Natural killer/T-cell lymphoma (NKTCL) is an Epstein–Barr virus (EBV)-associated, highly aggressive lymphoma. Treatment outcome remains sub-optimal, especially for advanced-stage or relapsed diseases. Programmed cell death receptor 1 (PD-1) and PD ligand 1 (PD-L1) have become promising therapeutic targets for various malignancies, but their role in the pathogenesis and their interactions with EBV in NKTCL remains to be investigated.Expression of PD-L1 was measured in NK-92 (EBV-negative) and SNK-6 (EBV-positive) cells by western blot, quantitative real-time PCR and enzyme-linked immunosorbent assay, and flow cytometry, respectively. Latent membrane protein 1 (LMP1)-harboring lentiviral vectors were transfected into NK-92 cells to examine the correlation between LMP1 and PD-L1 expression. Proteins in the downstream pathways of LMP1 signaling were measured in NK-92 cells transfected with LMP1-harboring or negative control vectors as well as in SNK-6 cells. PD-L1 expression on tumor specimens and serum concentration of soluble PD-L1 were collected in a retrospective cohort of patients with Ann Arbor stage I~II NKTCL, and their prognostic significance were analyzed.Expression of PD-L1 was significantly higher in SNK-6 cells than in NK-92 cells, at both protein and mRNA levels. Expression of PD-L1 was remarkably upregulated in NK-92 cells transfected with LMP1-harboring lentiviral vectors compared with those transfected with negative control vectors. Proteins in the MAPK/NF-κB pathway were upregulated in LMP1-expressing NK-92 cells compared with the negative control. Selective inhibitors of those proteins induced significant downregulation of PD-L1 expression in LMP1-expressing NK-92 cells as well as in SNK-6 cells. Patients with a high concentration of serum soluble PD-L1 (≥3.4 ng/ml) or with a high percentage of PD-L1 expression in tumor specimens (≥38 %) exhibited significantly lower response rate to treatment and remarkably worse survival, compared with their counterparts. A high concentration of serum soluble PD-L1 and a high percentage of PD-L1 expression in tumor specimens were independent adverse prognostic factors among patients with stage I~II NKTCL.PD-L1 expression positively correlated LMP1 expression in NKTCL, which was probably mediated by the MAPK/NF-κB pathway. PD-L1 expression in serum and tumor tissues has significant prognostic value for early-stage NKTCL.The online version of this article (doi:10.1186/s13045-016-0341-7) contains supplementary material, which is available to authorized users. NKTCLesponses –13. Howeesponses , 15. TheTumor immune escape is an emerging hallmark of cancer. Programmed cell death receptor 1 (PD-1) and PD ligand 1 (PD-L1) are important immune checkpoint molecules involved in T cell-mediated immune response and are key regulators of tumor immune escape –18. AberThere is a close correlation between Epstein–Barr virus (EBV) infection and NKTCL. Almost all cases of NKTCL exhibited a positive result of EBV-encoded RNA in situ hybridization in tumor samples. Additionally, pre- and post-treatment levels of circulating EBV DNA had significant prognostic implications for NKTCL patients –33. In p2 at 37 °C. The SNK-6 cell line was cultured in RPMI-1640  medium containing 2 mmol/l glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin, supplemented with 1000 U/ml interleukin (IL)-2  and 10 % human AB serum . The NK-92 cells were maintained in α-MEM  containing 20 % FBS , 2 mM l-glutamate, 100 mg/ml penicillin, and 100 mg/ml streptomycin (Life Technologies) and supplemented with 10 ng/ml IL-2 .The SNK-6 and NK-92 cells were routinely kept in Sun Yat-sen University Cancer Center (SYSUCC) and were incubated in a humidified incubator with 5 % COCells were washed with ice-cold PBS and were suspended in radioimmunoprecipitation assay (RIPA) lysis buffer  containing 1 % phenylmethylsulfonyl. After centrifugation at 14,000 rpm for 10 min at 4 °C, the protein content of supernatant was determined using the Pierce™ BCA Protein Assay Kit . Aliquots (20 μg protein per lane) were separated by 12 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes . The membranes were exposed to primary antibodies and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)  at room temperature for 1 h, followed by labeling with horseradish peroxidase-conjugated goat anti-rabbit IgG  for 40 min at room temperature. Signals were detected with enhanced chemiluminescence plus reagents . GAPDH was used as the internal control. The following primary antibodies were used: PD-L1, B-Raf, p-B-Raf, p38, p-p38, ERK, pERK , latent membrane protein 1 (LMP1), JNK, pJNK , and p65 .2O. The PCR was run on Stratagene Mx3000P Real-Time PCR system . The fluorescent quantity PCR conditions were as follows: pre-denaturation at 95 °C for 2 min, followed by 40 cycles of 94 °C for 20 s, 58 °C for 20 s, and 72 °C for 30 s. Primers were as follows: PD-L1 forward 5′-GAACTACCTCTGGCACATCCT-3′, PD-L1 reverse 5′-CACATCCATCATTCTCCCTTT-3′; LMP1 forward 5′-CAACAACGGCAAGACTCCC-3′, LMP1 reverse 5′-CCTCAAAGAAGCCACCCTC-3′. Each reaction was replicated three times. The fold changes in cDNA relative to the GAPDH endogenous control were calculated using the 2−ΔΔCt method [In order to quantify PD-L1 and LMP1 mRNA, total RNA was isolated from SNK-6 and NK-92 cells using TRIZOL Reagent  according to the instruction manual. One microgram of the total RNA was reversely transcribed into cDNA using Bestar™ qPCR RT Kit . The qRT-PCR reaction was prepared in a total volume of 20 μl containing 10 μl DBI Bestar® SybrGreen qPCR Master Mix , cDNA derived from 0.2 μg of input RNA, 5 pM each primer, and 7 μl double-distilled Ht method .The cell mixture was centrifuged at 1500 rpm for 5 min. The supernatant was collected and determined for the concentration of soluble PD-L1 using a sandwich enzyme-linked immunosorbent assay (ELISA) kit  according to the manufacturer’s instructions.Cells were labeled with anti-PD-L1 antibody  and then were assayed by flow cytometry using the Cytomics FC 500MPL cytometer. Data were collected and analyzed with the CXP version 2.2 software (Beckman Coulter Inc.).NotI and BamHI sites. The LV5-LMP1 vector and LV5 control vector (LV5-NC) were, respectively, cotransfected with packaging vectors pGag/Pol, pRev, and pVSV-G  into HEK-293T cells using Lipofectamine 2000 Transfection Reagent . After culturing for 72 h, the supernatants of the transfected cells were harvested. The lentiviral titers were determined by flow cytometric analysis for green fluorescence protein (GFP) expressed by viral vectors. 1 × 105/well NK-92 cells were infected with LV5-LMP1 and LV5-NC vectors, respectively, at a multiplicity of infection (MOI) of 300. After culturing for 48 h, western blot and ELISA were performed to determine the expression of proteins in NK-92 cells infected by LV5-LMP1 and LV5-NC, respectively.The LMP1-expressing lentivirus vector, LV5-LMP1, was constructed by the insertion of a full-length LMP1 cDNA into LV5 vector  at g and stored at −80 °C. The level of soluble PD-L1 was determined using a sandwich ELISA kit  as per the manufacturer’s protocol.Serum samples were collected before treatment from 77 patients with newly diagnosed NKTCL between 2008 and 2015 at SYSUCC and from 15 healthy volunteers. Serum was collected from the whole blood by centrifuging at 4000×Paraffin-embedded specimens were collected before treatment from the same NKTCL patients described above. Four-micrometer-thick sections were deparaffinized, rehydrated, and quenched. Immunohistochemical staining was performed using an anti-PD-L1 rabbit polyclonal antibody  and a two-stage immunohistochemical kit  according to the manufacturer’s instructions. The number of all tumor cells and those with membrane PD-L1 staining were calculated manually under high magnification (×200) using Image Pro Plus 6.0 software . Seven fields were calculated for each individual specimen to determine the percentage of tumor cells with membrane staining among all tumor cells. In order to minimize intra-tumor heterogeneity, two fields with the highest and lowest percentages were eliminated, and the average percentage of the remaining five fields was used to represent the level of PD-L1 expression for an individual.l-asparaginase, and oxaliplatin) and 33.8 % received CHOP-L  as induction chemotherapeutic regimen in dosages as previously reported [Seventy-seven patients with previously untreated NKTCL diagnosed at SYSUCC between 2008 and 2015 were enrolled in this study. The diagnosis was based on the WHO classification of hematopoietic and lymphoid tumors, and all patients had positive results for EBV-encoded RNA (EBER) in situ hybridization (ISH) , 41. Thereported . Complett test, and categorical variables were compared using the chi-square test or the Fisher’s exact test. Overall survival (OS) was measured from the date of diagnosis to the date of death or the most recent follow-up. Progression-free survival (PFS) was measured from the date of diagnosis to the date of disease progression, death, or the most recent follow-up. Survival data were calculated with the Kaplan-Meier method and compared using the log-rank test. Variables with statistical significance in univariate analysis were included in the multivariate analysis using a stepwise forward Cox regression model. Optimal cut-off values of serum and histological PD-L1 levels for predicting survival were determined using the receiver operating characteristics (ROC) curve analysis. The Spearman correlation test was used to explore the correlation between the serum and histological PD-L1 levels. Differences were considered statistically significant with a two-sided P value of <0.05. The statistical analysis was performed using SPSS version 17.0 software .Continuous variables were compared using a two-tailed Student’s P < 0.05). ELISA of cell culture supernatants found a remarkably higher level of soluble PD-L1 produced by SNK-6 cells than NK-92 cells . Additionally, expression of PD-L1 on cell surface was much higher in SNK-6 cells  and the NKTCL cell line SNK-6 (EBV-positive). The protein level of PD-L1 was remarkably higher in SNK-6 cell line than that in NK-92 cells Fig. . Consistlls Fig.  than in lls Fig. .Fig. 1ExP < 0.05) in NK-92 cells transfected with LV5-LMP1 vector, compared with those transfected with LV5-NC. Accordingly, remarkable upregulation of both PD-L1 protein  was observed in the LMP1-expressing NK-92 cells compared with the negative control. ELISA found a significantly higher concentration of soluble PD-L1 in cell supernatants produced by LMP1-expressing NK-92 compared with the negative control . Additionally, expression of PD-L1 on cell surface was much higher in LMP1-expressing NK-92 cells . Patients with NKTCL had a significantly higher concentration of serum soluble PD-L1 than healthy individuals .To explore the clinical significance of PD-L1 in NKTCL, we retrospectively examined the level of PD-L1 expression in paraffin-embedded tissues as well as the serum concentration of soluble PD-L1 in 77 patients with stage I–II disease. As shown in Fig. P = 0.020). After primary therapy, patients with a serum PD-L1 ≥ 3.4 ng/ml achieved a significantly lower CR rate than those with a serum PD-L1 < 3.4 ng/ml (61.5 vs. 84.3 %, P = 0.026, Table P < 0.001, Fig. P < 0.001, Fig. The optimal cut-off value of serum PD-L1 was 3.4 ng/ml based on the ROC analysis (see Additional file P = 0.026, Table P < 0.001, Fig. P < 0.001, Fig. Representative images for PD-L1 staining in tumor tissues are shown in Additional file Due to the relative rarity, sub-optimal current treatment strategies, and commonly observed chemoresistance of NKTCL, it is urgently warranted to identify novel therapeutic targets. Blockade of PD-1/PD-L1 interactions has emerged as a promising immunotherapy for cancer patients –27. PrevEBV plays a pivotal role in the pathogenesis of several hematopoietic malignancies . It has The underlying mechanisms of PD-1/PD-L1 activation vary between different types of cancers –47. ActiIn a retrospective cohort of 30 NKTCL patients reported by Han et al., PD-L1 was aberrantly expressed in nasal NKTCL specimens compared with the rhinitis specimens and PD-L1 expression closely correlated with some clinical and histopathological parameters . HoweverSeveral limitations exist in this retrospective study. The dynamic alterations of serum PD-L1 concentration after treatment and during follow-up, as well as their value in predicting relapse or prognosis, were not analyzed. Additionally, we did not analyze the prognostic impact of PD-L1 among patients with advanced-stage NKTCL due to the heterogeneous treatment delivered to those patients. Furthermore, we were unable to examine the levels of EBV mRNA and LMP1 expression in tumor samples due to the limited availability of paraffin-embedded tissues, which may provide useful information to understand the correlation between PD-L1 expression and EBV infection. Another issue that needs to be addressed is that the criteria of determining PD-L1 positivity varied among different studies. In Han’s study, the product of staining intensity and percentage of positive tumor cells was used to classify positive cases. However, in the present and some previous studies, only the percentage of cells with PD-L1 staining was used due to the heterogeneity of staining intensity, subjectivity of visional grading, and clinical feasibility –58. A unThe present study revealed a positive correlation between LMP1 and PD-L1 expression, which was probably mediated by the MAPK/NF-κB pathway in NKTCL. It also showed a significant prognostic value of PD-L1 expression level on tumor tissues and serum soluble PD-L1 concentration in early-stage NKTCL. Further studies are warranted to validate our findings in a prospective cohort and to explore the therapeutic value of PD-1/PD-L1 in NKTCL.


P = 0.032). Further subgroup analysis indicated that high PD-L1 expression in glioblastoma (GBM) was also associated with worse OS . Conversely, in index subgroup analysis, neither PD-L1 protein  nor gene  expression was significantly associated with OS. PD-L1 may represent a promising biomarker that predicts disease progression in patients with glioma or GBM. However, because of our limited sample size, further prospective or retrospective multi-centre, well-designed studies should be performed to verify this result.A large number of studies have shown that programmed death-ligand 1 (PD-L1) is abnormally expressed in gliomas. However, the prognostic significance of PD-L1 expression in glioma patients remains unresolved. Accordingly, we conducted a meta-analysis to determine the prognostic role of high PD-L1 in patients with glioma. Electronic databases were searched to identify studies evaluating PD-L1 expression and overall survival (OS) in these patients. A total of 6 studies (published in 4 articles) that involved 1052 patients were included. Pooled results showed that high PD-L1 expression was associated with worse OS in glioma patients (HR = 1.30, 95% CI: 1.02–1.65,  According to the World Health Organization (WHO) 2007 classification, gliomas are divided into grades I–IV, with each higher grade corresponding to an increased level of malignancy and poorer overall survival (OS). Glioblastoma  is the most malignant and aggressive form of primary brain tumour. Despite the use of a standard treatment regimen, including total surgical resection, radiotherapy, and adjuvant chemotherapy, GBM remains a major clinical challenge, with an overall 5-year survival rate of only 9.8%2. One significant challenge to treating GBM is the inevitable recurrence of these tumours. It is therefore very important to obtain an accurate prognosis while the tumour is in an early stage.Gliomas are the most common primary central nervous system (CNS) tumour, representing 40% of all brain tumours3. Observed in human malignancies, aberrant expression of PD-L1 is associated with clinicopathological features in several types of cancer, including colorectal4, breast5, and lung cancers6. Blocking PD-L1 or PD-1 has been demonstrated to be among the most promising immunotherapeutic strategies for boosting the immune system’s ability to combat cancer8. Although clinical anti-PD-L1/PD-1 therapies have been developed, data regarding the prognostic role of PD-L1 expression in glioma are limited, and findings to date are controversial. Several studies have found that high PD-L1 expression on glioma cells correlates with poor prognosis10, though not all reports support this conclusion12. Thus, it would be useful to determine whether PD-L1 expression is associated with the prognosis of glioma patients.Programed death-ligand 1 (PD-L1) is a T cell costimulatory molecule implicated in tumour immune escape mechanisms. Binding of PD-L1 to its receptor (PD-1) induces apoptosis or exhaustion of activated immune cellsIn this study, we conducted a meta-analysis to evaluate the prognostic role of PD-L1 in patients with glioma. To our knowledge, this is the first meta-analysis to quantitatively synthesize information from all previously published studies related to the potential prognostic value of PD-L1 in glioma patients.All relevant articles were retrieved from searches of the PubMed, EMBASE, Ovid and Cochrane Library databases. These searches were performed using the following keywords and text words:  and . The final search in this study was updated on October 15, 2016. References in eligible studies or textbooks were further manually reviewed to identify additional potentially eligible studies.Published studies were selected for analysis using the following inclusion criteria: (1) the study patients were confirmed by the department of pathology using diagnostic criteria for glioma, and gliomas were classified based on current WHO guidelines; (2) the study explored the prognostic significance of PD-L1; (3) the article was published in English. The exclusion criteria included the following: (1) duplicate studies; (2) letters, comments, meeting abstracts and reviews; (3) laboratory studies or animal studies or studies related to other types of CNS cancer.13 in Engauge Digitizer version 4.1 to estimate the logarithm-transformed HR and determined variance based on Kaplan–Meier curves.The following data from each enrolled study were extracted: the first author’s name, year of publication, country of origin, tests used for indexing, technique used to assay PD-L1 levels, cut-off value used to determine PD-L1 positivity and survival analysis method. Hazard ratios (HRs) for PD-L1 expression and OS and the corresponding 95% confidence intervals (95% CIs) were extracted from tables or Kaplan-Meier curves for both PD-L1 low/negative (control group) and PD-L1 high/positive  expressers. When both univariate and multivariate analyses of OS results were performed, HRs and 95% CIs were extracted preferentially from the multivariate analysis. When HRs were not reported, we used a previously published methodology14.Two reviewers (Song Xue and Ge Song) independently assessed the quality of the selected papers using a modified Newcastle-Ottawa Scale. The scale consisted of three items describing the subject selection criteria (0–4 points), comparability of subjects (0–2 points), and outcomes (0–3 points). The full score attainable was 9 points. In addition, articles with a total score of more than 5 points were considered to be of high quality2 metric. I2 > 50% or P < 0.10 in a Q test indicated large heterogeneity between studies, and we used random effects models to calculate the pooled HR and 95% CI. The fixed effects model was utilized for the remaining analyses. Subgroup analyses were applied to identify the sources of any observed heterogeneity. Such analyses were conducted for the GBM patients, index for PD-L1 protein and gene expression, cut-off value to indicate tumour PD-L1 positivity, and survival analysis method. All analyses were performed using Stata Version 12.0 (Stata Corporation) and Cochrane Review Manager version 5.3 (Cochrane Collaboration). All statistical tests were two sided, and P < 0.05 was taken to indicate statistical significance.HRs and 95% CIs were used to measure the effect size. An HR greater than 1 indicated a poor prognosis for patients with high/positive PD-L1 expression. The impact of PD-L1 on survival was considered to be statistically significant if the 95% CI for the HR did not overlap 1. Heterogeneity among studies was evaluated using Cochrane’s Q tests (Chi-squared tests) and the I12.Using a previously established retrieval strategy, 139 potentially relevant articles were initially identified from the databases searched. Eighty-six manuscripts were then excluded from the analysis for the following reasons: 9 were duplicate, 22 were reviews or letters, 32 concerned a carcinoma not related to glioma or GBM, and 23 were not related to PD-L1. Thus, 53 articles remained for further full-text assessment. After careful reading of the full text, 49 of the reports were excluded because they did not provide a prognosis (n = 41) or were based on an animal model (n = 8). Ultimately, 4 articles were included in this meta-analysis  involving 1052 patients were available for this meta-analysis. All of the relevant features in the 6 eligible studies are listed in Table Two of the selected articles each have enrolled two cohorts, and two articles each were selected to assess the prognostic value of expression of the PD-L1 protein and PD-L1 geneThe quality of the selected studies was assessed by using the Newcastle-Ottawa Scale and found to range from 6 to 8, indicating that the studies were of high quality Table .Table 2QP = 0.032), suggesting that high/positive PD-L1 expression is an indicator of a poor prognosis in glioma patients. Fig. P = 0.034, I2 = 58.4%), subgroup analysis of GBM patients was performed. A total of five studies were included in the GBM patient sub-group analysis . Our results revealed an HR for OS and positive PD-L1 expression of 1.40  , whereas that for PD-L1 gene expression was 1.20 . There was no statistically significant difference between these two groups (P for subgroup difference = 0.530)  . The results of our subgroup analysis of the analytical method were the same as those of the testing index subgroup analysis  with genetic deletions or mutations in phosphatase and tensin homologue (PTEN) had higher PD-L1 protein levels than cells wild-type for PTEN18. In addition, PD-L1 expression levels in cells in the tumour microenvironment exposed to cytokines were markedly higher, which has been termed an “adaptive immune resistance” mechanism. One example of adaptive immune resistance is an increase in the level of PD-L1 expression in glioma cell lines after the addition of IFN-γ16.Both glioma cell lines and glioma cells in tissue samples have been found to aberrantly express PD-L119, gastric carcinoma20, and lung cancer21. In contrast, there are no consistent results for the prognostic value of PD-L1 in glioma. In a study of 229 glioma patients (grades I–IV), Zeng et al. found no significant associations between PD-L1 and prognosis by univariate analysis. When the median survival time (12 months) was used as a cut-off value, they found that positive PD-L1 expression had a significant impact on poor OS in patients who survived or were followed up for 12 months12. Liu et al. first sought to determine the prognostic value of PD-L1 in tumour masses (TMs) collected from patients with GBM. A higher level of PD-L1 expression (>10 PD-L1+ cells) in a TM was significantly correlated with a poor prognosis, whereas no or low levels of PD-L1 (1–9 PD-L1+ cells) were not9. An increasing number of researchers have performed studies aimed at investigating the prognostic value of PD-L1 in GBM, but the results thus far have been inconsistent. For example, using IHC to analyse 135 GBM specimens, Berghoff et al. found no significant associations between a membranous staining pattern for PD-L1 expression and OS11. The same approach was taken by Nduom et al., who investigated the prognostic role of PD-L1 expression in 94 patients with GBM and found that high/positive PD-L1 expression was associated with significantly shorter survival (P = 0.0086). Additionally, by applying a multivariate Cox proportional hazards regression model, they found low/negative PD-L1 expression to be a protective factor for OS 10. Moreover, both of these groups analysed the GBM dataset at The Cancer Genome Atlas (TCGA) and reached contradictory conclusions regarding the prognostic value of PD-L1 gene expression. Berghoff et al. used a level 2 Agilent microarray to analyse a cohort of 446 patients, and the results showed no prognostic value of PD-L1 gene expression11. In contrast, Nduom et al. utilized level 3 Illumina RNASeq and found a significant correlation between high levels of PD-L1 gene expression and unfavourable prognoses. Multivariate analysis demonstrated that PD-L1 independently and negatively impacted survival 10. Therefore, meta-analysis of the data available regarding the prognostic significance of PD-L1 would be useful and is urgently needed.Increased PD-L1 levels have been associated with a poor prognosis in several cancers, such as nasopharyngeal carcinomaThe present meta-analysis is the first to estimate the correlation between PD-L1 overexpression and survival in patients with glioma. In this meta-analysis, we combined the statistics reported in 4 articles (6 studies) including 1052 patients with glioma, and the data indicated that PD-L1 expression plays a significant role in predicting OS in these patients. We found that the combined HR (1.40) for the GBM data alone was larger than that for all 6 eligible studies of glioma (1.30), suggesting that PD-L1 expression could serve as an important prognostic factor for malignant glioma. However, this result should be considered cautiously because heterogeneity was not eliminated in our GBM subgroup analysis of OS.P = 0.068 and P = 0.322, respectively). In addition, a strong tendency toward statistical significance was observed between positive PD-L1 expression and a worse prognosis in the cut-off subgroup analysis , suggesting that a cut-off value of 5% can be used to estimate OS. Nevertheless, several concerns should be addressed regarding these results. We could not eliminate heterogeneity in the testing index subgroup analysis of OS. There is a lack of validated assays for quantifying the level of PD-L1 expression, and no standardized cut-off criteria are currently applied when determining whether PD-L1 expression is positive. These factors may underlie the heterogeneity observed among the included studies22. In addition, the results of the subgroup analysis of cut-off values should be interpreted with caution because a relatively small number of studies (3 studies) used 5% as the cut-off value. Hence, additional analyses are needed to develop better sensitivity, and specific assays are needed to detect PD-L1 expression23.Based on subgroup analysis of the testing index, positive PD-L1 expression, at both the protein and gene levels, was associated with poor OS, but this relationship was not significant ; this likely introduced bias. Moreover, we could not extract HRs with 95% CIs from some of the studies; thus, we evaluated HRs using Kaplan-Meier curves. Finally, we were unable to evaluate the prognostic significance of PD-L1 by stratifying patients according to their clinical features because most of the primary studies did not provide sufficient information to analyse correlations between PD-L1 expression and clinical features, such as age, sex, Karnofsky Performance Status, and O-6-methylguanine-DNA methyltransferase status. Based on these observations, we believe that additional studies are needed and that our conclusions should be interpreted with caution.In conclusion, despite the limitations described above, our meta-analysis is the first report to focus on the prognostic significance of PD-L1 expression in glioma patients. Our study demonstrated high/positive PD-L1 expression to be associated with poor OS in patients with glioma, and this effect remained significant when the analysis was restricted to GBM patients. Furthermore, our sensitivity analysis did not indicate that alterations in the results (HR) were caused by any individual study, which strengthens the value of our findings. PD-L1 is therefore likely to be particularly useful as a prognostic marker, and it should be included in future validation studies.Supplementary Figure S1


Freestanding graphene/MnO2 cathodes were manufactured through a vacuum filtration process. The structure of the graphene/MnO2 nanocomposites was characterized using X-ray diffraction (XRD) and Raman spectroscopy. The surface and cross-sectional morphologies of freestanding cathodes were investigated by scanning electron microcopy (SEM). The charge–discharge profile of the cathodes was tested between 1.5 V and 4.5 V at a constant current of 0.1 mA cm−2 using CR2016 coin cells. The initial specific capacity of graphene/α-, β-, and γ-MnO2 freestanding cathodes was found to be 321 mAhg−1, 198 mAhg−1, and 251 mAhg−1, respectively. Finally, the graphene/α-MnO2 cathode displayed the best cycling performance due to the low charge transfer resistance and higher electrochemical reaction behavior. Graphene/α-MnO2 freestanding cathodes exhibited a specific capacity of 229 mAhg−1 after 200 cycles with 72% capacity retention.Different polymorphs of MnO Therefore, researchers have been developing different cathode materials such as LiMn2O4 and LiFePO4, which have a capacity of merely 150 mAh/g and 170 mAh/g, respectively [Nowadays low cost, clean and sustainable energy storage requirements with high performance are of great need because of rapid improvement of mobile and stationary electronic applications. Lithium-ion batteries have been one of the key energy storage devices to meet these energy demands since the last century . However2 , and Si 2 , the perectively .2) is one of the most promising metal oxide as a replacement for the Li-ion electrode material owing to its high theoretical capacity (308 mAh/g), environmental friendliness and low cost [2 has been fabricated and used with carbon materials to achieve excellent conductivity with a large specific surface area [Manganese dioxide  atomic-scale honeycomb lattice made of carbon atoms. Its unique properties such as high electrical and thermal conductivity, high chemical stability, large specific surface area and high mechanical strength have made graphene one of the most promising materials for energy storage devices . In recee cycles .2 has many crystallographic polymorphs including α-, β-, δ-, γ-, ε-, and λ-MnO2. The electrochemical characteristics of MnO2, such as electrocatalytic activity, specific capacity and oxygen reduction reaction, vary according to its crystalline structure and morphology [MnOrphology . However2  were produced by a microwave hydrothermal method. Freestanding graphene/MnO2 cathodes were manufactured through a vacuum filtration process without using any additional additives such as a binder. The effect of the different polymorphs, α-, β-, and γ-MnO2, on the structural and electrochemical properties of the manufactured, freestanding graphene/MnO2 cathodes was investigated. To the best of our knowledge, this study is the first to investigate the electrochemical performance of freestanding graphene/MnO2 cathodes for Li-ion batteries. The freestanding graphene/MnO2 cathodes exhibit a high specific capacity and excellent electrochemical cycling performance.In this work, different polymorphs of MnO2 phases were synthesized by a microwave-assisted hydrothermal method. α-MnO2 nanowires and β-MnO2 nanorods were prepared according to our previous report [2, 1.83 mg of (NH4)2S2O8, 1.35 mg of MnSO4 and 3 mmol were dissolved in 80 mL of distilled water. Then, the resulting solution was transferred into a Teflon (PTFE)-lined autoclave, sealed and placed in a microwave oven (Milestone ROTOSYNTH). The hydrothermal reaction was carried out for 60 min at 90 °C. Then the autoclave was cooled down to room temperature and the as-prepared black precipitate was filtered and washed several times with distilled water. γ-MnO2 was obtained after drying at 80 °C in a vacuum oven for 12 h.The α-, β-, and γ-MnOs report . To prep2 cathodes were prepared via a vacuum filtration technique. Firstly, 30 mg of GO was dissolved in 50 mL distilled water by ultrasonication for 1 h to obtain GO. Then 30 mg of as-synthesized MnO2 was added to the solution and ultrasonicated for another 1 h. The GO/MnO2 solution was filtered on a PVDF membrane by a vacuum filtration technique. In order to reduce the GO to graphene, the as-synthesized GO/MnO2 was subjected to a hydrazine solution after filtration of GO. 5.6 mL of a hydrazine solution was slowly poured onto GO/MnO2 and filtered. Then the obtained solid was peeled-off from the PVDF membrane and the freestanding graphene/MnO2 was obtained (approximate thickness is 15 μm). This process was carried out for all MnO2 phases.Graphite oxide (GO) was synthesized according to a modified Hummers’ method  by using2 composite cathodes was characterized using scanning electron microscopy (SEM). The structural and phase investigation of the freestanding cathodes was tested using X-ray diffraction (XRD) and Raman spectroscopy.The microstructural morphology of the freestanding graphene/α-, β-, and γ-MnO6) was dissolved in ethylene carbonate (EC) and dimethyl carbonate (DMC) , which was used as the electrolyte. In order to separate the electrodes, a microporous polypropylene membrane was used. Electrochemical tests of the cathodes were implemented between 1.5 and 4.5 V at a constant current density of 0.1 mA cm−2. The specific capacity of the freestanding graphene/MnO2 cathodes was calculated depending on the active mass of the graphene/MnO2 composite (about 20 mg) on Al foil. The resistance of the electrodes was evaluated via electrochemical impedance spectroscopy (EIS) using a Nyquist curve in the frequency range 1000 kHz–0.1 Hz with an AC amplitude of 10 mV with fresh electrode applied before the electrochemical cycling test.A CR2016 coin cell was used to investigate the electrochemical performance of the produced freestanding composite cathodes, assembled in an Ar-filled glove box. In this coin cell, the produced cathodes were used as a working electrode, and lithium foil was used as an anode. 1 M lithium hexafluorophosphate , cross-sectional characterization of graphene/MnO2 composite layers was implemented using SEM. As it can be seen from 2, β-MnO2 and γ-MnO2 structures were homogenously distributed and fixed between graphene layers.The surface morphologies of α-, β-, and γ-polymorphs of MnO. β-MnO2  shows the γ-MnO2  exhibits2. The typical reflection peaks of α-MnO2 are observed at 2θ values of 12.7°, 18.0°, 28.6°, 36.7°, 38.6°, 41.9°, 49.7o, 56.4° 60.2°, 65.4°, 69.6°, and 72.9° corresponding to (110), (200), (310), (400), (211), (420), (301), (600), (521), (002), (541), and (312) planes of α-MnO2 crystals [2, reflection peaks were observed at 2θ values of 28.7o, 37.4o, 41.0o, 42.9o, 46.1o, 56.7o, 59.4o, 65.0o, 66.8o, 67.3o, 72.3o, 79.7o and 86.6o corresponding to (110), (101), (200), (111), (210), (211), (220), (002), (310), (301), (202) and (321) planes of β-MnO2 [2, reflection peaks were observed at 2θ values of 22.0°, 34.8°, 37.0°, 38.5°, 42.2°, 57.0°, 65.4° and 68.6°, corresponding to (101), (130), (210), (400), (211), (402), (020), (421) planes of γ-MnO2 [2, graphene/β-MnO2 and graphene/γ-MnO2 composite structures. The graphene peak observed at a 2θ value of 25.8o indicates the (002) plane of carbon. However, there are still some remaining graphene oxide phases observed at 2θ values of 10.9o in graphene/α-MnO2 and graphene/β-MnO2, while almost all graphene oxide is transformed to graphene in the graphene/γ-MnO2 structure [o (110), 0, (310),, (400), 1, (420),, (211), 0, (002),f γ-MnO2 . Figure tructure –26.2, graphene/β-MnO2 and graphene/γ-MnO2 composites was performed via Raman spectroscopy using a 785 nm laser and the results are shown in 2 is generally used to characterize MnO2 structures, MnO2 structures may show different characteristic peaks due to different laser wavelengths and energy. Generally, in the Raman spectra of MnO2, the peaks between 500 and 700 cm−1 are attributed to the stretching mode of MnO6 octahedra [−1 originate from the formation of Mn2O3 or Mn3O4 and correspond to the bending mode of O–Mn–O [2 composite, α-MnO2 shows three weak peaks at 289 cm−1, 319 cm−1 and 376 cm−1 and one strong peak observed at 661 cm−1. In graphene/β-MnO2 composites, three weak peaks at 230, 330 and 385 cm−1 and two strong peaks at 562 and 648 cm−1 are observed. Graphene/β-MnO2 exhibited two weak peaks at 314 and 367 cm−1 and one strong peak at 658 cm−1. The observed peaks at around 1320 and 1590 cm−1 are related to the D- and G-bands of graphene [2 composite structures.Further phase characterization of graphene/α-MnOctahedra  and the f O–Mn–O . In the graphene  in the g2 in the graphene/MnO2 composites on the resistance of the cell, electrochemical impedance spectroscopy (EIS) measurements were performed and results are shown in Rct) of the graphene/α-MnO2, graphene/β-MnO2 and graphene/γ-MnO2 cathodes [2 composite cathode has the largest width, showing Rct = 102 Ω. Graphene/α-MnO2 with a Rct = 42 Ω has a smaller width than that of graphene/γ-MnO2 with Rct = 90 Ω. These Rct values indicate that the graphene/α-MnO2 composite cathode has better electronic contact and conductivity among the produced freestanding graphene/MnO2 cathodes [In order to investigate the effect of different crystal structures of MnOcathodes . As seencathodes .−1 in a voltage range from 1.5 to 4.5 V. In 2, graphene/β-MnO2 and graphene/γ-MnO2 cathodes are given for the 1st, 50th, 100th and 200th cycles. As shown in 2 cathode exhibited a specific capacity of 321 mAhg−1 upon first discharge with an open-circuit potential of about 3.2 V and an average voltage of approximately 2.25 V. It can also be seen that the capacity of the graphene/α-MnO2 cathode was sustained with a small amount of capacity loss. This could be attributed to the wire-like structure of α-MnO2 allowing ions to pass from the cathode. When the graphene-supported β-MnO2 cathode was investigated (2. While the first discharge capacity of graphene/β-MnO2 cathode was 198 mAhg−1, the graphene/γ-MnO2 cathode displayed a specific discharge capacity of 251 mAhg−1 (2 and γ-MnO2 electrodes decreased dramatically with increasing number of cycles. This could be attributed to the poor electrical conductivity and the textural modification during Li+ intercalation and de-intercalation processes. Cheng et. al. [2 cathodes and demonstrated a discharge capacity of 204.4 mAhg−1 for the first discharge with a constant current of 50 mAg−1. In our work, the as-prepared α-MnO2/graphene cathode was reached a specific capacity of 318 mAhg−1. This is probably due to graphene reinforcement, which increases the conductivity and electrochemical reaction of α-MnO2 with Li ions, as is reported in previous studies [The electrochemical performance of the as-synthesized cathodes was first evaluated by galvanostatic charge/discharge cycling at a constant current density of 254 mA gstigated , it can 1 mAhg−1 . The spe studies .2, graphene/β-MnO2, and graphene/γ-MnO2 cathodes. A remarkable result is obtained from the graphene/α-MnO2 cathode which has an initial capacity of 321 mAhg−1. It can be seen that there is no sudden loss of capacity and between cycles 2 and 44 it exhibits almost a stable capacity of 305 mAhg−1. The total capacity loss is 27% during 200 cycles. Graphene/β-MnO2 and graphene/γ-MnO2 cathodes were also cycled until the 200th cycle but they exhibited very poor capacity retention when compared with the graphene/α-MnO2 cathode. Although both of these cathodes display a high capacity during the first cycle, the capacity value decreases dramatically during the second cycle. While the total capacity loss for the graphene/β-MnO2 cathode was 61%, the graphene/γ-MnO2 cathode showed a 55% capacity loss after 200 cycles. Tu et al. [2-graphene cathodes and a γ-MnO2 cathode, and they observed huge capacity reduction due to the formation of Li2MnO3.u et al.  also rep2 phases. Freestanding graphene/MnO2 was successfully prepared with no further additives. The prepared nanocomposite samples were operated as positive electrodes for Li-ion batteries. The SEM images showed that α-MnO2 nanowires and β-MnO2 nanorods were homogenously dispersed not only at the surface, but also in the interlayer space of grapheme layers. Moreover, urchin-like γ-MnO2 microspheres were found wrapped by graphene nanosheets. The electrochemical cycling results demonstrated that the graphene/α-MnO2 cathode showed the best electrochemical performance among all prepared samples with an achieved initial capacity of 321 mAhg−1 and maintained its remarkable performance after many cycles. This study proved that α-MnO2 nanowires with graphene reinforcement could be promising cathodes for Li-ion batteries due to the high capacity and long cycle life.A facile and rapid microwave-assisted hydrothermal method was demonstrated to synthesize α-, β-, and γ-MnO


Furthermore, a significant increase of PD-1 in Treg cells was found inPIH compared with NP. In addition, PD-L1 Fc, an activator of PD-1/PD-L1 pathway,increased Treg cell percentage, enhanced Foxp3 mRNA expression, and elevatedlevels of TGF-β, IL-10, and IL-35 in PBMCs. However, anti-PD-L1 mAb exerted areverse effect. These findings revealed that PD-L1 Fc had a favorable effect onTreg cell differentiation, indicating a potential therapeutic value ofPD-1/PD-L1 pathway for PIH treatment.Pregnancy-induced hypertension (PIH) causes significant maternal and fetalmorbidity and mortality. A decreased number of regulatory T (Treg) cells isassociated with the pathogenesis of PIH. The programmed cell death-1(PD-1)/PD-ligand 1 (PD-L1) pathway is critical to normal pregnancy (NP) bypromoting Treg cell development. However, the relationship between PD-1/PD-L1and Treg differentiation in PIH has not been fully elucidated. In this study,venous blood was obtained from 20 NP and 58 PIH patients. Peripheral bloodmononuclear cells (PBMCs) were isolated from venous blood. The levels ofTreg-related cytokines  in serum and PBMCs weremeasured by ELISA. The percentage of Treg cells in PBMCs was assessed by flowcytometry. The mRNA levels of Treg-specific transcription factor Foxp3 in PBMCs,and PD-1 and PD-L1 in Treg cells were detected by qRT-PCR. The protein levels ofPD-1 and PD-L1 in Treg cells were evaluated by western blot. The serum levels ofTGF-β, IL-10, IL-35, and Foxp3 mRNA expression andCD4 Pregnancy-induced hypertension (PIH) syndrome complicates 6–10% of pregnancies andcauses significant maternal and fetal morbidity and mortality  andincl+ T lymphocytes, themajor cell population involved in the cell-mediated immune response, play a crucialrole in the development of PIH. Depending on the changes in differentcytokine-induced microenvironments, CD4+ T lymphocytes can differentiateinto T helper type 1 (Th1), Th2, Th17 or CD4+CD25+ regulatoryT (Treg) subsets performing inflammatory, regulatory or suppressor functions , indicating that Tregwas implicated in the development and progression of PIH. This provides insight intothe mechanism of PIH, providing Treg as a potential target for clinical treatment.However, the direct evidence of the role of PD-1/PD-L1 and the relationship betweenPD-1/PD-L1 and Treg in PIH requires further research. Accordingly, in this study, wehypothesized that PD-1/PD-L1 might alleviate PIH by regulating Treg differentiation.First, we explored cell quantities and function of Treg in PIH. Then, weinvestigated the relationship between the PD-1/PD-L1 pathway and Tregdifferentiation in PIH.The programmed cell death-1 (PD-1)/PD-ligand 1 (PD-L1) pathway is critical to immunehomeostasis. Recently, the relevance of Treg cells and PD-1/PD-L1 pathway incontrolling immune responses has been highlighted. Of note, PD-L1 enhances thestability of Treg cells and promotes Treg cell differentiation –15. ByAll study subjects were recruited from the Department of Obstetrics andGynecology at Anhui Provincial Hospital of Anhui Medical University. This studyincluded 20 normal pregnancies (NP) and 58 pregnant women suffering from PIHsyndrome including severe PE (n=20), mild PE (n=18), and HDCP (n=20). There wasno significant difference in age and gestational age between groups. Thepresence of PE was assessed according to ACOG guidelines . All pregnancies were singleton gestations, andnone of the participants had active labor at the time of enrollment and bloodsampling. Women with pre-existing renal diseases, chronic hypertension beforepregnancy, diabetes, recurrent miscarriage, disorders of the immune system, orusing immune suppressing medication were excluded from this study.This study was reviewed and approved by the Clinical Trial Ethics Committee ofAnhui Provincial Hospital of Anhui Medical University. All procedures werecarried out in strict accordance with the approved guidelines and regulations.Written informed consent was obtained from each participant prior to enteringthe study.Blood samples (5 mL) were obtained by venipuncture from severe PE, mild PE, HDCP,and NP women. Two milliliters was used for the preparation of serum, while theremaining 3 mL was heparinized for the isolation of peripheral blood mononuclearcells (PBMCs) by density gradient centrifugation. The serum was separated andstored at −70°C until required for cytokine determination using an enzyme-linkedimmunosorbent assay (ELISA). To illustrate the effect of the PD-1/PD-L1 pathwayon Treg cell differentiation, PD-L1 Fc  and anti-PD-L1 mAb  were added to the PBMCs from NPs and PIHpregnancies for incubation of 3 days. PBMCs were isolated for flow cytometry,quantitative polymerase chain reaction (qPCR), western blot, and ELISA. Allblood samples were obtained before the PE patients received treatments such assteroids or antihypertensive drugs.'sprotocol.The levels of transforming growth factor β (TGF-β), interleukin-10 (IL-10), andIL-35 in serum or cell culture fluid of PBMCs were measured with an ELISA kit, according to the manufacturer6PBMCs were resuspended and stained with anti-CD4-FITC monoclonal antibodies(mAbs) and anti-CD25-PE mAbs for surface antigens (eBioscience) according to themanufacturer's instructions. The cells were then permeabilized withpermeabilization/fixation buffer (eBioscience). The cells were resuspended in300 μL of PBS for subsequent flow cytometric analysis. Data were acquired usinga FACScalibur , and processed using the CellQuest program.Treg differentiation was performed by flow cytometry. In brief, 2×10+CD25+ Regulatory T Cell Isolation Kit, with a Midi&Mini MACS instrument (Miltenyi Biotec)following the manufacturer's instructions.Treg cells were isolated from PBMCs of the women by multi-step magnetic sortingusing a human CD4−ΔΔCT, andGAPDH was used for normalization.Total RNA was extracted and purified using TRIzol reagent , andan equal amount of total RNA (1 μg) was used for cDNA synthesis . The primer sets used in this study were designed using Primer-BLASTsoftware and were as follows: PD-1 Forward: 5′-ACCCTGGTGGTTGGTGTCGT-3′, Reverse:5′-CCTGGCTCCTATTGTCCCTC-3′; PD-L1 Forward: 5′-TTTGCTGAACGCCCCATA-3′, Reverse:5′-TGCTTGTCCAGATGACTTCG-3′; Foxp3 Forward: 5′-CACTGACCAAGGCTTCATCTG-3′, Reverse:5′-GGAGGAACTCTGGGAATGTG-3′; GAPDH Forward: 5′-GGACCTGACCTGCCGTCTAG-3′, Reverse:5′-GTAGCCCAGGATGCCCTTGA-3′. The cDNA (2 μL) was subjected to qRT-PCRamplification analysis using SYBR Green PCR mix . Theamount of target relative to a calibrator was computed by 2Protein was isolated from Treg cells that were lysed in radioimmunoprecipitationbuffer (RIPA) containing protease inhibitors at 4°C for 30 min. Cell lysateswere prepared with a RIPA lysis buffer kit , Treg cell:lysis buffer of 1:2 (v:v), and the protein concentrations werequantified using a Bio-Rad protein assay .Subsequently, equal proteins were separated by 10% SDS-PAGE gels and transferredonto PVDF membranes . After blocking with 5% fat-free milk,primary antibodies against PD-1  and PD-L1 were added, followed bysecondary antibody horseradish peroxidase-conjugated goat anti-rabbit IgG. GAPDHwas used as the loading control. The protein was detected with an enhancedchemiluminescence kit  and the band intensity wasquantified with Image-Pro Plus 6.0 software.'s t-testbetween two groups and reported as means±SD. All experiments were repeated atleast three times. P<0.05 was considered statistically significant.All statistical analyses were performed using SPSS 16.0 . Thedata were analyzed using StudentTreg cells maintain immune homeostasis and tolerance by cell-to-cell contact andby secretion of cytokines ,18. As sFlow cytometry analysis showed that, similar to the expression pattern ofTreg-related cytokines, the percentage of Treg cells in isolated PBMCs of HDCP,mild PE, and severe PE groups was significantly reduced compared with the NPgroup. Moreover, the percentage of Treg cells in isolated PBMCs of severe PEwomen was significantly decreased compared with that of HDCP pregnancies . FurtherThe mRNA and protein levels of PD-1 and PD-L1 were assessed by qRT-PCR andwestern blot, respectively. As shown in We found that compared with NP, HDCP showed significantly decreased Tregexpression in PBMCs. However, PD-L1 Fc elevated the reduced Treg expression inPBMCs of HDCP. In contrast, anti-PD-L1 mAb further reduced Treg expression inHDCP . SimilarIn line with the above results, HDCP showed reduced concentration of Treg-relatedcytokines in cell culture fluid of PBMCs compared with NP. Similarly, theseTreg-related cytokines in PBMCs were increased with PD-L1 Fc treatment anddecreased with anti-PD-L1 mAb .+CD25+ cells. With suppressive capacity and regulatoryfunction, Treg cells are major contributors to the tolerance maintenance of thefetus and the maintenance of normal pregnancy (Treg cells are a specialized subset of T cells characterized byCD4regnancy ,20,21. Tregnancy . TGF-β, regnancy .Accumulregnancy ,25. For regnancy . Consist+ Tregpercentage might account for the reduction of Treg cells in PE (PD-1 suppresses signaling through the T-cell receptor, resulting in reducedproliferation, cytokine production, and cytotoxic activation of T cells. PD-1 isconsidered a dominant responsive inhibitory receptor among all of the inhibitoryreceptors expressed in T cells . Apartfls in PE . Thus, w+ T cells in the presence ofanti-CD3 and TGF-β (As an activator of PD-1/PD-L1 pathway, PD-L1 Fc has been reported to significantlyalleviate symptoms and suppress disease progress in mice suffering from auto-immunedisorders by promoting the development of Treg cells . PD-L1 Fnd TGF-β . Furthernd TGF-β . As expe+ T lymphocytes can differentiate into the Th1, Th2, Th17 orCD4+CD25+ Treg subsets (+CD25+ T lymphocytes to specify Treg cells in thisstudy. Although we have detected mRNA expression of Treg-specific transcriptionfactor Foxp3 in isolated PBMCs of NPs, HDCP, mild PE, and severe PE groups, whichcould indirectly reflect the expression of Treg cells, other studies have usedCD4+CD25+ Foxp3+ T cells to specify Tregsubsets. Thus, this might be one of the limitations of this study. Furthermore,recent data showed that CD28 is a major target for PD-1 inhibition both invitro and in vivo (+ T cells, or Tregcells in PIH patients compared to NPs. These limitations should be investigated inour future study.As CD4 subsets ,7, we us in vivo ,32. In tIn summary, a reduction of Treg cells was associated with the pathogenesis of PIH.Interestingly, PD-1/PD-L1 pathway promoted Treg cell differentiation in PIH,indicating a potential therapeutic value of PD-L1 Fc for PIH treatment.


Have patience with everything unresolved in your heart and … try to love the questions themselves as if they were locked rooms or books written in a very foreign language. Don’t search for the answers, which could not be given to you now, because you would not be able to live them. And the point is to live everything. Live the questions now. Perhaps then, someday far in the future, you will gradually, without even noticing it, live your way into the answer.– Rainer Maria RilkeI entered university brimming with idealism and the desire to help. My premedical studies only partially satisfied this desire. I wanted to go beyond North America and do global health work; my heroes were Paul Farmer and other pioneers of social medicine. Motivated by their examples, I applied for some research funds from my university, and at 21 years of age found myself heading for Kampala, Uganda to gather data for an ambitious qualitative research project, on university students’ attitudes towards prevention of HIV/AIDS. Immediately after I touched down at Entebbe Airport I was confronted with some uncomfortable realities. I noticed the privilege and financial security I enjoyed compared to my Ugandan research subjects – who, as university students, were also my peers. I marveled at the incredible opportunity I had to do cross-cultural research as a mere university student, and realized my audacity in attempting this work despite my relative lack of experience and training. I was also acutely aware of the political and moral implications of my research topic. As I was embarking on this research, the reasons behind the success of Uganda’s HIV/AIDS prevention strategy were being hotly debated by the international academic community, large international funding organizations, and the Ugandan public.When, two months later, I unpacked my bags back home in Chicago, I found that my uncomfortable questions had travelled home with me as well. Ultimately, these questions left me so tangled in self-doubt and uncertainty that I was unable to finish the research, which then led to a sense of unfilled moral responsibility to my Ugandan friends. I feared that I had done more harm than good – primarily by wasting time and resources better spent elsewhere. I have since realized that my experience was not unique amongst learners from North America travelling to the Global South for research or educational opportunities. Hanson et al.So I packed my research notebooks away, and shelved my questions as well, apologizing to my Ugandan friends for leaving the project unfinished. I resolved to focus on local work and imagined that answers would come more easily in that setting. Eventually this work led me to medical school. Medical training promised a method of finding answers, through careful empirical observation and application of the best evidence to the patient problem at hand. I learned to search for randomized controlled trials or, better yet, meta-analyses, to aid my clinical decision-making. Yet, I remained perplexed. I encountered many patients that did not experience meaningful improvements in their health despite evidence-based care. Worse, there were many complex patients for whom no evidence seemed to apply.6My questions did not abate when I began my residency training in psychiatry. I learned many evidence-based interventions for psychotic disorders, but the problem of how to help a chronically homeless, socially isolated person with schizophrenia presenting to the ER was often more complicated than a reductionist meta-analysis could address. It seemed that the empirical evidence, while helpful, was not enough.Somewhat to my surprise, I found the questions that I faced daily in my practice drawing me yet again to global health. I was intrigued by reports of better outcomes of severe and persistent mental illness in lower/middle-income countries,Soon after I began residency, our department was invited by colleagues at Mbarara University of Science & Technology (MUST) in western Uganda to support their emerging postgraduate psychiatry program. This partnership, inspired by the educational collaboration between the psychiatry departments of Addis Ababa University and the University of Toronto,As I became involved in our partnership planning, all of my old questions and misgivings emerged: the fear of doing harm; mistrust of my own motives; concern about reinforcing existing unjust hegemonies. I realized that my questions about global health were intertwined with my questions about psychiatry. I wondered if the questions themselves could be a starting point for further engagement rather than a barrier to participation.I confess that I am still often confused about how to ethically engage in global health work, and find that over time my questions are only deepening and multiplying. The words of RilkeI am partially driven by a sense of moral responsibility and an awareness of my privilege in having received an expensive and lengthy education. I look to the available global health literature for guidance on ways to do more good than harm.In the end, however, the most persistent motivators are the same which fuel my clinical work in Canada: stories and relationships. The rich stories of our Ugandan colleagues, of Ugandans living with mental illness, and of our emerging partnership itself are a salve for the painful uncertainty of unanswered questions.


Raman spectroscopy, scanning electron microscopy, high resolution transmission electron microscopy, X-ray photoelectron spectroscopy and Near Edge X-ray-absorption fine-structure spectroscopy were used to probe the morphological, chemical and microstructural features of the produced material. The method described here is scalable and show a potential for controllable, large-scale fabrication of other graphene derivatives and promotes microwave plasmas as a competitive, green, and cost-effective alternative to presently used chemical methods.One of the greatest challenges in the commercialization of graphene and derivatives is production of high quality material in bulk quantities at low price and in a reproducible manner. The very limited control, or even lack of, over the synthesis process is one of the main problems of conventional approaches. Herein, we present a microwave plasma-enabled scalable route for continuous, large-scale fabrication of free-standing graphene and nitrogen doped graphene sheets. The method’s crucial advantage relies on harnessing unique plasma mechanisms to control the material and energy fluxes of the main building units at the atomic scale. By tailoring the high energy density plasma environment and complementarily applying  Research conducted both on fundamental and application levels of graphene and its derivatives led to an impressive number of scientific reports on the topic e.g., refs 8. Preserving graphene’s large electric double-layer capacitance, N-graphene offers improved wettability and pseudocapacitance7. Lower resistivity and a specific capacitance of 282 Fg−1 at 1 Ag−1 was reported for N-graphene based supercapacitor’s electrode with excellent lifetime , high power capability, and compatibility with flexible substrates9. By influencing atomic charge distribution N atoms create “activate sites” on the graphene scaffold that can participate in catalytic reaction directly, such as the oxygen reducing reaction in fuel cells or anchor metal nanoparticles used in the catalytic reaction7, making N-graphene a potential substitute for expensive metal catalysts. The use of N-doped graphene also improves the rate capabilities of graphene-based electrodes in lithium-ion and lithium-sulfur (Li-S) batteries. It exhibits strong-couple interactions for anchoring sulfur-containing species, thus achieving higher stability and reversibility when compared with pristine graphene11, with an expected specific energy density of ~600 W∙h∙kg−1. Additional reviews on the progress of graphene/N-graphene and graphene based materials for energy storage and conversion devices are presented in14.Carbon-based 2D nanostructures have attracted an outstanding research interest due to their extraordinary properties15. Graphene’s properties vary strongly as a function of its fabrication method and many of graphene and N-graphene’s outstanding properties are drastically dependent on its structural quality, i.e., sp3 carbons, defects, etc. Currently available commercial products (e.g.: https://graphene-supermarket.com/) exhibit a large variability of their physical properties, which substantially affects their usability.One of the greatest challenges on the path towards graphene commercialization is the production of high quality material on a large scale at low cost and in a reproducible manner3, wet chemistry reduction techniques that employ graphite oxide (GO)16, liquid-phase exfoliation17 and chemical vapour deposition (CVD)19. Generally, these methods allow the synthesis of large-area graphene films and micron/submicron sized graphene sheets. CVD allows the production of large area sheets , but its efficiency depends on the quality of the underlying polycrystalline metallic film  and it requires multiple processing steps to obtain transferable sheets19. Furthermore, CVD in the presence of carbon and nitrogen containing precursors  is the most common direct method to prepare N-graphene20 on a nickel or copper substrate at a temperature of 1000 °C. It is worth noting that the presence of metal impurities can affect the real performance of the synthetized material. N-graphene sheets can also be produced via solvothermal methods21 and using an arc discharge with graphitic electrodes22. Post-synthesis methods such as thermal and chemical reduction of GO, along with simultaneous N-doping25 and plasma treatment of graphene27 are also applied to produce N-graphene.Multiple processes have been reported for graphene synthesis, including mechanical exfoliation of natural or synthetic graphite30, N-graphene like films33 on various types of substrates , dielectric  and 2D substrates (h-BN)) at lower temperatures, nearly all of the current plasma techniques involve plasma enhanced chemical vapor deposition (PECVD)34. The presence of energetic electrons in plasma environment boost the ionization, excitation and dissociation processes of carbon precursors at relatively low temperatures36. The plasma systems comprise thermal and chemical reactor functions, as well as catalytic properties. Therefore, plasma assisted growth of nanostructures can be achieved without using catalysts at lower substrate temperatures due to the plasma’s unique ability to activate the surface, thus creating favourable conditions for nucleation and growth processes37. Unique aligned growth of vertical graphene on Ni foam and silicon support has been achieved39. However, PECVD methods typically require low-pressure environments (<10 Torr) with the synthesis and growth of the structures occurring as in a conventional CVD proceed via reactions on the surface which depend on the substrate properties, often with low growth rates34. These methods struggle in producing high quality continuous and uniform graphene/N-graphene films on large areas.While promoting controllable, catalyst-free growth of graphene13. Such self-standing graphene sheets are an alternative to surface-supported horizontal graphene because both surfaces can be utilized, while surface bound counterparts can effectively only use one, and allow the use of at least three open edges in applications. Some analysts foresee that free-standing sheets will dominate the market by 2025 (http://www.idtechex.com). The approaches commonly used to generate free standing graphene sheets are not focused on plasma methods, such as the ones based on colloids or thermal/chemical exfoliation of GO17. While being recognized as a versatile and low-cost method for the preparation of graphene sheets at a relatively large scale, chemical reduction of GO (widely used for batteries/supercapacitors’ electrodes preparation) requires pre-production of GO from natural graphite via Hummers method and relies on the use of toxic chemical agents. The final products also exhibit a moderate electrical conductivity 42. To the best of our knowledge, only a couple of examples in the literature focus on the production of isolated free standing graphene sheets using plasmas at atmospheric pressure conditions. In the work of Dato43 free-standing graphene sheets synthesis using microwave plasma is reported without justification for the process that is simply referred as gas-phase synthesis. Moreover, the synthesized carbon material contains a large amount of amorphous carbon along with some individual graphene sheets as described in a related patent44. Selective synthesis of only high quality free-standing graphene sheets has been reported in our previous works but this was at the expense of quantity, i.e. at very low yield of about 0.01 mg/min47.Many applications  require graphene nanosheets in the form of free-standing graphene/N-graphene sheets consisting of a few atomic monolayersin situ infrared (IR) and ultraviolet (UV) radiation are applied. Besides Hirracane Cyclone system (http://www.acsystems.pt) is used for graphene sheet collection. With the patent describing the method recently issued, it becomes necessary to further investigate the scale-up of the process as well as a direct, single step N-graphene synthesis. The possibility to tune the density and energy of the building units, i.e., C2 radicals, C and N atoms, in the high energy-density plasma environment, which translates in an effective control over the energy and material fluxes towards growing nanostructures in the assembly zone of the reactor, is the most crucial advantage of this method. The plasma properties have been tailored to achieve selective synthesis of free-standing graphene/N-graphene sheets. Scanning electron microscopy (SEM) and high resolution transmission electron microscopy (HRTEM) as well as X-ray photoelectron spectroscopy (XPS) and Near Edge X-ray-absorption fine-structure (NEXAFS) spectroscopy48 were used to study the morphological, chemical and microstructural features of the synthesized nanostructures. The effect of N doping on the sample structure was studied by Raman and Fourier Transform Infrared (FT-IR) spectroscopies and XPS. Van der Pauw method was also applied to determine the electrical conductivity of the assembled graphene sheets49.Here we intend to provide substantial evidence that microwave plasma technologies, and in particular these based on wave-driven plasmas, can be used as a competitive and disruptive alternative to chemical methods in the controlable fabrication of free-standing graphene sheets. The novelty of the plasma approach used is based on the process scale-up using large-scale configuration of microwave driven discharges and complementary engineering to control morphologies and structural qualities of targeted nanostructures. This is achieved via synergistic tailoring of the plasma environment and the “cold” outlet gas flow, where 46. The microwave power is provided by a 2.45 GHz generator, whose output power was varied from 500 to 2000W. The generated plasma has an extended active zone outside the wave launcher, as seen in Fig. http://www.acsystems.pt). A methanol glass container is connected to the exit of the Hurricane system to capture the smallest particles, capable to escape the system with the gas flow.A waveguide-surfatron based setup was used to create a surface wave (SW) induced microwave plasma at atmospheric pressure conditions 2H5OH, take place. The created radicals become precursors of chain reactions leading to creation of the main building blocks of carbon nanostructures, i.e. carbon atoms and C2 radicals50. It is worthy to note, that the assembling process requires very fast delivering and stacking of building units and equally fast supply of energy sufficient to overcome all the actual potential barriers. In the “mild” plasma zone, which includes also the “near” plasma afterglow, gas temperature drops from about 2000 K to 500 K. Note that the gas temperature changes also in radial direction, following closely a parabolic profile, due to the radial thermal losses. The transport of gas-phase carbon atoms/molecules into a colder zones through the so-called surface of vaporization  of the reactor results in formation of solid carbon nuclei that are gradually withdrawn in the outlet plasma stream where kinetic processes of assembly and growth of “flowing” carbon nanostructures take place. Nitrogen atoms can be incorporated within the carbon nuclei via careful plasma tailoring and their density can be effectively controlled by the percentage of N2 flux in the gas mixture51. Given the fact that the nucleation and growth processes are determined by the interplay of kinetic and thermodynamic factors, the engineering of structural qualities of targeted nanostructures was achieved via synergistic tailoring of the “hot” plasma environment and thermodynamic conditions in the “mild” zone of the plasma reactor. Furthermore, the flowing structures can be a subject of infrared  in the third, post-plasma zone and soft UV irradiation treatment, to “kick-off” the epoxy oxygen groups and sp3 carbons during the flight to a Hirracane Cyclone collecting system.The plasma reactor can be considered as the assemblage of three different zones, as presented in Fig. These three zones can be clearly distinguished in a photograph of the plasma reactor Fig. . The cor2, C) with sufficiently high energy that will then flow towards the “mild” plasma zone, i.e. the assembly zone of the reactor. Once in the larger volume, the departure from the supersaturation conditions of the environment, prone to foster nanosheets assembling, is preserved46. That is, the existence of a smaller amount of nuclei surpassing the critical size for nucleation and growth and conditions in which no supersaturation of species is possible promote the growth of existing nuclei rather than creation of new seeds. The reduction of Gibbs free energy of the system is behind the processes of nucleation and growth. This reduction of the Gibbs free energy of a saturated environment (e.g. plasma + solid phase) is proportional to the gas temperature. Lower gas temperatures, as well as larger densities of building units, foster supersaturation conditions in the environment and thus promote the synthesis of plenty nuclei and amorphous structures are more likely to be created. Contrarily, lower density of nuclei and higher temperatures lead to a deviation from supersaturation conditions, more prone to generate graphitic sheets. As expected, in the larger volume, the density of carbon nuclei decreases, and, as a result, the conditions favor the creation of planar nanostructures. Selective synthesis of graphene sheets only is achievable in a very narrow range of discharge operational conditions for the reactor design considered.Using a cylindrical reactor with expanding radius allows the injection of large power density in the smaller radius section to achieve larger fluxes of building units  and to determine important plasma parameters such as the gas temperature in the discharge “hot” zone, plasma emission spectroscopy has been applied. Optical emission spectroscopy permits to identify the active carbon species in the plasma that are the “building units” for the synthesis and growth of the carbon nanostructures in the mild plasma stream. The detected argon/ethanol plasma emission spectra in the visible range (240–750 nm) are shown in Fig. 2Σ → X2Σ), C2 , C atoms (247.9 nm), the hydrogen Balmer-alpha line Hα (6563 Å) and several Ar lines. These species are formed as a result of ethanol decomposition. As mentioned, the experiments were carried out in the open atmosphere so that some nitrogen containing species also appear. As it can be seen in Fig. 2 molecular band, i.e. the Swan system, with heads band at 473.7, 516.5 and 558.6 nm were detected. The typical C2 emission is generated by the radiative decay of the ext = 2.4 eV), ground state C2 molecules can easily be excited to this level either by electron impact or by three body recombination processes involving carbon (C) and argon (Ar) atoms50. CN species are formed in a three-body recombination reaction: C + N + Ar → CN*(B2Σ) + Ar. The inset in the Fig. To identify the species of interest  and precursor fluxes in the range 15–30 sccm, remains nearly constant in the central “hot” zone with variation from about 4000 K to 3000 K. It should be mentioned, that this temperature is associated with the axis of the “hot” plasma column, since the radiation collected originates mainly from this region. Having a much lower value close to the wall, the gas temperature exhibits a sharp radial gradient. The 2D map of the temperature as detected by an infrared-sensitive thermal imager (FLIR camera) is shown in Fig. The gas temperature in the “hot” plasma zone is a key parameter concerning ethanol decomposition. The collision rates in the plasma at atmospheric pressure are high enough to ensure local thermodynamic equilibrium. Therefore, the rotational temperature can be taken as a good approximation to the gas kinetic temperature. The emission band from the CN species corresponding to violet https://graphene-supermarket.com).The range of operational conditions, i.e. background and precursor flow, microwave power etc. fostering selective synthesis (only sheets) is very limited. Outside of this range different carbon allotropes, i.e. amorphous carbon, 2D sheets etc, at the same time can be synthesized. Pure graphene sheets collected at background argon flow of 1200 sccm, power of 2 kW and ethanol flow of 30 sccm are produced at a rate of 2 mg/min. Figure In Fig. −1 (in λL = 633 nm spectra) up to 1367 cm−1 (in λL = 458 nm spectra). Its relative peak intensity (according to the G line (−1 (in λL = 633 nm spectra) up to 2730 cm−1 (in λL = 458 nm spectra). Its relative peak intensity  the obtained results show that the samples contain graphene sheets with single or few mono-layers of carbon atoms56.Raman spectroscopy is the most efficient way to provide a quick and easy structural and quality characterization of the samples. Typical Raman spectra of the synthesized carbon sheets produced at 2 kW microwave power, 1200 sccm Ar flow and precursor flow 30 sccm are shown in Fig. In addition to the recorded Raman spectra, the powder deposited on TEM grids allow the identification of monolayer and multilayer sheets Fig. . Less tr2. Even though this is not the area ratio, the results give an indication about graphene sheets thicknesses and allow the calculation of the fraction of monolayers. For a set of randomly chosen samples, the percentage of counts of monolayers is about 40%. The investigated samples contained also bilayer, trilayer and higher order multilayer sheets. Approximately 15% of the counted multilayer sheets are composed by 5 to 10 monolayers while 10% contain 10 to 15 monolayers. The lateral size of the flakes is in the range 200–700 nm as seen from Fig. A number of HRTEM images obtained from different randomly chosen graphene samples regions were analyzed. Considering that graphene sheets are crumpled, overlapped and/or entangled, the area ratio between monolayers graphene and the total graphene area is very difficult to determine. Therefore, we counting the number of monolayers/bilayers/multilayers from visual observation of HRTEM images. Analyzing a number of graphene areas (at 5 different positions), we produced the statistics presented in Fig. 2 (284.4 eV), sp3 (285.2 eV) and C-O-C or C-OH (286.3 eV) bonds57, while the wide fourth peak represents π- π* shake up satellite (290.6 eV). Analysis of O 1s line fully support these findings, with the peak being well reproduced as the sum of aliphatic C-O-C bond (532.4 eV)57 and PbO (529.3 eV)58 contributions after taking into consideration different C 1s positions assumed for adventitious carbon . The representative XPS spectrum in Fig. bon Fig. . The latwall = 150 oC) was measured at the reactor’s outlet gas zone  and it is indicative of the amount of IR irradiation that is simultaneously applied . The combination of UV irradiation and modest heating (i.e. 150 oC) significantly increases sp2/sp3 ratio to about 15, while in the absence of both, annealing and UV irradiation, the same ratio is 10. This is probably due to the breaking of sp3 bonds by UV photons with an energy of about 3 eV (300–400 nm). The change in the amount of oxygen is, however, negligible (from 2.3% to 2.2%). At higher precursor fluxes (QEt = 36 sccm) and annealing temperature (260 oC), keeping the other operational parameters constant , the relative amount of oxygen is two times smaller (~1.1%), whilst the sp2/sp3 ratio (15) is preserved. Under these conditions, defects are activated and annealed out by reopening the bonds and letting them reorganize to the perfect “crystalline” structure. This can be achieved only for a limited range of temperatures. Thus, the control/tuning of the thermodynamic conditions in the third (gas phase) zone of the reactor results in additional structural quality improvements of “flowing” graphene sheets.The wall temperature  S/m, which is much higher than the reported values of graphene sheets powder obtained by chemical methods64 and comparable with a reported value65. This could be explained with highly conductive sheets having multiple interfaces between them.To measure the electrical conductivity of the graphene sheets, 0.1 g of the graphene powder was pressed into a disc of 8 mm diameter and 1.2 mm thickness. The electrical conductivity was measured applying Van der Pauw method at room temperature2 in respect to the background argon flow results in a high dissociation degree of N2 molecules therefore generating a significant amount of reactive nitrogen atoms (~1022 m−3) that can be incorporated into the growing carbon lattice structure. However, the dependence of N atoms density on gas mixture composition is to be noticed51. The rate of pure N-graphene sheets collected at a background argon flow of 1200 sccm, power of 2 kW, ethanol flow of 15 sccm and N2 flow of 5 sccm was about 0.5 mg/min. In the next Fig. In order to test the direct route for N-graphene synthesis as well as to demonstrate potential of the method, nitrogen gas flow (5–10 sccm) has been added to the ethanol flow. A small percentage of the N2 flow. Laser emitting at 633 nm has been used. As discussed, the three typical graphene peaks are attributed to the G band at ~1583 cm−1, 2D band at ~2658 cm−1, and D band at about ~1332 cm−1. The D band, i.e., “disorder” band, is related to a series of defects: bond-angle and bond-length disorder as well as hybridization that are caused by hetero atoms, i.e., nitrogen doping. The presented spectra are averaged over different locations. The most notable spectral change is the increase in the D band to G-band intensity ratio when N2 is included in the gas mixture. As seen from the figure, the D-peak intensity rises with the introduction of N2 gas. The nitrogen atoms constitute defects within the graphene lattice and consequently contribute to the D band intensity, yielding the observed increase of the D band to G-band intensity ratio.Furthermore, Fig. 2 carbon atoms bound to carbon or hydrogen atoms. The position of this peak was reported in our previous studies on free-standing graphene sheets synthesis induced by microwave plasma50 and was obtained with no need of binding energy (BE) correction since no charge accumulation exists in this sample. Peaks centered at higher BE are assigned to sp3carbon atoms bound to other carbons or hydrogen atoms (285.0 eV), carbon bound to nitrogen or singly bound to oxygen (peaks at 285.7 and 286.6 eV), carbon in epoxide and/or carbonyl groups (287.6 eV) and carbon in carboxylate groups (289.0 eV)57. The spectral features detected at BE >290 eV are energy losses due to π-π* excitations typical of carbonaceous systems with delocalized π electrons such as graphene. The peak centered at 289 eV may be overlapping some of these features. Peaks found around 286 eV, which are, in a first approach, identified as C-N and C-O, are most probably superposing some aliphatic carbon atoms (C) bound to other carbons (C-C<) from more electronegative vicinities, for instance C-COO− or C-(C = O) 57. XPS confirms undoubtedly the doping of graphene by nitrogen atoms. The N 1s region was fitted with two peaks, one centered at a binding energy equal to 399.4 ± 0.2 eV assigned mainly to pyridinic nitrogen57, and a minor peak centered at 401.2 ± 0.2 eV, corresponding to graphitic nitrogen66. Pyrrolic nitrogen, which is likely to exist in these structures, must be close to 399.9 eV67, i.e. between the two fitted components. The shoulder found around 398.6 eV (not fitted) can be attributed also to pyridinic nitrogen in a slightly different chemical environment than the N 1s main peak , and 1550 cm−1 in the spectrum of N-graphene, can be attributed to C-N stretching vibrations and N–H band, respectively72. Additionally, the most intense peak at 1390 cm−1 (C–O–H stretching vibrations) is detected along with a wider band at around 3000 cm−1 (C-H)76.FT-IR analysis was additionally employed to investigate the chemical bonding differences when graphene sheets are N doped Fig. 69. The Fsee Fig. 70, 71. IProcessing graphene sheets is a delicate task since the charged sheets can reply in an ambiguous way during their treatment. Two photos of graphene sheets suspended on a glass substrate are shown in Fig. −1 and slight decrease of the G and 2D peaks. Moreover, a small frequency shift of the D-line of about 2 cm−1 towards smaller wavenumbers along with widening of the line is observed. The enhancement of the D-line, both absolute and relative to the G and 2D lines, may be simply related to the cutting-up of graphene sheets. However, the process may also force the parallel orientation of the sheets in respect to the supporting dielectric surface and lead to the formation of a network of electrically-oriented connected graphene sheets. In this case, the reduced G and 2D peak intensity could be linked to an increase in the conductivity of the whole layer and a corresponding decrease of the scattering volume.The corresponding Raman spectra are shown in Fig. 77, is observed, and second dominant feature, the double-structured resonance, is observed at around 292 eV, corresponding to C 1s - σ*. This double resonance comes from excitonic , from defects in the top most graphene layers78 or O-containing functional groups, resulting from oxidation (as it is most probably in our case).To test this hypothesis, traces of graphene powder were mechanically smeared over silicon substrate (simple Si wafer as used for microelectronics) and analyzed by means of NEXAFS spectroscopy. Measurements were performed at different beam incident angles (θ) relative to the substrate surface as shown in Fig. 79. There is a clear angular dependence of the C1s → π* resonance peak at 285.2 eV, with its intensity increasing towards the plateau regions and towards the intensity of excitons, as the incident angle relative to the supporting surface decreases. The differences are strongly correlated with orientation of the bonds relative to the surface and therefore support the existence of self-organization of the graphene sheets with respect to the surface of the substrate. In graphene sheets, the π bond deriving from pz orbitals is oriented perpendicular to graphene plane (out-of-plane) and the σ bond is oriented along the intermolecular bonding axes (in-plane). Thus, as the angle between incident beam and surface increases out-of-plane excitation, i.e., π* resonance becomes weaker, and the ratio between σ* and π* resonances at 291.7 eV and 285.1 eV increases, as it is expected for highly oriented structures61 with a preferential orientation of the graphene sheets parallel to the substrate. It is to be noted that this alignment does not disappear over time or after functionalization by means of for example ammonia or nitrogen plasma.NEXAFS is a powerful tool to observe molecular orientations due to the high linearly polarized nature of the synchrotron radiation, specifically for 1s → 2p excitations, where the product of the orbital symmetry axis and the electric dipole leads to simple intensity correlations between incidence angle and molecular orientation of the radiation2/sp3 carbons ratio (~15) has been achieved. For a randomly chosen samples, the percentage of counts of monolayers in all graphene sheets is about 40%. The method is rapid, highly cost-efficient and environmentally friendly, since it does not require the use of catalysts and noxious chemicals. It is also customizable and versatile, allowing the synthesis of different types of 2D nanostructures  in the same reactor. Furthermore, the high energy density of the generated plasma environment allows the use of gaseous, liquid or solid precursors. Moreover, self-organisation of the graphene sheets in respect to the supporting surface after mechanical treatment was observed.In summary, microwave-driven plasmas were successfully applied for the first time in the selective synthesis of high quality graphene and N-graphene sheets via a single step process at atmospheric pressure. A high level of control over oxygen functionalities and spTo this end, our key enabling technology provides a rapid, single-step, cost-efficient and environmentally friendly method for selective synthesis of tailored graphene/N-graphene sheets at high yield and at atmospheric ambient. It is non-destructive, free of toxic chemicals, metal catalysts and substrates, and allows the use of carbon precursors in solid, liquid or gas state. The main advantage of our approach is the achivement of a very high and extremely controllable energy density in the plasma reactor, which allows effective control over the energy and material fluxes towards growing nanostructures at the atomic level via proper reactor design and tailoring of the plasma environment in a synergistic way. The ability to control the amount and localization of energy and matter delivered from the plasma bulk to the developing nano-structures is the key to achieve the desired morphological, structural and functional properties of targeted materials.Further scale-up along with better understanding of nucleation/growth processes in a multiphase environment will certainly lead to a substantial increase of the yield and a reduction of the production costs. Besides, mastering will be focused on reducing the multilayer (5–10 single layers) sheets percentage below 1%, controllable increasing of N-doping and optimization of collecting system for extraction of nanoparticles with dimensions less than 20 nm, i.e. graphene quantum dots. The use of large-scale configurations of wave-driven discharges paves the way for further scale-up and a higher level of customization. The exclusive plasma mechanisms that rule the distributions of energy and matter at atomic scale endow the method significant potential for the synthesis of other 2D materials . The method has also a potential for producing unique graphene-metal nanocomposities with well-designed nano-architecture. The expanding pool of prospect applications will most certainly continue to drive forward the research in this cutting-edge field.Supplemetary Material