Mitigating extreme heat in urban areas is beneficial and sometimes critical to human health. Thriving plant communities in community parks play an important role in mitigating extreme heat through providing cooling effect, while inevitably affecting how people perceive the benefits of using community parks for recreation. Thus, the impacts of plant communities on the thermal environment should be quantified to determine the optimal structure of the plant community. The goal would be to harmonize the functions of improving the thermal environment with the preferences people have related to the recreational benefits of plant communities with various levels of vegetation density. In this paper, the correlations between the structural characteristics of plant communities and their function in mitigating the thermal environment were investigated on calm summer days in Xincheng Central Park, Minhang District, Shanghai, China. In addition to analyzing the plant communities present and their effects on the park microclimate, a questionnaire was employed to determine the plant community preferences of recreational park users. The results showed that plant communities could reduce the air temperature by 1.23–2.42 °C and increase the relative humidity by 2.4–4.2% during the daytime. The microclimate conditions in plant communities with varying vegetation densities were significantly different. The canopy density and leaf area index primarily controlled the temperature reduction, while the canopy density and total canopy cover ratio primarily controlled the increase in humidity; meanwhile, these correlations varied at different times of the day. Moreover, most of the park users preferred a moderately dense plant community which met their environmental perceptions for recreation in parks. Age or education level variables of park users would also predict preferences for different plant community densities. Ultimately, one plant community pattern with appropriate canopy density (60%), leaf area index (≥3) and canopy cover ratio  was recommended, which would harmonize the functions of the mitigation of the thermal environment with most people’s perception of a desirable vegetation density. As urban populations grow and expand, the creation of urban heat islands (UHIs) have been aggravated mainly by the creation of impervious ground surfaces, the loss of vegetation, and generation of anthropogenic heat that is released from industrial and of human activities –4. Many Among all possible cooling measures, vegetation provides one of the most effective strategies used to mitigate the UHI effect directly through shading and indirectly through evapotranspiration –15. NumePlant communities in urban parks could not only mitigate the thermal environment, but also provide the chances for urban residents to get close to nature . Urban rThe structural characteristics of plant communities not only affect ability of the community to mitigate thermal environment but also influence the perceived appropriateness of park areas for outdoor recreation. However, previous studies mainly focused on quantifying the cooling and humidifying effects from plant communities or on comparing the preference of people for plant communities with different vegetation densities. Nevertheless, these studies did not consider the microclimate control and environmental perception of plant community together, especially on hot summer days.The present study was designed to both measure the climate control service provided by plant communities in a park and document the structural nature of these plant communities. The study simultaneously employed a questionnaire survey to determine the plant communities that park visitors prefer to enjoy when using the park for recreation such as walking, chatting, and dancing, etc. In the present study, we aimed to 1) find whether there was a significant difference in the microclimate conditions of plant communities varying in vegetation density; 2) quantify the ability of vegetation structure features on diurnal variation in cooling and humidifying effects; 3) survey the plant community scenes that most respondents preferred for outdoor recreation in urban parks; 4) recommend a plant community structure with high cooling ecosystem service on hot summer days that could also meet most respondents’ environmental perceptions of a desirable vegetation density for outdoor recreation. We selected the research sites in a community park in Shanghai where the surrounding air temperature often exceeds 35 °C in summer. This oppressive heat will often limit the outdoor recreation activities of park users. However, by providing plant communities that could effectively improve the microclimate of the park, land managers could provide relatively open fields at slightly cooler temperatures for potential outdoor recreation activities. This information will help land managers to understand the role of plant communities on mitigating UHIs in urban areas, and specifically, help to improve the thermal and recreational environments through improved design of the structural characteristics of plant communities.The field studies were conducted in a public park of Shanghai city where specific permits were not required to the experiments conduct. These field experiments did not involve any endangered or protected species. Shanghai Jiao Tong University and the Shanghai Urban Planning and Land Resources Bureau approved the questionnaire survey. All the contents in the questionnaire were not health-related and did not involve clinical research. The questionnaires were used anonymously, and did not cause any difficulties or adverse effects to the participants. Before the survey, the respondents were informed on the purpose of this investigation, ensuring the confidentiality of their answers. When the participants agreed to fill in questionnaires and checked the box stating their agreement, the interview began.Studies were conducted in Shanghai, China (30°82′30″–31°82′70″N and 120°85′20″–121°84′50″E), a city with a total population of 23.0 million. Shanghai experiences a subtropical oceanic monsoon climate with hot summers and cold winters. Based on meteorological data collected from 1952 to 2015, the Meteorological Bureau of Shanghai has recorded a minimum monthly mean air temperature of 4 C in January, and a maximum of 28 °C in July and August. The annual average precipitation was approximately 1200 mm, approximately 60% of which occurred from May to September.Fig 1). This site was chosen because 1) this community park can be regarded as a typical area currently experiencing urbanization in Shanghai, and is located in the suburbs; 2) this very level park has an average elevation of less than 10 m; therefore, temperature differences caused by topography are negligible and can be ignored; 3) densely spaced residential buildings and some commercial structures surround the park, and most residents visit this site for recreational activities including walking, chatting, and dancing, etc.; 4) approximately 90% of the total area is covered by vegetation, with different vegetation densities from very dense, especially the dense plant communities, to quite open; 5) compared with other community parks in the center of Shanghai or in new town, plants in this park were planted in 1990–1995, and consequently the plant communities have now stabilized.The present study was conducted in Xincheng Central Park, a community park covering 4.3 ha and located in the Minhang District in southwestern Shanghai . Each plant community occupied 500–600 m2 of land and these communities were uniformly distributed in this park . During the selection of these target sites, plant communities with steep slopes, which would complicate the field investigation and microclimate measurement, were eliminated. And the selected plant communities were chosen to be located 10 m away from hills and water bodies for avoiding the effects of surrounding elements on the experimental data. Furthermore, the areas within each selected plant community had similar or uniform structural characteristics within each community type, and they could be distinguished from surrounding plant groups. In addition, three unshaded paved squares in this park were selected as control plots with areas of 400–600 m2 each , moderately dense (sites XC1 and XC6), and dense plant communities . Furthermore, the leaf area index (LAI) and the canopy density  of plant community were measured with point transects. And the measuring points in each plant community were established uniformly on 5 m intervals between every two measuring points . The LAI at each measuring point was measured with an LAI-2200 plant canopy analyzer . Simultaneously, the CD was measured using a Sony DSC-RX100M2 digital camera  at the measuring points, where the camera lens remained horizontal at a height of 1.5 m from the ground. Adobe Photoshop CS5  was then used to change each photo into a gray two-tone image and to tally the image pixels using the following calculation [a and b are the number of hemisphere and gap pixels of the enclosed canopy district, respectively.During the field investigation of each plant community, the height  and diameter at breast height  of each tree were measured and recorded. The CC of trees and shrubs/woodland area in each plant community as well as their total canopy cover ratio  were also measured and recorded  from July 27 to August 7, 2015. The air temperature and relative humidity were measured at the established points in each plant community and control plots in Xincheng Central Park respectively at a height of 1.5 m above the ground. For measurements in each plant community, a corresponding measurement was made in the control plots. The measurements were conducted with a model NK5922 portable weather station  when the wind speed fell below 2.0 m/s . All meaTable 2), and an evaluation of 15 color photographs of scenes with various scenes of plant communities with three different vegetation densities taken from various community parks in Shanghai. One aim of the questionnaire survey was to identify the potential plant community scenes that met respondents’ environmental perceptions for outdoor recreation in a community park. The questionnaires were delivered randomly to visitors in Xincheng Central Park during the field measurements from July 27 to August 7 (excluding rainy days) in 2015. The questionnaire survey was carried out during 7:00 to 9:00 am and 5:00 to 7:00 pm, when relatively large numbers visitors could be expected on hot summer days. Respondents were asked to evaluate each of the 15 photos representing the three vegetation densities and consider their appropriateness for recreation; photos were scored with a Likert five-point rating scale from 1 (not appropriate for recreation) to 5 (absolutely appropriate for recreation). In total, 128 questionnaires were delivered and collected on site.The questionnaire mainly consisted of two parts: demographic variables  were followed by Duncan’s multiple comparison tests (p<0.05) to separate the means of the three plant community types. In addition, Pearson correlation matrix and linear regression were used to analyze and quantify the relative contributions of the structural characteristics of plant communities to temperature reduction (ΔT) and humidity increase (ΔRH). Correlation analysis was conducted separately for each measurement period with the data of each measurement day, and the regression analysis was done with the averaged data of each measurement day. For the survey data from the questionnaire, respondents’ perceptions on plant community scenes with three different vegetation densities were evaluated. And one-way ANOVA was performed for analyzing the relationship between plant community preferences and the characteristics of respondents. All these statistical analyses were performed using the SPSS software package .Firstly, the vegetation densities in plant communities were identified and categorized based on the field investigation. Subsequently, paired-sample Fig 3). Within this gradient from open to dense, the herbaceous layer of vegetation varied from an urban park lawn to a natural herbaceous layer of vegetation with grass and herbs. The shrub layer varied from being absent to a dense layer of natural shrubs and bushes. The tree layers varied from scattered individual trees in open areas to a dense mix of trees with different sizes. In plant communities with open vegetation density, people could enter the site for recreation and the views of the surrounding landscape were essentially not blocked at all. On the contrary, people could not penetrate the plant communities with dense vegetation, and the vegetation physically blocked views of the surrounding landscape.Based on the field investigation, vegetation densities in plant communities were identified and categorized .Air temperatures in the control plots ranged from 33.82±0.24 °C to 37.16±0.21 °C, and within the plant communities, the temperature ranged from 32.02±0.08 °C to 35.04±0.41 °C. Based on the results of paired-sample p<0.05). Moderately dense plant communities (XC1 & XC6) were the most effective, with average temperature reductions of 2.31 °C. Dense plant communities (XC4 & XC5) and open plant communities (XC2 & XC3) reduced the temperature by 1.95 °C and 1.25 °C, respectively. Plant communities with open structures were the least effective at reducing summer temperatures. The cooling effects from open plant communities were quite different with the moderately dense (p = 0.002) and dense plant communities (p = 0.005), while the difference in cooling effects between moderately dense and dense plant communities was not significant (p = 0.118).A further ANOVA revealed that there were significant differences for the air temperature reductions from plant communities with varying vegetation densities during the daytime . The presence of plant communities resulted in an average 3.4% increase in relative humidity, and the increase ranged from 2.4% in XC3 to 4.2% in XC5 .The relative humidity in the control plots ranged from 45.6±0.4% to 66.2±2.5%, and within the plant communities, it ranged from 46.9±1.5% to 72.7±0.6%. Based on the results of paired-sample p<0.05). The results suggested that the presence of dense plant communities (XC4 & XC5) with more complicated structures resulted in the highest increase in relative humidity (4.2%). Meanwhile, plant communities with open structures (XC2 & XC3) had the lowest humidifying effects (2.5%). The humidifying effect from open plant communities was significantly different from moderately dense (p = 0.034) and dense (p = 0.009) plant communities. Additionally, significant difference in the humidifying effects was not found between moderately dense (XC1 & XC6) and dense (XC4 & XC5) plant communities (p = 0.127).An analysis of variance was used to find the differences in the humidifying effects from plant communities with varying vegetation densities . It was found that the temperature reduction caused by the presence of plant communities had a significant positive correlation with LAI  and CD , while the increase in the humidity of plant communities had a significant positive correlation with CD  and total CC . In addition, these correlations may change during the daytime. Over the course of the measuring periods, LAI was found more often statistically significant for determining the cooling effect. While for the humidifying effect, the correlation with CD was more often statistically important. For the other structural characteristics of plant communities, no significant correlations with temperature reduction and humidity increase were found.To clarify the cooling and humidifying effects from plant communities, Pearson correlation analysis was undertaken to examine the relationship between plant community structural characteristics and cooling as well as humidifying effects with regards to the measuring time . The temperature reduction seemed to increase unevenly with increases in LAI and CD. Specifically, the incremental rate of temperature reduction was much higher when the LAI was above 3 and the CD was above 60%. Meanwhile, for the increase in humidity, the increment was much higher when the CD was above 50% and the total CC was close to 1.When considering the structural characteristics that correlated with the cooling and humidifying effects of the plant community, linear regression analysis was used to quantify the relationship between the cooling effect, LAI and CD as well as between the humidifying effect, CD and total CC  were significantly higher (p<0.05) than that of open (2.83) and dense (3.47) plant community scenes. By further investigating the plant community scenes that met most respondents’ environmental perceptions, it could be found that these plant communities could not only contain moderately dense vegetation but also provide some spaces for outdoor recreation.According to the results of questionnaire survey, respondents’ perceptions on the plant community scenes with three different vegetation densities could be found in Fig 3). One-way analysis of variance (ANOVA) found that demographic variables of the respondents would influence their perceptions on plant community density (Table 5). The results revealed that the age and education level of these respondents had a close relationship with this assessment (p<0.05). Respondents age 40–60 and 25–40 expressed a higher preference for moderate to dense vegetation density, compared with both younger and older subjects. The respondents age < 25 and >60 generally preferred the open plant communities which could provide the highest accessibility for recreation. For the education level, respondents with higher education level would prefer plant community scenes from moderate to dense vegetation density possibly due to their knowledge of the benefits from dense vegetation .Meanwhile, the open and dense plant communities in parks were also assessed as favorable for recreation to some extent (Table 4). A possible explanation for this finding on the cooling effect is that the interception of the tree canopy is diverse because of the incident angle of radiation changes throughout the day. When the angle is low and its intensity is weak, the cooling effect seems to largely depend on the CD. Whereas, the cooling effect may be more controlled by both CD and LAI when the angle slowly increases [Although many previous studies have investigated the cooling and humidifying effects of plant communities , 36, 37,ncreases . Howeverncreases , which aIn the present study, it was found that the microclimate conditions in plant communities with varying vegetation densities were significantly different. Furthermore, the study verified that both CD and LAI were better indicators than total CC (vegetation density) for predicting the cooling effects from plant communities. This result is consistent with the findings of Peters and McFadden in which the temperatures of plant communities were mainly controlled by differences in the stand-level LAI and CD . Most liTable 2) of the respondents had the age of 25–40 who expressed a higher preference for moderate to dense vegetation density.Plant communities in community parks are not only developed for their environmental benefits but are also designed to attract more visitors for recreation. According to many previous studies, plant community density is considered one of a number of environmental features contributing to the perceived appropriateness for outdoor recreation in park areas. Based on the opinions obtained through use of a questionnaire, the plant community landscapes that varied in three vegetation densities were all perceived as favorable for recreation, while the moderately dense scenes received the highest ratings. This finding confirmed some previous studies that defined the role of openness as an important environmental attribute of parkland and showed that moderately dense scenes were generally preferred by most respondents , 34. PeoGiven the results above, some recommendations could be given for the landscape design methods used in community parks. The plant communities within a park should harmonize the functions of mitigating the thermal environment with the environmental perceptions most people have of the preferred vegetation density. First, temperature reduction from plant communities was mainly controlled by differences in the stand-level LAI and CD. The CD should be no less than 60% and LAI should be no less than 3 to ensure plant communities provide the desired cooling effects. And the CC of trees should be no less than 0.60 to ensure the appropriate CD (60%) of one plant community. Further considering the environmental preference of most respondents for moderately dense plant community, the CC of trees should be 0.60–0.75 while the CC of shrubs/woodland area should be 0.20–0.45 . This type of plant community pattern could not only improve the thermal environment, but also meet the perceptions and demands of most respondents while providing recreation opportunities in a park. However, there are still some drawbacks in the recommended plant community pattern which should be further studied in the later research. Firstly, respondents with varying demographic variables may have different perceptions on plant community density and the respondents in the present study mainly had the age of 25–40, thus different plant community patterns should be further studied and developed for varying visitors in community parks. Furthermore, the present study mainly focused on the structural characteristics of plant communities without considering their species composition, which could influence the visual landscape  and climThe results presented in this study clearly showed that plant communities in community parks had an obvious potential to mitigate heat stress in hot summers, although the effects varied in different plant communities and time periods. Compared to the control plots, plant communities could reduce the air temperature slightly and increase the relative humidity. The temperature reduction from the plant community was most significant at 14:00–15:00, and the humidity increase was most significant at 8:00–9:00. Furthermore, our results indicated that even the microclimate conditions in plant communities varying in vegetation density were significantly different, both CD and LAI were better indicators than total CC for predicting the cooling effects from plant communities. The CD and total CC were effective in increasing the relative humidity. This study further clarified that these correlations appeared to change over time.In addition, moderately dense plant communities with some space for recreation in park areas were perceived most appropriate for outdoor recreation by respondents based on our questionnaire. And the demographic variables of respondents would have impacts on their perceptions of plant community density. All these survey results have important implications for the planting and landscape designs in community parks. This study suggests that adjusting the structural characteristics of plant communities based on the contributions of those communities to cooling and humidifying effects and simultaneously balancing the nature of the plant communities with the residents’ perceptions of the desired level of vegetation density required for recreation would facilitate the adoption of an improved planting design. Plant communities with appropriate canopy density (≥60%), leaf area index (≥3) and canopy cover ratio  were recommended in parks, that would harmonize the functions of the mitigation of the thermal environment with most people’s perceptions of a desirable vegetation density for outdoor recreation.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.


We propose a plasmonic Bragg reflector (PBR) composed of a single-layer graphene-based silicon grating and numerically study its performance. An external voltage gating has been applied to graphene to tune its optical conductivity. It is demonstrated that SPP modes on graphene exhibit a stopband around the Bragg wavelengths. By introducing a nano-cavity into the PBR, a defect resonance mode is formed inside the stopband. We further design multi-defect PBR to adjust the characteristics of transmission spectrum. In addition, through patterning the PBR unit into multi-step structure, we lower the insertion loss and suppress the rippling in transmission spectra. The finite element method (FEM) has been utilized to perform the simulation work. Surface plasmon polaritons (SPPs) are surface waves that propagate along the boundary surface between dielectric and metallic materials with fields decaying exponentially in both sides, thereby creating the subwavelength confinement of electromagnetic waves . These aGraphene, a single layer of carbon atoms densely arranged into a honeycomb pattern, has been widely explored as a newly alternative to plasmonic material , 19. GraIn this paper, we propose a PBR structure consisting of a single-layer graphene and silicon grating and numerically study its performance. We employ a silica spacer layer to separate the monolayer graphene and silicon grating and an external voltage gating to tune the surface conductivity of graphene. The finite element method (FEM)  has beenAs plotted in Fig. kGSPP >> ω), the Eq.  determined by Kobo formula  featuring the GSPP propagation loss. Throughout the paper, the influence of substrate silicon on the dispersion relation is negligible when the silica layer is above 100 nm [Re(NGSPP) and LGSPP on the gate voltage Vg and the thickness of silica layer h are illustrated in Fig. Re(NGSPP) shows a significant increase when the gate voltage Vg decreases or the thickness of silica layer h goes up. In Fig. Vg or a decreasing h. Hence, the two important factors should be both taken into consideration for our designs.Another important parameter derived from the above equation is e 100 nm . The depNGSPP. According to the aforesaid discussion, this can be realized by alternatively varying the thickness of silica layer yielding a silicon grating substrate, shown in Fig. Generally, for wavelength-sensitive operations, a plasmonic Bragg reflector is constructed by periodically modulating the effective refractive index of the waveguide. There are some popular accesses to achieve this such as width modulation  and refrλb is the Bragg wavelength and m is an integer assumed to be 1 in our discussion. NGSPP1 and NGSPP2 are the effective refractive index of GSPP on differently doped areas of graphene, respectively .In addition, we introduce a nano-cavity into the plasmonic Bragg grating causing a defect in the PBR’s periodicity. At some specific wavelengths, resonant modes will be formed around the nano-cavity, and accordingly, there will be a transmission peak inside the stopband . In Fig.As shown in Fig. wd = 90 nm and Dd = 100 nm. Through Fig. λ = 7.105 μm for these three cases. The red dashed circles denoting the resonant areas indicate that these defect modes from each nano-cavity will interfere with each other leading to these amazing features in transmission spectra. Besides, stopband can be greatly widened with multiple defect cavities. Then, we place one defect cavity in the center and place another one at different locations. Figure Since the defect cavity works differently when they are located at different sites, we further propose multiple defect cavities. At first, we compare the transmission spectra for three cases: one-defect, two-defect, and three-defect in PBR. All defect cavities are set at the same condition: NGSPP in the groove depth [In the end, we design the PBR unit into a multi-step pattern to deal with the high insertion loss and severe rippling in transmission spectra resulting from the abrupt change of ve depth . A threeIn conclusion, we design a PBR structure consisting of a single-layer graphene and silicon grating and numerically study its performance. We employ an external voltage gating to tune the surface conductivity of graphene. It is found that SPP modes on graphene exhibit a stopband around the Bragg wavelengths. Based on Bragg scattering condition, we discuss several modulation schemes to adjust the characteristics of transmission spectrum. Furthermore, we introduce a nano-cavity into PBR, consequently realizing a resonant defect mode. We further propose multi-defect PBR and achieve multiple peaks inside the stopband. At last, by designing the PBR unit into multi-step pattern, we lower the insertion loss and suppress the rippling in transmission spectra. We hope all the proposed designs above can help pave new ways in actively tunable modulation application.


These results suggest that such graphene/h-BN heterostructures on recyclable Pt foils grown by LPCVD are promising for high-performance graphene-based electronics.High-quality large-area graphene/h-BN vertical heterostructures are promising building blocks for many viable applications such as energy harvesting/conversion, electronics and optoelectronics. Here, we successfully grew high-quality large-area graphene/h-BN vertical heterostructures on Pt foils by one-batch low-pressure chemical vapor deposition (LPCVD). We obtained the high quality of about 200-µm-wide graphene/h-BN film having uniform layer thickness. Moreover, the obtained graphene/h-BN heterostructures exhibited field effect mobility of up to 7,200 cm Representing the thinnest and the most common 2D heterostructures, graphene/h-BN, have recently attracted great attentions due to their remarkable morphological, electrical, and thermal properties5. Such heterostructures have stimulated extensive interest for exploring some novel physics issues, such as fractional quantum Hall effect6, commensurate-incommensurate transitions7, Hofstadter’s butterfly behaviors8, and ballistic transport9. Thus, securing reliable methods for producing large-area, high-quality graphene/h-BN heterostructures becomes the most significant step for related fields. One plausible strategy for producing graphene/h-BN vertical heterostructures is based on layer-by-layer transfer of separately exfoliated 2D layers via a two-step process11. However, such a two-step process has some drawbacks during the transfer process, such as unwanted charge trapping that originates from any transfer-induced contaminants and defects, and poor surface flatness during the transfer; all of these are undesirable for obtaining high quality graphene/h-BN vertical heterostructures13. These drawbacks negatively affect the electrical and optical characteristics of graphene. Up to now, there have been some attempts for a direct CVD-based growth method of graphene on h-BN films to overcome these outstanding issues. Direct CVD-based growth of graphene on h-BN films may enable not only to obtain large-area uniform graphene films but also to reliably form versatile graphene/h-BN vertical heterostructures16. However, this approach still suffers from many technical issues such as poor quality of h-BN films, small size of graphene grains on h-BN, and h-BN film degradation that is caused by etching by H- and/or O-containing gaseous species during the growth of graphene17.Vertical heterostructures based on 2 dimensional (2D) layered materials such as graphene and h-BN have emerged as a new paradigm of functional materials4 gas as the substrate and the carbon precursor carrier, respectively. We found that using a single layer h-BN film as the substrate allows us to obtain graphene/h-BN heterostructures, because direct growth of graphene on h-BN can retain the pristine properties of graphene. Furthermore, facile transfer of graphene/h-BN vertical heterostructures (directly grown using the LPCVD method) onto arbitrary substrates via a straightforward electrochemical bubbling-based release method presents a unique recipe for fabricating many viable devices18. Our transfer method provides an additional advantage, i.e., it is characterized by a rapid process and extended use of recyclable Pt foils. The field effect mobility of the fabricated graphene/h-BN heterostructures based field effect transistors is as high as 7,200 cm2V−1s−1 at room temperature, suggesting that our approach of directly growing graphene/h-BN heterostructures on Pt foils using LPCVD can be quite promising for high-performance graphene-based electronics.In this work, we successfully grew continuous, large-area graphene/h-BN vertical heterostructures on recyclable Pt foils of 6 mm × 25 mm using one-batch LPCVD method. The growth process was implemented by employing a recyclable Pt foil and CH2/Si substrate, respectively (more details are provided in the Methods section).Figures −1, demonstrating that the h-BN film is a single layer20. Figure 2/Si substrate is continuous and large area. Figure 2/Si substrate along with the Raman spectrum of a bare SiO2/Si substrate. The two locations A and B, representing the regions of the bare SiO2/Si substrate and the graphene/h-BN film respectively as in Fig. −1 and 2,690 cm−1, respectively. The I2D/IG peak intensity ratio was well over 2, and the full width at half maximum (FWHM) of the 2D band was estimated to be 37 cm−1. This observation clearly indicates that the graphene film is a single-layer21. The scanning Raman mappings of the h-BN peak, G peak, 2D peak of the graphene and I2D/IG intensity ratio over a 75 μm × 75 μm area with a spot size of 2 μm and a step size of 2 μm were obtained, as shown in Fig. −1), 2D peak (the range from 2670–2690 cm−1), as well as I2D/IG intensity ratio (was close to 2), also revealed that the graphene film was grown on h-BN film  and H2 (from 5 sccm to 100 sccm) flow rates while keeping the growth time to 20 min, to assess the effects of these manipulations on the growth characteristics. Similar characteristics were found as a function of gas flow rates  and the FWHM values are quite similar (36–39 cm−1). These results also confirm that the grown graphene is a single-layer22. Furthermore, the Raman peak characteristics  were quite similar for different CH4 and H2 flow rates , owing to atomically flat and dangling-bond-free h-BN, significantly minimizing the interfacial charge trapping. Moreover, h-BN has a hexagonal structure that represents a typical sp2-hybridized material, quite similar to archetypal 2-dimensional graphene26 (the lattice constants are similar: 2.456 Å for graphene and 2.504 Å for h-BN). The van der Waals epitaxy growth of graphene on h-BN film has been reported by many researchers27. By taking advantage of the single-layer h-BN films grown on Pt foils, our approach to the epitaxial growth of graphene could be very effective for realizing uniform graphene on h-BN films in a controlled manner. Different from the LPCVD growth of graphene on metal foils such as Cu, Ni and Pt, the surface of a metal catalyst plays a key role in the decomposition of gas molecules and the nucleation of absorbed atoms during the growth process. Hence, with the help of catalytic Pt foil covered by the single-layer h-BN film, the decomposition and nucleation sites were effectively controlled. Hence, the CH4 gas must have been decomposed owing to the presence of underlying catalytic Pt foil during the growth process. As the growth time increases, the carbon atoms may suffuse swiftly over h-BN film. Additionally, because the h-BN film has a flat surface, with increasing the growth time, the uniform graphene film would be grown on h-BN film. Therefore, the high-quality graphene film can be grown on h-BN/Pt foil through the systematic optimization of growth conditions .  as the source and drain electrodes as shown in Fig. 20/cm3) substrate was used as a bottom gate that modulates the charge density in the graphene.To investigate the electrical transport properties of graphene/h-BN films, we fabricated graphene-based FETs (GFETs) with 40 μm long and 10 μm wide channels by transferring a graphene/h-BN film onto a 100-nm-thick SiO2 were prepared to evaluate their electrical characteristics following a standard photolithographic procedure. Figure vs. gate voltage (Vg) curves for both GFET devices, respectively. We obtained the hole mobility of up to 3,900 and 7,200 cm2V−1s−1 and the electron mobility of up to 3,600 and 7,000 cm2V−1s−1, respectively using the simple Drude model30. Note that the charge carrier mobility values of the graphene grown on h-BN is higher than those of the graphene transferred on SiO2. We believe that these observations are attributed to the fact that the intrinsically atomic-flat h-BN not only provides a low number of dangling bonds between graphene and h-BN, but also minimizes graphene’s structural defect density and the doping level owing to its excellent chemical stability and isostructural nature. Therefore, h-BN film is considered an ideal substrate to synthesize high-quality graphene. It should be noted that the mobility values observed in our devices are 2 to 3 times higher than those reported elsewhere for LPCVD grown graphene on h-BN film32. Moreover, the histogram of the carrier mobility values and the average Vdirac for up to 60 devices, shown in Fig. 2V−1s−1 range. It is important to note that such a high carrier mobility obtained for our graphene/h-BN heterostructures strongly suggests that a direct CVD-based growth of graphene on h-BN using Pt foils is very promising for constructing high-performance graphene-based FETs and tunneling devices, and is advantageous compared with two-step transferred graphene on h-BN film.Graphene field effect transistors (GFET) by using two different types of graphene grown on h-BN film and transferred on SiO2V−1s−1 range. Such a high carrier mobility obtained for our graphene/h-BN heterostructures strongly suggests that such graphene/h-BN heterostructures on recyclable Pt foils grown by LPCVD can be a promising approach for high-performance graphene-based electronics.We demonstrated direct growth of graphene/h-BN vertical heterostructures on Pt foils using the CVD method. Moreover, we found that using a single-layer h-BN/Pt foil as a substrate not only presents an ideal platform for obtaining continuous graphene/h-BN vertical heterostructures but also enables to realize desirable electrical characteristics of graphene. Direct growth of graphene on h-BN films retains the pristine properties of graphene by avoiding the interfacial contamination, thus yielding a clean interface between the graphene and the h-BN film. On the other hand, because the lattice structures of graphene and h-BN are very similar, during the growth procedure the carbon atoms will suffuse a large area on the h-BN film by diffusion, yielding large-area graphene/h-BN heterostructures. Our graphene/h-BN heterostructure exhibits high carrier mobility, in the 2,000–8,000 cm4 as the substrate and the carbon precursor carrier, respectively. A single-layer h-BN on the Pt foil was grown using the LPCVD method following our previously developed synthetic method that was reported elsewhere19. In addition, to avoid detrimental etching of h-BN films by H-containing gaseous species during the growth of graphene, a 1.5 m long quartz tube furnace with moving rails, allowing easy positioning of the furnace (i.e. heating zone) over some length of the quartz tube. Figure 2 gas flows from room temperature to 1000 °C for 25 min; when the temperature reached 1000 °C, the furnace was then repositioned from “A” to “B” to avoid H2 etching during the growth of graphene over the h-BN film. It should be noted that h-BN/Pt foil was kept at room temperature during the process of the furnace heating and was only exposed right after the temperature reaches 1000 °C. This is a key step for avoiding H2 etching as we can minimize the reaction time to etch away h-BN during the graphene growth. The Ar gas flow was then turned off and gaseous CH4 was introduced; the growth of graphene continued for 20 min; after the growth, H2 was turned off and the furnace was moved from “B” to “A” to allow a natural cool down to room temperature. The optimization of growth parameters for obtaining high-quality large-area single-layer graphene on the h-BN/Pt foil was systematically performed by varying the H2 and CH4 flow rate as well as the growth time.The growth process was implemented by employing a Pt foil and CH2/Si substrate. First, the graphene/h-BN/Pt foil was spin-coated with polyvinyl alcohol (PVA) and poly methyl methacrylate (PMMA) layers. The sample was then kept in vacuum for 24 hr. After establishing the vacuum, the electrochemical bubbling method was used for transferring PMMA/PVA/graphene/h-BN onto a 300-nm-thick SiO2/Si substrate. The PMMA/PVA/graphene/h-BN/Pt foil and a pure Pt foil were used as the cathode and the anode, respectively; a NaOH aqueous solution (1.0 M) was used as the electrolyte at room temperature. The bubbling transfer method was used with a stable current of 1 A, and the corresponding electrolytic voltage was run between 5–10 V and was applied for 2–5 min. After the PMMA/PVA/graphene/h-BN film was peeled off from the Pt foil, the sample was cleaned 3 times using deionized water to remove the residual NaOH solution, after which the sample was transferred onto a 300-nm-thick SiO2/Si substrate. Finally, the sample was dipped in acetone for 10 min to remove PMMA and then kept in hot water (100 °C) for 15 min to remove PVA.Figure Optical micrographs were acquired using optical microscope . The surface morphology was measured using field-emission scanning electron microscope (JEOL JSM7401F) and transmission electron microscopy . The film thickness and the surface topography were measured using an atomic force microscope . Raman spectra were measured using a micro Raman microscope  with a 532-nm-wavelength laser.Supporting Information


Sarcophilus harrisii) population. The first transmissible tumor, now termed devil facial tumor 1 (DFT1), was first discovered in 1996 and has been continually transmitted to new hosts for at least 20 years. In 2015, a second transmissible cancer [devil facial tumor 2 (DFT2)] was discovered in wild devils, and the DFT2 is genetically distinct and independent from the DFT1. Despite the estimated 136,559 base pair substitutions and 14,647 insertions/deletions in the DFT1 genome as compared to two normal devil reference genomes, the allograft tumors are not rejected by the host immune system. Additionally, genome sequencing of two sub-strains of DFT1 detected greater than 15,000 single-base substitutions that were found in only one of the DFT1 sub-strains, demonstrating the transmissible tumors are evolving and that generation of neoantigens is likely ongoing. Recent evidence in human clinical trials suggests that blocking PD-1:PD-L1 interactions promotes antitumor immune responses and is most effective in cancers with a high number of mutations. We hypothesized that DFTD cells could exploit the PD-1:PD-L1 inhibitory pathway to evade antitumor immune responses. We developed recombinant proteins and monoclonal antibodies (mAbs) to provide the first demonstration that PD-1 binds to both PD-L1 and PD-L2 in a non-placental mammal and show that PD-L1 is upregulated in DFTD cells in response to IFN-γ. Immunohistochemistry showed that PD-L1 is rarely expressed in primary tumor masses, but low numbers of PD-L1+ non-tumor cells were detected in the microenvironment of several metastatic tumors. Importantly, in vitro testing suggests that PD-1 binding to PD-L1 and PD-L2 can be blocked by mAbs, which could be critical to understanding how the DFT allografts evade the immune system.The devil facial tumor disease (DFTD) is caused by clonal transmissible cancers that have led to a catastrophic decline in the wild Tasmanian devil ( Sarcophilus harrisii   , 12. As in (B2m) . One potin (B2m) –15. Indein (B2m) . PD-L1 cin (B2m) ] on a vain (B2m) ], all ofin (B2m) , 19.Immunotherapy that blocks checkpoint molecule interactions, such as the PD-1:PD-L1 interaction, has achieved 10–87% response rates in a broad range of late-stage human cancers –22. Recein vitro and that PD-L1 was detected in the microenvironment DFT1 via IHC.The lack of devil-specific antibodies for flow cytometry and immunohistochemistry (IHC) has been a major obstacle in understanding the role PD-1:PD-L1 signaling, and immune function in general in devils. Here, we have developed a panel of α-PD-1 and α-PD-L1 monoclonal antibodies (mAbs) that are highly specific for devil proteins and are capable of blocking PD-1 binding to PD-L1 and PD-L2. We used these new mAbs to show that PD-L1 is strongly upregulated on DFT1 and DFT2 by IFN-γ Tasmanian devil PBMCs, tissues, and cell lines were collected with appropriate approvals from the Tasmanian Devil Captive Research Advisory Group, Tasmanian Government Department of Primary Industries, Parks, Water and the Environment (DPIPWE), Animal Ethics Committees from the University of South Australia (# U18-14), and the University of Tasmania (A0012513 and A0014976) and were performed in accordance with relevant guidelines and regulations. PBMCs were isolated by diluting 1:1 with PBS, layering on Histopaque at room temperature, and centrifuging at 400 g for 30 min. The PBMC layer was then collected and washed 2× with RPMI at 500 g for 5 min. Formalin-fixed paraffin-embedded (FFPE) tissue sections were collected and processed as reported previously .2 in complete RPMI supplemented with 10% FBS (cRF10), 2 mM l-glutamine, penicillin–streptomycin (100 U/ml and 0.1 mg/ml), 10 mM HEPES, and 0.5 mM β-mercaptoethanol. Chinese hamster ovary (CHO) cells were cultured at 35°C with 5% CO2 in 5% complete RPMI supplemented with 5% FBS (cRF5). For production of recombinant proteins, stably transfected CHO cells were cultured in suspension in spinner flasks in chemically defined, serum-free CHO Ex-Cell (Sigma # 14361C) media supplemented with l-glutamine and penicillin–streptomycin.Devil facial tumor 1 cell lines C5065, 1426, and 4096, the DFT2 cell line JV, and devil PBMCs were cultured at 35°C in 5% COFull-length translated gene sequences for Ensembl transcripts: PDCD1-201 ENSSHAT00000018586 (PD-1), CD274-201 ENSSHAT00000003059 (PD-L1), PDCD1LG2 ENSSHAG00000004176 (PD-L2), and IFNG ENSSHAT00000017850 (IFN-γ) were analyzed using the Phobius web server  and TMHMPrimers for PD-1, PD-L1, and PD-L2 were developed for inserting either full-length or extracellular domains (ECDs) into expression vectors . Total RNA was extracted from DFT1 cell line C5065 with or without 24 h stimulation with supernatant containing devil IFN-γ from CHO cells stably transfected with the pAF23 vector (devil IFN-γ). Devil PBMCs were stimulated overnight with ConA (5 μg/ml) prior to mRNA extraction. mRNA was reverse transcribed into cDNA using ProtoScript II First Strand cDNA Synthesis Kit (New England Biosciences). Qualitative assessment of PD-L1 expression in devil PBMCs and IFN-γ treated and untreated DFT1 C5065 was performed by amplifying a 280 bp segment of PD-L1 from cDNA. GAPDH was used as a reference gene and B2m was used as a positive control for IFN-γ effects. Primers used were as follows: cadeGAPDH_F1 and cadeGAPDH_R1 for GAPDH, B2Mex1F and B2Mex2R for B2m, and deB7H1_318_F and deB7H1_597_R for PD-L1. Reactions were performed following the manufacturer’s recommendations for Kapa HiFi HotStart ReadyMix (Kapa Biosystems # KK2602), and cycling conditions consisted of an initial denaturing step of 95°C for 3 min, followed by 30 cycles of 98°C for 20 s, 60°C for 20 s, and 72°C for 20 s, and a final extension step at 72°C for 5 min. Reaction products were then run on a 1% agarose gel at 100 V.BsaI for 1 h at ambient temperature and gel purified to create linear vectors for cloning. In order to expedite subsequent cloning and development of recombinant fusion proteins, we used overlap PCR extension and T4 ligase cloning techniques to create a modified pAF07 vector that contained bicistronic green fluorescent protein (GFP) and IRES-DsRed, which included additional restriction sites . All expression vectors included the Kozak consensus sequence GCCGCCACC upstream of the start codon to ensure efficient initiation of the translation process  were digested with  process .in vitro , we cre6 cells to be transfected in 150 mM NaCl or serum-free RPMI and incubated for 2 min. Twenty-five kilodalton linear polyethylenimine (Polysciences # 23966-2) was added to the DNA at a 3:1 ratio, and the solution was vortexed and incubated for 15–30 min at ambient temperature before the solution was added to the adherent cells. The cells were then incubated for 2–4 h before the media were removed and replaced with cRF10. The following day, the transfected cells were subjected to selection with hygromycin (1 mg/ml) for at least 7 days, and then maintained with 0.2 mg/ml of hygromycin.Stably transfected CHO cell lines expressing full-length surface proteins  and secreted proteins  were developed using a protocol modified from Matasci et al.  and Hsu 5 and 5 × 106 cells/ml by adding fresh media. Supernatant was collected and centrifuged at 3,200 rcf for 10 min at 4°C to remove cells and debris. The supernatant containing His-tagged proteins was then diluted 1:1 with 20 mM sodium phosphate buffer to a pH of 7.4. The diluted supernatant was then loaded onto HisTrap Excel columns , and the proteins were eluted using 20 mM sodium phosphate buffer with 0.5 M imidazole. Fusion proteins were then dialyzed in PBS using 6–8 kDa dialysis cassettes (Sigma # PURX60005) and then concentrated by evaporation in the dialysis cassette, aliquoted, and frozen until further use. The molecular weight and extinction coefficients for each protein were calculated using the ExPASy Bioinformatics Resource Portal tools  and cultured in magnetically stirred propeller flasks at 75 rpm. The density of cells was then maintained between 5 × 10l-glutamine, 50 μM β-ME, and 10 mM HEPES) with 20% FBS, and hybridoma fusion and cloning supplement (Roche # 11363735001). One day postfusion, HAT media supplement (Sigma # H0262-10VL) was added to the culture.Immunization of BALB/c mice for antibody production was approved by the University of South Australia Animal Ethics Committee # 139/13) and the University of Tasmania Animal Ethics Committee (# A0014680) and was performed in accordance with relevant guidelines and regulations. Mice were immunized subcutaneously on each flank and the back of the neck with a total of 100 μg of fusion proteins  in a SqualVax (Oz Biosciences # SQ0010) emulsion on days 0 and 14. At least 14 days after the day 14 booster, the mice were intravenously administered 100 μg of fusion proteins. Four or five days following the intravenous booster, the mice were humanely killed and the spleen and lymph nodes (LN) were harvested. Hybridomas were produced using SP2/0 mouse myeloma cells as the fusion partner and following a standard polyethylene glycol hybridoma fusion protocol , 39. Bri/13 and tvia ELISA, and results were confirmed via flow cytometry using a FACS Canto II (BD Biosciences) using stably transfected CHO cells . Hybridomas were then be subcloned by limiting dilution into 96-well plates complete IMDM with 20% FBS and hybridoma fusion and cloning supplement. Monoclonal cell lines were then screened by ELISA to select lines that produced the highest levels of mAbs. Anti-PD-1 and anti-PD-L1 mAbs were tested for their ability to block receptor:coreceptor interactions  using stably transfected cell lines  and PD-1 fusion proteins.Hybridomas were initially screened for antibody binding to fusion proteins 3) for 15 min on ice in u-bottom 96-well plates, and then incubated with 1 μg/sample of anti-PD-L1 clone 1F8 or anti-PD-1 clone 3G8 for 15 min on ice. The plates were then topped up with 150 μl with FACS buffer and centrifuged at 500 g for 3 min at 4°C. Samples were then incubated with 0.125 μg/sample of goat anti-mouse IgG-phycoerythrin (Abcam # AB98742) for 15 min. The plates were then topped up with 150 μl with FACS buffer and centrifuged at 500 g for 3 min at 4°C and then resuspended in FACS buffer prior to performing flow cytometric analysis using a FACS Canto II (BD Biosciences).DFT1.C5065, DFT1.1426, DFT1.4096, and DFT2.JV cells were cultured for 3 days with or without 5 ng/ml IFN-γ and then cultured for four additional days without IFN-γ. PD-L1 and PD-1 expression were quantified on days 1, 2, 3, and 7 by flow cytometry. Briefly, DFT cells were blocked with 5% normal goat serum in FACS buffer  from α-PD-L1 hybridoma clone 1F8 and α-PD-1 hybridoma clone 3G8 were used neat. The slides were then washed with PBS (2×) prior to adding Dako Envision + System HRP-labeled polymers and incubating for 30 min. The slides were again washed in PBS (2×). The Dako liquid DAB + substrate chromogen system (Dako # K3468) was used according to the manufacturer’s instructions. Finally, slides were counterstained with Mayer Hematoxylin for 30 s before washing in running water for 4–5 min, washing for 4–5 min in running water, and submerging in 95% ethanol for 2 min, 100% ethanol for 2 min, and xylene for 2 min (2×). Slides were then coverslipped and stored in the dark until examination.Immunohistochemistry staining methods for the tissue sections used here have been previously reported in Kreiss , Tovar eThe cases of DFT2 were previously diagnosed in Pye et al.  by histoMyotis davidii), cattle (Bos taurus), dog (Canis lupus familiaris), human (Homo sapiens), mouse (Mus musculus), opossum (Monodelphis domestica), and platypus (Ornithorhynchus anatinus)  with IFN-γ for 3 days and then used flow cytometry to quantify PD-L1 expression on days 0, 1, 2, 3, and 7 using α-PD-L1 (1F8). All cell lines expressed little or no PD-L1 on day 0, but upregulated PD-L1 following IFN-γ treatment   or experimentally inoculated (n = 5) with tumors as part of a different study (1) and found that PD-L1 was generally not expressed in DFT1  in the outer cortex of LN generally demonstrated positive cytoplasmic staining for PD-L1. A few germinal centers contained 5–10% PD-L1) Figure . PD-1 wa1 Figure  cells exThe transmissible DFTD represents an immunological enigma due to the fact that these transmissible tumor allografts are not recognized and rejected as foreign. This is despite genetic differences between the host and tumor and the high mutational load that has accumulated in the DFT1 in 20 years of host-to-host transmission . In humaAnalysis of the PD-1 protein sequence revealed putative ITIM and ITSM domains, which are critical for the inhibitory effects of PD-1 observed in other species. Their conservation suggests that PD-1 may serve as a negative regulator to immune cells in devils as it does in humans and mice. The devil PD-1 ITSM sequence was a perfect match to human, dog, bat, and cattle PD-1 ITSM, and the ITIM sequence varied by only a single amino acid, suggesting that strong selective pressure has maintained these motifs across the 160 million years since marsupials and placental mammals diverged . Furtherin vitro, which could have lasting effects on tumor phenotype, and also facilitates assessment of the roles of PD-1, PD-L1, and PD-L2 in tumor transmission, development, and metastases. Finally, recent evidence suggests that some devils can mount an immune response to DFT tumors   , 55. Unfin vitro results demonstrate that IFN-γ transiently upregulates PD-L1 on both DFT1 and DFT2 cell lines. Our IHC results are in agreement with the in vitro results showing that both DFT1 and DFT2 cells do not normally express PD-L1 or express very low levels of PD-L1. This is not surprising given that tumor-infiltrating lymphocytes (TILs) are rarely observed in DFT1 or DFT2 tumors, so local production of IFN-γ by T cells is likely absent in most cases (+ non-tumor cells in the tumor microenvironment of several metastatic tumors and scattered PD-L1+ tumor cells.Our st cases , 56, 57.In humans, the combination of α-CTLA4 and α-PD-1 has generally proven much more effective that either treatment alone –60. α-CTIn conclusion, this study is the first to show that receptor–ligand interactions between the potent cosignalling molecules PD-1, PD-L1, and PD-L2 are conserved in marsupials. Analysis of PD-1 protein sequence revealed putative ITIM and ITSM domains, which are critical for the inhibitory effects of PD-1 observed in other species. We report that PD-L1 expression is rare on both DFT1 and DFT2 cells, but that PD-L1 expression is upregulated by treatment with IFN-γ. Mapping the complex interactions and expression patterns of inhibitory checkpoint molecules will be critical for developing a DFTD vaccine and understanding how transmissible tumors evade host immune responses. Understanding the immune evasion mechanisms employed by these transmissible tumors could help to shed light not only on cancer immunology but also for transplant tolerance in many species including humans. Finally, the current iteration of the DFT disease vaccine uses DFT cells that have been stimulated with IFN-γ to upregulate MHC I prior to killing the cells and injecting with adjuvant. This treatment has proven effective at stimulating both humoral and cytotoxic responses against tumor cells and has provided short-term protection against tumor challenge . The resAF performed all lab experiments, developed proteins and antibodies, analyzed data, prepared the figures, and wrote the manuscript. AL, LC, JM, and AP provided intellectual input and revised the manuscript. GW provided intellectual input, laboratory support, and contributed to writing and revision of the manuscript. JH provided intellectual input, laboratory support, and contributed to the revision of the manuscript. GK reviewed the histopathology slides and contributed to writing and revising 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.


GFET channels were functionalised with a linker molecule to an immobile anti-hCG antibody on the surface of graphene. The binding reaction of the antibody with varying concentration levels of hCG antigen demonstrated the limit of detection of the GFET sensors to be below 1 pg/mL using four-probe electrical measurements. We also show that annealing can significantly improve the carrier transport properties of GFETs and shift the Dirac point (Fermi level) with reduced p-doping in back-gated measurements. The developed GFET biosensors are generic and could find applications in a broad range of medical diagnostics in addition to cancer, such as neurodegenerative (Alzheimer’s and Parkinson’s) and cardiovascular disorders.We report on the development of label-free chemical vapour deposition (CVD) graphene field effect transistor (GFET) immunosensors for the sensitive detection of Human Chorionic Gonadotropin (hCG), a glycoprotein risk biomarker of certain cancers. The GFET sensors were fabricated on Si/SiO In particular, its chemical stability, low electrical noise, significant surface-to-volume ratio, biocompatibility and field-effect make graphene potentially an ideal platform for a variety of biosensing applications [Since the discovery and isolation of graphene from graphite in 2004, the two-dimensional material has shown potential to revolutionise many technological areas owing to its remarkable electrical, optical, mechanical and thermal properties ,2,3. Graications . Whilst ications  and otheHere we report on the chemical vapour deposition (CVD) graphene field-effect transistor (GFET) biosensors aimed at detecting the presence of Human Chorionic Gonadotropin (hCG) protein biomarkers in samples from patients at different stages of disease progression. The proof-of-concept GFETs were functionalised with monoclonal anti-hCG antibodies for the detection of hCG protein biomarkers, label-free. hCG is a glycoprotein hormone 37 kDa) and its concentration level serves as a clinical marker associated with testicular, pancreatic and prostate cancer  kDa and ,9, as we2 wafer was purchased from Graphenea . The required chemicals applied for GFET fabrication were photoresist (PR), lift-off resist (LoR), Microposit developer and Microposit remover, all purchased from A-Gas Electronic Materials .Monolayer Graphene on a Si/SiOThe hCG antigen and complementary anti-hCG monoclonal antibody were purchased from Abcam . The stocks were stored at −20 °C. Linker molecule 1-pyrenebutanoic acid succinimidyl ester (Pyr-NHS), bovine serum albumin (BSA) blocking solution andPhosphate-Buffered Saline (PBS) tablets were purchased from Sigma Aldrich .. Current–voltage measurements (ID–VD and ID–VG) were acquired as a function of gate voltage (VG) from −100 V to +100 V with ID–VD curves from −100 mV to +100 mV. The ID–VD and ID–VG curves were measured for each separate functionalisation stage.Electrical characterisation and analysis were implemented under ambient conditions via a Keysight B1500A semiconductor device parameter analyser interfaced to aMPS150 probe station Graphene characterisation was performed using an XPLORA Raman spectroscopy system . The XPLORA system operated at a wavelength of 532 nm, with ~4 mW of incident power. The XPLORA Raman system was combined with an OLYMPUS BX41 microscope .To define and develop the necessary graphene channels and metallic contacts of Chromium (Cr) and Gold (Au), the Mask aligner J500/VIS  was deployed. A conventional fan oven  was employed to perform necessary annealing processes, as well as for pre- and post-baking stages during the photolithography processes. The Hotplate SH8  and Ultrasonic Cleaner  were used for pre- and post-baking procedures and PR removal, respectively. An Argon (Ar) plasma etching technique was used for graphene channel formation by means of a three-target, 6-inch sputtering machine . To aid in the formation of robust, metallic electrodes, an Edwards Thermal Evaporator was deployed for the evaporation of Cr.2 substrate. A conventional photolithographic patterning and metal lift-off technique was used to form the necessary graphene channels, as well as the source, drain and voltage sense electrodes. The fabrication process involved spin-coating the CVD graphene samples with Lift-off Resist (LoR) at 3000 revolutions-per-minute (RPM) for a few seconds. The samples were then placed in a fan oven for pre-baking. The purpose of the pre-bake was to densify the LoR to assist with the formation of clear, graphene channels. Subsequently, the samples were dried at ambient temperature. Next, the samples were spin-coated with a layer of positive Photoresist (PR). Immediately after PR deposition, the samples were post-baked on a hotplate. The hotplate solidifies the PR and removes any solvents remaining on the samples. This process generated ~500 nm of PR film on the surface of graphene. Successively, the coated samples were positioned in a mask aligner and exposed to ultra-violet (UV) radiation for lithographic patterning of graphene channels. The UV exposed samples were also submerged in a chemical developer before being placed in a vacuum chamber to dry. Alternatively, the samples can be left to dry at ambient temperature, outside of the vacuum chamber. An additional post-baking stage was applied to the samples following the drying and placed on a hotplate under an intermittent deep ultraviolet source (DUV). This exposure to DUV can reduce the quantity of PR/PMMA residue on the substrate surface prior to plasma etching, whilst reducing contact resistance.The GFETs were fabricated using high quality CVD graphene to form transducer channels on Si/SiOThe chemically developed and dried samples were transferred into a sputtering machine and exposed to Ar plasma etching for a few minutes. Throughout the process, the inert Ar gas functions as the gaseous etchant and removes the exposed regions of graphene, whilst leaving behind the shaped graphene channels (previously coated and protected with PR). Graphene shaping is effectively complete after an etching time of a few minutes at high power. The sputtering machine was also used to sputter and form the associated metallic electrodes, 30 nm of Au. Following successful shaping, the samples were transferred to a chemical remover to dissolve the LoR and PR remaining on the substrate to expose the graphene channels. The samples were immersed in the chemical remover, before being placed in an ultrasonic bath for ~2 h at ~60 °C. The samples were subsequently rinsed and bathed in de-ionised water and transferred into a vacuum chamber to dry for few hours.For the final stage of GFET fabrication, metallic Cr and Au electrodes were formed on the graphene channels. The samples were post-baked in a fan oven, instead of being irradiated with DUV. This post-baking stage is necessary to remove the solvents located within the PR and eliminate any additional water. Following photolithography, thermal evaporation of Cr was performed using an Edwards Thermal Evaporator for a few seconds. The Cr functions as an adhesive layer between graphene and the Au metallic contacts. The Cr target material was heated to ~2000 °C and evaporated within a high vacuum. The heated coil of Cr is applied to form a thin film of ~5 nm on the surface of the graphene samples. The metallic contacts are fully formed after sputtering 30 nm of Au on the Cr film. After the Au sputtering process, the initial chemical remover step is repeated and the final samples are dried in a vacuum chamber for a few hours, prior to electrical characterisation.2) in total, i.e., five GFET devices consisted of five asymmetric Cr/Au voltage sense electrodes with an associated graphene channel length of 720 μm, whereas the remaining ten GFET devices consisted of six symmetric voltage sense electrodes with a channel length of 300 μm, as shown in For this study, a number of CVD graphene samples were fabricated. Each sample consisted of fifteen GFET devices (with an area of ~1 cm2 samples) confirmed that the majority of the regions consisted of mono-layer graphene.Raman spectroscopy was performed on two representative CVD samples to analyse the quality of graphene and to estimate various properties, such as number of layers , doping D–VD characteristics of the fifteen devices on each 1 cm2 sample. A comparison between 2-probe, 4-probe and back-gated data enabled us to extract the contact resistance, mobility and sheet resistance of the graphene channels within the GFET devices. A typical 4-probe ID–VD and ID–VG measurement from a GFET sensor, using a Keysight parameter analyser interfaced to a Cascade probe station, is shown in D–VD data shows approximate linear response over the ±60 mV range and a slight non-linearity beyond this range probably due to Schottky contacts or charge traps at the interface with the Si/SiO2 substrate [D–VG data which shows a characteristic hysteretic behavior due to the presence of charge traps at the interface. The Dirac point is found to vary from 50 to 80 V depending on the voltage sweep and its positive magnitude indicates that the graphene channel is p-doped (at VD = 100 mV). The 4-probe resistance of the graphene channel is found to be ~700 Ω , whereas the mobility is approximately 1200 cm2/Vs.The GFETs were characterised using 2-probe, 4-probe and back-gated measurements under ambient conditions. The bare GFETs  were first measured to extract the Iubstrate . This isD–VG curve near its vicinity, a number of properties of the channel can be estimated, such as mobility, doping, defects etc.We have also explored the effect of annealing on our devices in order to improve the overall performance of the bare GFET sensors. 2) of the Ab without requiring a high pH [2 lattice. This type of immobilisation typically leads to a self-assembled monolayer (SAM) of molecules on the surface of graphene, although the Abs may remain randomly orientated.To facilitate antibody (Ab) immobilisation, we used a linker molecule Pyr-NHS ester. The linker retains a succinimidyl ester group, which interacts with the amino groups on the anti-hCG Ab surface to generate a stable, amide bond. In addition, the Pyr-NHS has an aromatic pyrenyl group, which can strongly interact with the graphene surface, via a non-covalent π–π stacking. Non-covalent binding is dependent on physical adsorption of the biomolecule, using hydrophobic, hydrophilic, electrostatic and van der Waals interactions. Thus, Pyr-NHS ester causes the pyrene group at one end of the linker to strongly bind to the graphene surface via π–π interactions and the succinimidyl ester group will covalently react with the amino group  and probe tips from Nano World, Neuchâtel, Switzerland, Multiple AFM images were generated in respect to the various necessary functionalisation steps. The difference in height and morphology at the hCG functionalisation stage is displayed in An SEM image (using a JOEL JSM-7001F) in 2 ~ 0.8. The corresponding sensitivity of the GFET biosensors is found to be ~(61.8 ± 16.7)%/decade of hCG concentration over the 1 pg/mL to 1 ng/mL range. This represents a significant sensitivity level for hCG antigen label-free detection. However, it is worth highlighting that our GFET biosensors have not yet been optimised for numerous parameters (mobility etc.) to achieve even higher sensitivity levels and lower detection limits. These aspects are currently being explored and will be reported on in subsequent future publications. The fabricated GFET sensors were characterised using 4-probe electrical measurements at each stage of the functionalisation and binding interactions of the anti-hCG antibody with hCG antigen at varying concentration levels of the latter. All measurements were performed in ambient conditions. The data in 2) leading to hysteretic effects. Therefore, a robust comparison of the resistive with back-gated GFET sensors will require careful device design and optimisation. The CVD graphene FET biosensors have been characterised at all stages of the fabrication process  and functionalisation  to establish a robust and reproducible process for the development of the proof-of-concept sensors. Our data presented in this study has demonstrated that CVD graphene FETs can sensitively detect hCG antigen with approximately three to four orders of magnitude lower concentration level compared to commercially available sensors. In addition, the GFET sensors can be operated in a resistive or back-gated mode and in the future we aim to compare the sensitivity of these two approaches. However, the data in Annealing of the GFET biosensors showed remarkable improvements in their electrical performance, in terms of enhanced carrier transport properties and Dirac point shift due to oxygen and water molecules being desorbed from the surface of graphene. Work is currently underway to compare annealed devices with those that have been fabricated in the same batch but not annealed. In addition, all the results reported in this study are based on direct current (DC) 4-probe resistance measurements. We therefore anticipate that further improvements in S/N can also be achieved using alternating current (AC) or radio-frequency (RF) detection techniques ,39.2 substrate as sensitive label-free immunosensors for the proof-of-concept detection of hCG antigen, with a LoD below ~1 pg/mL. Although characteristic current-voltage response curves of the GFETs have been established, the sign of the resistance change coefficient ΔR has a strong dependence on the potential environment of the graphene channels. In addition, we have also shown that annealing the GFET sensors can significantly improve their performance and potentially lead to even more sensitive devices. In the near future, the GFET biosensors will be explored for the detection of hCG biomarkers in case vs control samples from patients. Furthermore, in the next stage of development of the GFETs, we will also investigate the specificity of the sensors, optimisation of the quality of CVD graphene, contact resistance and novel AC and RF detection techniques to enhance the sensitivity of the biosensors.We have demonstrated the use of CVD graphene FETs on Si/SiO


Programmed cell death-ligand 1 (PD-L1) may be a useful molecule for targeted immunotherapy. Therefore, this meta-analysis aimed to investigate PD-L1 expression in breast cancer and its associations with clinicopathological factors and outcomes, which may help determine whether PD-L1 expression is a useful prognostic marker.The Medline Ovid, Cochrane, PubMed, Google Scholar, and Web of Knowledge databases were searched for studies that evaluated the prognostic or clinicopathological significance of PD-L1 expression in patients with breast cancer, and reported at least one survival-related outcome.Six studies that included 7877 cases were selected for the analysis. Higher PD-L1 expression in all cells was related to higher histological grade and lymph node metastasis. Higher PD-L1 expression in tumor cell was related to larger tumor size, estrogen receptor negativity, progesterone receptor negativity, human epidermal growth factor type-2 positivity, and triple-negative breast cancer. PD-L1 positivity in all cells was associated with poorer disease-free survival, although it was not significantly associated with overall survival.The present meta-analysis revealed that cases of breast cancer with PD-L1 positivity in all cells exhibited higher histological grades, lymph node metastasis, and poorer disease-free survival. Therefore, positive expression of PD-L1 may be a useful prognostic marker in breast cancer.The online version of this article (10.1186/s12885-017-3670-1) contains supplementary material, which is available to authorized users. Breast cancer is the most prevalent cancer among women, and is the second leading cause of cancer-related deaths. Molecular alterations are known to affect cancer occurrence and metastasis, which has led to the development of hormonal therapy that targets the estrogen receptor (ER), progesterone receptor (PR), or human epidermal growth factor type 2 (HER-2). However, up to 20% of patients with breast cancer experience disease progression and death, which highlights the need for more effective therapy .The efficacy of immunotherapy is clear for immunogenic tumors, such as malignant melanoma, non-small cell lung cancer, and urothelial carcinoma. Furthermore, programmed cell death protein-1 (PD-1) and programmed cell death-ligand 1 (PD-L1) may be useful molecules for targeted immunotherapy. PD-1 is a co-inhibitory receptor that belongs to the CD28/CTLA-4 family, and serves as a negative regulator of the immune system by inhibiting the function of T-cells in local tissues . PD-L1 , and found that PD-L1 positivity was associated with poorer overall survival (OS), compared to PD-L1 negativity . HoweverOn April 1, 2016, we searched several international databases  using the following terms: ‘breast cancer or breast carcinoma’, ‘PD-L1 or B7-H1’, and ‘prognosis’. Two independent researchers (JSK and HMK) reviewed the search results. The inclusion criteria were: (1) studies that evaluated the prognostic or clinicopathological significance of PD-L1 expression in patients with breast cancer, and reported at least one survival-related outcome ; (2) studies that used an anti-PD-L1 antibody for the immunohistochemistry; and (3) the specimens were obtained using core needle biopsy or from the postoperative specimen. The exclusion criteria were: (1) studies that included patients who had received neoadjuvant chemotherapy; (2) studies that included <50 cases; and (3) studies that were not published in English. The whole text was reviewed when the report fulfilled the inclusion criteria. In cases of disagreement, the reviewers discussed the report and tried to reach a consensus. A third researcher was consulted to provide a final opinion in cases where a consensus could not be reached.Data extraction was performed according to the Cochrane guidelines. The following variables were extracted for the present meta-analysis: first author’s name, publication year, patients’ nationality, number of patients, trial design, mean age, clinicopathological parameters, PD-L1 positivity, study end-points (DFS and/or OS), and hazard ratios (HR) and 95% confidence intervals (CI). All included studies indicated that written informed consent had been obtained from the included patients.p-value from the Q statistic was <0.1 and a fixed-effect model was used if the p-value was ≥0.1. The I2 value was also used to evaluate heterogeneity; I2 is defined as 100% × ([Q – df] / Q), and ranges between 0% (minor heterogeneity) to 100% (severe heterogeneity), where df = (the number of studies – 1). The standard cut-off values for I2 are 25% (low), 50% (moderate), and 75% (high) [http://www.r-project.org).Q statistics from the chi-square test were used to evaluate the presence of heterogeneity. However, as Q statistics are not very powerful for evaluating heterogeneity, a higher significance level is used to compensate for the low power of the test . The stu% (high) , 19. For% (high) . All anaThirty-two studies were identified from literature search and 17 studies were excluded after title and abstract reviewed. Nine studies were excluded for not meeting the inclusion criteria. Finally, this meta-analysis included 6 studies and 7877 cases –16 . Four studies evaluated PD-L1 expression in tumor cells, 1 study evaluated immune cells (lymphocytes), and one study evaluated both tumor and immune cells. The positive cut-off values for the immunohistochemistry varied between the studies, with some studies evaluating the proportion of cells with positive staining, and other studies using the H-score and Allred score to evaluate both staining intensity and staining percentage.The included studies evaluated various clinicopathological parameters, such as tumor size (≤2 cm vs. >2 cm), histological grade (1–2 vs. 3), lymph node metastasis, ER status, PR status, HER-2 status, Ki-67 labeling index, and molecular subtype (non-TNBC vs. TNBC). The studies all evaluated different cell populations for positive PD-L1 expression. Therefore, we analyzed PD-L1 positivity in all cells (tumor and immune cells) and in only tumor cells.p < 0.001; Fig. 2 = 0%, p = 0.53). The pooled RR for lymph node metastasis was 1.68 . Histological grade, lymph node metastasis, and Ki-67 labeling index were not significantly associated with PD-L1 expression in only tumor cells.Higher PD-L1 expression in only tumor cells was associated with larger tumor size . The combined HR for OS was 1.908 . When we re-performed the analysis after excluding the study by Baptista et al. [p < 0.001) and significant heterogeneity was detected in the included studies , although PD-L1 positivity now exhibited a significant association with poorer OS  and the appearance of the funnel plot revealed that publication bias existed , immune cells , blood vessel cells, fibroblasts, myofibroblasts, mesenchymal stem cells, adipocytes, and the extracellular matrix. This information has led to the development of immunotherapy as an option for cancer treatment. In this context, PD-1 and PD-L1 play roles in a typical immune pathway, and PD-L1 is expressed in 20–70% of patients with lung cancer , 21–24, Several studies have evaluated PD-L1 expression in patients with breast cancer, although their conflicting results necessitated a meta-analysis. Therefore, the present meta-analysis aimed to evaluate the clinicopathological and prognostic significance of PD-L1 expression in breast cancer. Our results revealed that higher histological grade and lymph node metastasis were associated with higher PD-L1 expression in tumor and immune cells, and that PD-L1 expression in only tumor cells was associated with larger tumor size, higher histological grade, ER negativity, PR negativity, HER-2 negativity, and TNBC. Previous studies have referred to the relationship between higher histological grade, lymph node metastasis, larger tumor size, and PD-L1 positivity as the ‘immune escape’ phenomenon. In this context, cancer cells often express tumor antigens that are identified by the host immune system, which results in clearance. However, an insufficient immune response reduces the anti-tumor reaction in most cases (the immune escape) , 28, 29.p < 0.001) with significant heterogeneity in the included studies . Thus, it remains possible that PD-L1 positivity is associated with poorer OS . Nevertheless, our findings should be interpreted with caution, based on their inherent limitations. First, there was strong publication bias among the included studies. This may have been caused by the heterogeneity of clinicopathologic characteristics, such as race, age, molecular genetic entities and tumor size, which resulted in a smaller effect in the meta-analysis. Second, as the clone and the manufacturer of the PD-L1 antibody that was used among the studies were different, this might have affected in different staining patterns and sensitivity. In particular, most studies included in this meta-analysis used rabbit anti-PD-L1 polyclonal antibodies . Compared to monoclonal antibodies, polyclonal antibodies have limitations that they could often show unspecific binding, high background staining and lack of reproducibility. Therefore, the difference in antibodies that were used might have influenced in the result of this study. Third, the cell components that were evaluated for PD-L1 staining and the thresholds that were used in the interpretation of PD-L1 positivity were different. Therefore, future studies are needed to prospectively evaluate a large group of patients using a standardized assessment of PD-L1 staining, which may help validate our findings.Our meta-analysis revealed that PD-L1 positivity in tumor or immune cells from patients with breast cancer was significantly associated with higher histological grade, lymph node metastasis, and poorer DFS. Therefore, positive PD-L1 expression may be useful for predicting prognosis among patients with breast cancer.


The prognostic role of programmed death-ligand 1 (PD-L1) in sarcoma remains controversial. We performed a meta-analysis so as to investigate the impact of PD-L1 on clinicopathlogical findings and survival outcomes in sarcoma.A comprehensive search in PubMed, Embase and the Cochrane Library was conducted for relevant studies. The odds ratios or hazard ratios, at 95% confidence intervals were used as measures for investigation of the correlation between PD-L1 expression and clinicopathlogical features or survival outcomes.p = 0.005), as was its association with event-free survival in bone and soft-tissue sarcoma patients . Additionally, the expression of PD-L1 was positively correlated with the infiltration of programmed death 1 (PD-1) positive T-lymphocytes .Fourteen eligible studies comprising 868 patients were selected for analysis. Pooled hazard ratios indicated that the association of PD-L1 expression with overall survival in bone sarcoma (osteosarcoma and chondrosarcoma) patients was statistically significant (1.987, 95% CI: 1.224–3.224, Our meta-analysis indicated that high PD-L1 expression is likely to be a negative factor for patients with sarcomas and that it predicts worse survival outcomes. Sarcomas are malignant tumors that are characterized by a wide diversity of subtypes with various cytogenetic profiles. Despite major treatment breakthroughs, standard treatment modalities combining chemotherapy, radiotherapy and surgery have failed to improve overall survival . TogetheImmune escape is regarded as an important biological process for primary cancer growth and metastasis. Programmed death 1 (PD-1) and programmed death-ligand 1 (PD-L1) have proved to play a predominant role in cancer immune surveillance , 5. In sIn the present study, a meta-analysis was conducted to evaluate the association between PD-L1 expression and clinicopathological characteristics or survival outcomes in bone and soft-tissue sarcoma patients.For primary retrieval, a total of 342 citations were identified by searching through three electronic databases, including 64 citations in PubMed, 154 citations in Embase and 124 citations in Web of Science. After removing 122 duplicates, the remaining 220 records were screened during the initial filtering, in which 169 records were excluded after viewing the titles and abstracts. From the remaining 51 articles a further 38 were excluded after viewing the full-text, including 29 articles with insufficient data, two with overlapping patients, six articles that were not relevant and one bioinformatics analysis. Eventually, 13 manuscripts –22 contaAll studies that were included were published between 2013 and 2017, the number of patients in each study ranging from 13 to 137. The proportion of patients expressing PD-L1 ranged from 6% to 75%. The basic characteristics of the 14 eligible studies are summarized in Table To detect the expression of PD-L1, all studies used immunohistochemistry, except for one study , which u2 = 67.5%, p = 0.001), subgroup analysis was performed to clarify the source. In subgroup analysis stratified by histological subtype, the pooled hazard ratio (HR) estimate for overall survival was 1.987  in patients with bone sarcoma (osteosarcoma and chondrosarcoma) with no heterogeneity , and 1.625  in patients with soft-tissue sarcomas with high heterogeneity    for non-Asian patients, with low heterogeneity , and 2.033  for Asian patients with high heterogeneity   for studies of which the patients included were less than 50, with no heterogeneity   Figure . As for ) Figure . In the ) Figure . When st) Figure . To clar) Figure . As for ) Figure , but the) Figure .2 = 65.6%, p = 0.012) in the group, a random-effects model was used to assess it, with a pooled HR of 1.943 . In subgroup analyses, whether stratified by histological subtype, country or cut-off, heterogeneity was not excluded  for group of which patients were more than 50, with medium heterogeneity   with no heterogeneity , and the HR of pooled RFS/DFS/PFS was 1.164  with no heterogeneity   Figure . However) Figure . Overall2 = 45.8%, p = 0.100). Using a fixed-effects model, pooled OR was calculated to be 4.012 , indicating that PD-L1 expression was significantly associated with the infiltration of PD-1 positive lymphocytes , indicating that such bias was not present within the studies.Sensitivity analysis was conducted to evaluate the influence of a unique study on pooled HR. As shown in Figure Sarcoma is a rare heterogeneous disease with more than 70 different subtypes, accounting for 1% of all cancers diagnosed in the United States each year . MetastaThe interaction between PD-L1 in tumor cells and PD -1 in T-lymphocytes negatively regulates the tumor-killing function of T-lymphocytes and protects tumor cells from the host immune system. Recently, much attention has been paid to PD-L1 expression in various solid tumors, due to the FDA's approval for anti-PD-1 antibodies in non-small cell lung cancer and melanoma with good efficacy and safety. Several clinical trials have reported that therapies targeting PD-1 and its ligand (PD-L1) improve patient outcomes, while tumor response has been related to PD-L1 expression , 24. RecIn the current meta-analysis, we combined 14 studies related to prognosis and clinicopathology of PD-L1 expression in sarcoma patients. For all sarcoma patients, we found that the expression of PD-L1 was significantly associated with poor event-free survival. In contrast, PD-L1 expression was significantly associated with overall survival in bone sarcoma (osteosarcoma and chondrosarcoma) rather than soft tissue sarcoma. As for patient ethnicity, there was a significant association between PD-L1 expression and poor overall survival in non-Asian patients, but one should draw conclusions carefully, in consideration of bias caused by the cut-off. Moreover, PD-L1 expression predicts poor event-free survival in bone and soft tissue sarcoma.We found that PD-L1 expression was significantly associated with the infiltration of PD-1 positive T-lymphocytes, indicating that an adaptive immune resistance mechanism may be occurring . In thisSignificant heterogeneity was noted in some analyses in the current study. This could have arisen from various sources. Firstly, the methods of PD-L1 measurement varied among these studies. Although the most common method was IHC, they did not use the same antibody and the dilution was also different. Because the type and the concentration of antibody can affect the sensitivity of IHC, differences also exist in the definition of PD-L1 expression. Secondly, the region where PD-L1 was expressed was not clear. Limited studies distinguished whether the PD-L1-positive cell region was from the tumor cells or the tumor microenvironment , 19, 20,p values using Tierney's methods [Another potential source of bias may be linked to the method of calculation of the HRs. When the data from multivariate or univariate survival analyses were reported, we used them directly. If the HRs were not provided explicitly, we calculated them from outcome data available in the articles. If this was impracticable, we extrapolated them from the Kaplan-Meier curves or quoted  methods , which w methods , of whicPublication bias is another concern in all forms of meta-analysis because only positive results tend to be published in journals. To minimize publication bias, we attempted to conduct literature searches as completely as possible, using Web of Science, PubMed, Embase and the Cochrane Library. No significant publication bias was found in this meta-analysis, except for the analysis of tumor infiltration of PD-1 positive lymphocytes.Additionally, the definition of bone sarcoma and soft-tissue sarcoma may bring heterogeneity to the whole meta-analysis. In other papers, osteosarcoma, Ewing sarcoma and chondrosarcoma have been included in the category of bone sarcoma . HoweverTherefore, the results of the current meta-analysis should be interpreted with caution and should be confirmed in well-designed prospective studies with appropriate multivariate analyses.In conclusion, this meta-analysis demonstrated that PD-L1 expression may be an effective predictive factor of poor prognosis and clinicopathological features for bone and soft tissue sarcomas. Furthermore, PD-L1 expression could be used to identify a subgroup of patients who would potentially benefit from targeted therapy against PD-1 or PD-L1.A systematic literature search of PubMed, Embase and the Cochrane Library was undertaken with no language restrictions . The strategy used was to search for the following words in relevant literature:  AND (“CD274” OR “B7-H1” OR “PD-L1” OR “programmed cell death 1 ligand 1 protein”).Patients and studies had to fulfill the following criteria to be included in the analysis: (1) Patients with pathologically confirmed sarcoma who underwent detection of PD-L1 in their tumor tissue; (2) Studies evaluating the relationship between PD-L1 and clinical features and/or survival outcomes. Studies were excluded if they: (1) were case reports, reviews or letters; (2) comprised overlapping sarcoma patients; (3) had insufficient information such that the correlation of clinical features or survival outcomes could not be extracted. When there were multiple publications regarding the same group of patients, only the most recent publication was included. Two researchers  screened the titles and abstracts of all the searched articles and verified that the studies met the inclusion criteria for subsequent analysis.p values and events; and (4) other variables including the methods of quantitative PD-L1 measurement, the definition of positivity  and the antibody's type, source and dilution used for immunohistochemistry (IHC). Disagreements were settled by consultation.The final articles that were included were assessed independently by two researchers . Data extracted included: (1) basic information including first author, year of publication, study period, follow-up duration; (2) information about the patients and tumors, including patient source, number of patients, gender, age, number of patients with PD-L1 expression, histological type of tumor, tumor site, tumor size, grade at diagnosis, tumor-infiltrating lymphocytes; (3) outcome measures including metastasis, recurrence, survival data, Kaplan-Meier curves, http://www.ohri.ca/programs/clinical_epidemiology/nos_manual.pdf), assessment of quality mainly focused on selection , comparability, and the outcome (assessment and follow-up) of the original studies. Assessment was carried out independently by two researchers .In accordance with the Newcastle-Ottawa Scale (Q test and Higgins I2 statistic. A fixed-effects model (Mantel-Haenszel method) was used to calculate parameters when no obvious heterogeneity existed among studies (I2 > 50% suggested high heterogeneity). Otherwise, a random-effects model was utilized. Publication bias was measured using funnel plots and Egger's test. All statistical analyses were conducted using STATA version 12.0 For the quantitative aggregation of the survival results, hazard ratios (HRs) and their 95% confidence intervals (CIs) were combined to give the effect size. Additionally, the pooled ORs and their corresponding 95% CIs were utilized to quantitatively determine the association between PD-L1 and clinicopathological characteristics of patients. Statistical heterogeneity among studies was assessed using Cochran's