Adrenocortical carcinoma (ACC) is a rare tumor in which prognostic factors are still not well established. Programmed Death Ligand-1 (PD-L1) expression in ACC and its association with clinico-pathological features and survival outcomes are unknown.Formalin-fixed paraffin-embedded (FFPE) specimens were obtained from 28 patients with ACC. PD-L1 expression was evaluated by immunohistochemistry (IHC) in both tumor cell membrane and tumor infiltrating mononuclear cells (TIMC). PD-L1 positivity on tumor cells was defined as ≥5% tumor cell membrane staining. TIMC were evaluated by IHC using a CD45 monoclonal antibody. For PD-L1 expression in TIMC, a combined score based on the extent of infiltrates and percentage of positive cells was developed. Any score greater that zero was considered PD-L1 positive. Baseline clinico-pathological characteristics and follow up data were retrospectively collected. Comparisons between PD-L1 expression and clinico-pathological features were evaluated using unpaired t-test and Fisher’s exact test. Kaplan-Meier method and log-rank test were used to assess association between PD-L1 expression and 5-year overall survival (OS).Among 28 patients with surgically treated ACC, 3 (10.7%) were considered PD-L1 positive on tumor cell membrane. On the other hand, PD-L1 expression in TIMC was performed in 27 specimens and PD-L1 positive staining was observed in 19 (70.4%) patients. PD-L1 positivity in either tumor cell membrane or TIMC was not significantly associated with higher stage at diagnosis, higher tumor grade, excessive hormone secretion, or OS.PD-L1 expression can exist in ACC in both tumor cell membrane and TIMC with no relationship to clinico-pathologic parameters or survival.The online version of this article (doi:10.1186/s40425-015-0047-3) contains supplementary material, which is available to authorized users. Adrenocortical carcinoma (ACC) is a rare and highly lethal malignancy arising from the adrenal cortex. In the United States, around 0.7-2 new cases per million are estimated every year ,2. OveraComplete surgical resection remains the only chance of cure for patients with early-stage disease (stage I and II). When feasible, resection is the mainstay of therapy, even for patients with locally advanced disease. Not without controversy, some studies suggest that adjuvant mitotane may improve clinical outcomes . HoweverThe management of metastatic disease is challenging and disappointing results have been reported with the few available systemic therapeutic options . DespiteThe biology underlying ACC is poorly understood . So far,The current progress in understanding how the immune system can modulate tumor progression or effective responses against cancer is unfolding . Immune In this study, our goal is to characterize PD-L1 expression in ACC tissues and to correlate levels of PD-L1 expression with clinico-pathological features as well as survival outcomes.A total of 28 patients with ACC were included in this study. Patient characteristics are summarized in Table In this cohort, 8 specimens were from metastasis and 20 from primary tumors. Among the metastatic specimens, only one had metastatic disease at diagnosis. Others had specimens collected at the time of relapse.Overall, PD-L1 expression on tumor cell membrane was considered positive in 3 of 28 patients (10.7%) . The extent of TIMC were recorded as focal in 7 patients (26%), mild in 9 patients 33.3%), moderate in 9 patients (33.3%), and high in 2 patients (7.4%). For PD-L1 expression in TIMC, scores greater than zero were identified in 19 patients (70.4%). There was no significant correlation between PD-L1 expression in TIMC and stage (UICC and/or ENSAT), grade, or excessive hormone secretion .p = 0.65)  Figure .Figure 2Multiple retrospective analyses described the correlation between levels of PD-L1 expression and prognosis in several malignancies . Some ofTP53 mutations, as predictors of poor prognosis and its value still needs to be confirmed [Tumor stage at diagnosis is the most important prognostic factor in ACC . Howeveronfirmed . From a Some tumors are infiltrated by immune cells and it can dynamically influence the host immune response against tumor . InteresWith the goal of restoring effective T cell responses, the inhibition of immune checkpoints such as PD-1 or PD-L1 has been considered attractive therapeutic targets using monoclonal antibodies. A set of well conducted clinical trials have reported encouraging clinical activity on PD-1/PD-L1 blockade across multiple tumor types. The first phase I clinical trial of nivolumab, an anti-PD-1 monoclonal antibody, showed significant clinical activity in RCC, melanoma, and NSCLC, leading to deeper investigations . Other aNo biomarkers have been established to precisely select patients for therapeutic strategies blocking the PD-1/PD-L1 axis. Moreover, while several studies have reported that PD-L1 expression in both tumor cell or tumor infiltrating immune cells is a potential predictor of response to immunomodulatory agents, the meaning and significance of PD-L1 expression in tumor cells or immune cells is still being investigated . Prelimi. Finally, we focused on the clinical significance of PD-L1 expression in either, tumor cells and immune cells. However, the criteria defining PD-L1 positivity (cut-offs) as well as the antibodies used to stain for PD-L1 have varied among different studies. Therefore, comparisons with other studies should be done with caution and a more specific definition of immune cells subtypes should be focus of further studies. Although the same methodology used in this analysis has been applied to other studies, the methodology for PD-L1 staining should be standardized to allow for reliable evaluation of PD-L1 expression and comparison among differing cohorts [Though this is the first study to date to evaluate the prevalence and prognostic significance of PD-L1 expression in ACC, there are several limitations. First, the retrospective nature of this analysis has led to missing data which may result in selection bias. In addition, considering that ACC incidence is low, it is difficult to perform studies with large and homogeneous cohorts. In our study, the sample size and the number of events in each group according to PD-L1 positivity were very small, limiting our ability to detect statistically significant differences between groups. At the same time, there may be a missing-variable bias, since other clinico-pathological features such as Ki67 were not available for the majority of patients in this cohort. Furthermore, tumor heterogeneity is a confounding factor and differences between of primary and metastasis still need to be investigated. In 2 patients from our series, we had the opportunity to review primary and metastatic sites of disease. In one case, the primary tumor and one met were evaluated; in the other case the primary tumor plus 3 independent metastases were compared. Interestingly, in both cases the same results regarding PD-L1 expression (positive or negative) were observed in primary and metastatic samples stained for PD-L1. In this study, we tried to minimize the selection bias due to tumor heterogeneity by analyzing whole tissue sections from surgical resections and excluding needle biopsies cohorts .In summary, ACC can express PD-L1 on both tumor cell membrane and immune cells and it may represent a potential target for therapeutic interventions. The current progress in cancer immunotherapy warrants prospective validation of our findings and further investigation of agents blocking the PD-1/PD-L1 pathway in this aggressive disease.Twenty-eight patients with ACC treated surgically at Dana-Farber Cancer Institute or Brigham and Women’s Hospital were retrospectively selected. Formalin fixed paraffin-embedded (FFPE) blocks from primary tumors or metastases were retrieved, and for each patient one representative tumor block was selected for analysis by a genitourinary pathologist. Baseline clinico-pathological characteristics such as age, gender, tumor size, grade, stage using UICC and ENSAT staging systems, hormone-related symptoms at clinical presentation, as well as follow up data were retrospectively collected for all patients. Patients who presented with Cushing’s syndrome, virilization, or feminilization were classified as functional tumors. This study received Institutional Review Board approval before tumor staining and data acquisition.PD-L1 expression was evaluated by immunohistochemistry (IHC) using a mouse monoclonal anti-PD-L1 antibody (405.9A11) developed in Dr. Gordon Freeman’s laboratory at Dana-Farber Cancer Institute. The immunohistochemical assay was validated using FFPE cell line controls known to be positive or negative for PD-L1 expression by flow cytometry . Four miCD45 immunostaining was performed on adjacent four micron-thick tumor sections, which were initially deparaffinized, rehydrated and heated with a pressure cooker to 125°C for 30 seconds in citrate buffer for antigen retrieval and then incubated with peroxidase  and protein blocking reagents (Dako #X0909) each for 5 minutes. Sections were then incubated with anti-CD45  antibody for 1 hour at room temperature followed by incubation with the Dako EnVision + System HRP labeled polymer anti-mouse (Dako #K4001) for 30 minutes. All sections were developed using the DAB chromogen kit (Dako K3468) for 2 minutes and then lightly counterstained with hematoxylin.For each sample, the percentage of tumor cells with PD-L1 expression on the cell membrane was estimated by two independent genitourinary pathologists who were blinded to clinical outcomes.TIMC were identified on the basis of IHC positivity for CD45, a pan-leukocyte marker expressed in lymphocytes, macrophages and dendritic cells ,43. The In this exploratory biomarker study, the pre-defined primary objective of this study was to characterize levels of PD-L1 expression on tumor cell membrane and TIMC in patients with ACC. Secondary endpoints were the correlation of PD-L1 expression with clinico-pathological features as well as 5-year survival rates. Five-year survival rate was defined as the time period between date of diagnosis and the date of death, or censored at 5 years after diagnosis. PD-L1 tumor positivity on tumor cells was defined as ≥5% tumor cell membrane staining. For PD-L1 expression in TIMC, any score greater than zero was considered positive.Statistical analyses were performed using SAS . Descriptive data are presented as mean and standard deviation (SD), or percentage. Comparisons between PD-L1 expression and clinico-pathological features were evaluated using Fisher’s exact test for categorical variables and unpaired t-test for continuous variables. Kaplan-Meier method estimated the distribution of 5-year survival rates by PD-L1 positivity. The association of 5-year survival rates with PD-L1 expression (negative vs. positive) was assessed by log-rank test and univariate Cox proportional regression analysis. Multivariate analysis were to be performed only if p-values were <0.05, considered statistically significant.


The Chilean National Examination of Orthopaedic Surgery (EMNOT) has been administered since 2009. It was developed to determine whether individual residents are meeting minimal knowledge standards at the end of their training programs.We performed a retrospective evaluation of the EMNOT for all years it has been administered (2009–2015). The test was analyzed for content, taxonomy of questions asked , residents’ performance, difficulty index and discrimination index.During the years of EMNOT administration, the most frequently tested areas have been pediatric orthopaedics (22.9 %), spine (13.8 %), general orthopaedics (13.8 %) and musculoskeletal trauma (9.9 %). A significant increase in questions with images was observed, as well as a significant decrease in the percentage of Type 1 and an increase in Type 3 questions. The Difficulty Index showed a medium level of difficulty for all years the examination has been administered. The Discrimination Index showed good discrimination in 2009, fair discrimination from 2010 through 2012, and excellent discrimination from 2013 through 2015.The EMNOT has evolved over several years to include better quality questions, better discrimination, and a more representative distribution of questions covering the different orthopaedic sub-specialties. This examination represents an effective instrument for quality assurance of orthopaedic residency programs in Chile. In recent decades, there has been a dramatic increase in the levels of scientific knowledge, diagnostic expertise and overall competence that orthopaedic surgery residents must master during their training. This growing amount of information creates a need for objective and standardized assessment methods to assess residents’ learning throughout their education, and especially at the end of their residency programs. In the United States of America, the Orthopaedic In-Training Examination (OITE), a national test taken annually by orthopaedic residents that evaluates knowledge of the most important subjects in orthopaedic surgery, has been assessed in several studies –4. HowevIn Chile, the rising expectations of patients, combined with the country’s economic growth and its aging population, have required that more new physicians be trained in different specialties. In response to this demand, the number of orthopaedic surgery residency programs throughout the country has grown during the last decade. As a result, the number of orthopaedic surgeons graduating each year in Chile has also increased significantly. The Chilean Society of Orthopaedic Surgery (SCHOT) plays an active role in the continuing education of orthopaedic surgeons and residents in Chile, and many of the Society’s active members serve as certifying experts to ensure the quality of training programs. This is similar to the other medical societies in Latin America, which have played an active role in quality assurance for specialty training –7.To determine whether individual residents are meeting minimal knowledge standards at the end of their training programs, the SCHOT developed the EMNOT, an acronym for “Examen Medico Nacional de Ortopedia y Traumatologia” . The EMNOT is a multiple-choice exam covering the areas of general orthopaedics (including basic sciences); musculoskeletal trauma; pediatric orthopaedics; spine; shoulder and elbow; hand; hip and pelvis; knee and sports medicine; foot and ankle; and musculoskeletal oncology. The EMNOT was first administered in 2009, and, to date, a total of 279 final-year residents have taken the test.In this study, we analyzed this educational tool by assessing the examination’s distribution of questions in the different subspecialties of orthopaedics, its question taxonomy, and its difficulty and discrimination indices.For this study, we first obtained institutional board approval from the President and the Board of Directors of the SCHOT and its ethics committee. The information we analyzed did not include the examinees’ and institutions’ identifying data.We retrospectively assessed the 7-year period during which the examination has been administered (2009–2015). The total number of residents taking the EMNOT (each year and during the entire period) was recorded. All questions were categorized using the taxonomic classification described by Buckwalter et al. . A questEach question was also classified into one of ten areas: general orthopaedics (including basic sciences); musculoskeletal trauma; pediatric orthopaedics; spine; shoulder and elbow; hand; hip and pelvis; knee and sports medicine; foot and ankle; and musculoskeletal oncology. The percentage of questions from each area as a fraction of the entire EMNOT was established. Residents’ performance on the complete examination, as well as on each taxonomic type of question, was recorded.P = R/T, where R is the number of correct responses and T is the total number of responses . Levels of P were established as proposed by Backhoff et al. [The Difficulty Index (P) was determined as described by Crocker and Algina . P referf et al. , with vaFinally, we calculated the Discrimination Index (D). D refers to the capacity of an item to discriminate between high-ability examinees and low-ability examinees. We first scored each examinee's test and rank-ordered the test scores; next, the top 50 % of students (high-ability examinees) and the bottom 50 % (low-ability examinees) were separated for analysis. D for each question is the number of examinees in the upper group who answered the item correctly minus the number of examinees in the lower group who answered the item correctly, divided by 50 % of the number of students taking the test each year. Levels of D were established as proposed by Ebel and Frisbie , with D Statistical analysis was conducted using Statistical Program for the Social Sciences (SPSS) version 18 . Categorical variables were expressed as percentages. Fisher’s exact test was used to analyze categorical variables. A p value less than 0.05 was considered statistically significant.A total of 279 residents finishing their orthopaedic surgery programs have taken the EMNOT since 2009; the number of examinees has increased each year, as shown in Table p < 0.01 , p < 0.05. Variations in the proportion of questions by taxonomy level are shown in Tables The proportion of Type 1 questions has decreased significantly (from 77.8 % in 2009 to 59.2 % in 2015). There has also been a significant increase in Type 3 questions (from 5.6 % in 2009 to 22.5 % in 2015), The most frequently tested areas have been pediatric orthopaedics (22.9 %), spine (13.8 %), general orthopaedics (13.8 %) and musculoskeletal trauma (9.9 %), as shown in Table P = 65.2; range 52.7 – 71.6) showed that the test has presented medium difficulty in each of the years it has been administered. The Discrimination Index  showed good discrimination in 2009 but only fair discrimination from 2010 through 2012; D values increased to excellent discrimination from 2013 through 2015, as shown in Table The Difficulty Index of the test  and an increase in the number of evaluation/decision-making questions (Type 3), the proportion of correct answers did not change during the administration period of the test Table . Therefo and an iThe EMNOT migrated from a paper-based exam to a computer-based exam in 2012. This change facilitated the inclusion of images, which resulted in an increased percentage of questions involving figures. There were no images in 2009 and 2010 and the test had a median of 34 questions with images from 2013 to 2015. This also helped with the development of type 2 and type 3 questions: the inclusion of radiographs, patient photos, and graphs has resulted in more intricate questions that require a more complex approach, requiring the examinee to provide diagnosis and treatment decisions.This examination was developed as a comprehensive examination testing core knowledge in all areas of orthopaedic surgery. In this analysis, we found that not all sub-specialties were proportionately represented by the test; pediatric orthopaedics and spine surgery were the most represented sub-specialties. Similar results were also found by Papp et al. in their report on the OITE exam . In our The test has evolved throughout the years, with an increase in the total number of questions, an increase in Type 2 and Type 3 questions, and a more homogeneous representation of the different sub-specialties. The Difficulty Index shows that the exam has had a medium difficulty level throughout all years of administration; the Discrimination Index shows that the test has increased its discrimination capacity in the last three years. While these results are encouraging, the EMNOT still faces the limitation that, as a multiple-choice examination, it only evaluates knowledge, diagnostic skills, and evaluation/decision-making skills. The assessment of surgical skills and attitudes of examinees still remains a challenge , 16.Although beyond the scope of this article, the results of these exams have also helped to identify learning needs for the development of a more relevant continuing education program. Moreover, yearly reports are given to Residency Program Directors in Chile to aid in curriculum development.In a recent review, Gurgacz et al. postulated that a socially useful credentialing process in surgical specialties should adhere to three main conditions: (a) only one institution should be responsible for the credentialing process, (b) best-practice standards for design, implementation, and monitoring of the examination should be used and (c) the organization should have a strong quality-improvement culture . The SCHThe EMNOT has evolved over several years to include higher quality questions, better discrimination, and a more representative distribution of questions covering the different orthopaedic sub-specialties. Thus, this examination developed and administered by the SCHOT represents an effective instrument for quality assurance of orthopaedic residency programs in Chile.


In addition, we systematically investigate their nonlinear optical responses. Our experiments indicate that the combined advantages of ultrafast relaxation, a broadband response from graphene, and the strong light-matter interaction from MoS2, can be integrated together by composition. The optical properties in terms of carrier relaxation dynamics, saturation intensity and modulation depth suggest great potential for the MoS2/graphene nanocomposites in photonics applications. We have further fabricated 2D nanocomposites based optical saturable absorbers and integrated them into a 1.5 μm Erbium-doped fiber laser to demonstrate Q-switched and mode-locked pulse generation. The fabrication of 2D nanocomposites assembled from different types of 2D materials, via this simple and scalable growth approach, paves the way for the formation and tuning of new 2D materials with desirable photonic properties and applications.Due to their relatively high compatibility with specific photonic structures, strong light-matter interactions and unique nonlinear optical response, two-dimensional (2D) materials, such as graphene and transition metal dichalcogenides, are attractive for ultrafast photonics applications. Here, we fabricate MoS Single- and few-layer 2D TMDCs materials have been successfully fabricated through different approaches, such as mechanical or liquid exfoliation12131416171822242) has been intensively studied. Unlike the semi-metallic graphene, MoS2 is a semiconductor with a band-gap that is dependent on the number of layers and varies from 1.29 eV1 (bulk) to 1.9 eV (single layer)272 has an indirect-to-direct transition when the thickness is reduced from bulk to monolayer12 shows strong light-matter interaction because of Van Hove singularities in the density of states2 a potential optical material for use in optoelectronics. Most recently, the fabrication of 2D heterostructures or nanocomposites assembled by graphene and other graphene-like 2D crystals (such as MoS2) has been demonstrated as a useful strategy for the realization of novel 2D materials with unique electronic and optoelectronic applications302/graphene nanocomposites were successfully synthesized322/graphene nanocomposites have also been reported for various applications of dye-sensitive solar cell fabrication322/graphene nanocomposites may have similar properties and comparable performance as materials containing composites2/graphene nanocomposites, their ultrafast carrier dynamics and nonlinear optical properties remain un-explored. However, understanding the ultrafast and nonlinear optical properties is critical for the use of MoS2/graphene nanocomposites in ultrafast photonics applications. Herein, we have investigated the carrier dynamics of the photo-excited states of MoS2/graphene nanocomposites by ultrafast pump-probe measurement with pump and probe wavelength in the near-infrared region (800 nm). Our results indicate that the carrier relaxation time of the MoS2/graphene nanocomposites is longer than that of graphene due to the existence of a covalent donor–acceptor structure between MoS2 and graphene. We have further studied the nonlinear absorption through the open aperture Z-scan system from the visible (400 nm) to the near-infrared (1560 nm) broadband frequency range. These findings suggest that MoS2/graphene nanocomposites could potentially be used as a passive Q-switcher and mode-locker in ultrafast lasers and ultrafast optical switches.Two-dimensional 2D) materials have received increasing attention in recent years because of their unique optoelectronic properties, which are a natural consequence of the quantum confinement effect associated with their ultra-thin 2D structure1D materia2Se32Te32Te340224Inspired by the excellent performance of graphene based saturable absorbers (SAs), many other graphene-like 2D nanomaterials, such as topological insulators , graphene (2) and MoS2/graphene nanocomposites (3) dispersed uniformly in N-methyl-2-pyrrolidone (NMP), whereas the inset shows the black solid MoS2/graphene nanocomposites. The transmission electron microscope (TEM) image of MoS2/graphene nanocomposites (2 homogeneously loaded on graphene. The MoS2 layers (black stripes) could be visibly identified and appear to be kept in close contact with graphene layers. The selected area electron diffraction (SAED) pattern is shown in the inset of 2 also confirmed the simultaneous appearance of both graphene and MoS2. The UV–Vis spectra were used to demonstrate that the MoS2/graphene nanocomposites had been successfully synthesized. As shown in 2/graphene nanocomposites (red solid line) at 610 and 670 nm correspond to the two characteristic absorption peaks of MoS2 (blue dotted line), whereas, the increase of light absorption at wavelengths shorter than 500 nm is due to the intrinsic absorption from graphene (black dashed line). Raman spectroscopy was carried out to further probe the internal structures of MoS2/graphene nanocomposites. The Raman spectrum , D and G peaks at 380, 407, 460, 1342 and 1593 cm−1, respectively. The in-plane E2g1 peak results from the opposing vibration of the two S atoms with respect to the Mo atom, whereas the A1g peak is associated with the out-of-plane vibration of only the S atoms in opposite directions2−1) between the E2g1g modes, the structure of the MoS2 in the nanocomposites has been suggested to consist of multiple layers462/graphene nanocomposites.The characterization of MoSmposites  reveals spectrum  shows thsolution , graphen2/graphene nanocomposites. The ultrafast carrier dynamics play a critical role in MoS2/graphene nanocomposites based optoelectronic applicationsT/T) of graphene and MoS2/graphene nanocomposites at room temperature are shown in T/T is defined as the relative change of the probe transmissivity caused by the pump:Ultrafast pump-probe and open aperture Z-scan measurements were used to investigate the ultrafast and nonlinear optical properties of the MoST/T is ascribed to the Pauli blocking induced by excitation based on hot carrier dynamics. Due to the identical wavelengths of the pump and probe pulses, upon the pump excitation, photon-induced carriers or excitons will occupy the probe transition states and reduce the probe photon absorption, leading to a positive ΔT. The time-dependent ΔT/T curve can be successfully fitted to a bi-exponential decay function:where, where, T/T was also observed in the MoS2/graphene nanocomposites. However, the carrier relaxation time of the nanocomposites is considerably longer than that of graphene (T/T) is proportional to the pump power intensity when the intensity is below 0.8 GW/cm2/graphene nanocomposites. Thus, the interaction between graphene and MoS2 is considered to play a significant role in their relaxation dynamics mechanism, as illustrated in A similar positive Δgraphene . The fas2/graphene nanocomposites with the covalent donor–acceptor structure, the Fermi energy of MoS2 (EF) is higher than that of graphene. Therefore, MoS2 can act as an electron donor through covalently bonding with graphene, thus facilitating the electron transfer from MoS2 to graphene. The details are described as follows. Upon photoexcitation with the 1.55 eV (800 nm) pump pulse, electrons can be rapidly transited from the valence band to the conduction band, in both graphene with its gapless band structure2 with its indirect gap energy of below 1.48 eV2 can be efficiently transferred to graphene, due to the slower excited state electron relaxation (hundred ps) of the pristine MoS25253542/graphene nanocomposites.The utility of graphene as a perfect electron acceptor has been proven in many systems49502O4 (BBO) crystal was used to double the frequency of the incident femto-second laser pulse at 800 nm through Second Harmonic Generation (SHG), allowing for the generation of laser pulses at 400 nm. In 2/graphene nanocomposites were moved along the beam focus, indicating the clear dependence of the transmittance peak on the input intensity.The open-aperture Z-scan measurements were also made under variable optical powers from the visible to the near-infrared broadband frequency range. A piece of β-BaB2/graphene nanocomposites show saturable absorption at near-infrared wavelengths, a home-made Erbium-doped femtosecond fiber laser  was also used as an excitation source. After being focused by a 10 cm focal-length lens, the laser beam was measured to be approximately 10 μm in diameter at the focal point. The saturable absorption curves were identified at different input intensities, as shown in To verify whether MoS2/graphene nanocomposites and graphene under the on-axis peak intensity I0 of 20.4 GW/cm2 at 800 nm. 2/graphene nanocomposites and MoS2 under 104.5 mW/cm2 at 1562 nm. In contrast with graphene and MoS2, the MoS2/graphene nanocomposites exhibit larger transmittance values with the same input intensity toward the focus, indicating an enhanced light-matter interaction compared to graphene or MoS2.The Z-scan measurement results are fitted by the equationT is the transmittance, L is the sample length, where, 2/graphene nanocomposites can be considered to be a broadband saturable absorber with an operation regime from the visible to the near-infrared. Meanwhile, compared with graphene and MoS2, the MoS2/graphene nanocomposites possess equivalent saturable intensity along with larger modulation depth. The larger modulation depth indicates that the MoS2/graphene nanocomposites may be a promising candidate for passive laser Q-switcher and mode-locker for the generation of ultrashort pulse.From these nonlinear optics fitting curves at different wavelengths, the saturable intensity and modulation depth are summarized in 2/graphene nanocomposites has allowed fabrication of a new type of 2D nanocomposites-based optical fiber saturable absorber device, as shown in The broadband saturable absorption of the MoSFurthermore, we have studied the evolution of the Q-switching parameters with an increase in pump power. The details are given in 2/graphene nanocomposites-based SA. Mode-locking started once the pump power exceeded a threshold of 70 mW. 2 pulse profile. The time-bandwidth product (TBP) of the pulses is ~0.405, indicating that the output pulses are slightly chirped. The above-mentioned experimental results also support the excellent performance of our fiber laser. Multiple soliton operations were observed when the pump power was increased to 120 mW.Aside from the passive Q-switching operation, we were also able to demonstrate the passive mode-locking operation with another relatively thin MoS2/graphene composites exhibit a fast delay time constant in the range of 1.3–1.4 ps and a slower constant in the 36–37 ps range. According to the slower delay time than that of graphene, we consider that graphene and MoS2 formed a covalent donor–acceptor structure. Indeed, during the excitation of the MoS2/graphene nanocomposites, there is an energy transfer from the excited states of MoS2 to graphene followed by the non-radiative relaxation of graphene’s excited state carriers to the ground state. Due to the excited state electron relaxation time of graphene and MoS2, we speculate that the carriers reach the ground state at picosecond relaxation times. Thus, the strong absorption of the nanocomposites and the immediate energy transfer from MoS2 to graphene could influence the excited state absorption cross section of graphene and further enhance the light-induced bleaching effect. Therefore, their covalent donor–acceptor structure is considered to be important in the mechanisms of the enhancement of nonlinear saturable absorption in the MoS2/graphene nanocomposites.In the pump-probe measurements, the MoS2/graphene nanocomposites are experimentally found to possess the enhanced and broadband saturable absorption. Taking advantage of their excellent saturable absorption property, a novel optical saturable absorber device based on MoS2/graphene nanocomposites was fabricated. The photonics application for ultra-short pulsed laser operation at the telecommunication band (1.5 μm) was also demonstrated. Our work provides a new type of tunable 2D photonics materials by combining the optical advantages of different 2D materials. We foresee that their widespread applications for ultrashort pulse generation or optical communications will be increasingly compelling and promising.According to the open aperture Z-scan measurements at various wavelengths from the visible to the near-infrared range, the MoS2SO4, NaNO3, and KMnO42/graphene nanocomposites were obtained via a hydrothermal method2MoO4·2H2O and 7.5 mmol L-cysteine (Lcys) with continuous stirring for 30 min. The suspension was then transferred into a 100 mL Teflon-lined stainless steel autoclave and tightly sealed at 240 °C for 24 h. Finally, after being washed several times with deionized water and ethanol, the as-synthesized product was collected by centrifugation, and then naturally cooled and dried in the vacuum oven at 80 °C for 12 h. The obtained black solid product was then annealed at 800 °C for 2 hours with 10% hydrogen in nitrogen flow at 200 sccm to yield the solid MoS2/graphene nanocomposites.As a precursor, graphene oxide (GO) was synthesized from graphite powder  by the modified Hummer’s method by using a mixture of H2 is a pristine nanoflake solution purchased from Graphene Supermarket. The graphene from Nanjing SCF Nanotech was dispersed in N-methyl-2-pyrrolidone (NMP) at a concentration of 2 mg/mL and the solid MoS2/graphene nanocomposites were dispersed in NMP at the same concentration.In our experimental measurements, MoSA full Ti: sapphire amplified laser system  with pulse repetition rate of 1 kHz, a center wavelength at 800 nm, and a pulse width of approximately 100 fs was used as the excitation source. The experimental setup of the pump-probe measurement is shown in 2/graphene nanocomposites. The incident laser pulses were also divided into two beams: the reflected beam was used as the reference, and the transmitted beam was focused by a lens as the excitation. The beam waist radius of the focused Gaussian beam was determined by a CCD camera. The pulse powers of the transmitted and reference beams were simultaneously measured by a dual-channel power meter (Newport).The Z-scan experimental setup is shown in 2 and the MoS2/graphene nanocomposites dispersion prepared in 1 mm-thick quartz cells. A comparison experiment on the pure solvents (NMP) was carried out, and no clear signal was observed for either pump-probe or Z-scan experiments. Meanwhile, the reproducibility of the transmission curves was tested by cycling the laser intensity up and down to further ensure that the detected materials were free of optical damage.Both pump-probe and open-aperture Z-scan measurements were performed on graphene, MoS2/graphene nanocomposites dispersion in NMP was drop-casted twice directly onto a fiber end-facet, which was then inserted in a standard FC/PC fiber connector for Q-switching operation. To achieve mode-locking operation, we drop-casted the dispersion only once onto another fiber end-facet so that the obtained SA was relatively thin. Allowing the fiber end-facets to dry at room temperature for approximately 10 h allowed us to obtain an even self-assembly of the MoS2/graphene nanocomposites molecules onto the fiber end-facets, as shown in 2/graphene nanocomposites-based SA device was successfully constructed for the fiber laser application by connecting the MoS2/graphene nanocomposites-on-fiber component with another clean, dry FC/PC fiber connector.The MoSHow to cite this article: Jiang, Y. et al. Broadband and enhanced nonlinear optical response of MoS2/graphene nanocomposites for ultrafast photonics applications. Sci. Rep.5, 16372; doi: 10.1038/srep16372 (2015).


Drosophila larva largely results from a sequence of runs and oriented turns. Thus, the larvae minimally need to determine (i) how fast to run, (ii) when to initiate a turn, and (iii) where to direct a turn. We first report how odor-source intensities modulate these decisions to bring about higher levels of chemotactic performance for higher odor-source intensities during innate chemotaxis. We then examine whether the same modulations are responsible for alterations of chemotactic performance by learned odor “valence” (understood throughout as level of attractiveness). We find that run speed (i) is neither modulated by the innate nor by the learned valence of an odor. Turn rate (ii), however, is modulated by both: the higher the innate or learned valence of the odor, the less often larvae turn whenever heading toward the odor source, and the more often they turn when heading away. Likewise, turning direction (iii) is modulated concordantly by innate and learned valence: turning is biased more strongly toward the odor source when either innate or learned valence is high. Using numerical simulations, we show that a modulation of both turn rate and of turning direction is sufficient to account for the empirically found differences in preference scores across experimental conditions. Our results suggest that innate and learned valence organize adaptive olfactory search behavior by their summed effects on turn rate and turning direction, but not on run speed. This work should aid studies into the neural mechanisms by which memory impacts specific aspects of behavior.How do animals adaptively integrate innate with learned behavioral tendencies? We tackle this question using chemotaxis as a paradigm. Chemotaxis in the  Drosophila melanogaster possess a brain of only 10,000 neurons , or by determining the preference via the proportion of time spent on either half of the dish throughout the entire 5-min testing period (PREFFILMED)  A. Both tFFILMED) B,B′. NexTo address this question it was important that larvae are capable of behaviorally expressing an associative memory—or not. That is, after paired odor–reward training odor preference was higher than after unpaired presentations of odor and reward , 2015. LGiven that the measurement of baseline preference involved testing the trained animals in the presence of the sugar reward, we asked whether the presence of the reward also had an influence on innate odor preference. We found this not to be the case , 2015. IDrosophila larvae orient in an odor gradient through a sequence of runs and turns, we considered the modulation of three general aspects of the orientation behavior: (i) how fast to run (run speed), (ii) when to initiate a turn (turn rate), and (iii) where to turn to (turning direction).We next asked by which particular behavioral processes learned preference comes about, and compare them to those behavioral processes underlying innate preference. Given that Regarding innate olfactory behavior, we found no systematic increase in run speed when using odor sources of increasing concentration B. Thus nkinesis , and more frequently when heading away from it (absolute bearing angle >90°) . Both thWhen heading toward the odor source, the larvae decreased turn rate after paired training, and increased turn rate after unpaired training . We devised a deliberately minimalistic model of larval chemotaxis in which run speed, turn rate and turning direction were parametrically estimated from the experimental data . Specifically, we assumed that turn rate and turning direction are functions of two variables, the current bearing angle toward the odor source and the current distance from it , as these appear to be the main determinants of sensory input during chemotaxis. Note that we estimated the parametric dependency of turn rate and turning direction separately for each of the 10 experimental groups, and that we used a constant run speed for each experimental group , both the turn rate and the angle of turning direction were estimated from the empirical data of the respective experimental condition. We found that such a model was largely sufficient to reproduce the pattern of odor preference across experimental groups , the empirical turn rate was substituted by random turn rates, while in the third mode , the empirical angle of turning direction was substituted by random angles of turning. We found that none of these simplified model modes could fully reproduce the across-group differences in preference scores obtained with the full model , and slow down when heading away.“Turn rate.” A larva may turn less often when heading toward the odor source , and more often when heading away from it.“Turning direction.” A larva may collect information about the direction of the odor source during its run and/or during the large-amplitude head casts flanking a turn, and use this information to turn more often into the desired direction.Regarding innate behavior, we confirmed that larval Drosophila modulate turn rate and turning direction, but not run speed , while after unpaired presentations of the odor and the sugar reward preferences are decreased (because the odor predicts where the sugar is not). These opposite, contingency-dependent modulations of preference are brought about by, respectively, opposite modulations of both turn rate and turning direction, but not of run speed , which were prefilled with 1% agarose  and stored at 4°C until used. To create a sweet, rewarding substrate, 0.2 M fructose  was added to the agarose. The odor  was presented by placing a 10 µL droplet within a transparent reinforcement ring that was fixed onto the inner side of the Petri dish lid . Dilutions were made in paraffin oil  as indicated in the Results. For all experiments, odor gradients were established for 1 min prior to the introduction of the larvae.Canton-S wild-type A group of ∼20 larvae was placed in the 4.50 × 0.85 cm starting zone of the Petri dish . To create a choice situation, the Petri dish contained only one odor source, which was placed 4.5 cm from the midline of the dish . The dish was closed with a lid and placed under a light pad . Larval behavior was filmed for 5 min from below for offline analyses  . After 5 min the dish was removed and the position of larvae was scored as being either on the odor side of the dish, the no-odor side, or in a 1-cm-wide neutral zone .n-amyl acetate . Both reinforcement rings on the lid were loaded with AM diluted 1:50 in paraffin oil to ensure that odor was present throughout the entire Petri dish. For the first training trial, larvae were placed into the starting zone of a fructose-containing Petri dish and covered with the lid that contained the two sources loaded with AM. After 5 min they were transferred to a fresh dish in which no odor was presented and no fructose had been added to the substrate. This training cycle was repeated two more times. Animals were then placed in the starting zone of a Petri dish with AM loaded on only one side of the Petri dish in order to create a choice situation. This test plate did not contain fructose, unless otherwise stated. Larval behavior data were then acquired as described above. The second group of animals was trained reciprocally (AM/+), i.e., odor and reward were presented in an unpaired way, and larvae were tested as mentioned.In half of the cases the sequence of training trials was as indicated , and in the other half of the cases the sequence of training trials was reversed . Note that the sequence of training trials does not have an effect on behavior at the time of the test .To characterize the impact of memory on olfactory behavior, a behavioral baseline is needed with respect to which memory effects can be assessed. Learned behavior after odor–reward training constitutes a search for reward that is suppressed if the sought-for reward is present during the test  innate . We thusDrosophila: We emphasize that experimentally naïve animals cannot be used to provide a reliable baseline to measure associative memories. This is because the nonassociative influences of animal handling, of odor exposure, and of sugar exposure in the trained but not the naïve larvae can confound such a comparison  , the number on the no-odor side (#noAM), the number of larvae on the middle stripe (#Middle) and the total number of larvae . From this, we calculated the odor preference  asAfter the 5 min test, we determined the number of animals on the odor side . Head casts that occurred within a time window of 5 sec before a turn to 0.5 sec after a turn were classified as flanking that turn, according to Runs are defined as the period between a turn and the first flanking head cast of the following turn.From the above variables we calculated the following measurements to describe chemotaxis behavior.For each larval trajectory  Choreography outputs time-series variables that describe larval movements and postures . From these, we calculated additional time-series variables  that allowed us to determine the distance of larvae from the odor source when turning, and the orientation and bearing of larvae before and after a turn :T) larvae spent on the odor side (AM) of the Petri dish  of the larval midpoint during runs, calculated per Petri dish N = 40, .2). We applied a sliding rectangular filter of 30 mm side length centered at each position (step width 2 mm).Larval density: defined as the number of animals per area (mmT):Turn rate: defined as the number of turns (NT) divided by the duration of time larvae were tracked (N = 40). To visualize how turn rate varies with bearing to the odor source, we calculated turn rate over all bearing angles , with data binned every 1° and a sliding filter of ±30° applied at each step. Data were pooled from all experiments to calculate a single value (N = 40). To visualize how turn rate varies over both distance to the odor source and bearing we pooled all data and applied a sliding box filter of ±30° and ±15 mm at each step (step width of 2° and 2 mm).The overall turn rate was calculated per Petri dish  was set to 0.0625 sec, corresponding to the sampling interval used for the experimental data. Model larvae started from positions drawn at random from the empirical start positions of the respective experimental group. Larval chemotaxis was modeled by a two-state Markov process (P(turn|run) = rturn · dt. The probability for remaining in the run state while running equalled P(run|run) = 1 − P(turn|run). At each time step, the turn rate rturn was set to the turn rate that was empirically observed for the respective experimental group, at the model's current bearing and distance from the odor source . The dependency of turn rate on the current bearing angle and the current distance to the odor source thus mimicked the effect of the actual sensory experience in real animals.Run and turn rate: while in the run state the model larvae moved in the direction of the head segment with constant run speed. Run speed was set to the median empirical run speed of the respective experimental group see . At eachP(turn|turn) = 1 as long as the absolute of the angle between the head and the tail segment was smaller than the absolute of the drawn turning angle. As soon as the designated turning angle was assumed, the state was switched to the run state with probability P(run|turn) = 1 and animals resumed their forward movement. In addition, the turn state was also switched to the run state with probability P(run|turn) = 1 whenever the head segment rotated into the border of the arena.Turning direction and turn actuation: whenever the model animal transitioned into the turn state, a turning angle was drawn at random from a subsample of the group's experimentally observed turning angles as follows: the preturn bearing angles at these turns were required to fall into the range , and the distance to the odor source at these turns into the range  . This procedure of drawing a turning angle from a subsample of experimental turning angles based on bearing angle and distance to the odor source  mimicked the effect of sensory input on the choice of turning direction experienced by real animals. While in the turn state the model larva rotated their head segments in the direction of the drawn turning angle with a constant angular speed of 53.7°/sec; this corresponds to the grand-average time derivative of the head angle at the onset of empirically observed events (onset was defined as the time-point where the head angle exceeded the 20° threshold). Animals remained in the turn state with probability To estimate the relative importance of turn rate and turning direction for overall differences between experimental groups, we ran our simulations in four different modes. In the first mode  the simulation was run as described above, such that both the turn rate and the turning angles were drawn from the empirical data. In the second and third mode either realistic turn rate or realistic turning direction was substituted with random behavior: for the second mode  turn rate equalled the average turn rates for a given experimental condition. For the third mode  for each turn the current bearing angle was substituted by a random bearing angle , and the current distance to the odor source was substituted with a random distance . A turning angle then was randomly drawn from a subsample of experimental turning angles around these constraints as described above for the fully realistic model. Finally, for the fourth mode (random turn rate and random turning direction) both turn rate and turning direction were chosen at random as described above.Larvae were modeled as a jointed, two-segment object consisting of head and tail segment A,B. Beca process :Run andFor each model mode and each experimental condition we simulated 1000 trials, where each trial consisted of a simulation of 13 animals over five minutes within a circular arena of 150 mm diameter. After the simulation, the trajectories in each trial were fragmented to match the fragmentation in the experimental data due to unresolved tracking at the boundaries of the Petri dish and during collisions: episodes of simulated trajectories were discarded at all time points at which the criteria (distance between arena center and midpoint >67 mm) was met, or the distance between the midpoints of any two model larvae was smaller than 5 mm. These settings reproduced the experimental constraints at the Petri dish boundaries, as well as the time course of the average number of tracked animals in the experiment . We did not test any of the simulated preference scores for statistical significance because at our sample size of 1000 trials the observed scores converge to their expected values.U-test; OSS, KW, MWU) were applied throughout the study, using Statistica  for the PC (the one-sample sign-test uses a web-based statistic tool provided on http://www.fon.hum.uva.nl/Service/Statistics/Sign_test.html). When multiple comparisons were performed within one analysis, a Bonferroni correction was applied to keep the experiment-wide error rate below 5% by dividing the critical P-value by the number of tests . When data are displayed as box plots, the middle line shows the median, the box boundaries the 25% (q1) and 75% (q3) quantiles, and the whiskers q1 − 1.5 × (q3 − q1) and q3 + 1.5 × (q3 − q1).Nonparametric statistics (one-sample sign test, Kruskal–Wallis test, Mann–Whitney The raw behavioral data set is available from the corresponding authors upon request.


We investigated if programmed death-ligand 1 (PD-L1) expression levels were prognostic of survival outcomes after intensity-modulated radiation therapy (IMRT) for non-metastatic nasopharyngeal carcinoma (NPC).104 patients with non-metastatic NPC treated with radical IMRT were investigated for their PD-L1 expression by immunohistochemistry (IHC) which were correlated with survival endpoints including locoregional failure-free survival (LRFFS), progression-free survival (PFS), distant metastasis-free survival (DMFS) and overall survival (OS).P = 0.042) and marginally longer PFS  compared to those whose tumors had PD-L1 IHC 0  or 1+ . PD-L1 IHC 2+ was independently prognostic of both LRFFS (P = 0.014) and PFS (P = 0.045) in multivariable analyses. Only induction chemotherapy followed by concurrent chemoradiation was prognostic of DMFS (P = 0.003) and no prognostic factor for OS was identified.After a median follow-up of 7.6 years, 21 (20.2%), 19 (18.3%) and 31 (29.8%) patients suffered from locoregional failure, distant metastases and overall disease progression, respectively, and 31 (29.8%) patients died. Patients whose tumors had PD-L1 IHC 2+ (moderate to strong membrane staining in ≥ 25% of tumor cells) enjoyed longer LRFFS (5-year 100% vs. 74.4%, Hazard ratio [HR], 0.159, 95% confidence interval [CI], 0.021–0.988; PD-L1 expression levels correlated with LRRFS and PFS in non-metastatic NPC treated with radical IMRT. It may play a role in radiosensitivity for NPC, which should be further confirmed in prospective studies using immunotherapy together with IMRT. Nasopharyngeal carcinoma (NPC) is an endemic malignancy in southern China, Hong Kong, Taiwan and Singapore where there is a strong genetic predisposition ,2. The u2 on day 1, 22 and 43 of IMRT. Patients who suffered from stage III to IVB diseases received concurrent chemoradiation as mentioned followed by adjuvant chemotherapy with cisplatin 80mg/m2 on day 1 and 5-FU 1000mg/m2 from day 1 to 4 every 4 weeks for 3 more cycles starting 4 weeks after completion of IMRT, according to Intergroup 0099 trial [2 on day 1 and 5-FU 1000mg/m2 from day 1 to 5 every 3 weeks) before concurrent chemoradiation due to close proximity of their tumors to the normal critical OARs including brainstem, spinal cord, optic chiasm/nerves, etc, in an attempt for good tumor shrinkage so that a radical radiation dose could be delivered to the tumors with relative sparing of these OARs.Prior approval from local institutional review board  was obtained before study commencement. All clinical investigations were conducted according to the principles of Declaration of Helsinki. All patients provided written informed consent before study recruitment. Patients with previously untreated non-metastatic NPC who were treated with IMRT between 2005 and 2009 were reviewed from a prospectively collected database. Formalin-fixed paraffin-embedded (FFPE) archived nasopharyngeal tumor samples were retrieved for PD-L1 expression analysis as described below. Pretreatment investigations and workup included blood tests for serum hematology, biochemistry, hepatitis B serology, serology for antibodies against EBV viral capsid antigen (VCA) and early antigen (EA), fabrication of customized thermoplastic head and neck cast for the following contrast-enhanced computed tomography (CT) scanning of the head and neck region in IMRT treatment position with 3mm slice thickness co-registered with the T1, T2 and gadolinium-enhanced magnetic resonance images (MRI) of the head and neck region acquired by a 3-tesla scanner for subsequent target and organs-at-risk (OAR) delineation and subsequent IMRT treatment planning and optimization and contrast-enhanced CT scan of the thorax and abdomen to rule out distant metastases. Patients who suffered from stage I to small stage II diseases received IMRT alone while those with bulky stage II disease received concurrent chemoradiation with cisplatin 100mg/m99 trial . Some paThe gross tumor volumes of both the primary tumor (GTV-P) and the radiologically involved cervical nodes (GTV-N) were outlined on the planning CT images with the aid of co-registered MRI images as we described before . SubsequA 9-field IMRT plan delivered by step-and-shoot technique was generated by Eclipse Treatment Planning System version 8.9, Palo Alto, CA, USA) using Anisotropic Analytical Algorithm . A totalImmunohistochemical staining of 5μm sections from FFPE archived tissue of nasopharyngeal biopsies or excision specimens of tumor was performed by in the diagnostic laboratory in Anatomical Pathology Unit of our hospital with the antibody 1:200 anti-PD-L1  using the standard protocol for routine diagnostic specimens. Hematoxylin and eosin (H&E) sections were also reviewed for the presence of tumors and TIL. Scoring of the intensity and percentage of tumor cells were done semi-quantitatively. The intensity of immunohistochemical staining with PD-L1 was scored by a 3-point scale. Score 0 meant minimal membrane staining with PD-L1 in less than 5% tumor cells, or no staining with PD-L1 was identified. Score 1 (1+) indicated minimal to moderate membrane staining with PD-L1 in between 5% to 24% tumor cells while score 2 (2+) comprises more positivity defined as at least 25% tumor cells demonstrating moderate to strong complete membrane staining with PD-L1. The same scoring system also applied to PD-L1 expression for TILs but their IHCs were reported separately from the IHCs for tumor cells.P < 0.05 (two-sided). All statistical analyses were performed by Statistical Package for Social Sciences (SPSS) version 20.0.Kaplan-Meier methods were employed for survival studies. Log-rank tests were used comparison of survival outcomes among patients with different stages of NPC and different expression levels of PD-L1 as defined above. Cox proportional hazard models with univariable and multivariable analyses were performed to identify prognostic factors of survival endpoints including locoregional failure-free survival (LRFFS), progression-free survival (PFS), distant metastasis-free survival (DMFS) and overall survival (OS) with age as continuous covariate and sex, use of IMRT alone, use of concurrent chemoradiation alone, use of induction chemotherapy followed by concurrent chemoradiation, use of concurrent chemoradiation followed by adjuvant chemotherapy, T-classification (T1/T2 vs. T3/T4), N-classification (N0/N1 vs. N2/N3), overall stage (I/II vs. III/IVA-B), expression levels of PD-L1 as categorical covariates. LRFFS was calculated from the starting date of IMRT to the date of occurrence of locoregional recurrence or death from any cause. PFS started from date of commencement of IMRT to the date of disease progression or death from any cause. DMFS was calculated from the starting date of IMRT to the date of occurrence of distant metastasis or death from any cause. OS was defined from the date of commencement of IMRT to the date of death from any cause. Receiver-operating characteristic (ROC) curves were constructed for the performance of the cut-off value of IHC scores of PD-L1 expression on the survival endpoints. Statistical significance was defined as Out of 146 consecutive patients with non-metastatic NPC treated with radical IMRT, only 104 patients were eligible with adequate archived tumor samples for IHC staining and subsequent analysis. The baseline characteristics of these 104 patients were displayed in After a median follow-up duration of 7.6 years (5.7–10.8 years), 21 (20.2%), 19 (18.3%) and 31 (29.8%) patients suffered from locoregional failure, distant metastases and overall disease progression respectively and 31 (29.8%) patients died during the study period. The median LRFFS, PFS, DMFS and OS were not reached. The mean LRFFS, PFS, DMFS and OS were 78.5, 71.7, 94.0 and 89.4 months respectively. The 5-year corresponding rates were 79.9%, 71.4%, 85.7% and 81.7% respectively.Seventy-eight patients (75.0%) had no membrane staining with PD-L1 for their tumor cells. Four (3.8%) and 22 (21.2%) patients had IHC 1+ and IHC 2+ for their tumor cells respectively . The basP = 0.042) compared to those whose tumors were PD-L1 IHC 0 or 1+ only (5-year 74.4%) (P = 0.067) compared to those whose tumors was PD-L1 0 or 1+ only (5-year 65.2%) (P = 0.048) and 0.590  respectively by using IHC 2+ as the cutoff  . Similarr 65.2%) . No signr 65.2%) , despiteP = 0.029) and PD-L1 IHC 2+ (P = 0.014) were independently and significantly prognostic of LRFFS in multivariable analysis. For PFS, T-classification, induction chemotherapy followed by concurrent chemoradiation and PD-L1 IHC 0 or 1+ and PD-L1 IHC 2+ were significant in univariable analysis. However only T-classification (P = 0.019) and PD-L1 IHC 2+ (P = 0.045) were significant as independent prognostic factors in the subsequent multivariable analysis. For DMFS, only induction chemotherapy followed by concurrent chemoradiation was prognostic in both univariable (P = 0.003) and multivariable analyses (P = 0.003). Only T-classification was prognostic of OS in both univariable (P = 0.031) and multivariable analyses (P = 0.026). Besides, no statistical analysis was performed for the correlation between PD-L1 expression of TIL and each survival endpoint because no definite PD-L1 staining was observed for all TILs.Univariable and multivariable analyses were performed for LRFFS and PFS Tables  and 4 asIn our study, PD-L1 was overexpressed in 25% (26 of 104 patients) of NPC patients and 21.2% of our patients whose tumors were IHC 2+ with PD-L1. Our result revealed that PD-L1 IHC 2+ was associated with better LRRFS and PFS. These advantages of PD-L1 expressed tumors have been observed in stage I resected pulmonary adenocarcinoma which was associated with a better relapse-free survival . HoweverThe role of TILs on survival outcome should be further explored. Hsu et al demonstrated that the presence of PD-1 expressing TILs was prognostic of locoregional relapse-free survival, disease-free survival and OS . HoweverThe prognostic role of PD-L1 in various types of malignancies remains to be deciphered. Though most studies delineated negative correlation of PD-L1 expression with survival in general –28, otheThe scoring system of IHC for PD-L1 has yet to be defined and it may vary for different types of malignancies. Traditionally, the median value of the percentage of tumor cells positive for PD-L1 in order to balance the patient groups is most often used. Alternatively, the semiquantitative H-score, obtained by multiplying the percentage of positively-stained tumor cells and the intensity of staining was also commonly used. In addition, definition of staining positivity  has not reached a unanimous consensus. For instance, positivity defined as ≥ 5% tumor cells positively stained with PD-L1 has recently gained popularity in lung cancer studies ,34. In fWe conclude that PD-L1 expression level can be used as a prognostic factor of LRRFS and PFS in non-metastatic NPC patients treated with radical IMRT. This has provided insight in designing immunotherapy targeting the PD-L1 pathway in combination with IMRT in the future, which should be validated in prospective randomized studies.


Adult neurogenesis, which is the continual production of new neurons in the mature brain, demonstrates the strikingly plastic nature of the nervous system. Adult neural stem cells and their neural precursors, collectively referred to as neural progenitor cells (NPCs), are present in the subgranular zone (SGZ) of the dentate gyrus, the subventricular zone (SVZ), and rostral migratory stream (RMS). In order to harness the potential of NPCs to treat neurodegenerative diseases and brain injuries, it will be important to understand the molecules that regulate NPCs in the adult brain. The genetic basis underlying NPC proliferation is still not fully understood. From our previous quantitative trait locus (QTL) analysis, we had success in using a relatively small reference population of recombinant inbred strains of mice (AXBXA) to identify a genetic region that is significantly correlated with NPC proliferation in the RMS.In this study, we expanded our initial QTL mapping of RMS proliferation to a far richer genetic resource, the BXD RI mouse strains. A 3-fold difference in the number of proliferative, bromodeoxyuridine (BrdU)-labeled cells was quantified in the adult RMS of 61 BXD RI strains. RMS cell proliferation is highly dependent on the genetic background of the mice with an estimated heritability of 0.58. Genome-wide mapping revealed a significant QTL on chromosome (Chr) 6 and a suggestive QTL on Chr 11 regulating the number of NPCs in the RMS. Composite interval analysis further revealed secondary QTLs on Chr 14 and Chr 18. The loci regulating RMS cell proliferation did not overlap with the suggestive loci modulating cell proliferation in the SGZ. These mapped loci serve as starting points to identify genes important for this process. A subset of candidate genes in this region is associated with cell proliferation and neurogenesis. Interconnectivity of these candidate genes was demonstrated using pathway and transcriptional covariance analyses.Differences in RMS cell proliferation across the BXD RI strains identifies genetic loci that serve to provide insights into the interplay of underlying genes that may be important for regulating NPC proliferation in the adult mouse brain. The persistent division of neural progenitor cells (NPCs) and the production of new neurons in the adult brain raise hope for potential therapies targeting the NPCs to compensate for neuronal loss in injured or disease brains. This process of continual neuron production, also known as adult neurogenesis, occurs in discrete brain regions that include the subgranular zone (SGZ) of the dentate gyrus, the subventricular zone (SVZ) of the lateral ventricle, and the rostral migratory stream (RMS) which is the rostral extension of the SVZ ,2. PreviThere have been considerable advances in knowledge concerning the regulation of adult neurogenesis over the past two decades ,7. AdultIt has also been previously shown that adult neurogenesis is significantly dependent on the genetic background, and the genetic diversity among inbred strains can serve as a reservoir for gene discovery -15. A wi60,204 = 4.77 , P < 0.0001), and heritability of cell proliferation in the RMS is estimated to be 0.58 (P < 0.0001).A substantial range of cell proliferation in the RMS, as detected by the uptake of BrdU, was identified in 61 BXD RI strains, with a 3-fold difference among the strains Figures . This raP = 0.4111; females = 63.97 ± 1.66; males = 65.83 ± 1.55) or body weight effect  on the number of proliferative cells in the RMS. We also examined the batch effect as the BXD panel has three epoch substructures: i) BXD 1–30 generated by Benjamin A. Taylor in the 1970s  to investigate expression co-variation among our candidate genes. The hippocampal transcriptome was selected based on findings from our earlier phenotypic correlation analysis where cell proliferation in the RMS is significantly associated with several hippocampal traits including hippocampus and dentate gyrus volumes and dentate gyrus granule cell number. The transcripts levels of each candidate genes were extracted from the BXD hippocampal expression database. A network graph Figure  was then60,170 = 2.88, P < 0.0001) . The number of BrdU + cells in the SGZ was 3.5 fold higher in C57BL/6 J compared to DBA/2 J, which was opposite to the parental strain differences observed in the RMS. However this reversal in phenotypic direction was not observed in the BXD RI panel. For example, BXD68 had both high numbers of BrdU + cells in the RMS and SGZ. Whereas, BXD55 had low numbers of BrdU + cells in the RMS and SGZ.The high correlation between the RMS linear density and the hippocampal traits such as volume and cell numbers, which harbours the SGZ of the dentate gyrus, led us to suspect common genetic determinants underlying these two neurogenic sites. We first assessed cell proliferation in the SGZ one-hour post BrdU injection. Similar to what was observed in the RMS, the number of BrdU + cells in the SGZ significantly differs among the BXD RI strains . Strain epoch, sex, body weight differences did not significantly influence the number of proliferative cells in the SGZ.The effects of confounding variables on cell proliferation in the SGZ were also examined. Age had a significant effect on the number of BrdU + cells in the SGZ .QTL mapping of cell proliferation in the adult SGZ revealed suggestive QTLs on Chr 1 (40-59 Mb), Chr 5 (104.5-119.5 Mb), and Chr 9 (71–83.7 Mb) and no significant QTL was identified Figure B. QTL maAdult neurogenesis is a multi-stage process that is complex in terms of regulation as each stage is modulated by different genetic and environmental factors ,9. The eTo probe the genetic architecture of a polygenic phenotype as complex as NPC proliferation requires a large genetic reference panel that can provide high resolution and power to identify genes with subtle but significant effects on NPC proliferation. In this study, we utilized the genetic diversity among the BXD RI strains as a tool to identify genes and pathways involved in NPC proliferation. Our BXD panel of 61 strains is the largest mapping panel employed thus far to study adult neurogenesis, and it is over twice the size the AXB/BXA RI set we have previously examined . In addiTo better understand the regulatory pathways modulating NPC proliferation in the RMS, we needed to first identify the molecular players participating in these pathways. It is presumed that the mapped loci harbor genetic modifiers of NPC proliferation. Our rather stringent candidate gene analyses revealed several genes in the significant Chr 6 QTL regions that are associated with neurogenesis and cell proliferation. One of them is Tgfa, which is a growth factor that binds to the epidermal growth factor receptors (EGFR). Upon binding to its receptor, Tgfa activates the ErbB signalling pathway and subsequently triggers the Mitogen-activated protein kinase (MAPK) signalling cascade. These pathways play crucial roles in regulating cell proliferation and differentiation. The absence of Tgfa resulted in attenuation of NPC proliferation in the dorsolateral corner of lateral ventricles and reduction of newborn neurons in the OB . AnotherCandidate gene pathway analysis further provided insights into the interconnection among the candidate genes. Both the Tgfa gene in the major Chr 6 QTL and the Camk2a gene in the secondary Chr 18 QTL are involved in the ErbB signalling pathway, which regulates diverse biological processes such as proliferation, differentiation, and survival. Tgfa serves as one of the extracellular ligands that bind to Epidermal growth factor receptors and subsequently triggers downstream intracellular signalling pathways, which is modulated by Camk2a. Camk2a also participates in the Wnt signalling pathway as well as Ppp2ca in the suggestive Chr 11 QTL. Ppp2ca is one of the major Ser/Thr phosphatases which has been previously implicated as a negative regulator of cell proliferation. Ppp2ca interacts with Axin, a scaffold protein that down-regulates the Wnt signalling pathway by destabilizing β-catenin . These fThe interconnectivity among candidate genes is further reflected at the transcriptional level. Because there is no BXD transcriptome data available for the RMS, we probed the hippocampal gene expression database for the transcript levels of our candidate genes. We later confirmed the expression of these candidate genes in the mouse RMS using the microarray data of laser microdissected RMS generated by Khodosevich et al. . Our genThe high correlation between cell proliferation in the RMS and several hippocampal traits such as the number of granule cells in the dentate gyrus led us to suspect shared genetic determinants underlying proliferation in RMS and dentate gyrus. We examined the QTLs modulating cell proliferation in SGZ of the dentate gyrus using our expanded panel of BXDs. The number of proliferative cells in the SGZ differed ~4 fold among the 61 BXD RI strains. A similar fold difference was reported by Kempermann and colleagues where they used Ki67 as a proliferative marker and quantified the number of dividing cells in the SGZ of 29 BXD RI strains . The proThe bottom-up approach, starting with the manipulation of a single gene to induce changes in phenotype, has yielded valuable insights into a gene’s role in adult neurogenesis. However, this approach is limited in capturing the full complexity of adult neurogenesis where several genes and signalling pathways can regulate neurogenesis in the adult brain. In this study, we gained insights into the genetic architecture underlying NPC proliferation using a top-down, phenotypic-driven approach.The rich genetic and phenotypic diversity in the expanded panel of BXD RI strains has allowed us to probe the complex networks regulating NPC proliferation in the adult mouse brain. Here, we performed genome-wide scans for QTLs associated with the differences in the number of NPCs among the BXD RI strains. Novel QTLs and significant loci-loci interaction were detected. Using bioinformatics resources and transcriptome databases, we further identified several candidate genes with correlating expression patterns and discovered the convergence of these genes on the same/parallel signalling pathways that are known to regulate adult neurogenesis. The combinatorial influence of these regulators on NPC proliferation remains to be elucidated. Nevertheless, our findings provide a firm starting point to unravel modifier genes of NPC proliferation and offer novel insights into the dynamic interplay of regulatory pathways controlling this process.BXD RI strains were obtained from two different sources. Parental strains C57BL/6 J, DBA/2 J, and BXD 1–42 RI strains were purchased from the Jackson Laboratory . BXD 43–103 RI strains were provided by Dr. Robert W. Williams and Dr. Lu Lu . Mice were housed at the University of Tennessee Animal Facility in a pathogen-free, ~23.5°C, and 45–50 humidity environment on a 12 h light-12 h dark cycle. A total of 61 BXD RI strains and 265 mice were used in this study. At least one male and one female per BXD strain were examined. Mice studied were between 50–85 days old. All experiments were conducted according to the Guide for the Care and Use of Laboratory Animals  and the Canadian Council of Animal Care. Approval was obtained from the Institutional Animal Care and Use Committee at the University of Tennessee Health Science Center and the Animal Care Committee at the University of British Columbia on all our animal protocols.-1) to label actively proliferating cells in their brains. One-hour post injection, mice were perfused with acetic acid: 95% ethanol (1:3) as previously described [th section was mounted on Superfrost/Plus slides for anti-BrdU immunohistochemistry. In brief, sections were deparaffinzed, and treated with 1 M HCl for 30 mins at 37°C to denature DNA. Slides were incubated with the mouse primary anti-BrdU monoclonal antibody  overnight at room temperature. The next day, secondary biotinylated horse anti-mouse IgG  was applied to the slides for 1 h at room temperature VECTASTAIN Elite ABC kit (Vector Laboratories) and 3, 3′-diaminobenzidine  were subsequently used to reveal BrdU immunoreactivity.All mice received a single intraperitoneal injection of BrdU  cells in the RMS was systematically quantified using the single best-section quantification method as previously described . BrieflyMice analyzed for RMS cell proliferation were also used to quantify cell proliferation in another active site of adult neurogenesis called the SGZ. The SGZ is located between the granular layer and hilus of the dentate gyrus of the hippocampus. The number of proliferative, BrdU + cells in the SGZ was counted throughout the left dorsal hippocampus for every tenth sagittal section. Counts stopped where the dorsal and ventral components of the hippocampus merged. The average number of BrdU + cells in the SGZ and SEM were determined for each BXD strain.P ≤ 0.05 were considered significant. Heritability was estimated in a broad sense where we calcualted the ratio of variance that is accounted for by the differences between strains over the total variance, which includes both between-strain variance and within-strain variance [One-way analysis of variance (ANOVA) was performed to detect significant inter-strain differences in the number of proliferative cells in the RMS . Tukey’s Honestly Significant Difference (HSD) post-hoc test was subsequenlty employed to determine signficant differences between two BXD strains. General linear modeling was used to determine the contribution of possible confounding covariates including age, sex, body weight, and strain epoch effects and their interaction effects on RMS proliferation. Residuals from regression fitting these variables for RMS linear density were obtained and subsequently used to adjust for the mean RMS linear density per strain . Analysevariance .P value of 0.05 and 0.63, respectively. A significant QTL is referred to as a chromosomal region with LRS score equal or above the genome-wide significant level (P = 0.05). A suggestive QTL is a region of the chromosome with LRS score equal or above the genome-wide suggestive level (P = 0.63). LRS scores of the mapped QTLs were converted to the likelihood of the odds (LOD) scores by dividing LRS by 4.61. The confidence limits of each QTL were defined by the 1.5 LOD support interval [Cell proliferation data collected from the 61 BXD RI strains was deposited into the GeneNetwork, which is an open-access online database that contains detailed genotype information of each BXD RI strain. Genome-wide interval mapping of QTLs regulating NPC proliferation was performed using WebQTL, a module of the GeneNetwork. The likelihood ratio statistic (LRS) was computed to assess the strength of genotype-phenotype association of the genome scans. Permutation test of 2000 permutations was computed to establish the significance and suggestive thresholds where the LRS values corresponded to a genome-wide interval .http://www.sanger.ac.uk/resources/mouse/genomes/) was used to confirm the presences of SNPs and indels in each of the candidate gene. The expression of each candidate gene in the adult brain is visualized using Allen Brain Atlas (http://www.brain-map.org). Microarray data on laser-microdissected NPCs in the RMS [http://david.abcc.ncifcrf.gov) and the QTLMiner module of the GeneNetwork (http://www.genenetwork.org/webqtl/main.py?FormID=qtlminer). Genes were prioritized according to the following criteria: 1) polymorphic (i.e. associated with ≥ 1 SNPs and/or Indels), 2) expression in the adult neurogenic sites including the RMS, and 3) associated with GO terms such as neurogenesis, cell proliferation, and cell cycle progression.An integration of bioinformatics strategies and gene expression data were employed to evaluate the underlying genes in the mapped QTL intervals. The genetic variation structure within identified QTL regions were examined using the single-nucleotide polymorphism (SNP) and insertion/deletion (indel) data available at the GeneNetwork SNP browser (genenetwork.org/webqtl/snpBrowser.py). The numbers of SNPs and indels that are associated with each candidate gene, and ones that differ between the two parental inbred strains  were determined. Sequencing data released by the Mouse Genomes Project ( the RMS  was usedhttp://www.genenetwork.org) under Record IDs: 13545 , 13586 (RMS linear density data corrected for age effects), 14786 , and 16187 (SGZ BrdU + cell counts corrected for age effects).BXD data supporting the results of this article are available in the BXD Published Phenotypes Database at the GeneNetwork  Whole-genome interval mapping of the old BXD strains 1–42 generated by Benjamin A. Taylor; 27 of the 61 BXD strains examined in this study belong to this group (B) Whole-genome interval mapping of the new BXD strains 43–100 generated at the University of Tennessee Health Science Center (UTHSC); 34 of the 61 BXD strains examined in this study belong to this group. Despite differences in LRS scores, the same Chr 6 QTL (76.8-88.8 Mb) is identified from mapping the two BXD sub-populations.Click here for fileCorrelation between the transcript levels of candidate genes and RMS linear density. Inter-strain differences in the expression of Anxa4 , Ppp2ca , Gnpnat1 , and Camk2a (a candidate gene in the suggestive Chr 18 QTL) were observed in the hippocampi of different BXD RI strains. (A) Scatterplot of the Anxa4 transcript levels negatively correlated with the mean RMS linear density. (B) Scatterplot of the Ppp2ca transcript levels negatively correlated with the mean RMS linear density. (C) Scatterplot of the Gnpnat1 transcript levels negatively correlated with the mean RMS linear density. (D) Scatterplot of the Camk2a transcript levels positively correlated with the mean RMS linear density. Each dot represents a BXD strain.Click here for fileCorrelation between the RMS linear density and total number of proliferating NPCs in the RMS. RMS linear density  was determined using the single best-section quantification method, and it is significantly correlated with the total BrdU + cell counts (y-axis) which was determined from surveying every 10th section throughout the medial to lateral extent of the RMS (P < 0.0001). Each data point represents counts obtained from a randomly selected RI mouse.Click here for file


Transparent and flexible electrodes are widely used on a variety of substrates such as plastics and glass. Yet, to date, transparent electrodes on a textile substrate have not been explored. The exceptional electrical, mechanical and optical properties of monolayer graphene make it highly attractive as a transparent electrode for applications in wearable electronics. Here, we report the transfer of monolayer graphene, grown by chemical vapor deposition on copper foil, to fibers commonly used by the textile industry. The graphene-coated fibers have a sheet resistance as low as ~1 kΩ per square, an equivalent value to the one obtained by the same transfer process onto a Si substrate, with a reduction of only 2.3 per cent in optical transparency while keeping high stability under mechanical stress. With this approach, we successfully achieved the first example of a textile electrode, flexible and truly embedded in a yarn. Electronic textiles offer a form of functional devices integrated in objects that are essential to human kind: clothes. Devices embedded in textiles would enable a form of electronics that is comfortable to wear, lightweight, easy to transport and imperceptible617Monolayer graphene offers transparency, flexibility and mechanical strength combined with electrical conductivity1819202123242729302 or Al2O3Large-area monolayer graphene films were synthesized by chemical vapor deposition (CVD) on copper foil using methane as carbon source. After growth, a thin poly(methylmetacrylate) (PMMA) film was spin-coated on the graphene/Cu substrate to support mechanically the graphene layer during the following copper etching process. Graphene monolayer sheets were transferred to polypropylene fibers after copper etching, with following hot acetone cleaning to remove the PMMA layer. −1) can be observed for both the PLA and PP fibers coated with graphene, which are assigned to the E2g vibrational mode of graphene. The 2D band (~2700 cm−1), also commonly used to identify graphene, overlaps with a region of intense and broad peaks intrinsic of the uncoated fibers. Nevertheless, the 2D band was observed on a control sample obtained by transferring a graphene film  onto a SiO2/Si substrate  was used to confirm the presence of graphene on the fibers. s) > 1 kΩ/sq due to topological defects such as dislocations and grain boundaries432 delocalization of π electrons in graphene and effectively scatter the charge carriers, forming highly resistive grain boundaries. Other defects such as wrinkles, folds, tears and cracks can also lead to a disrupted path of carrier transport, affecting the electrical resistivityvs. voltage characteristics is linear, as expected for ohmic conduction. Furthermore, for all the different fibers investigated here, the value of sheet resistance measured in air is typically lower than 12 kΩ/sq, with the best values reaching as low as 1 kΩ/sq  1 kΩ/sq . These v 1 kΩ/sq . FlexiblThe effect of bending on the electrical resistance of the graphene-coated fibers was tested to analyze its suitability as a flexible electrode in textile electronics . The folFinally to illustrate the high transparency, conductivity and flexibility of the graphene coated fibers, we show in Although most graphene derivative fibers have been presented as single components121314In summary, we have demonstrated that transparent monolayer graphene can coat textile fibers by wet transfer, forming a highly conductive and flexible thread with negligible aspect change. A surface treatment based on ultraviolet-ozone irradiation strongly promotes graphene adhesion to the fibers, leading to minor debonding upon on bending and high resistance stability. These first electrodes completely embedded in a textile fiber can pave the way for the development of devices for truly wearable, foldable, and transparent electronics.All the fibers have been fabricated by CenTexBel using a monofilament extrusion line.2 (0.4 sccm) for 10 min, the growth was performed under H2 (2 sccm) and CH4 (35 sccm) for 5 min, and then the chamber was rapidly cooled to room temperature for 10 min with a large Ar flow (50 sccm). The thickness and quality of the graphene film was first tested by transferring a CVD-grown graphene film onto a SiO2/Si substrate and examined by optical microscopy and Raman spectroscopy (see 2/Si or to the fibers (CenTexBel). In the case of the fibers, the graphene sheet was transferred by the scoop process with the fibers immobilized on a rigid PET support. Finally the PMMA was dissolved with acetone, leaving the graphene on the surface of SiO2/Si or of the fibers. The untreated fibers were used as received. UVO treatment of the fibers was performed using a UVO Cleaner® 144AX-220 (Jelight Company Inc.) for 11 min (6 min exposure time and 5 min exhaust).Single-layer graphene was grown by chemical vapor deposition using a Moorfield nanoCVD-8G with a cold-wall reaction chamber, using methane as a carbon source and 0.025 mm thick 99.999% pure copper foil . The copper foils were annealed at 1035°C under Hcopy see . For theca. 5 cm−1.The samples with and without graphene were characterized by AFM in multiple regions  Dimension 3100 with a Nanoscope IIIa controller) in tapping mode using commercial Olympus Si tips (OTESP), and by Raman spectroscopy . The collected Raman radiation was dispersed with a 600 lines/mm grating and focused on a Peltier-cooled charge-couples device (CCD) detector allowing a spectral resolution of 2p = RG + 2RC, where RG = ρL/W is the graphene resistance  and Rc is the contact resistance, we can estimate the contact resistance using samples with different lengths, L1 and L2 (W is the same). In this case, the contact resistance is given by Rc = (R2pDev1-ρG(LDev1/WDev1))/2 and ρG = (R2pDev1-R2pDev2)(LDev1/WDev1-LDev2/WDev2)−1. A contact resistance value of the order of 1 kΩ was found for our devices  with a resolution of 1.5 nm operating in the 200-950 nm range, coupled to a Deuterium Tungsten Halogen light source. Illumination and detection were done through optical fibers of 200 μm.H. A. and M. F. C. conceived the work. A. I. S. N. prepared the samples and performed the experiments. T. H. B. prepared the conductive graphene. L. V. M. carried out the AFM imaging. I. S. developed the polyethylene fibers. A. I. S. N., H. A., S. R., and M. F. C. carried out the analysis. H. A. and M. F. C. co-supervised the work and H. A. wrote the manuscript.How to cite this article: Neves, A. I. S. et al. Transparent conductive graphene textile fibers. Sci. Rep. 5, 9866; doi: 10.1038/srep09866 (2015).


Graphene-enhanced Raman scattering (GERS) spectra and coherent anti-Stokes Raman scattering (CARS) of thymine molecules adsorbed on a single-layer graphene were studied. The enhancement factor was shown to depend on the molecular groups of thymine. In the GERS spectra of thymine, the main bands are shifted with respect to those for molecules adsorbed on a glass surface, indicating charge transfer for thymine on graphene. The probable mechanism of the GERS enhancement is discussed. CARS spectra are in accord with the GERS results, which indicates similar benefit from the chemical enhancement. Surface-enhanced Raman spectroscopy (SERS) has become an efficient technique that enables detection and study of an extremely small amount of biochemical materials and single-molecule detection -4. SERS In the present paper, we report on a comparative study of the surface-enhanced Raman scattering and surface-enhanced coherent anti-Stokes Raman scattering for thymine (Thy) adsorbed on graphene layers.2 or 20 μg/cm2.In the Raman and coherent anti-Stokes Raman scattering (CARS) measurements, we use aqueous 1 mg/ml and 10 μg/ml solutions of commercially available thymine . The samples for optical experiments were prepared by depositing a drop of Thy solution on graphene-on-silica or glass substrates, respectively. The average surface density of Thy after water evaporation was either 200 ng/cm2:CH4 gas mixture for 10 min (15 mbar). Because of the self-limiting graphene growth on a copper substrate, almost single-layer graphene [The graphene-on-silica substrates were fabricated by depositing a graphene sheet on fused silica. Single-layer graphene was prepared by using chemical vapor deposition (CVD) of graphene on a copper foil described elsewhere ,14. Befor 10 min  mbar. Begraphene . After t3. The remaining PMMA/graphene stack was rinsed in deionized water for 30 min and then placed on a silica substrate in such a way that the graphene was facing the silica. In order to relax internal stress in the stack, another PMMA layer was deposited on top of the existing PMMA [The graphene sheet was spin coated by a 1-μm-thick polymethyl methacrylate (PMMA) layer, and then the copper foil was removed by wet etching in FeCling PMMA . After r−1 was used as the reference.The Raman spectra of the bare and Thy-adsorbed graphene layers were obtained using a Renishaw inVia Raman microscope  at an excitation wavelength of 633 nm and spot size of 1 μm. All Raman measurements were performed at room temperature. The WiRE 3.4 software (Renishaw) was used for Raman data acquisition and data analysis. The Si Raman band centered at 520 cmz) directions, respectively. The CARS images of the sample surface were composed of 250 × 250 pixels obtained with a 2-ms pixel dwell time using a piezo scanning system . CARS spectra were recorded with a scanning rate of 5 cm−1 s−1 in the frequency ranges of 1,200 to 1,700 cm−1 and 2,500 to 3,400 cm−1 with a resolution of about 8 cm−1.The experimental setup was based on a homemade CARS microscope equipped with a compact laser source  capable of providing pump and Stokes pulses with energies up to 10 nJ  [La ~ I (D)/I (G) where I (D) and I (G) are intensities of the G and D peaks in the Raman spectrum. In our case, the ratio La does not exceed 0.1 for single-layer graphene, indicating a good crystallinity of the fabricated graphene.Raman spectra of the fabricated graphene samples .The position equation : ωG = 1,ωG = 1,589 cm−1 corresponds to n ~ 1. It should be mentioned that this layer had defects that is proved by the presence of the weak D peak situated in the vicinity of 1,325 cm−1. The ratio I (G)/I (2D), which also can be used to estimate the number of graphene layers, does not exceed 0.4, indicating the presence of one to two layers of graphene. These results correspond well to SEM images of graphene-on-silica substrates that show gaps and defects in the graphene sheets.The obtained experimental value of −1 which originated from symmetric stretching vibrations of neighboring Si-O bonds [−1 is also associated with symmetric stretching vibrations of oxygen atoms, which involve also a substantial amount of surrounding Si atoms. The peaks at 485 and 600 cm−1 are attributed to the formation of four-membered (four oxygen atoms in the ring) and three-membered (three oxygen atoms in the ring) defects, respectively [−1 /I (G) for bare and Thy-adsorbed graphene-on-silica substrates are presented in Table I (2D)/I (G) is also sensitive to the doping effects [The observed shifts of the G and 2D peaks indicate that deposition of Thy results in doping of graphene ,30. Spec effects ,33.TableIglass) and graphene-on-silica (IGr) substrates  and mglass(Thy) are the Thy surface densities on relevant substrates. Under our experimental conditions, we can well register already 200 ng/cm2 of Thy adsorbed on a single-layer graphene  of about 5 × 102 was observed for the C(6)-H group and about 3 × 102 for NH and C = O vibrations of Thy molecules adsorbed on single-layer graphene.where 2 Figure b to get The assignments of Raman shift for thymine adsorbed on graphene and glass substrate are listed in Table −1 is attributed to the wag of N-H in the structure of Thy, 741 cm−1 is attributed to ring breathing and coupled to the out-plane wag of N-H, 804 cm−1 is attributed to the wag of C-H on C = C, 985 cm−1 is attributed to ring breathing coupled to in-plane -CH3 asymmetric stretching, 1,367 cm−1 is attributed to N-H and C-H in-plane bending, and 1,671 cm−1 is attributed to C = O stretching and coupled to N-H and C-H asymmetric bending. The bands of 1,410 and 1,491 cm−1 were also the characteristic peaks of GERS for ring modes of Thy, not visible on glass. The bands centered at 116 and 152 cm−1 in the Raman spectra deposited on the glass substrate indicate the presence of three-dimensional micro-crystallites of Thy due to the lower sticking probability on glass compared to graphene. These bands do not appear in the Raman spectra of Thy on the graphene-on-silica substrate. The microscopy images also show that Thy adsorbed on graphene seems to form flat flakes - the same observation we had in our previous work [−1 which is associated with the molecular vibrations in the substrate is absent in Figure −1 is available due to the very small thickness of flakes of Thy.The band observed for Thy adsorbed on single-layer graphene in Figure −1 in GERS spectra of Thy, which is attributed to the aromatic C(6)-H stretching vibration, is strongly enhanced, while the band at 2,932 cm−1, which is assigned to the symmetric CH3 stretching vibration, is decreased  mode at 3,055 cm−1 in the CARS spectrum. The aromatic C(6)-H stretching band of Thy in Raman spectra is observed at 3,062 cm−1, and in CARS spectra, this band shifts to 3,055 cm−1. In addition, we have to indicate that this band is indistinguishable for samples prepared on glass substrates  mode at 3,055 cm−1 in the CARS and at 3,066 cm−1 in Raman, respectively. The change in GERS compared to ordinary Raman strongly depends on the vibrational mode of adsorbed molecules and thus can provide an insight into the chemical mechanism.GERS was studied for the Thy adsorbed on single-layer graphene. The observed enhancement of the Raman signal of more than 100 times was observed for C(6)-H, NH, and C = O vibrations of Thy molecules adsorbed on single-layer graphene and was accompanied with a minor shift of these Thy bands. The highest enhancement in GERS effect was observed for ring modes of thymine. The CARS spectra of Thy adsorbed on single-layer graphene are in accord with GERS of Thy and demonstrated redistribution of the intensity of the Thy Raman bands in the 2,800- to 3,100-cm