in the graphene sheet due to the strong interaction between C and Mn. Depending on the relative orientation of graphene and BaMnO3, a quasi-half-metal or a magnetic semiconductor can be obtained. A remarkably large proximity induced spin splitting of the Dirac cones (~300 meV) is achieved. We also show how doping with acceptors can make the high-mobility region of the electronic bands experimentally accessible. This suggests a series of possible applications in spintronics  for hybrid organic-multiferroic materials and reveals hybrid organic-multiferroics as a new class of materials that may exhibit exotic phenomena such as the quantum anomalous Hall effect and a Rashba spin-orbit induced topological gap.Graphene and magnetoelectric multiferroics are promising materials for spintronic devices with high performance and low energy consumption. A very long spin diffusion length and high carrier mobility make graphene attractive for spintronics. The coupling between ferroelectricity and magnetism, which characterises magnetoelectrics, opens the way towards unique device architectures. In this work, we combine the features of both materials by investigating the interface between graphene and BaMnO Conventional III–V semiconductors were originally proposed as channel materials due to their high SOC and long spin lifetime. However, since the mean velocity of the carriers is quite small, the corresponding spin diffusion length is relatively short and results in weak magnetoresistance signalsμm) spin diffusion lengths57via an external applied field. In addition, to achieve graphene-based transistors one needs to open its vanishing electronic gap. Isolated pristine graphene is not optimal for spintronics applications: it is necessary to combine it with a material with high SOC, and either cut it into nanoribbons3O4proximity interaction)13magnetoelectric (ME) multiferroic material as substrate can satisfy all these points. ME materials are characterised by a coupling between ferroelectricity and magnetization, hence allowing one to control the magnetization via an electric fieldSpintronicsd electrons carry a strong spin polarization. First-principles calculations have shown that Mn adatoms interact strongly with carbon π orbitals inducing Rashba SOC and quantum anomalous Hall effect in graphene3 has been chosen as representative of this class of materials since it is ME203 is the same as for the class of RMnO3 hexagonal manganites (where R is a rare earth element). We find that the graphene–BaMnO3 (g–BMO) hybrid system is spin polarized with magnetization and charge transfer induced in the carbon atoms due to the strong interaction with the surface Mn atoms. The most stable reconstruction is quasi-half-metallic and acts as an injector of 100% spin polarized carriers. The electronic properties at the Fermi energy (EF) are governed by the details of the interface. The graphene Dirac cones for majority and minority carriers present a huge splitting of about 300 meV. Furthermore, we show that both the energy at which the band crossing occurs and the splitting of spin-up/spin-down bands can be tuned by doping of the graphene monolayer.This paper reports on the first principles investigation of the electronic and magnetic properties of the interface between graphene and ME multiferroics. We focus on manganite-based MEs, since the Mn 3, which is crucial in order not to short-circuit the graphene layer, ensure that electron transport will only occur through graphene and correctly model the spin polarization induced on the C network  with respect to the middle of the slab . The electrons at the surface give rise to the spin-polarized bands below EF  surface of BaMnOtion see . The slation see , charactbelow EF . A slighthe bulk .3, we can now address those of its interface with graphene. The g–BMO slab is constructed by placing a 4 × 4 graphene supercell on either side of the strained BMO slab. Atomic relaxations with different graphene offsets lead to the most stable structure shown in fat bands).Having clarified the electronic and magnetic properties of BaMnO5 m/s for the band crossing EF: only minority carriers can propagate at the interface. The majority band edge lies slightly above EF (78 meV), such that by applying a small gating potential, an injector of 100% spin polarised carriers is achieved (spin filter). The band contribution coming from each atom and orbital has been identified with projected bands. This technique  than that in the centre of the slab , as shown in 3 compounds as substrate. The magnetic moment on individual C atoms is at most As a consequence of this strong interfacial interaction, the surface Mn atoms induce spin polarization and charge transfer in graphene. The hybrid system is magnetic. As for the bare BaMnOolarized . These rF and, hence, the position of the Dirac cones. Several structures with one and two B substitutional dopants per graphene layer have been relaxed and analysed. The most likely site for a single dopant (3% p-type doping concentration) is sublattice “3”  is close to pristine graphene (8.5 × 105 m/s). Velocities up to 4.2 × 105 m/s (majority) and 6.6 × 105 m/s (minority) are reached for 2 Boron dopants.An important feature of the electronic structure is that the graphene Dirac cones are found at the K and K’ points of the 4 × 4 supercell. The latter are mostly contributed by C and, to a minor extent, by Mn and O. There is a remarkably large splitting of ~290 meV between spin up and down Dirac points. However, these high mobility regions occur at energies which are difficult to reach in experiments. Doping graphene with acceptors can shift Etice “3” . When a tice “3” . Inspecttice “3”  shows th3 interfaces have been checked. A metastable configuration is reached when one Mn lies in the middle of a C hexagon and two Mn are vertically aligned with C in the unit cell. This system is a magnetic semiconductor  effect and Rashba spin orbit splitting, analogously to graphene doped with Mn adatomsSince the electronic band structures exhibit quite different features for majority and minority spins, very different electronic transport properties are expected as well. A possible device exploiting these results is a spin–FET in which spin injection is obtained by graphene-BaMnO3. The strong interaction between graphene π electrons and the magnetic Mn d states induces a significant spin polarization in graphene. This interaction breaks the carbon network in six sublattices with different magnetic and charge transfer properties. The ground-state structure is a quasi-half-metal that allows for injection of 100% spin polarised carriers. Depending on the relative orientation of graphene and substrate, a magnetic semiconducting metastable state is also found. In each case, the graphene Dirac cones present a remarkably large spin splitting (300 meV). This high-mobility region (similar to suspended graphene) can be made accessible to experiment via substitutional doping of graphene with acceptors. The main features of the electronic structure do not depend on the Ba atom, and are expected to hold also for other hexagonal manganites RMnO3 or any magnetic insulator.In conclusion, first-principles calculations have been used to investigate the interfaces between graphene and a magnetoelectric multiferroic material, BaMnO3 is achieved for a real space grid cutoff of 1200 Ry, a shifted 6 × 6 × 12 k-point sampling of the Brillouin zone and a Fermi-Dirac smearing of 100 K in a 30-atom cell which is primitive for the ground state phase (space group P63cm).All calculations have been performed using the SIESTA implementationHow to cite this article: Zanolli, Z. Graphene-multiferroic interfaces for spintronics applications. Sci. Rep. 6, 31346; doi: 10.1038/srep31346 (2016).


An optimization method for condition based maintenance (CBM) of aircraft fleet considering prognostics uncertainty is proposed. The CBM and dispatch process of aircraft fleet is analyzed first, and the alternative strategy sets for single aircraft are given. Then, the optimization problem of fleet CBM with lower maintenance cost and dispatch risk is translated to the combinatorial optimization problem of single aircraft strategy. Remain useful life (RUL) distribution of the key line replaceable Module (LRM) has been transformed into the failure probability of the aircraft and the fleet health status matrix is established. And the calculation method of the costs and risks for mission based on health status matrix and maintenance matrix is given. Further, an optimization method for fleet dispatch and CBM under acceptable risk is proposed based on an improved genetic algorithm. Finally, a fleet of 10 aircrafts is studied to verify the proposed method. The results shows that it could realize optimization and control of the aircraft fleet oriented to mission success. Prognostic and health management (PHM) technology has a rapid development and been widely used in aeronautical equipment in recent years. The failure position and remain useful life (RUL) of equipment could be predicted by PHM. Further, it can be used in aircraft condition based maintenance (CBM) . HoweverThe aircraft usually performs mission in fleet manner and shares limited support resource. So, there will be a tradeoff range for fleet CBM. This means each aircraft can choose strategy among dispatching strategy, standby strategy, and maintenance strategy or their combination when the RUL has been obtained, and the synthetic strategy for fleet (combination of each aircraft's strategy) should meet the mission requirement.There are three forms of RUL in PHM, and each form includes some uncertainty. First is the point value of the time of potential failure. Second is the interval value of the time of potential failure –7. ThirdTwo methods can be used in reducing the impact of prognostics uncertainty on CBM decision. One is to reduce the uncertainty of failure prediction directly so that the decision risk will decrease –15. The Most research about RUL for CBM is about the life cycle maintenance optimization decisions on single aircraft and researchers would rather consider the maintenance decisions than think about the mission requirements and the dispatched strategy. The research on fleet CBM oriented to mission successes is few. Agent technology and the heuristic algorithm are used to fleet CBM in article , 23, butAn optimal aircraft fleet CBM method for aviation unit maintenance is proposed in the paper considering dispatch, mission, and resource constraints. Moreover, the RUL distribution of the key LRM has been transformed into the failure probability of the aircraft, and the calculation method of the costs and risks for mission is given. Then, an optimization decision making method for fleet dispatch and CBM under acceptable risk is proposed based on an improved genetic algorithm.m aircrafts and k integrated support stations (ISS) facing continuous combat missions (k < m), in which a single mission requires l aircrafts (l ≤ m). Each aircraft contains p LRMs of which RUL can be estimated. The mission preparation period starts at time t0, while the mission period is from time t1 to t2. The basic process of the fleet CBM decisions, which is mission success oriented, is given in Consider an aircraft fleet containing The aircraft fails when any key LRM fails.t0 is F(t) of which probability density function is f(t).The RUL distribution of LRM which is given at the time Assume the maintenance method of the LRM is renew, which means the LRM will be as good as new after maintenance, considering the field maintenance of aviation unit maintenance.t0 to t1.Only one aircraft can be repaired in each ISS simultaneously. But the total number of the aircraft maintenance may be more than one from the time Different LRM in the same aircraft can be replaced at the same time for renew is served as a maintenance method.j class is Cj.The maintenance cost of the different LRM varied while the same LRM cost is the same. The maintenance cost of the LRM on Each aircraft malfunction will cause the mission to fail when the fleet is on mission. The consequences of the economic loss will not be taken into consideration.Spare parts are plentiful.The basic assumptions of the problem are listed below in order to define the problem.The main work of the optimization decision making method for fleet CBM considering prognostics uncertainty includes the following steps: (1) the definition of the fleet initial health status based on the RUL prognostic, (2) maintenance program generating, (3) maintenance time and cost estimation, and (4) mission risk assessment. Based on the objects above, the optimal CBM and maintenance program through the rational optimization algorithm is obtained in the paper. The modeling framework is given in jth key LRUij on the ith aircraft is Fij(t). In the mission period (t1-t2), the probability of failure can be got byfij(t) is the probability density function of the Fij(t).Assume the distribution of the m aircrafts and each include n LRMs, the initial health status matrix of all LRM is P(a) that is given byConsidering an aircraft fleet containing Maintenance program considers whether a certain LRM should be maintained and the selection of the ISSs.U of fleet can be described asuij = 1 means that the jth LRM of the ith aircraft needs to be repaired; otherwise, uij = 0.The maintenance matrix S of the fleet is shown assiq = 1 means that the ith aircraft should be maintained at the qth ISS; otherwise, uij = 0.According to the assumption (4), the LRM can be maintained at the same place; however, many the LRMs fails. Therefore, the ISS matrix jth LRM spends time Tj and needs cost Cj. According to the assumption (5), the total maintenance time Tmi of the ith aircraft is given asAssume repairing the q, so the total maintenance time of all aircrafts can be calcluated asThere may be more than one aircraft that should be repaired at ISS The total maintenance cost of all aircrafts can be got asP(b) of the fleet can be built according to the Assumption (3). Consider pij(b) = 0 after the LRMij on the ith aircraft was renewed; otherwise, pij(b) = pij(a) without renew. The elements in the matrix can be obtained byWhether the aircraft is “dispatching” or “dispatching after maintenance” should be taken into consideration when calculating the mission risk of the fleet. The status of the aircraft should be updated if the single strategy of the fleet is “dispatching after maintenance.” Then, the modified health status matrix Pi(a) of the ith aircraft can be given asThe failure probability of the single aircraft could be estimated after modifying the health matrix of the fleet. According to the first assumption, “the aircraft fails when any key LRM fails”; the failure probability Formula  can be oi) = 1,2,…, m can be obtained by sorting the failure probability of single aircraft in ascending order. The ordered failure probability of the aircraft is given asThen, Pro(P2(a) is the smallest Pro(i). Then, set Pro(2) = 1, and let P1(b) = P2(a) after reordering. Pick up aircrafts of which Pro(i) = 1,2,…, l when the mission needs dispatch l aircrafts.Suppose PF is the mission risk.Assume the serious consequences of the mission that failed are similar without taking the economic losses into account. The failure probability of the fleet mission can be calculated by the following according to :(12)PF=The optimization problem in the paper is to find a fleet CBM strategy with acceptable risk and lowest cost considering prognostics uncertainty.MinC = ∑i=1m∑j=1n[uij × Cj].Thus, describe the objective of the optimization as RA of the site, the time constraint RB, the security risk constraint RC, the mission risk constraint RD, and the variable constraint RE.The constraints that should be considered about involve the maintenance ability constraint RA, set siq = 0   and uij = 0   if none of LRMs need maintenance. Else if any LRMij requires maintenance, then uij = 1 and the corresponding siq = 1 while the other siq = 0. Thus, the RA can be described asFor first constraint q should be finished before the mission starts. Thus, the RB can be given asAll maintenance of site q is ∑i=1msiq = x > 1 . If ∑i=1x−1siq × Tmi ≤ t1 − t0 < ∑i=1xsiq × Tmi, which means the maintenance for the xth aircraft could not be finished before the mission start time, this aircraft will not be taken into account when the maintenance decisions is “dispatching after maintenance.”Assume that the total number of the aircraft which maintained at site l aircrafts, and the RC can be described as  conditions, and the dimension of decision making variables which can be written as m × n + m × k is relatively large. So an improved genetic algorithm was proposed in this paper for the problem instead of traditional mathematical methods.The optimization problem cannot meet the KKT  = 1, the aircraft i need not be repaired and all siq  = 0; otherwise, the aircraft i needs to be repaired. Then, the determining condition is described as ∑q=1ksiq = 1 and siq ∈ {0,1}.According to formula , an inity aircrafts need not be repaired. Remove y rows which stand for these aircrafts. Then, a new (m − y) × k matrix S′ which represents the new relationship between the ISS and the aircraft that needs to be repaired can be built as the reduced cycle matrix of S.Consider that there areTmi of the aircraft needs to be repaired in matrix S′ which can be obtained by formula (i=1m−yTmi/k. It can be determined not meet the time constrain if max(Tmi) > t1 − t0 or AMT > t1 − t0, then turn to The maintenance time  formula . Then, tS′, and set siq = 0 .Initialize the matrix S′ from the first row to the kth row. The method of the qth is described as follows.Tmi − AMT|  . If the aircraft z makes the |Tmz − AMT | = min|Tmi − AMT|, then szq = 1. Furthermore, the aircraft can be selected in random if there is more than one aircraft that meets this formula.Calculate the value of |z is in to build a new reduced cycle matrix S′. Update the remaining maintenance time TGq = t1 − t0 − Tmz of the site q.Remove the line in which the aircraft TGq and the Tmi for the S′. If the formula “min(Tmi) | i = o ≤ TGq” can be met by a parameter o, form a new reduced cycle matrix and set soq = 1. Moreover, the remaining available reference time should be updated as TGq = TGq(b) − Tmo. This work should be repeated until the min (Tmi) > TGq; then, turn to the (q + 1)th row.Compare the Set the value of the matrix U should be adjusted if the constraints of resource maintenance cannot be met. Consider that the maintenance cost should be as low as possible and the requirement of the mission risk should be satisfied; the elements which uij = 1 should find pij(a) corresponded and the min{pij(a)}; then, set uij = 0. Return to U until meeting constrains RA and RB.The matrix U which is initial or got by adjusting, crossover, and mutation may not meet the requirement of the mission risk constrain. The penalty method can be used in the method followed to solve this problem.Some matrix U can be written asF is the vector of the penalty function and the Fi = max{0, gi}, while M, which is the penalty factor vector, is a large positive number.The energy function for every Emax and Emin are the maximum and the minimum values of the energy function in the population.The fitness function is given as follows in order to minimize the objective function:Proportional selection, single-point crossover, and the basic alleles can be used in solving this problem.This problem can be dealt with by some method written in the article –26 in orN is the size of the population and g is the genetic algebra, while T0 is the initial temperature and fi is the fitness of the ith individual.Simulate the annealing stretching for fitness before selecting the operator as follows:Pe and Pf can be defined as , and the mean μ and the variance σ2 are given in Consider a fleet containing 10 aircrafts and each aircraft includes 4 LRM  of which life can be predicted. Assume the RUL following Gaussian distributions Psl should be below the 10−8 while Psl should be below 10−6.The mission requires dispatch 8 aircrafts one hour later and lasting two hours. Assume there are 3 ISSs, of which ability of the maintenance are the same, being in charge of all aircrafts' maintenance. The maintenance time and cost of each LRM is given in Consider that there are 100 individuals in populations, and one of these individuals is described as follows:k1 = k2 = 0.97, k3 = k4 = 0.02. The result is described in Set up the −07 which meet the requirement.The total cost of the maintenance is 14439.3 and the mission risk is 8.95 ∗ 10Then, optimal maintenance program can be written as B/D are the LRMs that need to be repaired. There are six aircrafts and eight LRMs that need to be repaired, and the numbers of the aircrafts that need dispatch are 1, 3, 4, 5, 6, 7, 8, and 10.Then, the optimal scheme of aircraft CBM and dispatching are described completely in This paper researches optimization decision method for aircraft fleet CBM oriented to mission success considering prognostics uncertainty and the resource constrain. The CBM and dispatch process of fleet is analyzed; the modeling method and an improved genetic algorithm for the problem are given, and the method is verified by case about fleet with 10 aircrafts.The alternative strategy sets for single aircraft are defined; then, the optimization problem of fleet CBM is translated to the combinatorial optimization problem of single aircraft strategy. The relationship between maintenance strategy and mission risk is established, and the problem becomes easier to solve.This paper used the RUL distribution, which has the maximum information and the highest in prognostics. It has more accurate description of the uncertainty compared with others.The optimization decision with risk for fleet CBM is realized. The fleet mission risk is quantitatively assessed, and the optimal CBM strategy for fleet could satisfy the requirement of lowest maintenance cost and acceptable risk.The main advantages of this method are shown as follows.This paper presents a theoretical approach for fleet CBM considering prognostics uncertainty. Some factors have been simplified, such as the cost of risk, the consequences of risk mission, the effect of the CBM process form ability of maintenance personnel, and the effect of random failures. The focus of further work is a more detailed and comprehensive model considering all above factors.