Chitosan (CHT) is a polysaccharide with multiple claimed properties and outstanding biocompatibility, generally attributed to the presence of protonable amino groups rendering a cationic natural polymer. However, the effect of changes in CHT structure due to hydration is not considered in its performance. This study compares the effects on biocompatibility after drying at 25 °C and 150 °C scaffolds of chitosan, polyethylene glycol diglycidyl ether (PEGDE) crosslinked CHT  and glutaraldehyde (GA) crosslinked CHT. PEGDE crosslinked CHT showed a reduction in free amino groups and the amide I/II ratio, which exhaustive drying reduced further. In X-ray diffraction (DRX) analysis, PEGDE crosslinked CHT showed multiple peaks, whereas the crystallinity percentage was reduced with an increase in PEGDE concentration and thermal treatments at 150 °C. In a direct contact cell assay, high osteoblast viability was achieved at low and medium PEDGE concentrations, which was improved when the crosslinked scaffolds were thermally treated at 150 °C. This was attributed to its partial hydrophilicity, low crystallinity and low surface roughness; this in spite of the small reduction in the amount of free amino groups on the surface induced during drying at 150 °C. Furthermore, PEGDE crosslinked CHT scaffolds showed strong vinculin and integrin 1β expression, which render them suitable for bone contact applications. Tissue repair makes use of different strategies, including scaffold-free, cell-free and traditional tissue engineering, where cells are seeded on biodegradable scaffolds in the presence of bioactive molecules while being stimulated in a bioreactor. Therefore, scaffolds are an important element for tissue repair, because they not only provide mechanical support, but also facilitate cell proliferation and differentiation through their porous structure. The materials used to produce scaffolds are natural or synthetic polymers, ceramics and even biodegradable metals . Among them, natural polymers have caught the attention of several researchers due to their diverse and sometimes unexpected properties, such as reactivity, strength and toughness, when blended with other polymers ,3.Chitosan (CHT) is the N-deacetylated derivative of chitin with a typical DA of less than 0.35. It is thus a copolymer composed of glucosamine and N-acetylglucosamine. The physical properties of CS depend on several parameters such as the molecular weight , DD (in the range of 50–95%), sequence of the amino and the acetamido groups and the purity of the product .CHT has multiple claimed properties, such as biocompatibility ,6,7, antThe content of amino groups, degree of deacetylation and molecular weight are the main reasons for the diverse physicochemical properties of chitosan . In termIn addition, it is known that CHT is a semicrystalline material with a polymorphic crystalline structure that can be transformed from its hydrated forms to an anhydrous or “annealed” form. This quality of the CHT was reported for the first time by Sakurai . Surface properties are determinants of CHT cellular response ,29 but iIn this study, we argued that not only the presence of amino groups on CHT are responsible for its biocompatibility, but also that the existence of other factors, such as changes in crystallinity and surface roughness, played a significant role in its cellular response. Therefore, we proposed the use of polyethylene glycol diglycidyl ether (PEGDE) as a crosslinking agent in order to improve CHT mechanical properties, and to assess their biocompatibility with osteoblasts due to amino groups involvement in the crosslinking reaction. Furthermore, we assessed the effect of a high temperature drying treatment on PEGDE crosslinked CHT to fully appreciate if these structural changes will jeopardize CHT biocompatibility. −1, , while J.T. Baker provided acetic acid . MTS CellTiter 96® Aqueous Non-Radioactive Cell Proliferation obtained from Promega  Dulbecco’s modified Eagle’s medium (DMEM) from Biowest , human osteoblast hFOB1.19 from ATCC® CRL-11372™ . Integrin β1 (A-4): sc-374429, vinculin (7F9) sc-73614 and antibody  were purchased from Santa Cruz Biotechnology .Chitosan (CHT) with viscosity molecular weight of 223,332 gmolmentally ) and a dTg, which depended on water content and heat treatment,  was mixed with 32 μL PMS (phenazine methosulfate) after thawing. The entire procedure was performed at 25 °C and protected from light. The MTS solutions was used immediately after preparation.Cell viability studies were done using CellTiter 963 cells/scaffolds in a 96-well plate. The seeded cells were incubated at 37 °C, 5% CO2 and 95% humidity for 48 h. For the direct contact assays were used samples of 10 mg of weight. Scaffolds were sterilized by UV for 15 min. Osteoblasts, obtained from human osteoblast ATCC hFOB1.19, were seeded at 2 × 10® syringe filter of 0.22 μm. For an indirect contact test, extracts replaced the culture medium in a 96 well plate with 2 × 103 cells/well. The seeded cells were incubated at 37 °C, 5% CO2 and 95% humidity for 48 h.Extracts were obtained according to the practical guide for the preparation of samples and reference materials, ISO 10993-12 . 25 mg oFor both techniques (direct and indirect) a positive control was included which comprised cells with only culture medium (C+), with a negative control (C−), with hydrogen peroxide and with the target with MTS solution, as well.® was added to each well. This was incubated for 3 h and the absorbance was measured in a multi-well spectrophotometer (Thermo Scientific™) at 490 nm.After 48 h, 20 μL of MTS CellTiter 963 cells/mL (osteoblast ATCC hFOB1.19), and incubated at 37 °C, 5% CO2 and 95% humidity for 48 h. The samples were rinsed with PBS, fixed with 4% formalin and blocked with 1% ABS tween. The first antibody, Integrin β1 (A-4): sc-374429 was added (50 µL per plate diluted with PBS-tween 1:150) and incubated at 4 °C overnight. After washing with PBS, the second antibody  and left for 2 h. The samples were observed in a Leica fluorescence microscope equipped with a digital camera, using the LAS AF software . The same procedure was carried out for vinculin, where the antibody (7F9 sc-73614), was used. All antibodies were provided by Santa Cruz Biotechnology .Cytoskeletal alterations were measured by the expression of integrin and vinculin adhesion proteins. To carry out these tests, extracts obtained from chitosan scaffolds crosslinked with PEGDE were used. A 24-well plate with 120 µL of each sample was seeded with 20 x 10−1 and 3000 cm−1 (peaks at 3360 cm−1 and 3285 cm−1) corresponding to the O–H (from chitosan and water) and vibration of N–H in N–H2 groups. At 2920 cm−1 and 2850 cm−1 absorptions corresponding to stretching C–H in C–H2  were observed. While the absorption band at 1650 cm−1 was associated to C=O vibration in the amide I N–H (acetamide) group, the band at 1560 cm−1 was attributed to C–N and N–H2 bending in amide II region. At 1422 cm−1 bands related to CH2 bending were observed. FTIR spectra of chitosan films dried at 25 °C and at 150 °C are shown in −1 an absorption which can be related to CH2 wagging appeared; and finally, at 1319 cm−1, the amide III appeared. Amide III is commonly used for calculating the degree of acetylation. Skeletal vibrations characteristic of the chitosan structure appears at 1150 cm−1 (C–O–C), 1080 cm−1 and 1030 cm−1 (pyranose ring). At 1380 cm−1 became composed of the amide I and the corresponding imine bond [−1), however amide II was of higher intensity than amide I absorption. The Fourier-transform infrared spectroscopy (FTIR) spectra of chitosan films crosslinked with PEGDE showed a higher intensity of amide I in comparison with amide II. This difference was even more marked as the amount of PEGDE increased. The amino groups, now at 1577 cm−1, are more reactive than hydroxyl groups, and can react with the epoxy ring leading a crosslinked structure while showing lower intensity at the highest concentration of PEGDE  was reduced significantly, in comparison with the band at 1560 cm−1 (amide II), which shifts on drying. During GA crosslinking, amide II was smaller than amide I, and the absorption at 1380 cm−1 remained unchanged, but at a lower intensity as to the band at 1420 cm−1. In PEGDE-crosslinked CHT, a higher intensity of the amide I was observed again, in comparison with amide II. This indicates that either these bands are not a good reference to estimate the degree of crosslinking, or that the crosslinking mechanism is different for GA rather than for PEGDE crosslinking. However, the broad O–H and N–H bands located at 3360 cm−1 were of higher intensity compared to the vibration of C–H located at 2922 cm−1 in contrast to the observed behavior in CHT alone and GA-crosslinked CHT. Finally, absorptions at 1380 cm−1 and 1315 cm−1 were of higher intensity compared to the band at 1418 cm−1.The infrared spectra of the cross-linked films dried at 150 °C are presented in −1 to 3445 cm−1, and the shifting of the amide I band from 1635 cm−1 to 1655 cm−1. Our study shows that that drying at 150 °C reduces O–H intensity . This outcome shows that interaction of adsorbed water molecules is diminished not only in the 3400–3200 cm−1 region, but also in the carbonyl region, which also implies that dehydration takes place not only at the surface level, but also in the bulk of chitosan. Regarding, the amide I band, there was no apparent shift; nevertheless, it exhibited a significant reduction in intensity. This agrees with Zawadzki et al. , in whicThermal treatment at 150 °C yields an insoluble chitosan scaffold when placed in acetic acid. After thermal treatment at 150 °C it is possible that some chemical reactions involving amino groups lead to the crosslinking of the chitosan scaffold, for example oxidation of alcohols render aldehydes, which in turn reacted with the amino groups, leading to imine formation. This explain why the percentage of the amino groups quantified at 150 °C was very low. These results should be taken with caution, as it is possible that acetic acid was either not completely eliminated, not only from dissolution and film preparation, but also from purification of the raw product during manufacture; or because commercial chitosan employed was in partially protonated form. In addition, Balázs  mentionsThe binding and growth of cells in chitosan substrates has been attributed to the cationic nature of chitosan amine groups. Data suggest that, as the level of deacetylation is raising, the positive charge density of the chitosan increases; consequently, there is an increase in the attraction of negatively-charged cells. Factors such as the degree of deacetylation and chemical modifications influence the hydrophilic properties of chitosan films and their interactions with the biological environment ,41. ThesThermal stability can be evaluated as the onset temperature of the degradation step, the maximum of the decomposition temperature and the temperature corresponding to the 50% weight lost. In this study we use the maximum temperature and the 50% weight lost to compare the thermal stability.It is observed that the previously dried CHT at 150 °C shows a lower weight loss as a result of the drying process that removes part of the adsorbed water. However, the weight loss related to water above 100 °C is due to hydrogen bonds formation between functional groups in CHT molecules and water . In the The greater weight loss for CHT dried at 150 °C, compared to the dried at 25 °C, could be due to the fact that the second stage of chitosan degradation began around 150 °C , so the In relation to CHT crosslinked with PEGDE dried at 25 °C, a decrease in weight loss corresponding to adsorbed water is observed in comparison with pure chitosan. However, at higher PEGDE concentrations, adsorbed water remains the same. This could be associated with crosslinking between chitosan and PEGDE, which would decrease the absorption of water in the material . AdditioLikewise, more water was present in samples dried at 25 °C than in those treated at 150 °C. However, the mass loss in the second stage of degradation was similar. For CHT dried at low temperatures, the amount of retained water was higher in GA crosslinked CHT than in PEGDE crosslinked CHT, demonstrating its higher efficiency as a crosslinker for rendering a low water absorbing hydrogel. For pristine CHT, water is lost in the first stage; whereas, in the second stage, chitosan main chain degradation (depolymerization) and the elimination of volatile products, from pyranose ring destruction and deamination, is expected as reported by Zawadski et al. . NonetheFinally, for all CHT samples crosslinked with PEGDE and dried at 150 °C, it is generally observed that they exhibited better thermal stability than pure chitosan and chitosan crosslinked with PEGDE at 25 °C. This could indicate that the drying treatment at 150 °C promotes a better crosslinking between the chitosan and the PEGDE, which renders samples with higher thermal stability. In other words, CHT-PEGDE dried at 150 °C exhibited a smaller loss of mass (53–54%) at interval of 300–700 °C compared to CHT (72%). In the same way, all samples of CHT-PEGDE dried at 150 °C revealed a smaller loss of mass than CHT-PEGDE at 25 °C (58–70%).Petrova et al. reported the lost solubility of their samples, attributed to the formation of ionic crosslinks upon dehydration ; we obseThis highly ordered structure arises from the hydroxyl and amino groups which can form strong intermolecular and intramolecular hydrogen bonds. This structure was retained with a slight degree of crosslinking (CHT PEGDE1), but giving rise to additional peaks at 2θ = 5.6° and 2θ = 15° in more crosslinked samples (CHT PEGDE2 and CHT PEGDE3). Baroudi et al. attribute the 2θ = 15° to a hydrated chitosan polymorph crystal as a complex with water and acid .When GA was used as the crosslinking agent, the amorphous halo became more evident (peak at 2θ = 19.7°) which indicates the loss of the crystalline structure. Regarding the slightly PEGDE crosslinked CHT, while the peak at 2θ = 20.1° tended to disappear, the peak at 2θ = 9.58° not only reduced but shifted to lower angles—this outcome suggests an increase in chain separation. Interestingly, CHT PEGDE2 showed multiple peaks  denoting a heterogeneous structure; contrary to CHT PEGDE3, a more crosslinked sample, which clearly showed only two reflections at 2θ = 9.68° and 2θ = 20.2°.I) of each of the films. The table shows how crystallinity decreased with the degree of crosslinking and with drying at 150 °C.There are reports that suggested thermal treatments on chitosan can change its physical properties, affecting not only its appearance but also its aqueous solubility and rheology ,51,52. OIt has been reported that the anhydrous chitosan produced through exhaustive dehydration processes has an identical crystal structure to hydrated chitosan. This is, characterized by having an orthorhombic unit cell with a = 7.76 Å, b = 10.91 Å and c = 10.30 Å  as well as a = 4.4 Å, b = 10.0 Å and c = 10.3 Å . However, instead of containing four chains of chitosan with eight water molecules, only two chains were involved .I), as the water can easily penetrate amorphous regions. This was not observed, as Td1 was 15% when the sample was dried at 25 °C, and only 9% when the sample was dried at 150 °C for the pristine chitosan. This can be explained by the used formula and the presence of multiple peaks in the diffractogram. It was expected that more water was present in samples with a lower crystalline index , but only one peak at 399.5 eV was observed, and broadens during its drying at 150 °C.The XPS survey spectra of chitosan films dried at 25 °C and 150 °C are shown in GA crosslinked CHT exhibited three types of carbons at 25 °C, but they tend to disappear with drying at 150 °C, i.e., the C1s band broadens. GA crosslinked CHT showed also a symmetrical O1s peak for films dried at 25 °C and 150 °C. Finally, N1s become broader as thermal treatment increased; however, three peaks  at low temperature drying were observed. This is a clear evidence of the crosslinking reaction.In PEGDE crosslinked samples, three types of C1s were observed, but when PEGDE at medium and high concentration was used, C–O (286.4 eV) were clearer (more at.% contribution) in samples treated at 25 °C; nevertheless, it regained its original intensity when dried at 150 °C. PEGDE crosslinked CHT showed also a symmetrical O1s peak for films dried at 25 °C and 150 °C. For the case of N1s, no visible changes were observed at low concentrations (PEGDE1), at both drying conditions, but N1s became either broader or with two broad peaks (399 eV and 402.5 eV) at the higher concentration (PEGDE 3) when dried at 25 °C. Ra) of the films dried at 25 °C  are shown in Polar groups introduced by different procedures tend to be buried in bulk when they enter in contact with air for a prolonged period; however, they remain on the surface when they are in contact with water or any other polar environment . In our These results agree with oxygen surface composition obtained by EDX, as it was clear that oxygen-containing groups were at the surface rendering a more hydrophilic surface. Zhang et al.  report sIn general, surface wettability is strongly associated, not only with surface contact angle, but also with surface free energy, and it is affected by diverse aspects such as surface elemental composition and roughness. Wettability is perhaps the most important factor to determine the quantity and quality of adsorbed proteins, which in turn affects cell adhesion . Yinghui et al.  noted thChitosan scaffolds must be able to support cell adhesion, and the growth and development of the extracellular matrix, as well. Cell adhesion and proliferation in chitosan substrates has been attributed to the cationic nature of chitosan amine groups .This means that high cell viability in direct contact was achieved in pristine semicrystalline (CrI = 64.1%) CHT dried at 25 °C with 72.5% of free amino groups, Ra = 45.8 ± 4.6 and 72° of DMEM contact angle and in semicrystalline (CrI = 51.3%) PEGDE1 crosslinked CHT dried at 150 °C with 55.3% of free amino groups, Ra = 28.2 ± 7.9 and 57° of DMEM contact angle. This also means that, for a lower amount of amino groups, a higher hydrophilicity renders higher viability in the presence of a less semicrystalline structure. GA crosslinked CHT dried at 25 °C exhibited the lowest viability in direct contact with 51.5% of free amino groups, Ra = 26.3 ± 6.3 and a DMEM contact angle of 56°. Even when the contact angle is lower than pristine CHT, less amino groups are available, resulting in a lower viability. Furthermore, glutaraldehyde is considered as a toxic crosslinking agent that rendered an amorphous (CrI = 21.5%) and smooth CHT surface allowing little cell attachment.The distribution of the cells in scaffolds dried at 25 °C and 150 °C was observed after 48 h of incubation by either direct or indirect contact . A betteJayakumar et al.  evaluateVinculin is a cytoskeletal protein associated with cell-cell and cell-matrix junctions, and also to cell-biomaterial interactions. The ability to generate and transmit forces depends on the strength of the connection between the integrin adhesion receptors and the actomyosin cytoskeleton mediated by focal adhesion proteins. Vinculin connects the actomyosin cytoskeleton with β integrin subunits through binding to talin, or with the α4 and α9 integrin subunits through binding to paxillin, respectively. In the adhesion test, using the integrin antibody the obtained images are shown in The characteristics of the polymer surface could affect the amounts and types of bound proteins, as well as the conformation, orientation, or binding strength of the adsorbed protein. Zhang et al.  reports Although all PEGDE crosslinked CHT scaffolds support cell adherence, it is worth bearing in mind that that cell adhesion, as demonstrated in this study, is not indicative of how supportive a substrate is to cell spreading, and that cell spreading does not correlate with focal contact formation .The results obtained demonstrate that there are several chemical and structural differences between CHT scaffolds dried at 25 °C and 150 °C, as shown by various techniques. FTIR showed that amide II/amide I ratio changed as the drying temperature was increased in pristine CHT and GA crosslinked CHT, and that the formation of a more amorphous polymer or with reduced crystallinity was observed as demonstrated by XRD. In addition, the decomposition temperature (Td2) tends to increase as demonstrated by TGA. When the scaffolds were crosslinked with PEGDE at medium and high concentrations, a similar behavior was observed, i.e., a reduction in free amino groups, amine I/II ratio and crystallinity. However, osteoblast viability and adhesion was promoted by PEGDE crosslinked CHT at low and medium concentrations, mainly due to its hydrophilicity (low contact angles due to the presence of oxygen containing species), its low crystallinity and its low surface roughness, in spite of the small reduction in the amount of free amino groups on the surface induced during drying at 150 °C. Finally, these results suggest that PEGDE at low concentrations is a suitable biocompatible crosslinker agent for chitosan; and, consequently, these materials are can be used in bone contact applications


The significance of epithelial‐mesenchymal transition (EMT) and immune checkpoint proteins in thymic carcinoma remains unknown. We examined the clinical significance of EMT, tumor‐infiltrating lymphocytes expressing the immune checkpoint protein, programmed cell death 1 (PD‐1 + TILs), and the expression of PD‐1 ligand 1 (PD‐L1) in thymic carcinoma (TC). We also investigated the relationships between these immune checkpoint proteins and the EMT status and examined the impact of induction chemotherapy on patients with tumors that express these proteins.The relationship between PD‐1 + TILs/PD‐L1 and clinicopathological findings including EMT was investigated by immunohistochemistry (IHC) of surgically resected samples from 43 patients with TC. In 15 patients receiving induction therapy (IT), those factors were compared before and after IT.P = 0.0095, PD‐1; P = 0.001, PD‐L1; P = 0.0037). We found a significant relationship between PD‐L1 and EMT status (P = 0.01). In patients who received IT, PD‐L1 increased, and the change was strongly correlated with EMT status (P = 0.01).With IHC, 26 cases (60.5%) were positive for PD‐L1, and 19 cases were positive for PD‐1 + TILs (44.2%). The disease‐free survival rate in patients showing EMT and who were PD‐1/PD‐L1 positive was significantly worse compared to negative cases (EMT; Epithelial‐mesenchymal transition, PD‐L1, and PD‐1 + TILs have prognostic impact, and PD‐L1 is correlated with EMT status. PD‐L1 expression after IT was significantly higher compared to before IT and was correlated with the EMT change. Thus, PD‐L1 may be upregulated during EMT, and anti‐PD‐1/PD‐L1 immunotherapy may provide reliable treatment of TC in combination with chemotherapy. Because of the small number of cases, the biological and oncological characteristics of TC are poorly understood, and no standard treatment strategy has been established. Surgery, chemotherapy, and radiation therapy are performed to treat TC. Surgery is often the first treatment in clinical practice when complete resection appears possible according to preoperative imaging. However, tumor invasion of surrounding tissues and regional lymph node metastasis frequently occur. In patients with advanced clinical stages, multimodal therapy such as induction chemotherapy or chemoradiotherapy (followed by surgery) is performed. Despite those treatments, the recurrence rate and mortality rate remain high.The pathway involving the immune checkpoint proteins, programmed cell death 1 (PD‐1) and PD‐1 ligand 1 (PD‐L1), plays an important role in cancer progression and the cancer microenvironment.Epithelial‐mesenchymal transition (EMT) is a key process in cancer progression and distant metastasis inseveral malignancies including non‐small cell lung cancer (NSCLC).22.1This study was a retrospective analysis of 43 patients with TC whose tumors were completely resected between 1996 and 2017 at Osaka University Hospital . The Institutional Review Board approved this study, and written informed consent for the study and surgery was obtained from each patient. A computed tomography (CT) scan was routinely performed every 6 months as follow‐up.2.2Using clinical samples obtained from enrolled patients with TC, we performed IHC with several antibodies that recognize cancer‐associated proteins, including c‐kit (TC marker), PD‐1/PD‐L1 (immune checkpoint proteins), and E‐cad, N‐cad, and TGF‐β (EMT‐associated proteins). The primary antibodies used in this study are listed in the Data Immunohistochemistry staining for EMT evaluations was performed as in previous reports.PD‐L1 expression was evaluated as the percentage of cells staining positive for PD‐L1 , and PD‐L1 positive was defined as TPS ≥50%. In addition, we defined cases with an increase in PD‐L1 TPS after IT as PD‐L1 change‐positive by comparing PD‐L1 IHC results before and after IT. PD‐1 IHC was evaluated as the extent of TILs after hematoxylin‐eosin staining and PD‐1‐positive TILs (PD‐1 + TILs) using a visually estimated four‐point scale: 0 (absent), 1 (<30%), 2 (30%‐60%), and 3 (>60%). Samples with a score of 0 or 1 were considered negative, and those with a score of 2 or 3 were considered positive.2.3The correlations between clinicopathological factors and variables  were evaluated by Fisher's exact test. Categorical variables including sex, Masaoka stage, and histopathology and continuous variables including tumor size were evaluated with analysis of variance (ANOVA). The histological response to induction therapy was classified into four categories as follows: Ef0: no histological response, Ef1: more than one‐third of the tumor cells viable, Ef2: less than one‐third of the tumor cells viable, Ef3: no viable tumor cells according to General Rule for Clinical and Pathological Record of Lung Cancer.P values <0.05 were considered to be statistically significant.Disease‐free survival (DFS) and overall survival (OS) were analyzed with the Kaplan‐Meier method, and the log‐rank test was used to compare the survival distributions of subgroups. DFS was defined as the time from the data of surgery to the first event of either disease recurrence or death due to any cause. OS was defined as the time from the date of surgery to death due to any cause. The correlation between PD‐L1 IHC status (TPS) and variance was analyzed with ANOVA. The univariate Cox proportional hazards regression model was used to analyze DFS. All statistical analyses were performed with JMP version 13 for Windows . 33.1The median follow‐up time was 4.3 years : 3.0‐5.5 years). Patient characteristics are listed in Table 3.2P = 0.0037, and Figure P = 0.02). In addition, Kaplan‐Meier analysis showed that PD‐L1‐positive TPS patients had a significantly worse DFS rate compared to PD‐L1‐negative patients . A significant relationship between PD‐L1 expression and OS was also found . The relationships between PD‐L1 expression and clinicopathological factors are shown in Table We analyzed the clinical implication of PD‐L1 expression in TC. Figure P = 0.0056). We found no significant relationship for OS regarding PD‐1 expression by TILs . Univariate Cox proportional hazards regression models for estimating DFS showed that the presence of PD‐1 + TILs was one of the prognostic factors . No significant relationship was found between EMT‐positive and EMT‐negative patients for OS . Univariate analyses of DFS also showed that positive EMT status was a prognostic factor (Table P < 0.01). These results suggest that EMT is a potential prognostic factor for recurrence after surgical resection of TC.The clinical significance of the EMT status in TC remains unclear. Here, we investigated the clinical implication of EMT as a prognostic factor in TC and examined the relationship between EMT and clinical backgrounds including PD‐L1 expression with IHC staining of TC specimens. The relationships between the presence of EMT status and the clinicopathological background are shown in Table P = 0.002).Figure 3.4Next, to explore whether these relationships between PD‐L1 expression and EMT status after IT in TC are similar to our previous report of NSCLC,P = 0.004). We also evaluated IHC staining of PD‐L1 and EMT markers including TGF‐β before and after IT in the same patients and compared their status before and after IT. Upregulation of PD‐L1 TPS  showed a significant correlation with EMT positivity and with a change in TGF‐β . In addition, we found a significant relationship between the positive rate of EMT and the TGF‐β change .The PD‐L1 TPS of patients who underwent IT was significantly higher compared with the TPS of patients who did not undergo IT Figure A. Next, 3.5P = 0.03). Regarding OS, we found no significant relationship between the presence or absence of a change in PD‐L1 .Next, we examined the prognostic impact of the presence of a change in PD‐L1 TPS in 15 patients who underwent IT followed by surgery. Figure 4We analyzed IHC staining of EMT markers and the immune checkpoint proteins, PD‐1 and PD‐L1, in clinical samples of TC obtained by surgical resection. Our data suggest that the EMT status and immune checkpoint proteins have prognostic impact and that their expression is closely related. To our knowledge, our study analyzed one of the largest number of TC surgical samples regarding EMT status and PD‐1/PD‐L1 expression.To overcome advanced TC, surgery and chemotherapy are the maintherapeutic options. Despite those multimodal therapies, the outcome is far from satisfactory, and no standard therapy has been established. One reason is that biological research about TC has not made much progress compared to other carcinomas. Because TC is rare, our clinical experience has been insufficient, and oncological characteristics are poorly understood. In the present study, we revealed the microenvironment of TC from the viewpoint of cancer immunology. Recently, immunotherapy has been developed as another option for several malignancies. Agents that block PD‐1/PD‐L1 have exhibited dramatic antitumor efficacy in clinical trials for patients with a variety of cancer types.However, the clinical significance of PD‐L1 in TC is poorly understood. Although a few groups have discussed the clinical significance of PD‐1/PD‐L1 expression in TC with IHC analysis, the significance as a predictive factor is controversial. Katura et al showed that high PD‐L1 expression is correlated with unfavorable prognosis,Epithelial‐mesenchymal transition plays a crucial role in cancer progression, and this phenomenon can be found in the cancer microenvironment in several types of cancers. EMT is promoted by TGF‐β secreted from not only the cancer cell itself but also from other cell types. EMT confers invasive and metastatic abilities on cancer cells that are necessary for metastasis.We hypothesized that PD‐L1, N‐cad and TGF‐β expression was also upregulated after chemotherapy induction in TC. To test this hypothesis, we performed IHC for PD‐L1 and EMT markers including TGF‐β and compared the results before and after IT. Similar to our previous reports regarding NSCLC,In general use of immune‐check point inhibitor (ICI), PD‐L1 IHC assay is used and the results are biomarker for patient selection and therapeutic response of immune‐check point inhibitor (ICI). However, there are several problems such as sensitivity and appropriate cutoff in the IHC assays. From our results, EMT markers may be useful surrogate markers for case selection of ICI after anticancer agent.Our study has some limitations. The first limitation is the small sample size. Therefore, the predictive role of PD‐L1 expression is still controversial. Future studies should involve increased numbers of cases in collaboration with multiple institutions. As a second limitation, our IHC data provide supporting evidence that PD‐L1 expression is enhanced thorough the EMT process or TGF‐β signaling in TC. The mechanism of regulation of PD‐L1 expression needs to be elucidated by utilizing molecular biological techniques, although there are currently no commercially available TC cell lines.In conclusion, understanding the mechanism of PD‐L1 expression will yield important information regarding the role of immune checkpoint proteins in TC. Our results suggest that EMT markers may be informative for making decisions about induction of immunotherapy. In addition, our results suggest that the EMT status plays an important role in cancer progression and has a significant relationship with PD‐L1 expression. Our results also show that PD‐1/PD‐L1 blockade after chemotherapy or in combination with chemotherapy could be more effective than monotherapy and suggest another therapeutic option for treating chemo‐resistant cancer.The authors have declared no conflicts of interest. We state that the material has not been previously published or submitted elsewhere for publication. All authors have read and approved the manuscript and made a sufficient contribution to the work. We will transfer copyright to the Publisher. Click here for additional data file.


Pyrexia of unknown origin (PUO) is a common problem in day-to-day practice. FDG PET CT is an established investigation that aids in identifying the cause of PUO. Due to its high sensitivity PET detects an occult hypermetabolic focus in the body where CT helps in anatomical localization, vascularity, enhancement characteristics of the lesion detected on PET. It helps to differentiate benign versus malignant cause and target biopsy. Tuberculosis, lymphoma, pyelonephritis, thyroiditis appear hypermetabolic on FDG PET CT. Pericardial sarcoidosis is rare and not reported in literature as a cause of PUO. Presented here is a case of PUO secondary to pericardial granulomatosis diagnosed on PET CT. Cardiac MRI also helps in better tissue characterization and associated myocardial involvement of sarcoidosis. Histology confirmed the diagnosis of pericardial sarcoidosis in this case. With reducing cost, PET CT is being increasingly used in cases of fever of unknown origin. Common causes of PUO are bacterial infection as tuberculosis, hidden malignancies as lymphoma, autoimmune diseases as thyroiditis etc. High sensitivity of FDG PET enables early detection of lesions before morphologic changes set in. Other conventional imaging methods largely give anatomical information and depend upon manifestation of morphologic changes. FDG-PET CT is performed as a whole body procedure hence detects number and site of lesions not suspected clinically. We report a case of pericardial sarcoidosis suspected on PET CT and confirmed on histology. A 44 years old male presented with 4 weeks of fever, breathlessness. There was no weight loss (90 kg). Physical examination showed tachycardia 125 beats per minute, tachypnoea (36/minute), normal blood pressure (110/80 mmHg). Soft systolic murmur was heard in left parasternal space. 18F- fluorodeoxyglucose on 6 hours empty stomach. Scanning was done at 60 minutes using Siemens Horizon 16 slice PET CT system. The pericardium showed intense uptake of FDG in the anterior, inferior and right lateral walls. The anterior wall showed FDG avid thickening measuring 10×81mms standardized uptake value (SUV) 7.74. The inferior wall of pericardium showed thickening of 107×13mms with SUV value of 12.07. Few mediastinal lymph nodes were noted as follows: subcarinal node 17×13 mms SUV 3.86, left internal mammary node 17×6 mms SUV 2.58, right internal mammary node 8 mms SUV 2.81, left paratracheal 10 mms SUV 1.80, right paratracheal 10 mms SUV 3.24. Left supraclavicular node 19 mms SUV 2.53. Right level IV neck node 16 mms SUV 2.26 , WBC 7800/ µl (6000-10000/µl); platelets 414000/ µl (150000-450000/ µl); LDH (lactate dehydrogenase) 200 U/L (100-250); Blood Widal test excluded enteric fever. Sputum for AFB (acid fast bacilli) was negative for tuberculosis. Sonography showed bilateral pleural effusions, small pericardial effusion. There was no evidence of deep vein thrombosis on color doppler scan. FDG PET CT was performed using 7.7 mCi of SUV 2.26 . BilaterSUV 2.26 . CardiacSUV 2.26 . In view of these findings a diagnosis of granulomatous disease involving the pericardium was made. Serum ACE (angiotensin converting enzyme) was recommended. The value was 72 U/L . Tuberculin test was negative. Histology  showed non- caseating Granulomas, multinucleated Langhan’s giant cells and lymphocytic infiltrates . Steroids and empirical antitubercular treatment were initiated. Myocardial biopsy was not performed as FDG PET CT of myocardium was normal. The term sarcoidosis was introduced in 1899 by Caesar Boeck to describe skin lesions caused by epithelioid cells with pale nuclei and few giant cells. Due to its resemblance to sarcoma, he called these benign sarcoid of skin . Propionibacterial and mycobacterial DNA and RNA have been identified using PCR technique. Antibodies to mycobacterium tuberculosis have been detected in serum samples of patients with sarcoidosis . Sarcoidosis may affect any organ in the body. Involvement of lungs may cause dyspnea, cough, wheezing, chest pain. Uveitis may cause blurring of vision or blindness. Eye pain, photophobia, redness may also occur secondary to eye involvement. Neuro-sarcoidosis may present as cranial nerve palsy, headache, ataxia, cognitive dysfunction and seizures. Parotid gland enlargement in sarcoidosis is also known as Mikulicz syndrome. Musculoskeletal involvement includes sarcoid arthropathy, Lofgren syndrome . Heerfordt’s syndrome . Fever, weakness, weight loss may be general symptoms. Hypercalcemia and/or hypercalciuria is found in considerable number of newly diagnosed patients of sarcoidosis . Pulmona Myocardial sarcoid may present with acute left ventricular failure, tachyarrhythmias, conduction disturbances, valvular insufficiency or sudden death . Rarely  A study showed myocardial involvement in 25% of patients with sarcoidosis in the USA, and accounted for as many as 13–25% of deaths due to sarcoidosis . InvolveLeft ventricular free wall, septum and conducting system are involved in that order of frequency. Pericardial involvement results in restrictive pericarditis. It may rarely cause constrictive pericarditis . Negative Tuberculin test aids in distinguishing sarcoidosis from tuberculosis. Allergy to tuberculin skin test was observed in 87% of cases of sarcoidosis. A positive test requires extensive searched for tubercular focus . 67Ga citrate is an age-old radioisotope that localizes to the transferrin receptors of iron and lactoferrin of macrophages (67Ga citrate has been reported to localize in viral pericarditis, post aortocoronary bypass pericardial inflammation (rophages . As sarcrophages , 17. 67Gammation . There iammation .67Ga-citrate uptake are lymphoma, amyloidosis, atrial thrombus, myocarditis, systemic sclerosis (Other conditions with clerosis . FDG PET CT is a sensitive method to detect foci of inflammation. Sarcoid granulomas express glut-1 receptors. Consequently, there is increased FDG uptake . FDG PET PET has the advantage of imaging the whole body distribution of disease, which is difficult with MRI. Plain and dynamic contrast MRI provides non-invasive assessment of the heart for evaluation of cardiac sarcoidosis. MRI gives information about scar, oedema, perfusion defects and abnormal biventricular function. Late Gadolinium enhancement along the ventricular walls is the typical finding of myocardial sarcoidosis. MRI without PET risks failure to diagnose active myocardial inflammation seen early in the disease until it results in fibrotic changes which occur late. T2-weighted MRI shows inflammation of pericardium in form of edema but is less sensitive than PET, and also inflammation in areas of fibrosis is not easily detected . MRI has the advantage of no ionizing radiation, but it is contraindicated in patients with implanted pacemakers or defibrillators unless they are MRI compatible. Also care is required in the use of gadolinium contrast in patients with impaired kidney function. Pericardial involvement is seen as irregular and patchy pericardial thickening with enhancement on contrast MRI. There is a case report in a Japanese journal of pericardial tuberculosis presenting with fever .Pericard Corticosteroids is the mainstay of therapy for patients with cardiac sarcoidosis as it prevents left ventricular remodelling when myocardium is involved and improves survival . Pericar Primary pericardial sarcoidosis is a rare entity. PET scan aids in detection of pericardial inflammation to target biopsy. It also helps to detect inflamed lymph nodes that could be alternate site for biopsy to arrive at a diagnosis. Our case of pericardial sarcoidosis presented with PUO. Despite a diligent search, we were unable to find a report on pericardial sarcoidosis presenting with PUO.


Unfortunately, programs cannot prevent trainees from engaging in cheating behaviours such as content sharing, and little is known about the impact of re-using items.To alleviate some of the burden associated with the development of novel quality questions on a regular basis, medical education programs may favour the use of item banks. This practice answers the real pragmatic need of having to create exams We conducted an exploratory descriptive study to assess the effect of repeated use of banked items within an in-house assessment context. The difficulty and discrimination coefficients for the 16-unit exams of the past 5 years  were analyzed using repeated measure ANOVAs.M = 79.8% for the first use of an item, to a mean difficulty coefficient of 85.2% for the fourth use) and discrimination coefficients decreased significantly with repeated uses .Difficulty coefficients increased significantly (The results from our study suggest that using an item three times or more within a short time span may cause a significant risk to its psychometric properties and consequently to the quality of the examination. Pooling items from different institutions or the recourse to automatic generated items could offer a greater pool of questions to administrators and faculty members while limiting the re-use of questions within a short time span. The re-use of written exam questions answers pragmatic needs in many health professions educational programs. However, this practice puts programs at risk for questions and exam content being leaked from one cohort of examinees to the next cohorts. We examine here the concrete effect on question difficulty and discrimination of re-using written exam questions over a 5-year period. Our results suggest the practice is feasible when limiting the number of times a question is re-used within that time span.The increased interest in competency-based education (CBE) and programmatic assessment has had an effect on assessment practices within medical education. One of those consequences is the increase in assessment frequency to provide feedback regularly –4. A freThe development of assessment that appropriately and purposefully assesses students’ knowledge and skills, provides feedback, and that yields reliable scores, challenges even the best educators –7. Even To alleviate some of the burden associated with the development of novel quality questions on a regular basis, medical education program administrators and faculty members may favour the use of item banks . UsuallyThe use of banked items creates, however, a risk of item disclosure. The known use of banked items in assessment often leads to strategies such as content sharing –14—betweWhile there are limited studies about the impact of item disclosure in the context of classroom-based assessment, several studies have tackled the issue of item disclosure in the context of repeated exposure to the same items for repeat examinees on certification or licensure exams , 22–26. Park and Yang  argue agThe aim of this study was to assess the effect of repeated use of banked end of unit exam questions on their psychometric properties (difficulty and discrimination indices) over a 5-year period in a Canadian undergraduate medical education (UGME) program.We conducted an exploratory and descriptive study to investigate the effect of re-using written exam questions on their psychometric properties. The study was conducted within a Canadian undergraduate medical education program that uses a problem-based learning (PBL) approach. The curriculum has been organized in sequential blocks. During the first two years, students are exposed to 16 separated system-organ blocks in addition to a block dedicated to biomedical knowledge and a block serves as introduction to their future profession as physicians. These blocks are almost exclusively taught through PBL and mobilize 25–35 tutors per block. Each unit has a formal written and oral assessment.At the time when the study was conducted, the Canadian Tri-Council policy statement on ethical conduct for research involving humans stated that consent was not required from participants for a secondary analysis of anonymized data.Written exams for the 16 system-organ PBL blocks within the UGME program are comprised of some banked  and some new, MCQ and SAQ. New questions (or significantly modified questions) are written by a faculty member and are subsequently vetted by two faculty members for appropriateness, purposefulness and difficulty . Questions are not pretested. Questions are vetted after each administration, any significant change  would make that question ‘significantly modified’ and categorized as a new question. There is no washout period for questions, meaning a question could be used two years in a row. Block exams vary in length and ratio of new/used items, as there are no definite standards of practice. More specifically, the written exams can comprise about 50 to 90 questions, with 25 to 60% of re-used questions. MCQ answers receive either 0 or 1 according whether the candidate correctly identifies the appropriate answer. SAQ are marked on a scale of 0 to 1, given a weighted mark, depending on the number of sub-questions answered. For example, if the SAQ requires a list of three diagnoses, each correct diagnosis would be given a 0.33 mark.n = 972 students)). In that period, and for all cohorts and exams combined, 1,629 were identified as re-used questions meaning that they were used more than once in that period. These questions were thus included in our analysis. In other words, we pooled data from 90 different exams, with 16 exams administered to each of our five cohorts over the 2.5 years of their pre-clinical training.We collected data for the 16 different block exams administered to five different cohorts  for the different number of times used  as a within-subject comparison (independent variables). In other words, using the number of times a question was used as a within-subject comparison (where questions are treated as the subjects of interest), we investigated the impact on two dependent variables, that is, the difficulty and the discrimination coefficients. A total of six ANOVAs were computed. Analyses were done using SPSS 21 .Questions were only categorized in one of the three categories  as illustrated in Fig. M = 0.16, and 0.13, for 2nd and 3rd uses respectively).We observed an increase in question difficulty with repeated use, and independent of the number of uses. The overall mean difficulty coefficient of 79.8% for the first use of a question, to an overall mean difficulty coefficient of 85.2% for the fourth use of a question within a 5-year period . We observed a decrease in discrimination coefficients with repeated use, from an overall mean discrimination coefficient of 0.16 on the first use to an overall mean discrimination coefficient of 0.14 for the last use (p < 0.001), three times  or four times . The decrease in question discrimination was significant for questions that were used three times  or four times .Tab. de novo at each administration. Re-using items, however, comes with potential disclosure risks which can be a real threat to the validity of assessment data interpretation. This study analyzed the psychometrics properties of 1,629 re-used questions within a 5-year span in the context of a PBL UGME program at a Canadian University.The use of in-house item banks answers a real pragmatic need of having to create exams We observed a change in question property with an increased use of the question. More specifically, items which were re-used over a 5-year period became decreasingly difficult and less discriminating over time. The increase in difficulty coefficients, or the appearance of questions getting easier, may have several underlying contributors. As suggested earlier, item repeat increases the risk of item disclosure and there is evidence in the literature –14 for sWhether the observed change in psychometric properties is pedagogically significant—and can thus impact the validity of assessment data interpretation—is debatable. To assess the pedagogical impact of these results, one should consider what an overall 6% increase in difficulty coefficient and a 0.02 drop in discrimination coefficient represents. In most Canadian universities, the grading system translates a score (number) to a letter with an increase  at each 5% increments . Thus, creating an exam with frequently re-used banked questions could potentially increase students’ scores and consequently their final grade . The effect of decrease in discrimination may not be as impactful; it seems the discrimination coefficients were only minimally influenced by question re-use.n = 83) was used four times, and no question was used more than four times. We cannot posit on what happens when a question is used more than four times. We pooled data from 90 different exams (18 blocks × 5-year span) and thus had a large sample of re-used questions. Moreover, each exam is comprised of questions written by 2 to 3 different faculty members. We could not access frequency for MCQ options, which also would have been very interesting data to study. If options become less attractive through time, we could speculate about content/questions leakage from one cohort to the next. Although the results of this study cannot generalize beyond similar contexts, the significant changes in psychometric properties should not be overlooked.This study has limitations. The study was conducted within only one program, for a limited time span (5 years), where only a small sample of questions  can impact the question’s psychometric properties. Pooling questions from different institutions could be an interesting way to increase item-bank size and thus access to questions with known psychometric properties, while limiting the re-use of a specific question within a short time span, as also suggested by Gotzman and colleagues . Gierl a


We demonstrate a simple and highly sensitive optical fiber relative humidity (RH) sensor based on a long-period fiber grating (LPFG) coated with polyethylene glycol (PEG)/polyvinyl alcohol (PVA) composite films. The resonance wavelength of the LPFG is sensitive to environmental humidity due to the change in effective refractive index caused by the strong surface absorption and desorption of the porous PEG/PVA coatings. The sensor is sensitive in a wide range from 50% to 95% RH, with a highest sensitivity of 2.485 nm/%RH in the range 50–75% RH. The proposed RH sensor has the advantages of compact size, good reversibility, and stability, which makes it attractive for high-humidity environments. Relative humidity (RH) is an important magnitude in many fields, such as agricultural production, food storage, pharmaceuticals, health monitoring, and meteorological services. Traditional RH sensors are electric humidity sensors which consist of two main sensing parts: Electric capacity and electric resistance. They usually show hysteresis when placed in a high-humidity environment for a long time and could be affected by electromagnetic interferences. Therefore, much attention has been paid on optical fiber humidity sensors ,2 due toVarious types of optical fiber RH sensors have been proposed with different approaches. Firstly, the amplitude-based techniques detect the change of the optical intensity ,4,5. Sec2O3 nanofilm on the LPFG and packaged it with the polymer material. A temperature sensitivity of up to 0.77 nm/°C was achieved. LPFG is usually insensitive to surrounding RH. Different coating methods have been studied to combine LPFG with RH sensitive films as an RH sensor. Alwis et al. [As an optical fiber sensor, LPFG has been widely used in many areas, for the measurements of SRI  and temps et al.  have pres et al.  have fab2 laser writing technique is a flexible technique for the fabrication of LPFGs thanks to its advantages such as low cost, high grating temperature stability, etc. The method can be applied to practically any fibers, and the writing process can be computer programmed to generate complicated grating profiles without using any masks [2-laser-written LPFGs can be attributed to the residual stress relaxation and glass structure changes due to laser heating. The LPFGs written in a commercial boron-doped fiber can give a clean transmission spectrum, just like UV-written LPFGs [There are different techniques for the fabrication of the LPFGs. The UV light writing technique is the common one adopted to fabricate LPFGs in photosensitive fibers by lots of groups . The CO2ny masks . The mecen LPFGs .2-laser-written LPFG coated with polyethylene glycol (PEG)/PVA composite films. The films are coated on the LPFG by spontaneous evaporation of the PEG/PVA water solution. The coated films improve the interaction between the LPFG and the external humidity environment based on the adsorption and desorption of the PEG/PVA thin films. The coated LPFG humidity sensor shows a high RH sensitivity in a wide range from 50% to 95% RH. The experimental results show good repeatability, reversibility, and stability. The sensor structure has the advantages of small size and simple fabrication, and the thickness of the film can be controlled by changing the concentration of the solution. The PEG and PVA materials have good hydrophilicity and high molecular stability. The polymer composite film has a refractive index close to that of fiber cladding, which makes the sensor highly sensitive to RH changes. Therefore, it is possible to accurately measure the external RH based on the wavelength shift of the LPFG.In this paper, we demonstrate a highly sensitive humidity sensor based on a COAccording to the phase-matching condition, the relationship between the fundamental core mode and the forward propagated cladding modes of LPFG can be expressed as follows :λm= with a period of 300 μm and a length of 1.8 cm. The CO2 laser pulse had a frequency of 5 kHz and a wavelength of 10.6 μm. The spot beam diameter of the CO2 laser focused on the fiber was approximately 30 μm. The output power of the laser was around 0.6 W. The LPFG had a resonance wavelength of ~1560 nm. The mode patterns of the LPFGs were observed using a Charge-coupled Device (CCD) camera  [0,8 cladding mode. The LPFG was fixed in the glass U-groove (50 mm × Φ2 mm × 1 mm) with the heat-curing adhesive. The U-groove was fixed on the slide with the heat-curing adhesive, as shown in The LPFG was written in boron–germanium co-doped single-mode fiber  by CO, Japan) ,21. The PEG : 25322-68-3) and PVA (CAS: 9002-89-5) solid powder reagents were used in the experiments. The organic solvents alcohol was of analytical grade, and deionized water was used throughout the experiments. In our experiment, relative humidity sensing is based on the wavelength shift measurement of the coated LPFG. Therefore, higher resolution can be achieved when the refractive index of the coating materials is near the refractive index of the fiber cladding. The refractive index of hydrophilic materials is generally higher than that of fiber cladding, it is very unusual for the material to have both higher hydrophilicity and lower refractive index. PEG is a polar material and was adopted to reduce the refractive index of the coating PVA films. The refractive index of PEG can be close to the index of the fiber cladding when the external humidity rises to a certain degree. The refractive index of the coating materials decreases due to absorption of water molecules. During the process of humidity change, the film swells and shrinks. As described in , as the According to the theoretical and experimental results, changes in the thickness of the nanofilms coated on the LPFGs could change the refractive index sensitivity of the LPFGs dramatically when the thickness of the nanofilms is a few hundreds of nanometers  ,25. In oBoth PVA and PEG materials are water-soluble polymers and have many OH and O groups bonded in the backbone chain. The 15% (wt./wt.) PEG solution was prepared by dissolving the PEG powder in deionized water. The solution was prepared at room temperature and stirred until it became clear and transparent. Then, the PVA and PEG solutions were mixed together. To obtain a uniform dispersive solution, the PEG/PVA solution required an hour of ultrasonic processing. The PEG/PVA films were coated on the LPFG by evaporation of the water molecules of the PEG/PVA solution in a dry oven. Before the coating process, the LPFG sample in the glass U-groove was cleaned repeatedly with deionized water and alcohol and dried with a vacuum oven. We used a high-precision pipette to inject the mixed solution into the glass U-groove, ensuring that the same volume of mixed solution was injected every time during the process. After the mixture solution was dropped into the glass U-groove, the coated LPFG was placed in the drying oven for about 12 h to ensure enough water evaporation. The PEG/PVA films were then coated on the LPFG surface. Since the glass U-groove filled with the PEG/PVA mixed solution was horizontally placed in the drying oven and the solute was uniformly dispersed in the solution, the evaporation of water molecules was a slowly and evenly changing process. Therefore, we believe that the film was uniformly coated on the surface of the LPFG after the water molecules were completely evaporated. The thickness of the polymer film was ultimately determined by the concentration of the mixed solution . Figure The scanning electron microscopy (SEM) image of the coated LPFG is shown in We measured the refractive index sensitivity of the LPFG before it was coated with polymer, as shown in During the experiment, the temperature of the RH chamber was fixed at 30 °C. The RH was changed from 50% to 95% with a step of 5% RH. When the humidity value is less than 50%, the number of water molecules absorbed by the materials is not large enough to decrease the refractive index of the coating materials to fall below the refractive index of the grating cladding, so the resonance dip of the LPFG will not shift with the change of the humidity. By finding coating materials with a lower refractive index, the dynamic range of the humidity measurements could be increased. The change of humidity was observed by measuring the wavelength shift of the resonance dip when the humidity reached a stable value at the set value. The refractive index change of the coating polymer could be estimated by comparing the wavelength shifts of the bare LPFG  and coatTo investigate the temperature cross-sensitivity of the proposed humidity sensor, the temperature sensitivity of the sensors was measured when the sensors were set at different temperatures from 20 to 50 °C with a step of 5 °C. The RH was fixed at a specific value when the temperature was changed. For the humidity of 66% and 70% RH, the bare LPFG showed a temperature sensitivity of −0.43 nm/°C. When the coated LPFGs were set under different relative humidity environments, the temperature sensitivity of the sensor was much lower than that of the bare LPFGs. Moreover, when the humidity was in the range of 66–70% RH, the temperature cross-sensitivity was almost negligible, as shown in We coated the LPFGs with films consisting of different compositions of PEG/PVA. The experimental results of the humidity sensing measurements suggested that the LPFGs coated with the composite polymers with different combination ratios have different humidity sensitivities.A comparison of wavelength-based fiber optic humidity sensors coated with different types of films is shown in The resolution of the humidity measurements is dependent on the wavelength resolution of the wavelength shift measurements. In our experiment, we used an OSA with a wavelength resolution of 0.02 nm to measure the wavelength shift of LPFGs. The resolutions of the sensors could be improved by using equipment with higher-wavelength resolution. Since the temperature and humidity test chamber  used in our experiment was quite large, it took about 5 min for the instrument itself to rise from one set RH value to another one. We did not have equipment to measure the response time of the RH sensor. The response time of the sensor is related to the film thickness and the coating materials. Our sensor has almost the same film thickness as the one reported in the reference . The senIn summary, a high-sensitivity optical fiber humidity sensor based on PEG/PVA film-coated LPFG was fabricated. Experimental results show that the sensor with a coating thickness of 906.3 μm has a high humidity sensitivity in a wide range of 50–95% RH. The measured results suggest that the proposed sensor has lower temperature cross-sensitivity. The sensor exhibits good linearity, high stability, and reversibility in the wide working range, which makes it a better choice for RH sensing measurement.


Graphene grown directly on germanium is a possible route for the integration of graphene into nanoelectronic devices as well as it is of great interest for materials science. The morphology of the interface between graphene and germanium influences the electronic properties and has not already been completely elucidated at atomic scale. In this work, we investigated the morphology of the single-layer graphene grown on Ge substrates with different crystallographic orientations. We determined the presence of sinusoidal ripples with a single propagation direction, zig-zag, and could arise due to compressive biaxial strain at the interface generated as a result of the opposite polarity of the thermal expansion coefficient of graphene and germanium. Local density of states measurements on the ripples showed a linear dispersion relation with the Dirac point slightly shifted with respect to the Fermi energy indicating that these out-of-plane deformations were n-doped, while the graphene regions between the highs were undoped. These properties include high carrier mobility, transparency, and thickness of only one-atom2. They are important for several applications: data communications3, high-performance light emitting diodes (LEDs)4, low-power photonics5 and ultrafast- and broad-band photo-detection from ultraviolet to the terahertz range6. However, all of these properties were obtained by using exfoliated samples, a process that cannot be scaled for large scale device production. For this purpose, graphene must be defectless and produced over a large area at low cost7.Since the pioneering work of the Manchester’s group8. There are many reports about the synthesis of large area single-layer graphene samples. They represent the state of the art of the synthesis by CVD processes with the production of highly crystallographic oriented single-layer graphene with sample area as large as one square meter with good electronic properties9, such growth techniques apparently overcome the challenges of large area graphene production. However, this method is still based on the use of conductive substrates which turn it non-viable to be used for the fabrication of electronic devices. In this way, to obtain graphene by CVD, the film must be transferred to a dielectric or semiconducting substrate. The most commonly used technique is the transfer of graphene by applying a sacrificial layer of polymer over the sample and removing the copper foil using a suitable acid solution10. The transfer process usually leaves some polymeric residues that are responsible for the deterioration of graphene’s transport properties11. Also, since it is a delicate procedure it can generate defects in the transferred layer. Besides, it was demonstrated that metallic contamination levels on the graphene layer are much higher than the accepted in the fabrication of electronic devices12. One alternative route is the direct synthesis of single-layer graphene on dielectric or semiconducting substrates14.Today, chemical vapour deposition (CVD) is the main growth technique used and is the biggest bet for achieving this goal15. The growth of single-layer graphene on Ge(100) substrate was successfully achieved by both atmospheric- and low-pressure CVD 24. Although this orientation (100) is really interesting for implementation into Si technology, it undergoes reconstructions into the [107] facet in both the graphene growth and/or after post-growth annealing23, which would limit its use in possible applications. Nevertheless, there are several works about other atomically flat orientations such as (110) and (111) that arise as models to future applications as these surface orientations are expected to facilitate the high electron and hole mobility for n- and p- field effect transistors (FETs), respectively25. In the last few years, the study of the graphene/Ge interface was focused on the surface reconstruction of germanium26, the interaction between graphene and germanium substrate27, the effect of doping of the substrate on graphene growth28, and graphene as a protective layer of germanium substrate29. However, a more complete investigation that also considers the role of crystallographic orientation and the morphology of graphene is still needed.Germanium is an intrinsic semiconductor with higher carrier mobility than silicon, and its integration with graphene sheets is of high interest for both fundamental materials science and electronic device applications32. Ripples in graphene films have been observed and studied on both freestanding and supported single-layer graphene35. These results revealed that the interaction between graphene and the substrate is an important factor to be taken into consideration for the formation of those surface corrugations. In particular, the opposite polarity of the thermal expansion coefficient - negative for graphene and positive for metals – was suggested to be responsible for the wrinkles formation by CVD36. Epitaxial graphene grown on silicon carbide (SiC) shows the presence of ridges at the surface due to the relaxation of compressive stress generated during the cooling step of the synthesis because of the negative thermal expansion coefficient of graphene37.One important aspect is the morphological and electronic characterizations at nanoscale of the ripples formed in the graphene grown by CVD on Ge substrates, since they can have a significant influence on the electronic and magnetic properties of the graphene filmIn this work, we have studied the morphology and electronic structure of graphene films on Ge substrates. In order to do that, we have synthesized single-layer graphene by CVD using methane as precursor on germanium (undoped-Ge(110) and p-type doped with Ga Ge(100)) substrates. The films were characterized by Raman spectroscopy and mapping, while the surface topology was investigated in atomic-scale by scanning tunneling microscopy (STM) in ultra-high vacuum at room temperature conditions as well as by atomic force microscopy (AFM). The local electronic changes were determined by scanning tunneling spectroscopy (STS). Our Raman results revealed that the graphene layer was submitted to a biaxial compressive strain. The morphology of the graphene films was characterized by the presence of sinusoidal ripples with a single propagation direction (zig-zag).22. More information about preparation and growth of the samples such as temperature, environment, CH4:H2 flow ratios and time are in the method section and Supplementary Information, Fig. To study the morphology and electronic structure of graphene films, two sets of samples were synthesized by CVD. The first one was obtained on Ge(110) surface and the second one was prepared on Ge(100) surface as described elsewhere−1), G- (~1600 cm−1) and D- (~1350 cm−1) bands. In these spectra the O2 (~1557 cm−1)- and N2 (~2329 cm−1) peaks due to atmospheric oxygen and nitrogen were fitted and removed  and (33 ± 4) cm−1 for the growth on Ge(100), and the 2D- and G-bands intensity ratio (I2D/IG ≥ 2.0), see Figs D/IG ratios are around 0.20.We could determine that the graphene films were single-layer, by taking into account both the symmetry and Full Width at Half Maximum (FWHM) of the 2D-band fitted with a single Lorentzian curve with FWHM (39 ± 6) cmThe STM images shown in Figs The deformations observed in the Figs D) shifted around 50 meV, standard deviation of 20 meV, to the left from the Fermi level (EF), indicating that, at the top of the ripples, graphene films are slightly n-type doped.Local density of states (LDOS) of graphene/Ge interface was studied by STS at room temperature on every sample. Figures −1graphene (~3.9 nm−1) which is the lattice constant of graphene and second one is λ−1ripples (~0.27 nm−1) which is the wavenumber of the ripples. The two-dimensional-FFT of the same image is shown in Fig. The morphology of the ripples was determined through STM images and by means of analysis of profiles of the Fig. 18. The roughness of this sample was determined over an area of 20 × 20 µm2  and its value was of (0.8 ± 0.2) nm, i.e., the graphene/germanium interface is flat throughout the scanned area. Similar roughness was obtained from the surface of Ge (110) submitted to the same thermal cycle used for graphene growth, as can be seen in the Supplementary Information, Fig. 23. The formation of the Ge(107) facets was determined by different microscopy techniques and the roughness values are in the order of ~70 nm for bare Ge(100) and ~30 nm for graphene/Ge(100)22. On the other hand, for graphene grown on Ge(110) there is no such rearrangement (facet) of the Ge surface, so the system is quite flat.Although the synthesis procedure was performed close to the melting point of germanium, it was possible to obtain uniform and high-quality graphene films. Figure 22, we can estimate the type of strain  of the sample. The neutrality point (zero point) was taken from literature38, and corresponds to the expected 2D and G positions for suspended freestanding single-layer graphene. The experimental data are positioned in the region of neutrality of charge between the black and blue lines. We can say that the strain (ε) was compressive biaxial in the range between −0.2% and −0.6% for graphene but centred at −0.4%. These results were similar to those reported in20 for Ge (110). The origin of this compressive strain in graphene can be attributed to the opposite polarity of the thermal expansion coefficients between graphene (−8 × 10−6 K−1) and germanium (6 × 10−6 K−1) in the cooling step of the synthesis by CVD.Raman spectroscopy may be used to provide an estimate of the strain in graphene by analysing the positions of the 2D and G peaks. Figure F and ED coincide in the same energetic point . This result was different of what was observed in the Fig. Knowing that out-of-plane deformations present in the graphene films can be enough to cause distortions of the atomic bonds on ripples and promote displacement in ‘π’ orbitals, we analysed the Fig. 39. Our STS results showed that the ripples on graphene layer are slightly n-doped, while the valleys between two of them were undoped, in good agreement with the theoretical conclusions. These results can be compared with STM/STS results obtained from CVD graphene on copper, where the authors suggested that the constraint imposed at the boundaries between the intrinsic and the n-doped regions plays a vital role in creating these 1D ripples40. In this case, the n-doped region is generated by the introduction of nitrogen atoms, creating a defective zone that relaxes the ripples while the undoped region remained strained and with ripples near the boundaries. Recently, it was suggested that applied strain along the zig-zag direction results in the band gap opening in graphene32. However, since our STS results were obtained at room temperature, in this experiment we were not able to determine the opening of the band gap if it is below 50 meV.Recently, theoretical calculations using a molecular dynamics approach to model the formation of ripples in graphene have shown that sinusoidal ripples appear to arise in the presence of compressive biaxial strain, as indicated by Raman results, and the presence of these ripples induces spontaneous n-dopingIn this work, we have performed surface characterization of single-layer graphene grown on Ge substrates with orientations (110) and (100) by CVD. STM images clearly show the presence of sinusoidal ripples with ~3.6 nm wavelength and ~0.3 nm in height, independently of the crystallographic orientation of the substrates. Those deformations have the same direction of propagation (wave front), the zigzag direction, and are aligned with the armchair direction independently of the randomness of its distribution. We suggested that these sinusoidal ripples appear to arise in the presence of compressive biaxial strain, as revealed by Raman results, due to thermal expansion coefficients with different polarities of graphene and germanium substrates.The STS measurement results on the ripples showed a linear dispersion relation with the Dirac point, slightly shifted with respect to the Fermi energy, indicating that these out-of-plane deformations were n-doped, while the graphene regions between the highs were undoped. These periodic alternances between undoped and n-doped regions throughout the entire surface of graphene films must be taken into account in the future development of electronic devices.2) from 2.5 cm and 5.1 cm wafers, respectively , and cleaned with acetone, isopropyl alcohol, deionized water, respectively, and finally dried under pure N2 flux. After that, the Ge substrates were placed into the reactor chamber that was evacuated down to a base pressure of approximately 1.0 mPa. The growth environment was a mixture of Argon  and Hydrogen  gases at 100 and 50 standard cubic centimetres per minute (sccm), respectively. Two sets of samples were prepared as summarized in the Supporting Information and following the step-by-step from22. Once the deposition temperature and pressure were reached, methane gas was introduced in the reactor, and its flux was of 1.5 and 0.5sccm for Ge(110) and Ge(100) respectively. The synthesis was carried out at 910 °C with 5 × 104 Pa for both substrates, but the times were 60 and 120 minutes for Ge(110) and Ge(100), respectively. After the graphene deposition, the system was cooled to room temperature at a rate of 50 °C/s, under the same atmosphere used during the synthesis.Graphene films were synthesized on undoped Ge (110) and p-doped Ge(100) substrates by CVD at semi-atmospheric pressures. The substrates were cut into small pieces  chamber. These measurements were simultaneously performed at room temperature using an electrochemically etched W tip.Raman spectroscopy and AFM were performed using the integrated system from NT-MDT. Raman measurements used a micro-Raman spectrometer (NTEGRA SPECTRA), equipped with a CCD detector and a solid-state laser, which produced excitation energy of 2.62 eV. The Raman maps were taken over a 30 × 30 µmSupplementary Information


Objective: To investigate the association between tumor PD-L1 expression and patient survival to determine whether PD-L1 represents an independent prognostic feature for patients with non-metastatic clear cell renal cell carcinoma (RCC).Patients and Methods: The tissue bank of the Fudan University Shanghai Cancer Center was queried to identity tissue samples of patients treated with radical nephrectomy, for non-metastatic sporadic clear cell RCC (ccRCC) between 2008 and 2015. Real-time polymerase chain reaction and immunohistochemistry staining was performed to detect the expression level of PD-L1 in paired cancer tissue and paracancerous tissue.Results: Three-hundred-and-thirty patients were enrolled in this study, with a mean age of 55.0 years at surgery and a mean tumor size of 5.2 cm. Two-hundred-and-forty-two (73.3%) and 88 (26.7%) patients showed a high and low expression of PD-L1 mRNA, respectively, while 254 patients had positive PD-L1 immunohistochemistry staining. Two-hundred-and-ninety-two patients had consistent results for mRNA and the PD-L1 protein based on these different detection methods. Patients with high PD-L1 expression were more likely to exhibit adverse pathologic features including an advanced T stage (P = 0.002) and lymph node metastasis (P = 0.044). The Kaplan–Meier curves of PFS and OS stratified by PD-L1 expression had a statistically significant difference. PD-L1 expression maintained a significant predictive role for PFS and OS in the multivariate cox model.Conclusions: Our data suggests that PD-L1 correlates with prognosis in RCC and targeting the PD-1/PD-L1 pathway should be considered in the treatment of RCC patients. Immunotherapy plays an important role in the treatment of renal cell carcinoma (RCC) , 2. CytoProgrammed death-1(PD-1) and its ligand programmed death-ligand 1 (PD-L1) have recently become research hotspots in immunotherapy. New drugs targeting the PD-1/PD-L1 axis were reported to give very promising clinical responses in patients with various types of cancer –6. EarliThe tissue bank of Fudan University Shanghai Cancer Center (FUSCC) was queried to identity tissue samples of patients treated with radical nephrectomy for sporadic non-metastatic ccRCC, between 2008 and 2015. The institutional ethical committee has approved the informed consent provide for collecting the tissue into tissue bank. Tumor tissue samples paired with adjacent normal tissues were collected at the time of surgery and preserved under appropriate conditions only after informed consent was signed by individual patients. Clinical and pathological information were collected, including age at surgery, gender, clinical manifestation, laterality, tumor size, and the International Society for Urologic Pathology (ISUP) grade and tumor-node-metastasis (TNM) stage groupings , 15. CliPD-L1 were 5′-TGGCATTTGCTGAACGCATTT-3′ and 5′-TGCAGCCAGGTCTAATTGTTTT-3′. For the β-actin gene, the sense and antisense primers were 5′-GCCGACAGGATGCAGAAGGAGATCA-3′ and 5′-AAGCATTTGCGGTGGACGATGGA-3′, respectively. For each assay, a total of 10 μl reaction mixture was prepared using SYBR Green PCR master mix (Applied Biosystems) according to the manufacturer's instructions. Specific cycling conditions for β-action and PD-L1 were carried out as follows: denaturation at 95°C for 3 min, followed by 45 cycles of denaturation at 95°C for 20 s, annealing at 60°C for 20 s, extension at 68°C for 20 s, and measurement at 80°C for 20 s, followed by a final extension at 72°C for 5 min. To confirm the specificity of amplification, melting curve analyses were performed and PCR products were sequenced and resolved in a 1% agarose gel. The PD-L1 mRNA expression was represented as ΔCt = Ct(PD-L1) – ΔCt(β-actin), and the relative expression of PD-L1 in ccRCC was measured using the ratio of PD-L1 expression in ccRCC/matched normal tissues. “Low PD-L1 expression” denotes the ratio of PD-L1 mRNA expression in ccRCC/matched normal tissues of <1.5; “high PD-L1 expression” denotes the ratio of PD-L1 mRNA expression in ccRCC/matched normal tissues of >1.5.Total RNA was isolated using the TRIzol reagent , followed by reverse-transcribed reaction using the SuperScript First Strand cDNA system . Real-time PCR was performed using the ABI Prism 7900 sequence detection system (Applied Biosystems) with β-actin as an internal reference. The forward and reverse primers for 2O2 for 15 min at 37°C, and non-specific binding was blocked by incubation in 10% normal goat serum for 60 min at room temperature. For antigen retrieval, sections were autoclaved in 0.01 M sodium citrate buffer, pH 6.0 (at 20 psi) for 10 min. Tissue sections were then incubated overnight with primary antibody at 4°C. Chromogenic antigen detection was carried out using a peroxidase-conjugated secondary antibody (60 min incubation) and DAB reagents . Tissue sections were counterstained with Meyer's hematoxylin . All sections were examined and scored by two experienced pathologists blinded to all clinical data in an open discussion. The percentages of tumor cells with positive PD-L1 staining were quantified. In accordance with previous studies, PD-L1 tumor positivity was defined as membrane staining of ≥5% tumor cells  and the Envision detection kit . Tissue sections (4 μm thick) obtained from archived formalin-fixed, paraffin-embedded tissue blocks were deparaffinized in a xylene series and rehydrated with a graded ethanol series. Thereafter, endogenous peroxidase was quenched by incubation in 0.3% Hor cells , 18.PD-L1 mRNA expression or IHC expression with clinical and pathological features were evaluated using the Student's t-test for continuous variables and the χ2-test for categorical variables. Progression-free survival (PFS) and over survival (OS) was measured as the time from the date of surgery to the corresponding endpoints or until the most recent follow-up visit for censored (living) patients. Kaplan–Meier method was used for estimating PFS and OS, and a Log-rank test was taken to compare different Kaplan–Meier curves. Clinical outcomes were evaluated using Cox proportional hazards regression models univariately and multivariately, between groups with different levels of PD-L1 mRNA or IHC expression. The level of significance was defined as p < 0.05. SPSS software V13.0  was used for all statistical analyses.The associations of In total, 330 patients were enrolled in this study, with a mean age of 55.0 years at surgery and a mean tumor size of 5.2 cm. Males (66.4%) were approximately twice as many as females (33.6%). The demographics and disease characteristics were summarized in PD-L1 mRNA, respectively, while 254 patients showed positive PD-L1 IHC staining. Different methods might have controversial results for detection of PD-L1. As shown in PD-L1 mRNA expression had positive PD-L1 IHC expression, however, there were still some cases with negative PD-L1 IHC expression. On the other hand, most cases with low PD-L1 mRNA expression had negative PD-L1 IHC expression, while a small percentage of cases had positive PD-L1 IHC expression. The subgroup of 292 cases was then established which had consistent results for PD-L1 mRNA and IHC expression.Among 330 ccRCC patients, 242 (73.3%) and 88 (26.7%) showed a high and low expression of PD-L1mRNA or IHC expression is also shown in PD-L1mRNA expression were more likely to exhibit advanced pathologic features including poorer tumor grade (P = 0.010), advanced T stage (P = 0.034), and lymph node metastasis (P = 0.005). There was no statistically significant difference in age and sex among patients with different PD-L1 mRNA expression . Patients with positive PD-L1 IHC expression had almost the same clinical and pathologic features as patients with high PD-L1 mRNA expression, except for tumor size.The comparison of the clinical and pathologic features correlating with P < 0.001). The median PFS and OS for patients with low PD-L1 mRNA expression was longer than the subgroups with high PD-L1 mRNA expression (The Kaplan–Meier curves of PFS and OS stratified by PD-L1 expression are shown in pression , while tpression .PD-L1 mRNA expression may be associated with lower PFS. In the multivariate analysis, most of these factors remained statistically significant except for the age at surgery. PD-L1 mRNA expression. Multivariate analysis revealed that most of these factors maintained a significant predictive role for shorter OS. Notably, our study indicates that the expression stratification of PD-L1 based upon qRT-PCT or IHC staining may yield a different prognostic value for OS, that is, high PD-L1 mRNA expression was an independent prognostic factor for OS, however, positive PD-L1 IHC expression nearly lost significance after adjusting for covariants.The results of univariate and multivariate Cox regression analyses for PFS were described in PD-L1 is a cell surface glycoprotein, which was found in 1999 and belonged to the B7 family of T cell costimulatory molecules , 21. OveRCC is an immunogenic tumor associated with high levels of infiltrating immune cells, mainly mononuclear cells. These mononuclear cells are predominantly composed of T cells. As previously reported, PD-L1 expressed in tumor cells of RCC, influencing the biological behavior and correlating independently with poor cancer-specific survival rates, while PD-1 expressed in infiltrating mononuclear immune cells within RCC , 11. MosThis study included 330 cases of non-metastatic Chinese ccRCC patients and explored both qRT-PCR, which is highly sensitive and objective, and IHC to detect the PD-L1 expression in paired tumor tissues and adjacent normal tissues. Our results revealed that although high PD-L1 mRNA expression was an independent prognostic factor for OS, positive PD-L1 IHC expression nearly lost significance after adjusting for covariants, which indicated that IHC staining alone was not accurate enough to define the PD-L1expression level. To overcome the challenge of normalizing the expression levels, we used absolute values of cancerous tissue normalized to the values of corresponding paracancerous tissue to establish a measurable method of PD-L1 expression. With this approach we defined a 2-tier system for expression evaluation. The results of qRT-PCR in the present study should be more quantitative and reliable. It may be more persuasive to provide results based on two detection methods used together.Our results showed that the high expression of PD-L1 in tumor cells correlated to poorer pathological features, including a greater tumor size, high nuclear grade, lymph node metastasis, and more advanced stages. In addition, patients with high PD-L1 expression had a significantly increased risk of disease-specific mortality and overall mortality. Our work provides further evidence in the Chinese population that the functional level of the PD-L1/PD-1 pathway weakens the body's immune surveillance, thereby contributing to tumor progression.However, the present study also has some limitations. First, the expression of PD-L1 is not uniform even in the primary tumor. Near the margin of the tumor, the PD-L1 expression was induced by external factors produced by juxtaposed T lymphocytes. The PD-L1 expression of other parts is more likely to be affected by intrinsic factors and the molecular mechanism. The expression pattern of PD-L1 in different tumor locations could not be distinguished by qRT-PCR in the present study. Second, due to the heterogeneity of the tumor, the expressions of PD-L1 in primary and metastatic tumor cells were not the same. In some cases, the expression of PD-L1 in the primary tumor is negative, while it is positive in the metastatic tumor , 26. ThaOur data thus suggests that PD-L1 correlated with the prognosis in ccRCC and targeting the PD-1/PD-L1 pathway may be therapeutically efficacious and should thus be considered in the treatment of RCC patients.The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.This study was carried out in accordance with the recommendations of the local ethics committee with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Fudan University Shanghai Cancer Center ethics committee.WX, YQ, and JZ conceived the idea. YQ, HZ, GS, BD, YZ, and YS provided patient data and material. YQ, FX, YPZ, and XZ contributed to the sample-preparations. FX, XZ, and SZ carried out the laboratory analyses. WX and YQ performed the statistics. WX, FX, and JZ analyzed and interpreted the data. YQ, JZ, and DY were involved in the planning and supervising. WX, FX, and YQ drafted the manuscript and designed the figures. WX, FX, YQ, and DY wrote the manuscript, with contributions from the other authors. All authors read and approved the final 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.


Transparent conducting electrodes (TCEs) are the most important key component in photovoltaic and display technology. In particular, graphene has been considered as a viable substitute for indium tin oxide (ITO) due to its optical transparency, excellent electrical conductivity, and chemical stability. The outstanding mechanical strength of graphene also provides an opportunity to apply it as a flexible electrode in wearable electronic devices. At the early stage of the development, TCE films that were produced only with graphene or graphene oxide (GO) were mainly reported. However, since then, the hybrid structure of graphene or GO mixed with other TCE materials has been investigated to further improve TCE performance by complementing the shortcomings of each material. This review provides a summary of the fabrication technology and the performance of various TCE films prepared with graphene-related materials, including graphene that is grown by chemical vapor deposition (CVD) and GO or reduced GO (rGO) dispersed solution and their composite with other TCE materials, such as carbon nanotubes, metal nanowires, and other conductive organic/inorganic material. Finally, several representative applications of the graphene-based TCE films are introduced, including solar cells, organic light-emitting diodes (OLEDs), and electrochromic devices. Anotherf 550 nm . The sta−1, requiring further improvement by additional acid treatment or doping. The method of LB assembly was also employed to deposit GO and SWNT in a layer-by-layer manner a. Zheng −1 at 90% transmittance cyclohexane (TAPC) (30 nm)/1,4,5,8,9,11-hexaazatriphenylene hexacarbonitrile (HAT-CN)(10 nm)/TAPC(30 nm)/HAT-CN(10 nm)/TAPC(30nm)/4,40,400-tri(N-carbazolyl)triphenylamine:iridium(III)bis picolinate (TCTA:FIrpic)(5 nm)/2,6-bis[30-(N-carbazole)phenyl] pyridine:iridium (III) bis picolinate (DCzPPy:FIrpic) (5 nm)/1,3-bisbenzene(BmPyPB) (40 nm)/lithium fluoride (LiF) (1 nm)/aluminum (Al) (100 nm), showed an electron quantum efficiency (EQE) of 15.6% and a power efficiency (PE) of 24.1 lm/W, comparable to the EQE of 18.5% and PE of 28.5 lm/W obtained with an ITO-based OLED [At first, a basic OLED structure of anode/PEDOT:PSS/lectrode . The grased OLED . Flexible OLEDs that were fabricated with graphene-based anode shown in Electrochromic devices are widely used in display devices, including smart windows and mirrors, for improving indoor energy efficiency or personal visual comfort. Recently, rigid electrochromic devices have advanced, and they now offer flexibility, stretchability, and foldability for deformable devices  119,133,141. PolSmart windows that are based on electrochromic devices were demonstrated by Kim et al., who investigated the electrochemical and electrical characteristics of the PEDOT:PSS polymer-based electrochromic devices s of the different number of graphene layers used as electrodes . The fou3-CH3NO2 and HNO3 to obtain a low sheet resistance, was provided for flexible heaters, and it was mechanically stable after bending 1000 times with 1.1% strain [High performance, transparent, and flexible heaters have been fabricated using graphene-based electrothermal films that are suitable for automobile defogging or deicing systems and heatable smart windows, as shown in % strain . AdditioA new strategy of reduced large-size graphene oxide (rLGO)/silver nanowirehybrid film has been reported to design high-performance transparent film heaters. The thin rLGO provided a protective effect for the silver nanowire network against oxidation as well as the low sheet resistance of rLGO .New TCE materials, including carbon nanotubes, metal grids and nanowires, conductive polymers, and recently graphene, have appeared over the past decades in many fields of application. In particular, these new materials offer prospective advantages of flexibility, bendability, and even stretchability, enabling them to be applied as TCEs in wearable optoelectronic devices and displays. Among them, graphene has attracted special attention due to its superior electrical conductivity and optical transmission when compared to other TCE materials. Additionally, the excellent moisture barrier and mechanical flexibility of graphene allows it to be hybridized with other TCE materials to further improve the properties of TCE films.In the past decades, a number of methods for synthesizing high-quality graphene at low cost have been extensively studied, and the development of a large-area synthesis and transfer method using a roll-to-roll process has been a viable step in the practical application of graphene TCEs. Despite the tremendous advances in the graphene synthesis process and quality improvement, TCE films that are based on graphene still have issues that need to be improved, such as poor adhesion to substrates, low abrasion, and poor electrical conductivity as compared to conventional ITO.