The LNDR strongly depends on temperature (T) as well as on SQD size, and the T dependence is consistent with photocurrent (PC)-decay behaviors. With increasing light power, the PC-voltage curves are more structured with peak-to-valley ratios over 2 at room temperature. The physical mechanism of the LNDR, governed by resonant tunneling of charge carriers through the minibands formed across the graphene/SQDs:SiO2 MLs and by their nonresonant phonon-assisted tunneling, is discussed based on theoretical considerations.One of the interesing tunneling phenomena is negative differential resistance (NDR), the basic principle of resonant-tunneling diodes. NDR has been utilized in various semiconductor devices such as frequency multipliers, oscillators, relfection amplifiers, logic switches, and memories. The NDR in graphene has been also reported theoretically as well as experimentally, but should be further studied to fully understand its mechanism, useful for practical device applications. Especially, there has been no observation about light-induced NDR (LNDR) in graphene-related structures despite very few reports on the LNDR in GaAs-based heterostructures. Here, we report first observation of LNDR in graphene/Si quantum dots-embedded SiO Their device performances, however, are still far below commercial standards. For example, the quantum efficiency, on/off ratio, and responsivity of SQD PDs are currently as low as ∼1% at 530 nm/5 V23567Si is a principal material in semiconductor industries, but is of limited use in its photonic device applications due to its small- and indirect-bandgap nature. Si quantum dots (SQDs)235679102 (SQDs:SiO2) multilayers (MLs)-heterojunction tunneling diodes2 MLs heterojunction diodes.Since the advent of graphene in 2004, its high optical transparency, large carrier mobility, and easy tuning of work function have made it play key roles as transparent electrodes and others in various kinds of graphene-based device structures such as heterostructures with two-dimensional materials1315172 MLs heterojunction tunneling diodes. The LNDR behaviors strongly depend on SQD size (d), temperature (T), and irradiance power (P), and are well explained by resonant tunneling of charge carriers through the miniband formed across the graphene/SQDs:SiO2 MLs and by their nonresonant phonon-assisted tunneling. The T dependence of the LNDR properties is consistent with that of the lifetimes found in photocurrent (PC)-decay curves. As P increases, the photo I-V curves are more structured with the peak-to-valley ratios from ∼1.5 to ∼2.2 at room temperature, possibly originating from electric-field screening due to space charge buildup and state filling.Negative differential resistance (NDR) has a long history as one of the important tunneling phenomena not only under dark202122242527282931342 MLs tunneling diodes, composed of SQDs:SiO2 MLs with a total thickness of ∼100 nm, between monolayer graphene sheet and n-type Si wafer  SiO2 barriers, and broaden into minibands (extended Bloch-type states) if the mean free path of the carriers exceeds the ML period, as shown in 2 MLs become localized in the SQDs along the direction perpendicular to the layers and conduction proceeds by phonon-assisted tunneling (hopping between adjacent wells)V > 0 on graphene), the barrier oxide field increases linearly with the applied electric field2 MLs from the substrate (n-Si) to graphene by electron tunneling through minibands, thereby producing a dark current (DC). The rapid increase of current at larger d indicates the current is mainly governed by the tunneling probability that is enhanced at larger d due to the reduced barrier (SiO2) thickness, as shown in V < 0) the hole carriers give rise to the main current through phonon-assisted-tunneling conduction  curves under reverse bias for various SQD sizes. Note that the negligible PC is observed for the forward bias. This can be understood in terms of the slow transit of the electrons in the region of the SQDs:SiO2 MLs 2122T as well as d. In contrast, such RTD behaviors are not observed in dark I-V curves under forward bias as well as under reverse bias, irrespective of T and d  until the voltage reaches the valley voltage VV, as indicated in VV, the current through the device increases again as in conventional diodes. The T dependence of the peak voltages for each d are summarized in The most striking feature of the measured PCs in  T and d , possiblI-V curves more structured with peaks and valley were obtained for d = 2.5 nm, as shown in P-dependent monotonic shift of VP, as shown for d = 3.4 nm in d = 2.5 nm, the 1st peak voltage VP1, the 2nd peak voltage VP2, and VV are indicated in P-dependences are summarized in The NDR behaviors seem to strongly depend on the density of charge carriers, judging from no finding of the phenomenon without illumination. And there have been several reports on the dependence of the NDR behaviors on the carrier density in III-V superlattices25To probe the charge-transfer dynamics, transient PCs were recorded for various bias voltages and temperatures, as shown in 2 MLs structure, as explained above. Since at the valley voltage VV the electron wave functions become strongly localized within each SQD region, the overlapping of the wave functions decreases and the current is therefore reduced, which results in NDR. The threshold condition for the decrease in the overlapping of the wave functions is given approximately by eVd ~ ΔE. Here, Vd is the voltage drop across the ML period  and ΔE is the width of the miniband. Thus, the peak voltage VP ≈ ΔEN/e. As a result, the NDR in the SQDs:SiO2 MLs occurs when the potential energy difference between two adjacent SQDs in the MLs exceeds ΔE, which corresponds to the transition from band-like conduction to phonon-assisted-tunneling (hopping) conduction between the localized states of the SQDs. Clearly, this gives rise to NDR because the spatial overlap between the states of neighboring QDs decreases with increasing bias voltage. For example, no NDR is observed at temperatures below 200 K for the smallest SQD system (d = 2.1 nm), as shown in V curves show well-defined NDR behaviors. This phenomenon can be explained by the decrease of the recombination lifetime in the non-resonant tunneling regions at high temperatures.The observed NDR phenomena can be understood by considering the miniband formation in the SQDs:SiOE(k) = E0 [1 − cos(kds)], where 2E0 (=ΔE) is the band width and ds is the average distance between SQD layers . E0 (=ΔE/2) is proportional to the tunneling probability (TP) through the barrier which can be approximated by TP ≈ exp Vb is the barrier height and w is the width of the potential barrier. For larger w, TP is exponentially suppressed, as described in E becomes smaller. As summarized in VP increases with increasing temperature T for the four different SQD sizes. The current increases with increasing the bias voltage up to VP by the miniband conduction, as shown in VP to VV, the current decreases through the phonon-assisted tunneling. The resonance through the miniband occurs again at a higher bias voltage, resulting in a current increase above VV, consistent with the results in T is increased, the phonon scattering becomes stronger due to larger population of optical phonons at higher T. The phonon-assisted tunneling gives rise to the shift of VP, which is more sensitive to T at smaller d, as shown in d. The PC decay time or the life time decreases with increasing the temperature due to the enhanced phonon-assisted tunneling, as shown in T-dependent variations of the peak voltages, as shown in The energy dispersion relation of the miniband is given by the simple one-dimensional model as P, the plateau between VP1 and VP2 shrinks, and finally, it disappears as the two peaks merges at P = 3.0 mW. In contrast, the slope between VP2 and VV expands as P increases. The peak (VP2)-to-valley (VV) ratio varies from 1.57 to 1.84 in the range of P between 0.5 and 2.5 mW. These evolutions suggest that a blocking process in carrier transport exists around extrema VP1, VP2, and VV under high carrier densities. The shape of the photo I-V curve as a function of P is thought to be strongly affected by electric-field screening due to space charge buildup and state filling25VP1corresponding to the resonant tunneling through the miniband moves toward a higher voltage as the excited carrier density is increased. Simultaneously, valley VV, which is thought to correspond to increasing nonresonant phonon-assisted tunneling, also moves towards higher bias voltage. These behaviors can be explained by the electric-field screening generated by the space charges. Even when the carrier transport may be dominated by the resonant-tunneling-transfer path, it is considered that the carriers cannot enter into the miniband states in adjacent SQD filled by the optical excitation at point VP1. This will saturate the current up to VP2. Note that the position of VP2 is almost invariant. This means that the state filling is reached faster when the carrier density is higher, thereby causing such a pinning of VP2 for various light powers. The P-dependent monotonic shift of VP for d = 3.4 nm, as shown in VP)-to-valley (VV) ratio varies from 1.53 to 2.21 in the range of P between 0.5 and 3.0 mW.As shown in In these tunneling diodes we use graphene as an electrode. In addition to the electronic/optical advantages of graphene, graphene as a transparent electrode plays an important role in the observation of NDR in photo I-V curves. Unlike the devices with metal electrodes, the tunneling diodes with graphene electrodes give rise to the NDR behaviors in the wide range of light wavelength if the photon energy is greater than the band gap of SQDs. We also mention that the NDR behaviors can be adjusted by tuning the Fermi energy of graphene, which can be achieved by doping (or gating). The density of states of graphene affects the peak to valley ratio because the density of states of graphene is zero (or minimum) at the Dirac point. The shift of Dirac point with varying the applied bias gives a strong effect on the observed current because the current directly depends on the density of states.The achievements in this work  will enhance the possibilities that the heterojunction diodes can be employed in optoelectronic devices such as optical modulators, optical switches, optical memories, and photodetectors for imaging, sensing, recording, and communications.x/SiO2 MLs with 50 periods of 2 nm thin layers were grown on n-type (100) Si wafers at room temperature (RT) using an Ar+ beam with an ion energy of 750 eV and a Si target under oxygen atmosphere in a reactive ion beam sputtering system with a Kaufman type DC ion gun . HRTEM proved regularly-distributed SQDs within SiO2 matrix  in a graphite-heater-based chemical-vapor-deposition quartz tube furnace at a growth temperature of 1000 oC with 10-sccm H2 and 20-sccm CH4 flowing at a pressure of 3 Torr412 MLs/n-type Si wafers and blow-dried with dry N2. The PMMA/graphene/SQDs:SiO2 MLs/n-Si stack was then heated on a hot plate in air at 180 oC for 2 h to cure the PMMA. After the samples were cooled to RT, the PMMA was stripped by soaking them in acetone for 1 h at RT. Subsequently, the graphene/SQDs:SiO2 MLs/n-Si stack was put in isopropyl alcohol for 10 min and dried by blowing N2 to minimize the water traps possibly present at the graphene/SQDs:SiO2 MLs interface, and annealed at 400 oC for 1 h in vacuum to remove the surface adsorbates2 MLs and the graphene/SQDs:SiO2 MLs, respectively by AFM height profiles (G/I2D) of the G and 2D bands peaked at ~1585 and ~2683 cm−1, respectively, was about 0.47 on the graphene/SQDs:SiO2 MLs  was used to measure the average power of the laser pulses. The laser pulse temporal profiles and transient PC were monitored with the 50-ohm-terminated, 500-MHz–bandwidth digital oscilloscope (Tektronix DPO 4054)How to cite this article: Lee, K. W. et al. Light-induced negative differential resistance in graphene/Si-quantum-dot tunneling diodes. Sci. Rep.6, 30669; doi: 10.1038/srep30669 (2016).


Programmed cell death-ligand 1 (PD-L1) and driver mutations are commonly seen in non-small-cell lung cancer (NSCLC). However, the prevelance of PD-L1 over-expression and its prognostic value in Epstein–Barr virus (EBV) associated pulmonary lymphoepithelioma-like carcinoma (LELC) remains poorly understood.A total of 214 NSCLC patients and 113 surgically treated pulmonary LELC patients were included. Paraffin-embedded tumor sections were stained with PD-L1 antibody. Correlations between PD-L1 expression and clinicopathological features as well as survival outcomes were analyzed.p = 0.008). Multivariate analysis shows that PD-L1 expression level, N stage and M stage were independent prognostic factors for DFS. N stage and M stage but not PD-L1 expression level were significantly associated with overall survival (OS).The frequency of PD-L1 over-expression in NSCLC was 51.4%. No significant association was observed between common driver mutations and PD-L1 over-expression. Remakably, the positive rate of PD-L1 in pulmonary LELC was 74.3%. High PD-L1 expression was associated with impaired diseas-free survival (DFS) compared with low PD-L1 expression (PD-L1 over-expression was not related to common driver mutations in NSCLC. Pulmonary LELC have remarkably high incidence of PD-L1 expression. PD-L1 was a negative prognostic factor for DFS in surgically resected pulmonary LELC. These findings may provide a rationale for immunotarget therapy in this virus-associated lung cancer. Lung cancer is a leading cause of cancer-related mortality worldwide . Non-smaAnother less understood etiology of NSCLC is Epstein–Barr virus (EBV) infection . This raIn vitro studies have shown that driver mutations not only directly promote the proliferation of cancer cells but also indirectly induce immune evasion via the up-regulation of PD-L1 [Recently, immunotherapy has intensively been studied in a variety of cancers and is emerging as a promising treatment option . Evolutiof PD-L1 . Howeverof PD-L1 –12. Alsoof PD-L1 –16. HoweTherefore, the present study aimed to prospectively explore the association between PD-L1 expression and common driver mutations in NSCLC. Moreover, we investigated the prevalence and prognostic role of PD-L1 in a large cohort of surgically resected pulmonary LELC.To avoid selection bias, the first cohort prospectively enrolled 214 non-selective NSCLC patients. Baseline characteristics of these patients are presented in Table p = 0.034), tumor differentiation (p < 0.001) and gender (p = 0.010). However, no significant association was observed between PD-L1 expression and age (p = 0.398), smoking status (p = 0.372), stage (p = 0.548), EGFR mutations (p = 0.611), ALK rearrangements (p = 0.099) or KRAS mutations (p = 0.199). The most striking phenomenon was the PD-L1 expression in pulmonary LELC. In the 11 pulmonary LELC patients enrolled, 10 (90.9%) of them demonstrated PD-L1 positivity with a median H-score of 150 . Pulmonary LELC showed 9 times higher chance of having PD-L1 over-expression than non-LELC did . The remarkable phenomenon led us to expand this cohort of patients to study the overall prevalence and prognostic role of PD-L1 in pulmonary LELC.For PD-L1 immunohistochemical staining, we defined cases with more than 5% expression as positive ones. Thus, a total of 110 (51.4%) patients were positive for PD-L1 with a median H-score of 15 . The correlation between PD-L1 expression and patients' characteristics is shown in Table The second cohort involved 113 consecutive pulmonary LELC patients who were surgically treated in Sun Yat-sen University Cancer Center. The baseline characteristics of these patients are shown in Table p = 0.004). T stage was also significantly associated with PD-L1 over-expression. Other clinicopathological variables, including gender, smoking history, lymph node stage (N stage), metastasis and pathological stage were not significantly associated with PD-L1 over-expression. Due to the rare mutation rate of EGFR, ALK and KARS, the assessments of the association between PD-L1 over-expression and driver mutations are infeasible.As is shown in Table p = 0.008)   and those with high PD-L1 expression (H-score > 30). Kaplan–Meier analysis revealed that patients with high PD-L1 expression had shorter DFS than those with low PD-L1 expression . For OS, advanced N stage and advanced M stage remained the independent risk factor for poor prognosis Table .p = 0.008). The mutation rate in common driver genes including EGFR, KRAS and ALK was extremely low in pulmonary LELC. These results imply that pulmonary LELC is a very distinct subtype of lung cancer in terms of genetic aberrations and immune status.The present study reveals that the overall incidence of PD-L1 over-expression in non-selective NSCLC patients was 51.4% (110/214). There was no significant association between common driver mutations  and PD-L1 over-expression. However, PD-L1 over-expression was significantly associated with gender, tumor differentiation and pathological types. Remarkably, in pulmonary LELC, a rare subtype of primary lung cancer that is closely related to EBV infection, we found a very high incidence of PD-L1 over-expression . This phenomenon led us to expand this cohort of lung cancer and to investigate the clinical significance of PD-L1 in pulmonary LELC. In the expanded cohort involving 113 surgical LELC samples, the incidence of PD-L1 over-expression was 74.3%, which was consistent with the study by Chang et al. . High PDThe incidence of PD-L1 over-expression in NSCLC has been reported to vary from 19.6% to 65.9% and its association with clinicopathological features as well as patients' prognosis is very controversial –21. In Cin vitro studies provide solid evidence that PD-L1 could be regulated by oncogenic pathways. However, in tissue level, the association between PD-L1 and driver mutations is rather controversial. In the present study, we found no significant association between PD-L1 expression and EGFR mutations, KRAS mutations or ALK rearrangements, consistent with a previous study by Yang et al [in vivo studies on the association between oncogenes and PD-L1 regulation. In addition, the expression patterns of PD-L1 deserve deeper investigation. Yet some studies have shown that PD-L1 was positively associated with driver mutations in NSCLC samples [PD-1/PD-L1 pathway has been recognized as a key mediator of immune suppression . Cancer ng et al . Howeverng et al . One pos samples , 30. TheAnother mechanism of PD-L1 up-regulation is called adaptive immune resistance. In cancers with chronic virus infection, the anti-cancer and/or antiviral immune response could induce the secretion of important inflammatory factors like interferon gamma (IFN-γ), which may be utilized by cancer cell itself to maintain immune suppressive milieu. Our previous study have indicated that the up-regulation of PD-L1 in EBV infected NPC was partly mediated by IFN-γ. Pulmonary LELC is a rare subtype of primary lung cancer that is morphologically indistinguishable from NPC. It is also closely related to EBV infection indicated by positive in situ hybridization of EBERs and is characterized by intensive lymphoid infiltration. Unlike lung adenocarcinoma, pulmonary LELC rarely presents with EGFR mutations, KRAS mutations or ALK rearrangements. In a study by Liang et al. , they foAll in all, we show here that PD-L1 expression was not related to common driver mutations in NSCLC tissues and PD-L1 over-expression is a hallmark of pulmonary LELC. We further propose that the PD-1/PD-L1 axis might play a critical role both in the persistent infection of EBV and the resistance to immune elimination during malignant progression in pulmonary LELC. These findings extend those recently reported in NPC, hepatocellular carcinoma, which are virus-associated cancers and has also been considered as “immunogenic.” Our studies support a rationale for administering anti-PD-1 and anti-PD-L1 therapies to the EBV infected pulmonary LELC population. Future studies will need to characterize the genomic signatures of this tumor and identify additional factors responsible for inducing PD-L1 expression and hence immunosuppression within the tumor microenvironment.This study enrolled two cohorts of NSCLC patients. The first cohort prospectively enrolled newly diagnosed NSCLC patients in Sun Yat-sen University Cancer Center (SYSUCC) from January 2014 to December 2014. Patients who were pathologically diagnosed as NSCLC and provided sufficient tumor tissue for genomic analysis were included. The second cohort focused on surgically resected pulmonary LELC. NSCLC patients who were surgically treated in SYSUCC between January 2008 and December 2012 were retrospectively reviewed. Those who were diagnosed as pulmonary LELC with positive EBER results were eligible. All patients underwent endoscopic examination of the nasopharynx to rule out metastatic NPC. Detailed process of patient selection is shown in Patients' medical records were reviewed for the collection of clinicopathological information including gender, age, smoking status, tumor size, staging and treatments. Pathologic or clinical staging was determined according to the seventh edition of American Joint Committee on Cancer (AJCC)/International Union against Cancer (IUCC) staging system, which is based on tumor size, location, and the extent of lymph node or distant metastases. OS was defined as the time between surgery and death from any causes. Disease-free survival (DFS) was defined as the time from surgery until recurrence or death. Patients were censored at their last known alive date. Follow-up information was obtained from patients' medical records or by telephone interview.H = 7.4) and cooled slowly at room temperature for 20 minutes. After blocking the activity of endogenous peroxidase with 3% hydrogen peroxide for 8 minutes, the sections were treated with anti-PD-L1 antibody and incubated overnight. Subsequently, the slides were rinsed in PBS three times and incubated in HRR-linked secondary antibodies. After incubation, slides were washed again with PBS and then visualized using diaminobenzidine. The sections were counterstained with hematoxylin and then mounted.Immunohistochemical staining was performed using rabbit monoclonal anti-PD-L1 antibody  to determine the expression of PD-L1. Four-μm-thick sections from formalin-fixed, paraffin-embedded (FFPE) tissue blocks were de-waxed and rehydrated accordingly. For antigen retrieval, slides were heat-treated for 30 minutes in citrate buffer solution . Cases with more than 5% expression of PD-L1 were considered positive according to previous reports.EGFR mutations and KRAS mutations were detected using PCR-based direct sequencing. Briefly, genomic DNA was extracted from tumors embedded in paraffin blocks. PCR amplification was done using HotStarTaq DNA polymerase  using specific primers. PCR products were sequenced directly using Applied Biosystems PRISM dye terminator cycle sequencing method  with ABI PRISM 3100 Genetic Analyzer . ALK rearrangements were detected by Fluorescence in situ Hybridization (FISH) using a break-apart probe to the ALK gene  per manufacturer's instructions. At least 100 representative tumor cells were counted and then analyzed by using an Olympus fluorescence microscope equipped with orange, green, and 4′, 6-diamidino-2-phenylindole filters. Images were captured using the Video Test Image Analysis System. FISH-positive cases were defined as ≥ 15% of the tumor cells that showed a split red and green signal and/or an isolated (single) red signal. Otherwise, the specimen was classified as ALK FISH negative.p-value < 0.05 was deemed significant. Optimal cutoff value of PD-L1 H-score for survival discrimination was determined by X-tile software  as previously described [All the statistical analyses were performed using SPSS version 22.0 . Pearson's chi-squared test or Fisher's exact test was used to assess the correlations between PD-L1 expression and categorical variables. Median H-scores of PD-L1 was compared between groups using Mann-Whitney U test. The survival analysis was performed with Kaplan–Meier method and log-rank test was used to evaluate survival difference. Subsequently, the PD-L1 expression status was entered into multi-variate Cox regression analysis. Hazard ratios (HR) and 95% confidence intervals (CI) were used to determine the prognostic value of PD-L1 expression. A two sided escribed .The sample size of the first cohort was calculated prior to the initiation of the present study. Sample size was calculated to obtain an accurate estimate of the proportion of patients with PD-L1 positive tumors. Assuming a percentage of 50%, more than 193 samples were required to ensure a 95% CI of less than ± 7% (Wilson score method). Taking into account patients with tumors of undetermined status, an overall sample size of 214 was chosen.


Chordomas are primary malignant tumors of the notochord that are resistant to conventional chemotherapy. Expression of programmed cell death ligand 1 (PD-L1), prevalence of tumor-infiltrating lymphocytes (TILs), and their clinical relevance in chordoma remain unknown. We evaluated PD-L1 expression in three chordoma cell lines and nine chordoma tissue samples by western blot. Immunohistochemical staining was performed on a chordoma tissue microarray (TMA) that contained 78 tissue specimens. We also correlated the expression of PD-L1 and TILs with clinical outcomes. PD-L1 protein expression was demonstrated to be induced by IFN-γ in both UCH1 and UCH2 cell lines. Across nine human chordoma tissue samples, PD-L1 protein was differentially expressed. 94.9% of chordoma samples showed positive PD-L1 expression in the TMA. The expression score of PD-L1 for metastatic chordoma tumors was significant higher as compared with non-metastatic chordoma tumors. Expression of PD-L1 protein significantly correlates with the presence of elevated TILs, which correlates with metastasis. In summary, our study showed high levels of PD-L1 are expressed in chordoma, which is correlated with the prevalence of TILs. The current study suggests targeting PD-L1 may be a novel immunotherapeutic strategy for chordoma clinical trials. Chordoma, an extremely rare cancer presumably originating from notochord, accounts for 20% of primary spine tumors and 1- 4% of primary malignant bone tumors . The staThere is growing interest in the immunoregulatory receptor programmed cell death 1 (PD-1) and the corresponding B7 family of ligands as a pivotal mechanism of tumor immune tolerance and escape in cancer. PD-1 is a member of the B7-CD28 family that co-regulates T-cell receptors and contributes inhibitory signals to mediate physiological immune escape and tolerance , 7. PD-1Tumor-infiltrating lymphocytes (TIL) in many cancers express PD-1 and have been shown to be correlated with high PD-L1 expression in different cancers -15. In tThe aim of our study is to investigate the expression level of PD-L1, TILs, and clinicopathological parameters in chordoma, with a particular focus on any potential correlation with prognosis.Morphology of chordoma cells usually consist of small non-vacuolated cells, intermediate cells with a wide range of vacuolization, and large heavily vacuolated  cells , 25. UCHTo validate the chordoma cell line data, the PD-L1 western blot assay was performed on total protein isolated from 9 chordoma human tumor samples Figure . BrachyuP = 0.0057; Figure PD-L1 immunoreactivity was found in the cytoplasm of tumor cells. PD-L1 staining was detected in 78 TMA samples (including recurrence or metastasis) from 56 patients. The median age was 62 years (range: 24 - 85), and predominantly male (69.6% of patients). Of the 78 chordoma samples, 94.9% were positive for PD-L1 expression. Based on PD-L1 staining intensities in tumor samples, no staining (0) and weak staining (1+) specimens were classified as PD-L1-low patients (57.1%); moderate staining (2+) and intense staining (3+) specimens were classified as PD-L1-high patients (42.9%) Figure . We evalP = 0.0532; Figure P = 0.0325).The median overall survival for patients with low PD-L1 expressing tumors was 145 months compared with 81 months for patients with high PD-L1 expressing tumors, exhibiting a borderline trend, but not achieving statistical significance .Representative pictures of chordoma cases showing different levels of TILs (scores 0 - 2+) were depicted in Figure P = 0.0415, Figure We evaluated the clinicopathologic features of chordoma tumor samples and found no significant relationship between PD-L1 expression and age at surgery, gender, or tumor location. The presence of increased TILs was not significantly associated with survival. The median overall survival for TILs-few patients compared with TILs-prominent patients exhibited a borderline trend, but did not achieve statistical significance Figure . There wPD-L1 has been shown to be expressed in different solid tumors -23. Our Loss of tumor suppressor phosphatase and tensin homolog (PTEN) had been shown to increase PD-L1 expression and immunoresistance in glioma . In primIn order to characterize the relationship between the expression level of PD-L1 protein and clinical behavior, we used a chordoma TMA that allowed the simultaneous characterization of the expression status of PD-L1 in 78 chordoma samples. Our study showed that 42.9% samples were classified as staining highly for PD-L1. In our analyses, there was no correlation of PD-L1 expression with age at surgery, gender, or tumor location. Our study showed the median PD-L1 expression score for the group with metastsis was significantly higher compared with the PD-L1 expression score for the group without metastasis. Furthermore, previous studies have demonstrated a strong correlation between the expression of PD-L1 and poor prognosis in different solid tumors, including glioblastoma, pancreas, ovarian, breast, renal cell carcinomas, head and neck squamous cell carcinomas, esophageal, and non-small cell lung cancers , 37-41. TILs induce expression of PD-L1 through up regulation of cytokines, such as IFN-γ . In accoMultiple agents targeting the PD-1/PD-L1 system in cancer are currently at different stages of clinical development -46. Thesin vitro [Since these initial reports of clinical activity of PD-L1 blockade therapy in several tumor types, including advanced melanoma, lung, and renal cell carcinomas in 2012, there has been a growing interest in immunotherapy to see if these agents could be applied to other tumor subtypes. Furthermore, PD-L1 silencing with siRNA has been shown to inhibit colon cancer proliferation, migration, and invasion in vitro . Increasin vitro . On the in vitro . In conc2-95% air atmosphere at 37°C and passaged every 3 - 4 days using trypsin-EDTA when cell confluence reached 90–100%.UCH1 and UCH2 are established human chordoma cell lines and were kindly provided by Dr. Silke Bruderlein  , 50. Ano5 cells were incubated at 37°C for 48 hours in medium supplemented with 100 U/mL, and then washed with excess culture medium.Cells were treated with recombinant human interferon IFN-γ , as previously described . BrieflyExpression of PD-L1 protein was evaluated by Western Blot analysis. Protein lysates from chordoma cell lines were extracted using 1× Cell Lysis Buffer . The protein concentrations were determined using Protein Assay Reagents  and a SPECTRAmax Microplate Spectrophotometer from Molecular Devices . The primary antibodies for PD-L1 (1:1000 dilution), brachyury (1:1000 dilution), and β-actin  were purchased from Abcam, Santa Cruz, and Sigma-Aldrich, respectively. The secondary antibodies were bound to IRDye1 800CW or IRDye1 680LT . Western blots were carried out as previously described . NormaliNine of the chordoma tissue samples Tissue1–Tissue9) were obtained from the Massachusetts General Hospital Sarcoma Tissue Bank and were used in accordance with the policies of the institutional review board of the hospital (IRB protocol # 2007P-002464). Written informed consent was obtained from all patients whose specimens and clinical information were used for this research study. All tissue diagnoses were confirmed histologically, and proteins were extracted from these frozen tissues. The data of each patient's age, gender, metastasis, recurrence, tumor location(s), follow up months, and disease status were collected . A retrospective study of 78 samples (including recurrent and metastatic) from 56 chordoma patients was identified for the tissue microarray (TMA) immunohistochemical staining as previously reported . The cliThe expression level of PD-L1 was determined based on the Immunohistochemistry Protocol (Paraffin) from Cell Signaling Technology . Briefly, 5-μm-thick array sections were baked at 60°C for 1h, dewaxed with xylene (three times for 5 minutes each), transferred through 100% ethanol (twice for 5 minutes each), rehydrated through graded alcohol, and then immersed in deionized water for 10 minutes. Antigen retrieval was processed with Target Retrieval Solution . After antigen retrieval, the slide was washed with PBS twice for 5 minutes. Following the process of antigen retrieval, endogenous peroxidase activity was quenched by incubation in 3% hydrogen peroxide. After protein blocking with blocking solution  for 1 hour at room temperature, primary antibody  was applied at 4°C overnight in a humidified chamber. Primary PD-L1 antibody was probed at 4 °C overnight. Each step was succeeded by three Tris-buffered saline (TBS) rinses, and the bound antibody on the array was detected by using SignalStain® Boost Detection Reagent  and SignalStain® DAB . Finally, sections were counterstained with Hematoxylin QS (VectorLaboratories) and the slide was mounted with VectaMount AQ (Vector Laboratories) for long-term preservation. The immunostaining intensity pattern of PD-L1 was assessed on a scale semi-quantitatively as follows: 0, no staining; 1+, weak staining; 2+, moderate staining; and 3+, intense staining. Scoring was calculated from the mean of the two independently conducted assessments. PD-L1 staining images were obtained by using a Nikon Eclipse Ti-U fluorescence microscope (Nikon Corp) with a SPOT RT digital camera (Diagnostic Instruments Inc.).The TIL analysis were performed on the hematoxylin and eosin-stained chordoma TMA slide. Scores of the TMA were evaluated for the presence of TILs at 200× magnification and semi-quantitatively as follows: score 0, no TILs; 1, rare/few TILs; 2, brisk/prominent TILs.P values < 0.05 were considered as statistically significant.All statistical analyses were performed using GraphPad Prism software 5.0. Kaplan-Meier survival curves were generated to examine the relationship between the expression levels of PD-L1 and patients' survival rate. Survival time was calculated from the date of tumor diagnosis to the date of death or last follow-up. The statistical significance between two groups was determined using unpaired Student's t-test. Data were expressed as mean ± standard error of the mean (SEM). For comparison between expression of PD-L1 and immune infiltrates, a one-way ANOVA analysis was used. The statistical significance is described in figures and in legends.


Stacking various two-dimensional atomic crystals is a feasible approach to creating unique multilayered van der Waals heterostructures with tailored properties. Herein for the first time, we present a controlled preparation of large-area h-BN/graphene heterostructures via a simple chemical deposition of h-BN layers on epitaxial graphene/SiC(0001). Van der Waals forces, which are responsible for the cohesion of the multilayer system, give rise to an abrupt interface without interdiffusion between graphene and h-BN, as shown by X-ray Photoemission Spectroscopy (XPS) and direct observation using scanning and High-Resolution Transmission Electron Microscopy (STEM/HRTEM). The electronic properties of graphene, such as the Dirac cone, remain intact and no significant charge transfer i.e. doping, is observed. These results are supported by Density Functional Theory (DFT) calculations. We demonstrate that the h-BN capped graphene allows the fabrication of vdW heterostructures without altering the electronic properties of graphene. Low-dimensional materials have been emerging recently as new systems with unique structure and size-dependent properties. While having lateral dimensions typically at the micron scale, the thickness of layers is at the atomic scale, rendering them “true” nanoscale materials with significant anisotropic properties. While graphene is undoubtedly the archetypical 2D material, inorganic layers derived from layered transition metal oxides or dichalchogenides have the potential to increase as well as diversify the portfolio of 2D systems available to date2), allowing the use of h-BN as an alternative gate dielectric. Moreover, the surface optical phonon modes of h-BN have energies double that of similar modes in SiO2, offering the possibility of improving the high-temperature and high-electric field performances of h-BN/graphene based devices compared to the usual oxide/graphene counterparts.Hexagonal boron nitride (h-BN) promises to be an ideal dielectric substrate for improved graphene-based devices24et al. have so far focused on 2D materials  on epitaxial graphene/SiC112 on epitaxial graphene by CVD and performed TEM and Raman spectroscopy to investigate the interface of this heterostructure. Very recently, Yuan et al.h-BN has proved to be an excellent substrate for monolayer graphene by improving the performance of the quantum yield, for example6782 and ii) a continuous h-BN layer on the graphene surface. The h-BN/graphene heterostructure was also studied by Scanning and High-Resolution Transmission Electron Microscopy (STEM/HRTEM), Near Edge X-Ray Absorption Fine Structure (NEXAFS) and X-ray as well as angle-resolved X-ray Photoemission Spectroscopy (XPS and ARPES). Measurements of electronic structure were corroborated by Density Functional Theory (DFT) calculations.Here, we study the interface properties of h-BN on epitaxial graphene on 4H-SiC(0001). We demonstrate that the h-BN flakes can be deposited on the graphene in various sizes. Due to the inertness of both surfaces, van der Waals forces play a major role in the h-BN layer organization. Two distributions were observed using micro-Raman mapping: i) isolated h-BN thin flakes of 2×2 μm3N) micropowders to liquid-state polyborazylene (PBN)3N as a crystallization promoter allows h-BN to crystallise at a lower temperature (1200°C) than under classical conditions (1800 °C)ex situ from the growth chamber. After ultrasonication of the h-BN suspension, a few drops of solution were deposited on the epitaxial graphene. Raman spectroscopy was carried out to characterize the morphology of h-BN on graphene. −1. Several intense peaks are visible in this range; these correspond to second-order Raman bands originating in the SiC substrate−1, ii) the G band at 1595 cm−1 and iii) the 2D band at 2720 cm−1; the small D peak indicates the high quality of the sample. In the case of h-BN/graphene, we note a new peak at ∼1367 cm−1 originating from the E2g mode of h- BN vibration192g vibration mode for two representative areas of the h-BN/graphene heterostructure. As it is shown by the different colour scale in 21Thermal decomposition of undoped 4H-SiC(0001) can be used to produce large graphene films with a long-range order and high electron mobilitynditions 00 °Cex serature 10°C than s, Si 2s, and Si 2p peak intensities were not notably affected by the h-BN deposition (s and N 1s core level peaks observed on the h-BN/graphene spectrum. The different contributions to the spectra were extracted by a curve fitting procedure (see Methods). The depth position of the corresponding species across the h-BN/graphene interface was identified by varying the incident photon energy (hv = 510 and 825 eV) and thus changing the surface and bulk sensitivity. The experimental data points are displayed as dots. The solid line is the envelope of the fitted components. The C 1s and Si 2p spectra of the h-BN graphene heterostructure are shown in s spectra show the conventional deconvolution expected for epitaxial graphene on SiC(0001)232426p doublet was reconstituted with a 2p1/2:2p3/2 ratio of 0.5 and a spin-orbit splitting of 0.6 eVp peak at 101.5 eV. The small shoulder at 102.1 eV was attributed to an interface layer area of the SiC(0001) substrate, while the component at 100.9 eV was attributed to the presence of Si clusters formed when Si-C bonds were broken during graphitization31s and N 1s are shown in s and N1s spectra exhibited three peaks fitted with Gaussian components . The main peak at binding energy BE ~ 191 eV for the B 1s and BE ~ 398.6 eV for the N 1s were attributed to B-N bonding, matching those reported for bulk h-BN333435et al.i and the nitrogen vacancy. Interstitial nitrogen binds strongly with a N atom of the lattice forming a N-N pairet al.2O and B-NO2, respectively, (one and two N vacancies are replaced by one and two oxygen). In the case of N 1s, the components at higher BE ~ 399.8 eV and ~401.2 were attributed to O-B-N40s, B 1s and N 1s NEXAFS spectra were measured for different incidence angles of the linear polarized synchrotron light. As shown in s NEXAFS spectra. These spectra were characterized by two sharp resonances at 285.3 eV and 291.7 eV and a broad structure at 292.7 eV. These features were characteristic of pristine graphene on SiC24*, π* resonance intensity was strongly enhanced. This feature decreased with an increased incident angle and was almost suppressed for normal incidence (θ = 85°). As expected for a planar π conjugated system such as graphene, the σ* resonances revealed the opposite angular dependence behaviour. The sharpness of the NEXAFS features indicated a well-defined bonding environment and a long-range periodic order in the electronic structure. Thus, the NEXAFS spectra unequivocally proved the conservation of sp2 bonds between carbon atoms in the h-BN/graphene heterostructure. The fact that no modifications were induced on the C 1s NEXAFS spectra with respect to pristine graphene, confirmed the dominant role of van der Waals interactions between graphene and h-BN layers. s and N 1s NEXAFS spectra. In addition to the characteristic B(1s → π*) excitonic transition of h-BN at ~192.0 eV  and two σ* peaks at ~198.2 eV and ~199.5 eV, two other excitonic π* peaks were observed in the B 1s x-ray absorption edge at ~192.7 eV and ~193.3 eV3 and B-O3 environments, i.e., B-N2-O and B-NO2, respectivelys NEXAFS spectra displayed an excitonic peak with a maximum centred at ~401.5 eV and two σ* transitions at ~405.8 eV and ~408.1 eV, as found in the literature for a bulk h-BN43i at around 0.6 eV above the valence band maximum (VBM) of h-BN, i.e. just above 397 eV for the 4 eV energy gap of h-BN37s XPS spectra. For the orientation of the h-BN flakes on graphene we clearly saw that the flakes were not all perfectly flat on the graphene substrate, as observed in the HRTEM image. In fact, for normal incidence of light and a polarization vector almost in the plane of the sample (θ = 85°), the π* resonance was still visible, even if attenuated with respect to the grazing incidence (θ = 18°).To investigate the atomic composition, as well as the chemical bonding environment of our samples, XPS measurements were carried out for the pristine graphene and the h-BN/graphene heterostructure, as shown in position , whose pθ = 18°, ° and 85°K point of the first graphene Brillouin zone, for the pristine graphene layer and the h-BN/graphene heterostructure. On the pristine graphene layer  at −0.3 eV below the Fermi level (EF). This n-type doping is typical of epitaxial graphene and is due to the charge transfer from the SiC substrate. After h-BN deposition  of the Dirac cone branches probably increased because of the interaction between the graphene and h-BN layers. It has been proposed that a bandgap would be induced in graphene aligned in a commensurate way to a h-BN substrateIn order to clearly highlight the effect of h-BN on graphene’s electronic structure, ARPES measurements were carried out. ne layer , the linposition , these π3N) micropowders in order to obtain highly crystallized material. The epitaxial h-BN/graphene domains were shown by micro- Raman spectroscopy. More importantly, the STEM/HRTEM results showed that the h-BN layers are highly crystalline without any interdiffusion. XPS and ARPES spectroscopy shows a sharp interface between graphene and h-BN layers. This demonstrates that epitaxial graphene may be a suitable template to combine with alternative vdW solids due to its lack of dangling bonds, chemical inertness, and ability to remain intact under high stress. This study demonstrates a simple and efficient route for the controlled fabrication of multilayered graphene/h-BN heterostructures with promising applications in nanoelectronics.In summary, we are able to synthesize a high-quality multilayer h-BN/graphene heterostructure. The h-BN is synthesized by using the PDCs route, from a liquid-state polyborazylene as polymeric precursor additivated with lithium nitride (Li−5 mbar) and then further heated to 1550 °C in an Ar atmosphere for 10 mn. It was then cooled down to room temperature and transferred ex-situ from the growth chamber to the analysis chamber. The h-BN layers were synthesized using the Polymer Derived Ceramics route by adding lithium nitride (Li3N) micropowders to liquid-state polyborazylene (PBN), as recently describedThe h-BN on graphene/4H-SiC(0001)  (Si-face) was created in two stages: first few-layer graphene was produced on a 4H-SiC(0001) commercial wafer off-axis. The sample was heated to 1100 °C in a UHV  and α = 0 eV (SiC an IL). The graphene thickness was calculated from the ratio between the intensity of the G and SiC components48IG and ISiC represented the area of the G and SiC components of the C 1s peak extracted from the deconvolution. For epitaxial graphene on SiC, the areal density of the C atom in graphene NG was about three times that of C atoms in SiCd = 14.9 Å corresponding to about 6 ML of graphene. The Si 2p spectra were fitted by sums of Voigt curves, that is, the convolution of a Gaussian  by a Lorentzian . Where the LW are fixed at 45 meVs and N 1s, the peaks were fitted by the sum of Gaussian functions . For ARPES measurements, the photon energy (hν = 60 eV) and sample orientation were set in order to explore the k-space region around the K point in the ΓK direction of the Brillouin zone. The spot size was 100 × 40 (H × V) μm.XPS/ARPES experiments were carried out on the TEMPO beamlineAb initio calculations to describe the electronic structure of the h-BN/graphene van der Waals heterostructure were performed using a very efficient DFT localized orbital molecular dynamic technique (FIREBALL)sp3d5 for B, sp3 for C, N and Si, and s for H were used with cutoff radii (in atomic units) s = 4.5, p = 4.9, d = 5.2 (B), s = 4.5, p = 4.5 (C), s = 4.2, p = 4.2 (N), s = 4.8, p = 5.4 (Si) and s = 4.1 (H)et al.How to cite this article: Sediri, H. et al. Atomically Sharp Interface in an h-BN-epitaxial graphene van der Waals Heterostructure. Sci. Rep.5, 16465; doi: 10.1038/srep16465 (2015).


Genomes of higher eukaryotes have surprisingly long first introns and in some cases, the first introns have been shown to have higher conservation relative to other introns. However, the functional relevance of conserved regions in the first introns is poorly understood. Leveraging the recent ENCODE data, here we assess potential regulatory roles of conserved regions in the first intron of human genes.We first show that relative to other downstream introns, the first introns are enriched for blocks of highly conserved sequences. We also found that the first introns are enriched for several chromatin marks indicative of active regulatory regions and this enrichment of regulatory marks is correlated with enrichment of conserved blocks in the first intron; the enrichments of conservation and regulatory marks in first intron are not entirely explained by a general, albeit variable, bias for certain marks toward the 5’ end of introns. Interestingly, conservation as well as proportions of active regulatory chromatin marks in the first intron of a gene correlates positively with the numbers of exons in the gene but the correlation is significantly weakened in second introns and negligible beyond the second intron. The first intron conservation is also positively correlated with the gene’s expression level in several human tissues. Finally, a gene-wise analysis shows significant enrichments of active chromatin marks in conserved regions of first introns, relative to the conserved regions in other introns of the same gene.Taken together, our analyses strongly suggest that first introns are enriched for active transcriptional regulatory signals under purifying selection.The online version of this article (doi:10.1186/1471-2164-15-526) contains supplementary material, which is available to authorized users. Drosophila, and C. elegans[C. elegans  and Drosophila  to humans , while the fraction of non-coding DNA including introns greatly varies up to several folds [Recent complete sequencing projects have confirmed that almost all eukaryotes have introns –6. Diffe elegans, 7, 8. T elegans, as well elegans–14. Notial folds , 16, somal folds , 18.D. melanogaster and D. simulans, Haddrill et al. [The mere existence of introns in the eukaryotic genome is intriguing, given the cost of transcription and the cost of maintaining a splicing regulatory system that ultimately eliminates the introns from the functional product of the gene. In particular, whether introns have evolved under selective constraints, and the extent thereof, are not entirely clear; while some studies suggest that introns evolve largely free from selective constraints –22, othel et al.  demonstrl et al.  showed tl et al. , 28, 29,l et al. . Besidesl et al. –33. ThusDrosophila[Tetrahymena, the most conserved introns are found closer to the 5’ end of genes. Furthermore, introns harboring regulatory elements tend to be the first introns [The 5’-most “first” intron differs from the other introns in several aspects in terms of processing, epigenomic marks, length and evolution. First, in terms of processing, despite an overall 5’-to-3’ trend in splicing during transcription, the first intron is removed (on average) somewhat later . Second,osophila, 37. Zheosophila have als introns –40, and, introns . OverallPrevious investigations of potential regulatory role of first intron were mainly performed in Drosophila or plants. The extent to which the previous conclusions hold true in mammals is not known. Moreover, the recent explosion of human epigenomic data via the ENCODE project Table  providesAs a proxy for purifying selection, we compared evolutionary conservation across introns grouped by their position in the transcript structure. Conservation in an intron was estimated as the fraction of intron sites that were conserved based on a PhastCons score threshold. Three different multiple alignments  and different PhastCons score thresholds were used. Our conclusions hold for all choices of alignment and threshold; here we only present the results based on mammalian conservation with PhastCons score threshold of 0.5. The primary focus of our investigation was transcriptional regulatory elements in introns. Therefore, to exclude the possibility of splice site signals biasing the conservation score, especially for short introns, the sequences within 300 bps of the splice junction, which are considered to harbor splicing regulation signals , were exWe found that the median conservation in first introns (3.3%) was significantly higher than all other groups using Wilcoxon rank sum test p < 2.2e-16)  in which a gene is expressed. Genes that expressed in a very few or most tissues have low complexity whereas the genes expressed in an intermediate number of tissues have the highest expression complexity , 62. It Next we assessed the correlation between the regulatory signals and the number of exons. As shown in Figure We next investigated whether or not conservation in first introns is related to the level of gene expressions. For this, the degrees of conservation were plotted against the levels of gene expressions in several human tissues. Gene expression data sets were obtained from RNA-seq atlas constructed by  from 11 Although the analysis thus far was based on all ‘transcripts’, we repeated the analyses by selecting a single representative transcript per gene, resulting in ~16,000 transcripts. As shown in Additional file Our analysis thus far is based on grouped data. Next we assessed for each gene whether the regulatory signals in the first intron preferentially occur within conserved region. We partitioned each first intron into conserved and non-conserved regions, obtained percent coverage by regulatory signals in each part and calculated the log-odds ratio with 95% confidence intervals (see Methods)  each intron was binned into five equal-sized bins, and we estimated for each bin, the fraction of introns in which the highest signal in the intron was in the particular bin. As shown in Additional file Despite an enrichment of signals toward 5’ as well as the fact that the general patterns of enrichment in the first introns also hold for the 5’ flanking regions . Thus we investigated the trends in the first introns compared to that in the second introns when controlled for their distance from the TSS. We categorized first and second introns into bins corresponding to distance of the 5’ end of the intron from the TSS and compared the first and the second intron trends within each bin. Note that because first introns are much longer than the second intron  the presence of active regulatory chromatin marks, 2) higher expression levels of genes, and 3) a greater number of exons within genes. Overall, our results strongly suggest that first introns in human are enriched for evolutionarily selected active transcriptional regulatory signals that are likely to be important for regulating complex gene expression patterns of large multi-domain genes.The precise mechanism by which individual conserved, putative regulatory regions in the first intron, regulate gene expression, as well as other potential functions of conserved regions in the first intron, are unclear. The extreme lengths of mammalian first introns represent another enigma. The evolutionary path leading up to long introns as well as whether the first intron length is under selective constraint are not known. Finally, whether it is beneficial for the regulatory elements to reside within the first intron, as opposed to, say, the upstream region of the gene, or whether evolution is neutral to this outside-inside choice, is another open and interesting question. To ultimately resolve the mystery of introns, these are some of the questions that will need to be addressed.The exon-intron position information of 36,024 Refseq mRNA (KnownGenes) without duplicates was downloaded from the UCSC Table Browser (January 2013). Genomic sequences for each chromosome were obtained from the primary GRCh37/hg19 assembly, and were used for retrieve intron sequences. Very short introns (less the 1 kb in length) were excluded, as well as very long introns (greater than (third quartile + (interquartile range × 1.5)) in length to minimize the outlier effects. To minimize interference from splicing signals in interpreting intronic conservation, we excluded 300 bps from both the 5’ and 3’ ends of each intron . Then rePhastCons scores were used to estimate position-wise sequence conservation . The PhaGenome positions of peaks  for DHSs, TFBSs and chromatin marks measured in the ENCODE Tier-1 cell lines  were downloaded from the ENCODE Project from UCSC genome browser (March 2013). The specific download links are provided in Table 2 (RPKM + 1) to report expression levels of mRNAs for 11 different human tissues  among the variables representing evolutionary conservations of introns or flanking regions, regulatory signals mapped onto introns or flanking regions, and mRNA expression levels. Introns or flanking regions with no conservation or regulatory signal were excluded; we ascertained that this only makes our results more stringent because the fraction of first introns that were eliminated were smaller than those for other introns and including this with “0” value would make the results even stronger.To test whether the regulatory signals and chromatin marks are enriched in conserved regions compared to non-conserved regions in each gene, odds ratio of the signal estimates between the two regions was estimated. Briefly, first introns were divided into two regions, conserved first introns and non-conserved first introns by the criteria of Phastcons ≥ 0.5, and the chromatin signals and regulatory signals described in the main text were overlaid into each region, generating a 2×2 contingency table. Odds ratios were then estimated for each gene from the contingency table. The odds ratio is the ratio of the proportion of regulatory signals in conserved sequences to that in non-conserved sequences. The odds ratios were thus computed using the “Text::NSP::Neasures::2D::odds” Perl module, and the 95% confidence intervals associated with odds ratios were calculated using the formulas, d sites) .Figure S2. Proportions of regulatory chromatin marks in intron ordinal groups in H1-hESC and K562. Figure S3. Correlation between regulatory signals and conservation in first introns in H1-hESC and K562. Figure S4. Correlation between regulatory signals and conservation in the upstream flanking regions in three different cell lines. Figure S5. Relationship between flanking region conservation and the numbers of exons. Figure S6. Relationship between the proportions of regulatory signals in introns of each ordinal position and the numbers of exons. Figure S7. Analysis based on a single representative transcript for each gene. Figure S8. Enrichment of regulatory marks in the first intron in two additional cell lines. Figure S9. Five prime to three prime biases in signal density along the first intron. Figure S10. Excluding spillover of signals s from the promoter. Figure S11. Excluding genes whose first introns overlapped with exons or flanks of another genes. Figure S12. Analyzing the effect of proximity to the TSS. (PPTX 936 KB)Additional file 1: Figure S1: Comparison of conservations in first introns with those in the other introns using an alternative grouping strategy.


IDO1 and CXCL9) was associated with MPDL3280A response in melanoma but not NSCLC or RCC. On treatment, responding tumors showed increased infiltration of Th1-dominant immune infiltrate and evidence of adaptive PD-L1 up-regulation. In contrast, progressing tumors displayed the following patterns of tumor-infiltrating lymphocytes (TILs) and PD-L1 expression: (1) few/no TILs and absent PD-L1 expression (immunologic ignorance), (2) TILs present with minimal/no PD-L1 expression , or (3) TILs residing solely around the tumor cell mass outer edge (excluded infiltrate), suggesting that resistance to MPDL3280A may be associated with impaired T cell trafficking and/or function.PD-L1 expressed in the tumor microenvironment regulates Th1 immune responses and mediates cancer immune evasion through interactions with PD-1 or B7.1 receptors on activated T cells. MPDL3280A, an engineered human monoclonal antibody, targets PD-L1 and inhibits its function. To identify immunologic predictive and pharmacodynamic biomarkers of MPDL3280A treatment, we performed a comprehensive analysis of tumors and blood samples collected at baseline and/or on treatment from ≈280 patients with locally advanced or metastatic solid tumors, including NSCLC, RCC, melanoma and bladder cancer. Regardless of tumor type, clinical responses were characterized by PD-L1 expression, the presence of markers of T cell activation (Th1 gene signature and CTLA4), and the absence of fractalkine at baseline in the tumor microenvironment. Elevated baseline expression of IFN-γ and IFN-γ-inducible genes  increased in all patients following MPDL3280A treatment, representing a pharmacodynamic measurement of PD-L1 inhibition. In addition, analysis of PBMC showed an increase in T cell activation, as measured by IFN-γ and granzymes A and B gene expression in responders following MPDL3280A treatment, consistent with the observations in responding tumors. Baseline soluble PD-L1 was not associated with response. Some indication-specific biomarkers, such as plasma VEGF, decreased in responders with RCC but not with other indications. In NSCLC, a decrease in tumor burden markers, CA-125 and CEA, was associated with response. Similarly, IL-6 and IL-8 were differentially expressed on treatment in responders vs non-responders. Additionally, responders exhibited a decrease in circulating tumor DNA (ctDNA) in plasma, suggesting that ctDNA may be used to monitor MPDL3280A clinical activity in NSCLC.In conclusion, these data provide general and indication-specific mechanistic insights into immune checkpoint inhibition, potential mechanisms of response and resistance, as well as identification of potential predictive and pharmacodynamic biomarkers of anti-PD-L1/PD-1 clinical activity across multiple tumor types.


Motor neuron disease (MND) is a neurodegenerative disease of mid-life, with average survival of 3-5 years after diagnosis. The only approved treatment, riluzole, is only moderately effective and so there is a great need for novel therapeutics. TAR DNA binding protein 43 (TDP-43) has held a particular centrality in MND research since its discovery as the principal protein component of the characteristic protein aggregates found in the disease. Subsequent work has investigated its role as an RNA-binding protein and regulator of approximately 3000 RNA transcripts. In the majority of disease cases, both familial and sporadic, normally nuclear TDP-43 is sequestered in the cytoplasm in protein aggregates. Using differentiated motor neuron-like cells (NSC-34) and primary motor neurons, we have treated cells with the disease-relevant ER stressor tunicamycin. Overnight treatment with a low concentration of tunicamycin resulted in the formation of aggregates immunoreactive for endogenous TDP-43, as visualised by immunocytochemistry and Western blotting of the RIPA-insoluble fraction. We have found that these aggregates do not co-stain for TIAR, a well-known marker of stress granules. However, using the oxidative stressor sodium arsenite, we are able to induce formation of stress granules, though they do not co-stain for TDP-43. The tunicamycin treatment also led to a moderate decrease in cell viability, as shown by MTT and Trypan Blue Exclusion assays. In this study, we have recapitulated a known pathological process (ER stress) of MND in vitro, which resulted in the formation of TDP-43 aggregates. The signalling pathways driving TDP-43 aggregation are unknown, hence work detailing these mechanisms is likely to present wide scope for therapeutic intervention.


Biomarkers in easily accessible compartments like peripheral blood that can predict disease progression in idiopathic pulmonary fibrosis (IPF) would be clinically useful regarding clinical trial participation or treatment decisions for patients. In this study, we used unbiased proteomics to identify relevant disease progression biomarkers in IPF.Plasma from IPF patients was measured using an 1129 analyte slow off-rate modified aptamer (SOMAmer) array, and patient outcomes were followed over the next 80 weeks. Receiver operating characteristic (ROC) curves evaluated sensitivity and specificity for levels of each biomarker and estimated area under the curve (AUC) when prognostic biomarker thresholds were used to predict disease progression. Both logistic and Cox regression models advised biomarker selection for a composite disease progression index; index biomarkers were weighted via expected progression-free days lost during follow-up with a biomarker on the unfavorable side of the threshold.A six-analyte index, scaled 0 to 11, composed of markers of immune function, proteolysis and angiogenesis  predicted better progression-free survival in IPF with a ROC AUC of 0.91. An index score ≥ 3 (group ≥ 2) was strongly associated with IPF progression after adjustment for age, gender, smoking status, immunomodulation, forced vital capacity % predicted and diffusing capacity for carbon monoxide % predicted .This index, derived from the largest proteomic analysis of IPF plasma samples to date, could be useful for clinical decision making in IPF, and the identified analytes suggest biological processes that may promote disease progression. CO) or 10% in forced vital capacity (FVC)]  , 7 over y (FVC)] , 8, 9.Many groups have reported on biomarker analysis in IPF using approaches such as genomic or transcriptomic profiling, microbiome analyses and candidate biomarker analyses in blood, bronchoalveolar lavage fluid and lung tissue specimens –14. BlooSimilarly, studies have explored the contribution of angiogenesis to fibrogenesis with mixed results . Some huIn terms of protease imbalances in IPF, several studies have identified alterations and these have been the subjects of several review articles. Alterations in metalloproteases are the most commonly noted , 35, butCOMET (Correlating Outcome Measures to Estimate Time to progression in IPF) ; howeveneumonia , 53, sugpatients . Lower ll models , suggestCath-S is a single chain, non-glycosylated cysteine protease ubiquitously distributed in the lysosome. It is expressed mainly in lymphatic tissues and is characterized as a key enzyme in class II-mediated antigen presentation . LGMN is-/- murine models demonstrate elevated expression of type 1 collagen and alpha-smooth muscle actin, with abnormal accumulation of autophagosomes in macrophages and impaired clearance of damaged mitochondria [Inhibition of Cath S can promote autophagy and apoptosis . Thus, lchondria . Intereschondria . Thus, iLGMN was identified as diagnostic and prognostic liver fibrosis biomarker . LGMN-deProtease imbalances have been noted in IPF , 35. FouVEGF is a potent and specific endothelial cell mitogen that regulates blood and lymphatic vessel development and homeostasis. The VEGF receptor family consists of three members, VEGFR1 (FLT1), VEGFR2 (KDR/FLK1) and VEGFR3 (FLT4) . Among t® validation on another cohort, we chose to address sensitivity and specificity of the index using bootstrap analysis. The index performs well across all 100 different bootstrap analyses performed  in a very small volume. Comparable proteomic assays would include mass spectrometry and ELISA platforms. Mass spectrometry is somewhat cumbersome and requires improved reproducibility and cost before it reaches mainstream clinical applicability. ELISA platforms are highly sensitive and useful for identifying single to multiple targets. However, it is now clear that these assays cannot be multiplexed above approximately 70 analytes without multiple measurements secondary to cross reactivity , 70.This study is only capable of describing associations between the selected analytes and disease progression. As discussed above, we feel these analytes may be indicative of pathobiologic processes that could be aberrant in IPF, but it is also possible that these analytes are changed for reasons unrelated to disease. Unraveling this question will require future mechanistic work in animal models and using IPF tissues and cells.Together, our results demonstrate a 6-analyte SOMAmer panel measuring circulating markers of immune function; proteolysis and angiogenesis can be used to create a simple index with excellent sensitivity and specificity for predicting progression-free survival in IPF. Use of this index on easily accessible plasma should be further validated to offer a simple way of predicting IPF patient outcomes. In addition, this analysis serves to highlight aberrant biological pathways associated with IPF disease progression, providing rationale for increased study of these biological pathways in lung fibrosis.S1 FileTable A) Immunodulation Therapy in the COMET Cohort. Fig A. Kaplan-Meier curves showing progression free survival for IPF patients with baseline biomarker levels above or below the identified thresholds (A) Carbonic Anhydrase XIII, (B) Granulin(GRN) and (C) Nascent polypeptide-associated complex subunit alpha (NACA). RMST indicates the comparison of restricted mean survival time (i.e. area under the K-M curves) between the two groups. Fig B. ICOS is shed by activated T cells. A million CD4 positive splenocytes were stimulated with CD3 + CD28 then  or TH1  for 24 hr. Cell free supernatants were collected and concentrated using Amicon Ultra Centrifugal filters . Equal amounts of protein from each sample were separated on a 4–20% gradient SDS-polyacrylamide gel and transferred to a PVDF membrane . PVDF membrane was probed with rabbit monoclonal ICOS (Abcam). Fig C. Periostin levels correlate on SOMAscan and ELISA. The same plasma samples were run on a periostin ELISA developed by Abbot Pharmaceuticals and were compared to measures of periostin made by SOMAmer. The correlation was significant, Spearman r = 0.44; p<0.0001.This file contains the following: (DOCX)Click here for additional data file.S2 File(XLSX)Click here for additional data file.


We propose a novel mechanism to construct a tunable and ultracompact refractive index sensor by using the Fano resonance in metal-graphene hybrid nanostructure. Plasmon modes in graphene nanoribbons and waveguide resonance modes in the slits of metal strip array coexist in this system. Strong interference between the two different modes occurs when they are spectrally overlapped, resulting in a Fano-type asymmetrically spectral lineshape which can be used for detecting the variations of ambient refractive index. The proposed sensor has a relatively high figure of merit (FOM) over 20 and its sensing performance shows a good tolerance to roughness. In addition to the wide range measurement enabled by the electrical tuning of graphene plasmon modes, such ultracompact system also provides an angle-independent operation and therefore, it can efficiently work for the detection of gas, liquid, or solids. Such optical nanostructure may also be applied to diverse fields such as temperature/pressure metering, medical detection, and mechanical precision measurement. Exhibiting an asymmetric profile of spectral lineshape, Fano resonance (FR) was first experimentally observed by BeutlerFano-type transmission/reflection based on localized surface plasmon resonance (LSPR)45611In this paper, we propose an ultracompact FR-based metal-graphene hybrid nanostructure for detecting the refractive index variations of surrounding dielectrics. The sensor operates at mid-infrared wavelengths and its performance is electrically tunable and angle-insensitive. A schematic representation of the metal-graphene hybrid nanostructure is depicted in WM), period (p), heights of the upper (h1) and and bottom (h2) silver strips are set as 358 nm, 410 nm, 180 nm, and 40 nm, respectively. To achieve optimal transmission performance, other structural parameters are d1 = 55 nm, d2 = 35 nm, t = 20 nmWe first consider the metallic nanostructure without introducing the graphene ribbons. As mentioned before, TM-polarized normal incidence can excite two different resonant modes in metal strip array, the coupled SP mode at strip surface and the WR mode in the slits of strips, at rather different frequenciesεg = 2.5 + iσg/ε0ωΔ, where ε0 is the permittivity in vacuum, ω is the circular frequency of incident light, and Δ is the thickness of graphene (Δ = 0.5 nm). Under the conditions of low temperature and high doping (i.e. Ef represents the Fermi energy of graphene), the graphene conductivity is dominated by intraband transition and thereby can be simplified as e is the electron charge, τ is the relaxation time. In our considered wavelength range, the relatively low photon energies (compared with Ef) means that the graphene conductivity obtained from the simplified semiclassical model should agree well with that calculated from random phase approximation (RPA) which can give the accurate expression of the graphene dielectric constantIt has been demonstratedWG) of the graphene ribbons is set as 42 nm, which is achievable via current fabrication technologyWhen graphene ribbons are placed inside the slits of the metal strip array, GSPs may be excited by the WR and then interact with the WR. In particular, the spectral overlap of these two plasmon resonances may result in a Fano-type lineshape. To realize such a spectral overlap, the width  of GSPs, another component of wave vector is produced in the graphene nanoribbons by the oscillation modes of free carriers with restricted motions: kx ≈ mπ/WG, where m represents the number of nodes.To reveal the physical mechanism for the excitation of graphene surface plasmons (GSPs), we study the dispersion relation of the GSPs. Assume that the graphene ribbons have a finite width of The thickness of the graphene ribbons is too small and are neglected in the following derivation for the dispersion relationship of GSP in a graphene ribbon. According to the Maxwell’s Equationsρext and the current densities the macroscopic quantities ω is the circular frequency of incident light, μ0 and ε represent permeability and permittivity of the dielectrics. As a characteristic of SPP, the electromagnetic field has a form of β represents the propagating constant on graphene and ky indicates the exponential amplitude decay along y direction. xE component is neccessary for the exctiation of GSPP, and we assume a TM-polarized incidence to the considered system along y direction. where Ex, Ey, Ez, Hx and Hz are included in Ex, Ey and Hx. This is the result that the charge distribution on graphene, which is regarded as graphene surface charge, changes the characteristic of the electromagnetic field near the graphene layer. For example, the non-homogeneous electron distribution on graphene along z-axis produces the Ez component of the electromagnetic field. However, Hy remains zero because it is independent of the surface charge. Then combining It should be noted that Ez can be written asSince Combining y < 0 andfor the half space with y > 0. ks ≡ ky,s is the wave vector component perpendicular to the interface of the two dielectric media. According to boundary relations, when crossing an interface, the tangential component of electric field is continuous while the transverse component of magnetic field is affected by the surface current as the following equation:for the half space with Note thatg). By applying the boundary relations to and the surface conductivity σ is provided by the graphene sheet  and kx = mπ/WG.where Such a complicated structure is experimentally achievable. How to cite this article: Pan, M. et al. Tunable angle-independent refractive index sensor based on Fano resonance in integrated metal and graphene nanoribbons. Sci. Rep.6, 29984; doi: 10.1038/srep29984 (2016).


Understanding the underlying mechanisms involved in graphene growth via chemical vapour deposition (CVD) is critical for precise control of the characteristics of graphene. Despite much effort, the actual processes behind graphene synthesis still remain to be elucidated in a large number of aspects. Herein, we report the evolution of graphene properties during in-plane growth of graphene from reduced graphene oxide (RGO) on copper (Cu) via methane CVD. While graphene is laterally grown from RGO flakes on Cu foils up to a few hundred nanometres during CVD process, it shows appreciable improvement in structural quality. The monotonous enhancement of the structural quality of the graphene with increasing length of the graphene growth from RGO suggests that seeded CVD growth of graphene from RGO on Cu surface is accompanied by the restoration of graphitic structure. The finding provides insight into graphene growth and defect reconstruction useful for the production of tailored carbon nanostructures with required properties. The growth of graphene on copper (Cu) substrates via chemical vapour deposition (CVD)1345789101178910111213142) surfaces serve as templates for the new growth of defective graphene during ethanol CVDCVD growth of graphene on Cu is a surface-mediated process131415161718171722Here we report the variation of graphene properties during lateral growth of graphene from RGO flakes on polycrystalline Cu foils by methane CVD. A combined microscopic and spectroscopic study correlated the growth length of CVD-grown graphene from RGO, reflecting the stages of in-plane graphene growth, with the corresponded structural quality of the graphene. The correlation demonstrated that graphene exhibited substantial enhancement in structural quality while it was laterally grown from RGO flakes on Cu surfaces up to a few hundred nanometres by the CVD process. The monotonous improvement of the structural quality of the graphene with increasing extended length of the graphene grown from RGO suggested that seeded CVD growth of graphene from RGO as low-quality seeds on Cu substrates was accompanied by the restoration of graphitic structure.RGO  were directly measured using a scanning electron microscope (SEM). The GO flakes, instead of RGO flakes, were prepared on Cu foils before CVD because they were naturally reduced  upon hea131626RGO and GGCN on the same Cu surface with the increase of CVD growth time was examined and compared. The GRGO growth length was defined as the distance between the GRGO growth front and the corresponding RGO edge  substrate , RGO (red circle), GGCN (blue hexagon) and GGCN edges (green pentagon) were measured at several positions on the sample. Thick RGO  and thin CVD-grown graphene (purplish area) on the SiO2/Si (pink area) were clearly discernible in an optical microscope image37382/Si substrate was indicated by a green-dotted line in RGO  of GRGO varied widely from 0.55 to 0.80 . The ID/IG values of GRGO were mostly larger than those of GGCN and GGCN edges, whereas these values were substantially smaller than those of RGO, indicating that the structural quality of GRGO was lower than that of GGCN and GGCN edges, but it was higher than that of RGO. Similar to the distribution of the ID/IG values, the intensity ratio of the 2D peak to the G peak (I2D/IG) of GRGO also scattered over relatively wide ranges compared with those of RGO, GGCN and GGCN edges  substantially smaller those of high-quality graphene monolayerD/IG and I2D/IG of GRGO has yet to be specified.Graphene edge structures can govern the structural properties of subsequently grown graphene from the graphene edge structures2930SiO2/Si, . EspeciaCN edges . The I2DRGO during seeded CVD growth has been specified, little is known about the evolution of the structural quality of graphene subsequently grown from the initially-formed GRGO during the later stages of CVD growth. To understand the evolution of the structural quality of GRGO with the increase of growth time, we measured Raman spectra on graphene samples in a direction away from the RGO flakes because the growth front of GRGO moved with time in such a direction during the CVD process, as already demonstrated in RGO, the graphene samples were grown on the Cu with GO flakes by CVD for 900 s. Large-scale characterization424344RGO and GGCN on the Cu completely coalesced into a single large-area RGO and graphene hybrid film after CVD for 900 s, suggesting the scalability in this study  compared with the thin CVD-grown graphene specimens (GRGO and GGCN)  . For a pN)  and width (Γ2D) do not show a significant variation until the defect density increases up to the certain value2D considerably decreases when the defect concentration further increases over the certain value, while Γ2D strongly increases. As shown in D/IG increased until the position moved from x1 to x4, however, it decreased when the position further moved from x4 to x8 (black square). In addition, I2D/IG  patterns varied from fully amorphous diffraction rings to the inner diffraction spots exhibiting streaking. This TEM observation suggested that graphene showed improvement in the structural quality while it was grown from RGO on the Cu by CVD for 100 sTransmission electron microscopy (TEM) is complementary to Raman spectroscopy, ideally suited for more detailed microanalysis of graphene atomic structure and its derivatives2223RGO and GGCN coalesce into a single large-area film as a RGO-graphene hybrid film  was added to a round-bottom flask  containing H2SO4  while stirring in an ice bath. KMnO4 (22.5 g) was added slowly to keep the reaction temperature of the suspension below 20 °C. Next, the flask was placed in the oil bath at 30 °C. The oil bath was removed completely at the end of the 2 days. After air cooling, diluted H2SO4  was added slowly to the flask, and the stirring was maintained for 2 h. To this mixture, H2O2  was added slowly and then the colour of the suspension turned dark brown to yellow, and the stirring was continued for 2 h. The obtained graphite oxide was purified with distilled water repeatedly by centrifugation. GO sheets were exfoliated from graphite oxide by ultra-sonication. The products were re-dispersed in distilled water.GO was arranged by the exfoliation and oxidation of natural graphite flakes (Sigma-Aldrich) according to the modification of Hummers method. A mixture of natural graphite flake (5.0 g) and NaNOAfter exposure to UV, a surface of Cu foil turns into hydrophilic surfaces and it was dipped in mixture of toluene (100 ml) and (3-aminopropyl)triethoxysilane  to ensure a uniform GO distribution on the Cu foil. The 3-APTES treated Cu foil was rinsed sequentially with ethanol after toluene and dried in a 60 °C atmosphere for 20 min. At the end of the process, the Cu foil was immersed in the GO solution (1.5 mg/1 ml) for 1 min and dried at room temperature.2) was introduced. The growth of graphene was then performed at 1060 °C for a certain time under a gas mixture of 2 s.c.c.m. methane gas (CH4), diluted in 300 s.c.c.m. argon gas (Ar) and 60 s.c.c.m. of H2. Finally, as prepared, the sample was cooled down to room temperature with Ar and H2 after turning off the flow of CH4. After all the process was over the Cu foil was removed from the furnace for further characterization. For Raman spectroscopy and its correlated studies, the graphene samples on the Cu foil were transferred to SiO2/Si wafers (Si(100) covered by 300-nm-thick SiO2) using a poly(methyl methacrylate) (PMMA) assisted process. Briefly, the PMMA dissolved in chlorobenzene was spin-coated onto the graphene samples at 2,000 r.p.m. for 30 s. The PMMA-coated samples were placed in a Cu etchant  to remove the Cu foil. After complete etching of Cu foil, the PMMA-coated samples were scooped out of the etchant using the SiO2/Si substrates. Finally, the PMMA layer was then removed with acetone and the surface was further rinsed several times with deionized water.Following GO deposition onto a Cu foil , the Cu foil was loaded into a CVD furnace and heated up to 1000 °C under a pressure of 1.0 Torr while 100 s.c.c.m. hydrogen gas (H−1 (using a grating with 600 grooves mm−1). The intensity of each Raman peak was extracted from the maximum value without any data processing over the corresponding spectral range .Spatially resolved Raman spectroscopy was performed using a Raman microscope  at Korea Basic Science Institute, equipped with a piezoelectric sample scanner. The wavelength of the excitation laser was 473 nm, and the power of the laser was kept below 0.3 mW without noticeable sample heating. The laser spot size was approximately 0.32 μm with a 100× objective lens . The spectral resolution was 2.0 cmHow to cite this article: Chang, S.-J. et al. Graphene growth from reduced graphene oxide by chemical vapour deposition: seeded growth accompanied by restoration. Sci. Rep.6, 22653; doi: 10.1038/srep22653 (2016).


Arabidopsis TPP riboswitch as an elaborated k-turn. The Escherichia coli TPP riboswitch contains a related k-junction, and analysis of >11 000 sequences shows that the structure is common to these riboswitches. The k-junction exhibits all the key features of an N1-class k-turn, including the standard cross-strand hydrogen bonds. The third helix of the junction is coaxially aligned with the C  helix, while the k-turn loop forms the turn into the NC  helix. Analysis of ligand binding by ITC and global folding by gel electrophoresis demonstrates the importance of the k-turn nucleotides. Clearly the basic elements of k-turn structure are structurally well suited to generate a three-way helical junction, retaining all the key features and interactions of the k-turn.The k-junction is a structural motif in RNA comprising a three-way helical junction based upon kink turn (k-turn) architecture. A computer program written to examine relative helical orientation identified the three-way junction of the  The kink turn (k-turn) is an extremely widespread structural motif that generates a tight kink in duplex RNA ,2, thereThe standard k-turn comprises duplex RNA with a three-nucleotide bulge followed by G•A and A•G pairs . The nucHaloarcula marismortui Kt-7 or the human U4 snRNA k-turn. Non-standard simple k-turns have a substitution in one of the G•A pairs. For example, in Kt-23 sequences of 30S ribosomal subunits of different species the 2n position has a frequency U>C>G>A, although examples analysed can form normal k-turn structures despite the departure from the standard sequence  riboswitches contain a hitherto unsuspected motif that is a k-turn that has been elaborated into a three-helix junction. This nevertheless retains all the key interactions found in a simple k-turn. We term this structure the k-junction, adding another branch to the scheme shown in Supplementary Materials. Visual analysis of structures and the computational results was performed in PyMOL 1.5.3. The software was implemented on a standard desktop workstation running Windows 7. RNA-containing PDB files were downloaded from the Protein Data Bank (pdb.org) and stored on the local hard drive. Non-RNA atoms were removed from these files and only files containing at least ten complete nucleotides were retained.RNA structures were analysed using software written in Python 2.7 together with the BIOPYTHON package. The scripts are available for download in All standard Watson–Crick base pairs, including the G–U wobble pair were identified, and for each structure minimal helical segments consisting of two base pairs in close proximity and suitable relative orientation were identified, and abstracted to sets of four vectors. The first vector defines the location of the helix segment, while vectors 2–4 define the orientation of the helix segment and are orthonormal. The second vector defines the main helix axis, the third the direction of the Watson–Crick hydrogen bonds projected to the plane perpendicular to the main helix axis, and the fourth is defined by being orthonormal to the second and third vectors. Therefore, these vectors define a coordinate system, with its origin located at this particular base pair and its coordinate axes aligned with the main features of the helix segment.The k-turn motif search pattern was defined by calculating the relative coordinates of helix segments from the C and NC helices of known k-turn structures. To do this, helix segments from the NC helix were expressed in terms of the coordinate system defined by a helix segment from the C-helix and the relative location and orientation was recorded. For each structure the identified helix segments were checked against pairs matching the relative location and orientation of helix segments from the search pattern. Positively scoring helix segments were written to output files. Helix segments from each were saved as residues consisting of pseudo-atoms into a PDB file. These structures were then analysed by visual inspection.Arabidopsis thaliana TPP riboswitch (3D2G) and E. coli TPP riboswitch (2DGI) were used to define secondary structure masks for the sequence alignments. All sequence composition and covariation analysis was calculated using a modified version of Jalview which was kindly provided by Dr James Procter (University of Dundee).A full alignment of 11 197 TPP riboswitch sequences taken from the Rfam database , accessi2, 50 μg/ml T7 RNA polymerase, 1 U/ml inorganic pyrophosphatase (Sigma), for 3 to 3.5 h at 37°C. The products were purified by gel electrophoresis under denaturing conditions. RNA was extracted from gel slices by electroelution and exchanged five times into 40 mM K-Hepes (pH 7.5), 100 mM KCl, 10 mM MgCl2 for ITC analysis, or 25 mM Tris, 192 mM glycine (pH 8.3), 1 mM MgCl2 for analysis by gel electrophoresis. Concentrations were measured by absorbance at 260 nm using extinction coefficients calculated from the nucleotide composition and a hypochromic effect correction factor.Transcription templates were prepared by PCR amplification from a plasmid-borne gene for the TPP riboswitch using primers hybridizing to the T7 promoter and the 3′-end of the riboswitch. RNA was transcribed from 75 μg/ml DNA template in 30 mM Tris–HCl (pH 8.0), 10 mM DTT, 0.1% Triton X-100, 0.1 mM spermidine–HCl, 4 mM each NTP , 40 mM MgClE. coli TPP riboswitch was:The sequence (written 5′ to 3′) of the unmodified GGACUCGGGGUGCCCUUCUGCGUGAAGGCUGAGAAAUACCCGUAUCACCUGAUCUGGAUAAUGCCAGCGUAGGGAAGUUCE. coli TPP riboswitch and variants were performed by isothermal titration calorimetry (ITC) at 303.15 K as described by Kulshina et al., 1 mM MgCl2 with or without TPP for 5 min at 20°C before addition of glycerol to 25% and loading onto the gel. Electrophoresis was performed either at 4 W for 2 h or 3 W for 2.5 h without or with (respectively) TPP added to the running buffer, such that the temperature remained below 20°C. The contents of anode and cathode chambers were mixed to prevent depletion of Mg2+ ions. After electrophoresis RNA was visualized by UV shadowing.The electrophoretic mobility of TPP riboswitch RNA and sequence variants was analysed in 10% polyacrylamide gels in 25 mM Tris, 192 mM glycine (pH 8.3), 1 mM MgClA. thaliana TPP riboswitch (PDB code 3D2G) contained two helices with a strong structural similarity to the k-turn geometry.We wrote a computer program in Python 2.7 to analyse relative orientation of segments of double helix in coordinate files of RNA structures downloaded from the PDB. A search pattern was defined by calculating the relative coordinates of helix segments from the C and NC helix of known k-turn structures. This structural criterion was then applied to other RNA structures to search for sections in which two helical segments had a relative orientation that was similar to those of a conventional k-turn. For all helix segments we searched for nearby candidate helical segments that matched the search pattern. Using this approach we observed that the A. thaliana was solved at 2.25Å resolution by Ban and colleagues •Hoogsteen (A) pairs just as found in a standard k-turn structure, although the G1b•A1n pair is less buckled than is normally observed in a standard k-turn. Unlike a simple k-turn, an additional adenine is inserted between the G1b and A2b. This is directed away from the helix, re-entering the major groove of the NC helix and making a triple-base interaction with the U5b•A5n base pair via the Hoogsteen edge of A5n.The secondary structure and conformation of the junction are shown in junction . This hajunction , beginnijunction . Both G•Upon an initial inspection it seemed that the UUU sequence that precedes the 1b position should be the loop of the k-turn, where the central U is stacked upon the end of the NC helix in the manner of a normal L2 position. Nevertheless, further analysis of the detailed interactions in the core (see below) led us to extend the formal definition of the loop sequence in the 5′ direction to include the preceding C, so that we now define the loop as CUUU, labelled L1, L1.1, L2 and L3 sequentially, whereby L1, L2 and L3 are in positions equivalent to those in the simple k-turn see B. In conThus all the global features of a k-turn occur in the TPP junction, and the structure can be superimposed with a standard k-turn such as Kt-7 in the ribosome, using the backbone atoms for nucleotides –1n, L1, 2b, 1n, 2n D with anA. thaliana TPP riboswitch k-junction to see if corresponding interactions could be identified.The core of the k-junction and key hydrogen bonding interactions are shown in H. marismortui Kt-58. This k-turn has a two-nucleotide AG loop opposed by a single A nucleotide loop; the A is stacked onto the NC helix, thus taking the normal role of L2, and the preceding base paired nucleotide provides the O2′ that donates its proton to A1n (O2′ of the cytosine designated as L1 (see above) is hydrogen bonded to A1n N1 (O-N distance 2.7Å), and is thus performing the role of L1 in a simple k-turn E. Althoun to A1n .The nucleotide 5′ to L1 is designated –1b, and that to which it is base paired on the opposite strand is –1n. In a simple k-turn the O2′ of –1n donates a hydrogen bond to either N3 or N1 of the conserved A2b nucleobase . This –1A. thaliana k-junction is a standard k-turn structure, with all the key hydrogen bonding interactions.Thus once we have set the component nucleotides in the correct structural reference frame we see that in most respects the E. coli solved by Serganov and coworkers •G(sugar) base pair, the 1b nucleotide is an adenine so that the 1b•1n pair is a trans A(Hoogsteen)•A(Hoogsteen) base pair that is achieved by placing the A1b in a syn conformation. This contrasts with the 2b•2n base pair of Thelohania solenopsae Kt-23 for example, which is A(Hoogsteen)•A(sugar)  that do not interact elsewhere within the same molecule. The loop is identified as the CGU sequence (i.e. L1 through L3), but the 5′ C and G are base paired with the opposing CG sequence again making a continuously stacked C-T helix.The observation of the k-junction as the major structural component prompted us to examine carefully the structure of the corresponding TPP riboswitch from oworkers  at 2.05Åoworkers A. We finA(sugar) . Like thA. thaliana riboswitch, has all the critical features of a k-turn. Like the A. thaliana riboswitch, there is an additional hydrogen bond between G-2n O2′ and A3b O2′ (O–O distance = 2.8Å).The standard k-turn cross-strand hydrogen bonds accepted by the conserved adenine nucleobases are present; CL1 O2′ to A1n (O-N distance 2.5Å) and the G-1n O2′ to A2b (O–N distance 2.9Å) B. The acE. coli TPP riboswitch using two distinct methods. We used gel electrophoresis under non-denaturing conditions to follow changes in the global conformation of the riboswitch RNA. In addition, we have studied the binding of TPP to the RNA using isothermal titration calorimetry (ITC); Ferré D’Amaré et al.  leads to a lower affinity of TPP binding by a single order of magnitude.Studied by ITC, the unmodified riboswitch sequence exhibits an exothermic reaction on binding of TPP . The titd for the natural-sequence riboswitch, 2+ ions were present. In agreement with previous observations . The k-E. coli sequence) in 16.89%. The analysis also shows that 21.53% of sequences have an A•A pair at the 2b•2n position. The 3b•3n CG base pair is strongly conserved at 99.10%. An analysis of the full riboswitch is presented in Supplementary Figure S1. This reveals that the most conserved nucleotides are those contributing to the ligand binding pocket, and those of the k-junction.How general is the k-junction in RNA structure? Analysis of 11 056 TPP riboswitch sequences suggests that the essential features of the k-junction is preserved in this large sample . The mosH. marismortui in the J4,5 region. Interestingly, this was previously identified as a k-turn . There is a standard G1b•A1n base pair, but a trans A2b(Hoogsteen)•A2n(Hoogsteen) pair. In contrast to the riboswitch k-junctions there are no nucleotides separating G1b and A2b, and the G1b•A1n pair is significantly buckled. The loop is clearly identified as the preceding GUA sequence and designated L1 through L3 with their usual structural functions. The C helix begins with the uridine immediately 5′ to GL1. The structure of the non-bulged strand of the NC helix does not map linearly onto the sequence. In terms of the sequence the nucleotides run 5′ to 3′ as G3n A1n A2n U; the final uridine is base paired with AL2, requiring an S-shaped turn to form in the backbone. A similarly ‘out of sequence’ non-bulged strand is found in the NC helix of the complex k-turn Kt-11 of T.thermophilus, and we may therefore regard this as a complex k-junction. As previously, we designate the nucleotides in terms of their role in the structure, so that A1n is paired with G1b for example. With the components of the k-junction assigned we find that all the standard hydrogen bonding interactions are present. L1O2′ donates a proton to A1n N1 (O–N distance = 2.7Å), and A-1n O2′ to A2b N1 (O–N distance = 2.7Å). Thus this is another N1 class structure, and the A2b N6 to A2n N7 distance is 3.5Å. Like the simple k-turns, the L3 O2′ to L1/L2 phosphate proS O is found in this structure (O–O distance = 2.7Å), and like the riboswitch k-junctions one further hydrogen bond forms between G-2n O2′ and A3b O2′ (O–O distance = 3.3Å).The k-junction is not restricted to the TPP riboswitches, and we have found further examples in other RNA species. A k-junction is found the large ribosomal subunit of a k-turn , but cloH.marismortui 50S ribosomal subunit, at J94/99 . Although the T helix is only partially defined by the available structure this is plainly a three-helix junction that is similar to those of the TPP riboswitch. The three-base GCC loop can be defined according to function as L1 (stacked onto the C helix), L1.1 and L2 (stacked onto the NC helix). The NC helix begins with a standard trans G(sugar)•A(Hoogsteen) pair at the 1b•1n position. Once again this is almost planar, in contrast to those of the simple k-turns. A1n N1 accepts the standard hydrogen bond from L1 O2′ (O–N distance = 2.7 Å). The 2b•2n position is a trans A(Hoogsteen)•A(sugar) pair connected by a single hydrogen bond. A2b N1 accepts a hydrogen bond from –1n O2′ (O–N distance = 2.9 Å). Thus both the standard cross-strand hydrogen bonds are present, and once again this is an N1-class structure.The importance of the k-junction motif is further underlined by finding a second example in the H. marismortui ribosome. Given that the k-junctions are not immediately easy to recognize, and indeed have been overlooked in a number of structures, it is likely that they are relatively widespread and that more examples remain to be discovered.Our analysis shows that the basic elements of k-turn structure are well suited to accommodate a three-way helical junction, preserving all the key features and interactions of the k-turn. This has been extensively exploited in the TPP riboswitches, but clearly occurs more widely as demonstrated by the two examples found in the Supplementary Data are available at NAR Online.