Graphene is a flat monolayer of carbon atoms, arranged in a two-dimensional hexagonal structure, with extraordinary electrical, thermal, and physical properties. Moreover, the molecular structure of graphene can be chemically modified with molecules of interest to promote the development of high-performance devices. Although carbon derivatives have been extensively employed in industry and electronics, their use in regenerative medicine is still in an early phase. Study prove that graphene is highly biocompatible, has low toxicity and a large dosage loading capacity. This review describes the ability of graphene and its related materials to induce stem cells differentiation into osteogenic, neuronal, and adipogenic lineages. An outburst of research on regenerative medicine has recently emerged to develop nanostructured materials as smart interfaces to be used for cellular studies and regenerative medicine. Tissue regeneration is a demanding field in terms of development of biomaterials since it requires a variety of fabrication scales, ranging from signal transduction levels to macroscopic tissue recapitulation. Current nanoscale research is focusing primarily on new materials that might be manufactured at high production volume and thus be associated with significant human application. The final goal is a better understanding of the complexity that entails the native extracellular matrix (ECM) through the creation of in vitro models, leading to engineered scaffolds specifically designed to modulate cells differentiation. Many authors have demonstrated that cell shape, morphology, attachment, proliferation, and migration can be controlled by cell-material interactions –4. The rIn the search for brand new factors which may have an influence on cell behavior, much attention has been recently given to environmental components ,7, and pAlthough carbon derivatives have been extensively employed in industry and electronics, their use in regenerative medicine is still in an early phase . The strThe exceptional properties of graphene and its potential different applications have led to the development of composite materials that include few-layer-graphene (FLG), ultrathin graphite, graphene oxide (GO), reduced graphene oxide (rGO), and graphene nanosheets (GNS), comprising a broad set of “graphene-family nanomaterials” (GFNs) Figure  15–17].–17.15–17Some of them, like GO substrates, have been already demonstrated to stimulate human MSCs to differentiate into adypocites ,18, to iBone tissue engineering promises to restore bone defects that are caused by severe trauma, congenital malformations, etc. Many researchers are studying the ways to confer a pro-osteodifferentiation or osteoinductive capability on implants or scaffold materials, where osteogenesis of seed cells is promoted. Graphene provides a new kind of coating material that may confer the pro-osteodifferentiation capability on implants and scaffold materials by surface modification. Here, we review recent studies on the effects of graphene on surface modifications of implants or scaffold materials. The ability of graphene to improve the biological properties of scaffold materials, and its ability to promote the adhesion, proliferation, and osteogenic differentiation of MSCs or osteoblasts have been demonstrated in several studies .2) and graphene-coated SiO2 [2 surface. The results were supported by fluorescent imaging, revealing that in the graphene substrate the initial number of osteoblasts almost duplicated, compared to a 1,5 increase factor in the SiO2 film. Nonetheless, these initial observations tended to be controversial, considering the study of Nayak et al. [2, it did not demonstrate to influence the shape or structure of seeded cells compared to uncoated surfaces. Furthermore, these authors investigated the osteogenic potential of graphene on MSCs. MSCs cultured on uncoated Si/SiO2, PDMS and PET surfaces showed a CD-44 positive staining and a complete negative osteocalcin (OCN) staining. On the other hand, OCN was significantly represented when cells were cultured on the same graphene-coated surfaces  in loading and releasing bone morphogenic protein type 2 (BMP-2) . The ostThe osteogenic differentiation potential of MSCs on graphene substrates was confirmed also by the study of Crowder et al., where a 3D graphene structure was used to induce osteogenic differentiation . The autin vitro co-culture of hBMMSCs and in vivo implantation. The nanofibrous structure itself demonstrated significant enhancement in cell adhesion, proliferation and osteogenic differentiation of hBMMSCs, and with the incorporation of CNMs, the composite nanofibrous scaffolds further promoted osteogenic differentiation of hBMMSCs significantly. Between the two CNMs, graphene showed stronger effect in promoting osteogenic differentiation of hBMMSCs than CNT. The results of in vivo experiments revealed that the composite nanofibrous scaffolds had both good biocompatibility and strong ability in inducing osteogenesis. CNMs could remarkably enhance the expression of osteogenesis-related proteins as well as the formation of type I collagen. Similarly, the graphene-containing composite nanofibrous scaffolds demonstrated the strongest effect on inducing osteogenesis in vivo. These findings demonstrated that CNMs-containing composite nanofibrous scaffolds were obviously more efficient in promoting osteogenesis than pure polymeric scaffolds [The effect of graphene on MSC osteogenic commitment has been also studied by Duan et al.  who compcaffolds .The same positive results have been observed by Tavarty et al.  that hypIn vitro cytocompatibility of the GC1,5 coating was evaluated using a hBMMSCs culture system. The proliferation and ALP, osteopontin and OCN osteogenesis-related gene expression of the cells on the GC1,5 coating did not deteriorate with the adoption of graphene. Conversely, even better adhesion of the hBMMSCs was observed on the GC1,5 coating than on the pure CS coating. All of the results indicate that the GC1,5 coating is a good candidate for load-bearing implants [Owing to the superior mechanical properties and low coefficient of thermal expansion, graphene has been widely used in the reinforcement of ceramics. Xie et al.  studied implants .Inducing human NSCs to differentiate into neurons is a critical challenge to reach an important biomedical goal, since a promising opportunity in therapies for neural regeneration could arise ,27.Park et al. tested the graphene substrate as a promoter of human NSCs differentiation into neurons . The mosOther results indicating that graphene-based substrates can promote neural differentiation came from the study by Hong et al. . After cIn the attempt of influencing in a significant way stem cell differentiation, several declination of graphene scaffolds were studied. Wang et al. tested MSCs on fluorinated sheets of graphene, observing a strong enhancement of neuronal differentiation when compared to cells seeded on graphene . This inThe study by Yang et al. investigated the capability of GO, graphene and CNTs to induces dopamine neural differentiation of mouse embryonic stem cells (ESCs) . ESCs weAnother important characteristic of graphene could be represented by its capability to form a functional neural network. In the study by Tang et al., neurospheres were seeded on graphene substrates and after a 14 days culture the process of network formation was made clear by beta-tubulin immunostaining . Newly f2, GO/TiO2, and rGO/TiO2), with and without flash photo stimulation [2 substrate, by a rate of 48% in the GO/TiO2 substrate and by a rate of 24% on the TiO2 substrate. No differences were detected without flash stimulation. When the differentiation rate was analyzed it was found that the rGO/TiO2 substrate was the more beneficiary of the flash photo stimulation: an 88% decrease in the ratio of glial cells and an 81% increase in the ratio of neuronal cells. In the other 2 substrates the difference was less evident: 25% of neuronal cells increase rate on the GO/TiO2 substrate and 15% on the TiO2 substrate. Overall, flash photo stimulation of graphene-based substrate provide both a greater cell proliferation and a greater human NSCs differentiation into neurons rather that glia cells. However, these results could be obtained when the flash photo stimulation of human NSCs was at an optimum concentration of a biocompatible hole scavenger, and at an optimum flash intensity.Stimulating human NSCs to differentiate into neurons rather than glia is a another key point in order to obtain a neural regeneration result . The stumulation . When thFor central nervous system (CNS) regeneration, the selective differentiation of NSCs into either neurons or oligodendrocytes (as opposed to astrocytes) is highly desirable. Several approaches have been employed to guide differentiation into neurons, however, oligodendrocyte differentiation has proven to be much more elusive, resulting in only a small percentage of the differentiated cell population. The primary approach to guide oligodendrocyte differentiation has focused on either developing culture media containing a combination of growth factors or the forced expression of key oligodendrocyte-promoting transcription factors. In this view the developing a biomaterials-approach to achieve efficient differentiation of NSCs into mature oligodendrocytes, while eliminating the potential adverse or variable side-effects from growth factors and viral gene vectors, would be highly beneficial.Shah et al.  developeGO based nanoparticles has been used also to drive the commitment of ESCs into dopamine neurons. Yang et al.  studied in vitro and in vivo, then lead to other biological effects, as expected of any foreign particulate. Current studies mostly focus on the pulmonary inflammation caused by nanomaterials insults, while little is known about the neuroinflammatory effects. Given that some nanomaterials  have been attempted to be used in neuroscience, the neuroinflammation should be considered. External insults range from hypoxia and ischemia to a number of bacterial and viralinfections, all of which elicit a characteristic neuroinflammatory reaction in the brain. Microglia, astrocytes, and peripheral macrophages are key players mediating this response. In the brain, most of the damage caused by nanomaterials is mediated by the microglia, a macrophage-like, phagocytic cell that is normally inactive unless confronted by potentially damaging xenobiotics. In response to certain cues such as brain injury or immunological stimuli, however, microglia are readily activated. Graphene has been at the forefront of nanotechnology and advanced materials sciences due to its intriguing physical and chemical features. Especially, it has been utilized in a variety of biomedical applications. Recently, Li et al. [in vitro culturing and justify whether this graphene-induced neuroinflammatory effects are detrimental or beneficial to the neural cells. The importance of this work is the elucidation of the pro- and/or anti-inflammatory effects of graphene and pave the way for the applications of graphene in biomedicine. The graphene, especially 3D graphene, supported microglia growth and showed comparable biocompatibility to the commercial tissue culture polystyrene substrates. Despite of the similar proinflammatory responses in the microglia without lipopolysaccharide (LPS) activation, 3D graphene evoked much milder neuroinflammation in the microglia after LPS activation in comparison to 2D graphene, suggesting that the topographical structures of the materials might affect the inflammatory behaviors. Furthermore, the unique topographical structures of 3D-graphene may restrict the morphological transformation of microglia under over-activation, leading to the anti-inflammatory effects.Novel important applications of graphene in neuroscience has been found in the end by Song at al. , who stui et al.  discoverGraphene and GO substrates have been also used for investigating their effects on the adipogenic differentiation of MSCs. In particular, in the study of Lee et al., MSCs were plated on graphene or GO sheets in presence of adipogenic differentiation medium for 14 days . The celKim et al. evaluated GO potential in differentiating human adipose-derived stem cells (ADSCs) into adipocytes, demonstrating a higher adipogenesis on the GO substrate when compared to the control (tissue culture polystyrene) . This stIn regenerative dentistry, stem cell-based therapy often requires a scaffold to deliver cells or growth factors to the injured site. GO and silk fibroin (SF) are promising biomaterials for tissue engineering as they are both non toxic and promote cell proliferation. A field that could be improved by the availability of an effective film scaffold would be the reparation of periodontal tissues. The periodontium, which is composed of four dental tissues , is constantly maintained by periodontal ligament stem cells (PDLSCs) owing to their great capacity of differentiation into cementoblasts, odontoblasts, and fibroblasts. PDL plays a key role in the attachment of teeth to the jaw; in the most drastic cases of periodontitis, which is associated with a chronic inflammation process, PDL destruction could lead to the loss of the tooth . With thAs well reported above, graphene has drawn attention as a substrate for stem cell culture and has been reported to stimulate the differentiation of multipotent adult stem cells. Recently, Lee et al.  reportedThe same results have been reched by Park et al.  that demThe successful reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) by Takahashi and Yamanaka in 2006  was seenIn an interesting study by Yoo et al. , the autThe generation of ESCs-like cells from somatic cells by ectopic expression of defined factors is an approach commonly known as cell reprogramming. Moreover, because iPSCs generation is known to be a multiple-step process mediated by overexpression of the transcription factors Oct4, Sox2, Klf4, and cMyc, the generation of iPSCs is very inefficient, and cell reprogramming is considered a stochastic process in which successive barriers must be overcome to reach a state of pluripotency. In particular, one of the first noticeable changes during the reprogramming of somatic fibroblasts is their transformation into tightly packed clusters of rounded cells in a process that resembles a mesenchymal-to-epithelial-transition (MET). More interestingly, recent studies found that microtopography substrate affects the MET, improving reprogramming efficiency. The authors examinated whether somatic fibroblasts should be efficiently reprogrammed into the pluripotent state on graphene-based substrate. Fitstly they carachterised the monolayer graphene film by Raman spectroscopy, then they studied whether the graphene substrate enhances cell reprogramming by seeding Oct4-GFP knock-in (KI) mouse embryonic fibroblasts (MEFs) onto both the control and graphene-coated substrate in MEF medium. One day after plating, MEFs were transduced using specific vectors expressing Oct4, Sox2, Klf4, and cMyc transcription factor. The MEFs plated on the graphene substrate began to form colonies 10 days after viral infection, exibiting a significant increase in colonies undergoing reprogramming on graphene substrate. To quantify the reprogramming efficiency, they performed FACS analysis for Oct4-GFP-positive iPSCs derived from Oct4-GFP KI MEFs in both the control substrate and graphene-coated substrate. Fifteen days after doxycycline (dox) induction, the graphene substrate cultures had a significant increase in the number of GFP-cells and quantitative real-time PCR analysis showed that pluripotency marker genes, including Oct4, Nanog, Sox2 and Esrrb were markedly elevated in graphene-coated substrate cultures compared to uncoated substrate cultures. The pluripotent state of graphene-induced iPSCs was assessed by immunostaining of pluripotency marker. Consistent with these results, they observed that the graphene-coated substrate significantly increased the number of Oct4, SSEA1 and Nanog colonies. Moreover, in order to analyze the differentiation potential of graphene mediated iPSCs, their capacity for direct differentiation into the cell types of the three germ layers has been tested. Immunocytochemical staining and real-time PCR showed that the differentiated cells were positive. In the end the further developmental potency of the graphene induced iPSC lines was investigated by SCID mice. Four weeks after injection, teratomas were readily visible and histological analysis confirmed the presence of cell types derived from all three embryonic germ layers validating the pluripotency of these graphene induced iPS cells. In order to examine whether graphene substrate affects chromatin state during reprogramming, they measured the enrichment level of histone modifications that mark active  in the graphene induced cell reprogramming. Surprisingly, they found a dramatic increase of H3K4me3 expression on graphene substrate and that the transcription start sites of Oct4 and Nanog were enriched for H3K4me3 in graphene surface-induced reprogramming cultures. Taken together, these results suggest that the graphene-coated substrate specifically promotes the MET process in cell reprogramming without affecting the EMT process. In this study, the autors showed that the epigenetic reprogramming of somatic fibroblasts is enhanced on graphene substrate providing a potential method for the efficient generation of iPSCs. Interestingly, they found a specific induction of MET by the graphene substrate during reprogramming and an accumulation of H3K4me3, which facilitates increased Oct4 and Nanog occupancy. These data indicate a unique role for graphene substrates in facilitating cell reprogramming .Carbon nanoparticles such as zero dimensional (0D) fullerenes, one dimensional (1D) CNTs, and recently two dimensional (2D) graphene have been investigated for applications in therapeutics, bioimaging, and regenerative medicine. MSCs are currently being widely investigated to repair, regenerate, and restore damaged tissues. Nanoparticles have been employed to deliver growth factors or genes into MSCs to manipulate their differentiation . The devThe literature on the biological interactions of graphene family is growing rapidly, and includes studies primarily motivated by biomedical applications, and environmental health and safety. As with other biomaterials, the issue of potential toxicity arises not only in biomedical applications, but also in non-biomedical products where unintended occupational, consumer, and environmental exposures can occur. The exact mechanism of GO and the derived composites in SCs differentiation is still unresolved. It has been generally hypothesized that the surface characteristics of graphene family nanomaterials such as nanotopography, surface stiffness, and large absorption capacity influence the molecular pathways that control the fate of stem cells. Both G and GO were reported acting as preconcentrators for chemicals, proteins and growth factors on their surface to promote cell differentiation.In this view, we have reported a review of the international literature to produce a state-of-the-art update of graphene applied to tissue engineering and stem cells.Many studies confirmed that graphene and its related materials are able to induce human stem cells differentiation into specific lineages. Materials coated with graphene or GO or even 3D graphene foam were capable to guarantee viability and to induce osteogenic differentiation of stem cells when compared with traditional substrates or scaffolds ,14,21,22Even if we are still uncertain about potential in vivo applications, mostly due to open questions around toxicity, graphene clearly appears to be a step forward in the field of tissue engineering.In this light it is interesting the results found by Chen et al. that express an expert opinion based on scientometric patterns revealed by CiteSpace without prior working experience in the regenerative medicine field. Emerging trends and new developments have been identified based on structural and temporal properties derived from the relevant publications. The detected surge of the keyword graphene in the literature of regenerative medicine led the authors to investigate the nature and context of its use in regenerative medicine. The investigation revealed a rapidly increasing number of studies that specifically used graphene and GO in regenerative medicine research because of their desirable surface properties for, among other applications, culturing and maintaining SCs. Similarly a detected burst of citations provides insightful guidance for navigating through the fast-changing .ad hoc sample names.There is also an emerging literature on potential health risks. Despite the popular image of graphene as a large-area substrate coating, many graphene-family materials are dry powders at some point in their processing, and in this form pose the most significant exposure risk through inhalation. Of particular concern are FLG samples directly following the thermal exfoliation step or after washing and drying. There is need for measurement of airborne dust levels in research laboratories, and in pilot and full-scale manufacturing facilities. GFNs are high-surface-area materials with corresponding high potential to cause adsorptive and quenching artifacts in biological assays . Adsorpt


In this study, a CMOS compatible capacitive humidity sensor structure was designed and fabricated on a 200 mm CMOS BEOL Line. A top Al interconnect layer was used as an electrode with a comb/serpent structure, and graphene oxide (GO) was used as sensing material. XRD analysis was done which shows that GO sensing material has a strong and sharp (002) peak at about 10.278°, whereas graphite has (002) peak at about 26°. Device level CV and IV curves were measured in mini-environments at different relative humidity (RH) level, and saturated salt solutions were used to build these mini-environments. To evaluate the potential value of GO material in humidity sensor applications, a prototype humidity sensor was designed and fabricated by integrating the sensor with a dedicated readout ASIC and display/calibration module. Measurements in different mini-environments show that the GO-based humidity sensor has higher sensitivity, faster recovery time and good linearity performance. Compared with a standard humidity sensor, the measured RH data of our prototype humidity sensor can match well that of the standard product. With the consumer market growth, requirements for high performance and low cost MEMS/sensors are increasing, especially for digital multimedia products [etc. Recently, humidity sensors have been successfully integrated into high-end mobile phone products including the Samsung Galaxy S4, and their market will increase dramatically with the growing consumer electronics demands.With the developing of the Internet of Things and sensor networks, MEMS/sensor “consumerization” has begun to emerge, and more and more new applications of MEMS/sensors have begun to be found in consumer products such as mobile phones, PAD, products ,2. HumidBased on their measurement units, humidity sensors can be divided into two types: relative and absolute humidity sensors. According to the operating principles, these are three main types: Capacitive, resistive, and thermal conductivity humidity sensors. Most humidity sensors are capacitive RH sensors because of their low cost, linearity, minimal long-term drift and hysteresis .2 and sp3 hybridized carbon atoms which can be considered as insulators [etc.Graphene is one of the most promising materials developed in this century with its exceptional mechanical, thermal and electrical properties ,5,6,7. Gsulators . GO is ssulators ,10,11,12sulators ,15,16,17In this work, a capacitive humidity sensor was designed and fabricated with GO as sensing material. The capacitance of the sensor device was increased about four times from ~22.5% RH to ~85% RH, which shows excellent sensitivity. After integrating the sensor device with a dedicated readout ASIC, a prototype humidity sensor was built and its measured data was compared with that of a standard humidity sensor.The sensor structure was designed and fabricated with a comb/serpent capacitor structure by a standard 0.35 μm CMOS compatible process, as shown in After wafer dicing was finished, and the sensor was fabricated by filling GO material in the structure. In this post CMOS process, a GO-based water dispersion solution was used as filling material, and after the filling process the sensor was thermally treated to remove the water. XRD was used to check the sensing material properties, and CV/IV data was measured in different environments to check the leakage and capacitance properties.A prototype humidity sensor was designed and built by integrating the sensor with a dedicated readout ASIC and display/calibration module at the PCB level. A standard frequency-voltage convertor  was used as readout circuit to extract the capacitance variation by the RC oscillation mechanism. The prototype sensor was then evaluated in different environments, and all the measured data was compared with a standard humidity sensor  as shown in All the measurements were done at a room temperature of 25 °C. CV was measured by a 4284A instrument  and IV was measured by an Agilent 4156C device. This study adopted a very convenient way to measure and calibrate humidity sensors by using saturated salt solutions. As shown in The RH of the atmosphere above the saturated salt solution in the sealed chamber was fixed at a given temperature, and it can cover almost the entire range of relative humidity, as shown in A standard 0.35 μm CMOS BEOL process was used to fabricate the comb/serpent capacitor structure. A BEOL Al metal layer was used as capacitor electrode with line/space/height = 0.78 μm/0.54 μm/0.8 μm. The structure was designed with consideration of least fringing field and parasitic capacitance, and PAD was formed by the same metal layer. A top-view photo of the structure can be seen in −11 f, and a very good uniformity can be obtained with a standard deviation of ~1.76E−13 f and NU% of ~ 1.41%. From the IV data, the structure shows a very good leakage control with a max leakage current of ~5E−14 A.After wafer fabout, a CV and IV mapping test was done to check the uniformity and leakage performance of the structure. As can be seen in After wafer dicing, the sensor was fabricated by filling GO material in the sensor structure. A GO-based water dispersion solution was used as filling material, and after the filling process the sensor was treated in a baking oven at a temperature of 60 °C for about 50 min to remove the water. Cross-sectional SEM was done to check the filling performance, and the GO filling performance can meet the sensor requirements as can be seen in 3 structure network. Because of the existence of oxygen functional groups and some other structural defects, the interlayer distance of GO is greatly increased [GO film was prepared on a blanket substrate to do XRD analysis. Compared with graphite, GO has a variety of reactive oxygen groups, such as carboxylic acids (COOH), carbonyls (C=O), epoxides (–O–) and hydroxyls (–OH), which are linked to a C-spncreased ,13,14,15−11 A with a comb/serpent electrode length of ~165 mm at about 50% RH level, which can meet well the sensor application requirements.Leakage is very critical to any sensor application with a capacitive sensing mechanism, because more leakage current will make capacitance measurement inaccurate and cause the device to malfunction. Leakage of the sensor device was measured in voltage sweeping mode from 0 V to 5 V. As shown in Capacitance was measured at frequency of 100 kHz by an Agilent 4284A instrument. C/C0 ratio is plotted in To evaluate the potential value of GO material in humidity sensor applications, a prototype humidity sensor was designed and built by integrating the sensor with a dedicated readout ASIC and a display/calibration module on the PCB level. A standard Analog Microelectronics CAV444 frequency-voltage convertor was used as a readout circuit to extract the capacitance variation caused by the RC oscillation mechanism. A schematic circuit block of the prototype sensor and block diagram of the frequency-voltage convertor are shown in To build the prototype sensor, the sensor device was firstly wire-bonded with the CAV444 on the PCB level, which can convert the change of capacitance to voltage. As can be seen in After that, the prototype humidity sensor was built by further integrating a LCD display and calibration module, which can be seen in After linear calibration, the prototype sensor was used to measure the RH value in environments at different RH levels. The measured data was then compared with that of a standard humidity sensor, as shown in Response/recovery time was defined as the time (τ) required to achieve ~63% value of a humidity step function. Recovery time was measured by a simple method as shown in In this work, a CMOS compatible capacitive humidity sensor structure was designed and fabricated on a 200 mm CMOS BEOL Line. BEOL Al with a comb/serpent structure was used as electrode, and GO was used as sensing material. The sensor device shows excellent sensitivity and faster recovery time than a standard sensor. A prototype humidity sensor was designed and built by integrating the sensor device with a dedicated ASIC at the PCB level, and the output value of the prototype sensor was shown to match well that of a standard humidity sensor.


Streptococcus pneumoniae infection are key in devising strategies towards the development of better vaccines and drugs. To that end, in this study we investigated the role of S. pneumoniae and its surface antigen Pneumococcal surface protein A (PspA) in modulating the expression of co-stimulatory molecule Programmed Death Ligand 1 (PD-L1) expression on dendritic cells (DCs) and the subsequent effects of increased PD-L1 on key defence responses. Our data indicate that stimulation of DCs with PspA increases the surface expression of PD-L1 in a time and dose dependent manner. Characterization of mechanisms involved in PspA induced expression of PD-L1 indicate the involvement of Toll-Like Receptor 2 (TLR2) and calcium homeostasis. While calcium release from intracellular stores positively regulated PD-L1 expression, calcium influx from external milieu negatively regulated PD-L1 expression. Increase in PD-L1 expression, when costimulated with PspA and through TLR2 was higher than when stimulated with PspA or through TLR2. Further, knockdown of TLR2 and the intermediates in the TLR signaling machinery pointed towards the involvement of a MyD88 dependent pathway in PspA induced PD-L1 expression. Incubation of DCs with S. pneumoniae resulted in the up-regulation of PD-L1 expression, while infection with a strain lacking surface PspA failed to do so. Our data also suggests the role of PspA in ROS generation. These results suggest a novel and specific role for PspA in modulating immune responses against S. pneumoniae by regulating PD-L1 expression.Pneumonia leads to high mortality in children under the age of five years worldwide, resulting in close to 20 percent of all deaths in this age group. Therefore, investigations into host-pathogen interactions during  S. pneumoniae is the number one cause of bacterial pneumonia and can also cause meningitis, sepsis and otitis media. In some developing countries Streptococcus pneumoniae can account for over 50% of all pneumonia deaths [S. pneumoniae based on the structure of the polysaccharide capsules [S. pneumoniae also possess various surface associated proteins that contribute to its virulence; many of which are able to elicit measurable protection in mice e. g. Pneumococcal surface protein A (PspA) [Pneumonia and pneumonia related illnesses are a major cause of mortality in children under the age of five years worldwide, with close to 20 percent of all deaths in this age group . S. pneua deaths . There acapsules . A 7-valcapsules . The capA (PspA) , 8, 9.PspA, a serologically variable, cross-reactive, cross-protective protein is present on almost all strains of pneumococci and has been shown to be a promising candidate antigen for a protein-based vaccine –12. Anti+ and CD8+ T cells [Staphylococcus aureus triggered conversion of neonatal CD4+ T cells into FOXP3+ CD25+ CD127low T cells [S. aureus modulates T cell responses via TLR2 and PD-L1 [-/- mice respond inadequately to Mycobacterium tuberculosis infection [Burkholderia pseudomallei impairs T cell function [The costimulatory molecule Programmed Death Ligand-1 (PD-L1) is constitutively expressed and its surface expression is upregulated on murine hematopoietic cells  and non-hematopoietic cells  upon stimulation. It has been documented that PD-L1 interacts specifically with B7-1 to inhibit T cell proliferation . The int T cells . The PD- T cells . S. aurend PD-L1 . T cellsnfection . PD-L1 efunction .S. pneumoniae to escape phagocytic killing by DCs and for eliciting optimal proinflammatory cytokine production [S. pneumonniae induces apoptosis in bone marrow derived DCs via two mechanistically different pathways. Pneumolysin dependent but caspase independent and delayed onset but caspase dependent mechanism accompanied with DC maturation [S. pneumoniae during pneumococcal pneumonia [S. pneumoniae by DCs [S. pneumoniae or its antigens in modulating PD-L1 expression on DC is not reported. In this study we investigated the role of S. pneumoniae and its antigen PspA in regulating the surface expression of PD-L1 on DCs. As DCs are known for their critical role in regulation and modulation of the host immune response against bacterial infections, in this study, we characterized the role of S. pneumoniae infection in regulating PD-L1 expression. Our data indicate that PspA induces the expression of PD-L1 on mouse bone marrow derived DCs. Results also point towards a role for the route of calcium entry and Toll-Like Receptor (TLR) 2 in mediating PD-L1 expression.DCs are crucial for innate and adaptive immune response against bacterial pathogens. In recent years, several groups have studied how pneumococcal virulence factors modulate DC function. The precise role of PD-1:PD-L1 pathway in dampening the T cell response is still unclear in pneumococcal infections. The role of PavA during pneumococcal infection has been investigated and it was demonstrated that it is essential for oduction . S. pneuturation . Furtherneumonia . Cao et e by DCs . Clp coue by DCs . HoweverAnimal experiments were done with approval and in strict accordance with the guidelines of the National Institute of Immunology Institutional Animal Ethics Committee (IAEC#327/13). The procedures used in the study were carried out by well-experienced competent workers trained to perform them. All efforts were made to minimize suffering and pain.S. pneumoniae strain D39 (capsular type 2), R6 (ATCC BAAA-255) and JY2008 (ATCC 55143) were used in the study [S. pneumoniae for 3h followed by addition of 40 μg/ml of gentamycin  and DCs were incubated at 37°C for an additional 24h.Virulent he study . JY2008 he study  and mainPhycoerythrin-tagged antibody against mouse PD-L1 were procured from BD Biosciences USA. Recombinant mouse Granulocyte macrophage colony stimulating factor (GM-CSF) was procured from R & D Systems (USA). Antibodies to pAkt, Bcl2, Bax and β-actin, specific and control siRNAs against mouse genes, and Luminol kit for chemiluminescence detection were purchased from Santa Cruz Biotechnology, USA. Chemicals U0126, 3, 4, 5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), ethyleneglycol-bis(2-aminoethylether)-N, N, N', N'-tetraacetic acid (EGTA) and diphenyleneiodonium (DPI) were purchased from Sigma Chemical Co., USA.+ cells were depleted following Magnetic Assisted Cell Sorting. Cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum, 0.05 M 2-mercaptoethanol, 1 mM sodium pyruvate plus 15 ng/ml GM-CSF for 72h. Recombinant PspA was expressed and purified as described before [3Csk4, TLR4 was stimulated with 0.1 μg/ml lipopolysaccharide (LPS) and TLR7 was stimulated with 1.0 μg/ml immiquimod) in the presence or absence of 15 μg/ml PspA for 24h. For some experiments, DCs were incubated with biopharmacological inhibitors to various molecules. For example, DCs were incubated with 100 μM TMB-8 for inhibiting calcium release from intracellular stores, 3 mM EGTA for inhibiting calcium influx from external medium, 10 μM U0126 for inhibiting MAPK-ERK and 10 μM diphenyleneiodonium (DPI) for inhibiting reactive oxygen species (ROS). For some experiments, DCs were transfected with siRNAs to various genes as described before prior to stimulation with 15 μg/ml PspA [6/ml) were transfected with 60 pmol of siRNA for 72h using the Hiperfect transfection reagent  in OPTIMEM medium . GM-CSF was added 5h following transfection, and incubation was continued for 72h for DC differentiation. Knockdown was verified by semi-quantitative reverse transcription-polymerase chain reaction. Six to eight week old female BALB/c mice reared in a pathogen-free environment were used throughout the study. DCs were differentiated from mouse bone marrow as described previously . Brieflyd before . The purAt the end of the incubation period, DCs were first incubated with Fc-Block  to mask CD16/CD32 Fc receptors and stained for the surface expression level of PD-L1 using phycoerythrin-tagged monoclonal antibody to PD-L1 and analyzed by flow cytometry  as described previously , 40. The6/ml) were incubated with recombinant PspA3–286 from R36A [DCs , Mitogen Activated Protein Kinase-Extracellular-signal Regulated Kinase (MAPK-ERK), PhosphatidylInositide-3 Kinase (PI-3K), Protein Kinase A (PKA), Calcium/calmodulin dependent protein Kinase II α (CAMKIIa) during PspA induced PD-L1 expression. To inhibit calcium homeostasis, we treated DCs with EGTA to chelate extracellular calcium and TMB-8 to inhibit intracellular calcium release from the endoplasmic reticulum. Interestingly, inhibiting intracellular calcium burst completely inhibited PspA induced PD-L1 expression, while inhibiting calcium influx from external milieu further increased PspA induced PD-L1 expression . This inM. tuberculosis infection [Inhibiting ERK-MAPK with U0126 increased PspA induced PD-L1 expression suggesting the involvement of this pathway in PD-L1 expression . On the nfection . As show3R with TMB-8 completely abrogated PspA induced PD-L1 expression, while inhibiting calcium influx from extracellular medium using EGTA increased PD-L1 expression  proteins STIM1 and STIM2, the molecular sensors that regulate Store Operated Calcium Entry (SOCE) via the Calcium Release Calcium Activated Channel (CRAC) ORAI1. As shown in the S. pneumoniae. To that end we incubated DCs with wild type D39, strain R6 (derived from D39 that lacks capsule but has PspA) or JY2008 (lacks capsule and surface PspA). As shown in S. pneumoniae infection.We next investigated whether the effects observed with PspA would be replicated during infection with 3R) and EGTA  followed by incubation with S. pneumoniae D39 and PD-L1 expression was monitored. Similar to the data obtained with PspA Click here for additional data file.S2 FigMouse bone marrow derived DCs were transfected with siRNAs directed against the indicated gene for 36h as described previously . Total R(DOC)Click here for additional data file.S3 FigMouse bone marrow derived DCs were stimulated with 15 μg/ml PspA for 24h and PD-L1 expression was monitored by flow cytometry. Thick line represents PspA stimulated cells stained with isotype matched control monoclonal antibody. Dotted line represents unstimulated cells stained for PD-L1. Thin line represents PspA stimulated cells stained with antibody specific to PD-L1.(DOC)Click here for additional data file.S4 FigMouse bone marrow derived DCs were transfected with siRNA against TLR2 for 36h, followed by stimulation with PspA for 24h. PD-L1 levels were monitored using flow cytometry. In Panel A, dotted line represents unstimulated cells transfected with control siRNA. Thin lines represent cells transfected with control siRNA followed by stimulation with PspA for 24h. Bold line represents the cells transfected with siRNA specific to TLR2 followed by stimulations with PspA. Data from one of three independent experiments is shown. Panel B, shows the relative expression of PD-L1 represented as relative MFIs. Panel C shows the knockdown efficiency of siRNA against TLR2 in DCs.(DOC)Click here for additional data file.S5 FigMouse bone marrow derived DCs were stimulated with PspA for 2h. 30 minutes prior to the incubation period cells were loaded with 10 μM DCFH-DA. At the end of incubation period, cells were quickly and thoroughly washed with culture medium and immediately analyzed for ROS level by flow cytometry. Thin line represents unstimulated cells stained for ROS. Bold line represents cells stimulated with PspA (15 μg/ml).(DOC)Click here for additional data file.


Digital game-based learning environments provide emerging opportunities to overcome learning barriers by combining newly developed technologies and traditional game design. This study proposes a quantitative research approach supported by expert validation interviews to designing a game-based learning framework. The goal is to improve the learning experience and decision-making skills of soccer referees in Turkey. A serious game was developed and tested on a group of referees (N = 54). The assessment results of these referees were compared with two sample t-test and the Wilcoxon signed-ranked test for both the experimental group and the control group. The findings of the current study confirmed that a game-based learning environment has greater merit over the paper-based alternatives. Football is the world’s most popular sport with approximately 33.4 million individuals involved   Laws of the Game (LOG) book consists of the rules of the game of football  was essential where it was important to assess their requirements and monitor their progress. In addition, it was important to include a qualitative aspect in this study as the viewpoints of referees were crucial to address the design challenges during the development of such an interactive software-intensive system.For the qualitative part of the study, expert reviews were conducted on five experienced referees who had been working in the education department of the Turkish Football Federation for at least 10 years when a database of 700 questions for the game platform was prepared. The protocol used for the expert reviews were based on the following questions “Do you think this question is suitable for assessing a novice referee?”, “Can you please rate this question between 1 and 3 in terms of its difficulty level?”, “What would be learning outcome for this question?”, “Can the referees reach this question in the other platforms?” and “Is this question similar to the questions that are used in your training programs?”.Q1: Do you think you can improve your knowledge level and decision-making skills by playing the game?Q2: Which question category/categories are more useful for you?Q3: Do you prefer to play the game or study from the FIFA book?Q4: What kind of features do you think are missing in the training framework?Q5: Do you like to have a competition between the other players? If your answer is yes, is it useful or not?For the final qualitative phase of the study, validation interviews were conducted with the participants so as to explore the benefits and potential issues encountered during the interactive assessments by asking these questions:The answers were evaluated and categorized as positive, negative or neutral by the five retired first class football referees (who had managed matches in the Super League for at least 7 years).For the quantitative part of the study, an interactive assessment (i.e. pre-test and post-test) was conducted in the form of a game-like structure to a group of football referees, namely those who have joined the referee training program. The aim was to explore the benefits of the proposed platform in improving the knowledge level and decision-making skills of these football referees by measuring the differences between the referees’ pre-test and post-test scores. The numerical results of both tests were analyzed by using statistical techniques to understand whether or not a significant difference between the groups exists.A serious game is a special type of game which is used to educate (or sometimes inform) participants about a selected topic Abt . Here, aTrue/False Questions and Multiple Choice Questions categories are selected from the actual questions that are administered every year to measure a referee’s level of knowledge of the rules of football while the questions in the Video Questions category consist of videos of critical positions that occur in real matches that were played during past football seasons.The game has three different types of questions related to the laws of the game of football, which are: (1) True/False Questions, (2) Multiple Choice Questions and (3) Video Questions. The questions in the In addition to the question categories, a virtual game board was designed. The game board consists of a set of steps as shown in Fig. Using a traditional perspective, the participants use a virtual dice with three faces which are corresponding with three numbers. Each number on the die represents each question category as follows: (1) represents True/False Questions, (2) represents Multiple Choice Questions, and (3) represents Video Questions.First, the player throws the die, and produces a one, two or three. Based on the number thrown, he takes a question from one of the corresponding categories above. For example, if the die shows 3, the player takes a question from the video questions category.If the player answers correctly to the question, he moves one step forward. Otherwise, he moves one step backward (unless the player is at the starting point or at a bonus step). Before going one step backward, the correct answer to the questions and a detailed explanation of the correct answer is shown to the player.As a simple reward structure, there is a group of checkpoints in the game known as bonus steps. The bonus steps are designed to keep the player in the game. This also prevents players from answering incorrectly to a group of questions and returning to the starting point. In addition, this mechanism incrementally provides the player with small rewards thereby achieving motivation.The bonus steps also offer additional time for correct answers to a question being asked in this step. The first bonus step gives 5 s of bonus time, the second bonus step offers 10 s, the third bonus step offers 15 s, and finally 20 s are added as bonus time for the fourth bonus step.The timer of the game starts with the first question and it stops when a player answers the final question.Finally, each player’s racecourse completion time is compared and placed in a table which sorts the players according to their completion time. The player at the top of the leader-board is the winner. The flow of the game is illustrated in Fig. The mechanics of the game can be identified as follows:To evaluate the effect of our game correctly, we conducted experiments in which we allowed participants play the game. Following this, we analyzed the results of these experiments. Creating a sample is necessary in order to obtain valid results. The members of the sample group are selected to have a similar level of knowledge at the beginning of the training program. In order to ensure this similarity, 54 football referees, all of whom had completed the referee course at the same time, were selected. These referees were randomly separated into a control group (N = 27) and an experimental group (N = 27). The referees in the control group are not allowed to log in to our game system. They were only able to learn the rules of the game from the LOG book. The referees in the experimental group were allowed to log in to our game system. However, they were not permitted to use LOG book.Multiple Choice QuestionsVideo QuestionsIn order to evaluate the level of knowledge of the referees regarding the game rules, a pre-test was administered to a selected group of referees for the training program at the beginning of the training period. This test consisted of 50 questions, which were separated into two sections thus:From a group of questions, 35 multiple choice questions and 15 video questions were selected.To build a proper assessment tool, an expert from the Turkish Football Federation was interviewed. The number and content of questions in the test was determined by him. This expert indicated that there are 50 questions in the real exam administered by the Turkish Football Federation to measure a referee’s level of knowledge of the rules of the game. Hence, the number of questions determined in both tests was 50.The pre-test was administered on 9 December 2014, at which there were 54 football referees as participants. The pre-test results of both groups are shown in Fig. Important points were considered during the data analysis. The first important point is that the average scores of both groups were very near to each other in every part of the exam. In other words, in both groups, the referees’ level of knowledge of the game was nearly identical at the beginning of the training program. The second important point was the number of successful referees in the exam. In real exams, 70 out of 100 is the threshold for referees to be successful. When the pre-test results were examined, from 27 individuals, 4 were found who were able to pass the exam in the experimental group .To improve the quality of training, further research should also be conducted to provide a more immersive environment with computer-generated landscapes with a 3D virtual soccer field that could be based on virtual reality or augmented reality. Such a system would provide full simulation of a match in a virtual football field, which can provide the trainee a real life like a learning experience. A further study with more focus on referee training on such a medium that is based on the tracking of the positions and actions of a participant with continuous feedback on an artificial interactive computer-generated football field is therefore suggested.


Cases with IHC score of 1, 2, 3 all underwent apparent decrease (p = 0.007). However, no significant changes were observed on tumour-infiltrating immune cells (ICs) (p = 0.337). Subgroup and semiquantitative analyses all presented similar results. Moreover, patients with response to NACT presented significantly reduced PD-L1 expression on TCs (p = 0.004). Although it was not confirmed by the Cox proportional hazard regression model, there was an apparent difference in disease-free-survival (DFS) between negative-to-positive switch of PD-L1 status and the contrary group . Our data revealed that antecedent chemotherapy for NSCLC may results in inconsistency of PD-L1 expression. PD-L1 expression is suggested to be monitored around treatment and on serial samples, at least, on the latest tumor specimen.The effects of treatments to programmed death ligand-1 (PD-L1) expression is unknown. The aim of this study was to investigate the impact of neoadjuvant chemotherapy (NACT) on PD-L1 expression in non-small cell lung cancer (NSCLC) patients. PD-L1 expression was detected by immunohistochemistry (IHC) method in 32 paired tumor specimens pre and post-NACT. The positivity of PD-L1 on tumor cells (TCs) changed from 75% to 37.5% after NACT ( Global cancer statistics indicate that lung cancer remains the leading cause of cancer-related mortality2Recent insights into the molecular mechanisms governing the host response to cancer cells has led to the identification of important signaling pathways and checkpoint molecules involved in the anticancer immune response456789101012131012141516181920222425Based on above data, the prognostic and predictive value of PD-L1 IHC was assumed to temporally dependent on the antecedent treatment and time of biopsy. We therefore conducted this exploratory analyses with paired NSCLC specimen pre and post-NACT to explore the impact of chemotherapy on PD-L1 expression. The association between PD-L1 change patterns and prognosis were also analyzed.2, days 1 and 8 plus cisplatin 75 mg/m2, days 1, every 3 weeks). The median tumor size at baseline was 35.0 mm (IQR: 26.3–40.0). After the NACT, the median value decreased to 22.0 (IQR: 18.0–30.3). This change was significant according to the result of Wilcoxon test (p < 0.001). Finally, partial response were found in 18 (56.2%) patients, while 50% patients (n = 16) achieved down-staging after NACT.Finally, 32 patients  were included for analyses. Baseline demographics and PD-L1 expression, as presented by H-score, are summarized in p = 0.003). However, a contrary result was found in ICs, whose PD-L1 positive rate increased from 43.8% to 56.2% after NACT, although no statistical significance was reached (p = 0.319).p = 0.007). Subgroup analyses also supported the finding, as summarized in p = 0.337).Another perspective is, as shown in p = 0.005, p = 0.474, p = 0.004, p = 0.378, p = 0.441, 1.000 separately, Semiquantitative analyses demonstrated that the H-score of PD-L1 on TCs was significantly decreased . Besides, no significant differences were detected among four change patterns of PD-L1 on ICs  months. As shown in The PD-1/PD-L1 axis is one of the crucial mechanisms underlying immune escape of tumor cells. These pathways are currently attractive therapeutic targets for human cancers, including NSCLC. One of the provocative findings accompanied with anti-PD-L1/PD-1 exciting efficacy involves the potential predictive value of PD-L1 expression on tumor cells27Our result demonstrated that the expression of PD-L1 on tumor cells is not consistent for patients with NSCLC. The expression profile was correlated with antecedent chemotherapy. Therefore, the dynamic property of PD-L1 expression demonstrated by our study may provide a possible reason to why negative status from chemo-naive sample finally confer certain responses when PD-1/PD-L1-directed therapy are given in second or later lineset al. reported that cytotoxic agents, specifically paclitaxel, etoposide and 5-fluorouracil, could induce PD-L1 surface expression in breast cancer cells, which lead to promoted PD-L1-mediated T cell apoptosiset al. have revealed doxorubicin-dependent down-regulation of cell surface PD-L1p = 0.004) on TCs, but not in those not responded (p = 0.196). To verify the hypothesis, we analyzed the difference of PD-L1 expression on tumor-infiltrating lymphocytes (ICs). No significant alteration was observed, neither in the response group nor non-response group. Of note, whether drug resistance induces PD-L1 expression is largely unknown. In present study, no significant induction of PD-L1 on tumor cells was detected in population exerted no response to NACT. We thereby proposed non-monotonic mechanisms underlying the effect of chemotherapy on PD-L1 expression, including both cytotoxic-related PD-L1 reduction and other signal-associated PD-L1 regulation. It has been identified that constitutive IFN-gamma secretion or oncogene activation will stimulate PD-L1 expression. Besides, PD-L1 up-regulation mediated by IFN-gamma is commonly considered as a classically adaptive way for immune resistance31Many anticancer agents exert immunomodulatory effects on host system in addition to their cytotoxicityet al. reviewed that PD-L1 and PD-L2 did not correlate to the prognosis of NSCLC, nor did they relate to other clinically pathological factorset al. reported that high expression of PD-L1 in the primary foci of NSCLC was an independent predictor of poor prognosiset al. revealed that high PD-L1 expression is independently associated with longer overall survival and correlated with high tumor grade and younger patient ageThe prognostic value of PD-L1 has also been well-discussed among various cancers. PD-L1 expression on tumor cells correlates with poor clinical prognosis of renal, ovarian cancers, breast cancers151617For patient with pathological N2 NSCLC, the most menacing problem is postoperative recurrence. Thus new therapeutic targets for NSCLC are urgently needed. Alternative strategies such as immunotherapy or combination strategies involved of immunotherapy are now being considered for NSCLC treatment. CheckMate 012 investigate the efficacy and safety of combining anti-PD-1 antibody nivolumab with platinum doublet (n = 56), which achieved ORR as 33–50%, and remarkable 1-year survival rate as 59–87%This study is limited by retrospective feature and relatively small samples. Althouth we present an interesting and relevant finding, further research are warranted to explore the intrinsic mechanism of chemotherapy-induced alteration of PD-L1 expression. Considering the complexity of tumor micro-environment, deep illustration about effects of chemotherapy on tumor infiltrating lymphocytes patterns may provide better interpretation. The alteration of PD-L1 expression confers potentially impact on future treatment decision. Despite certain limitations, our data was the first to demonstrate the inconsistance of PD-L1 around chemotherapy and highlight the need to monitor PD-L1 biological changes around treatment and on serial samples, at least, the latest biopsy.Based on our result, the expression of PD-L1 on tumor cells is not consistent around chemotherapy for patients with NSCLC. The value as a predictive biomarker for anti-PD-1 efficacy and future immunotherapeutic decision should be cautious about the inconstancy of PD-L1. PD-L1 expression is suggested to be monitored around treatment and on serial samples, at least, on the latest tumor specimen.All patients were evaluated in a multidisciplinary meeting for treatment strategy setting. Patients pathologically diagnosed with NSCLC and received NACT prior to surgical resection at Sun Yat-sen University Cancer Center  from January 2010 to March 2014 were screened for eligibility. We only included cases met all of the following conditions: 1). diagnosed as stage IIIA (N2)/IIIB (T4N0-1) by invasive mediastinoscope; 2). received 1-4 cycles neoadjuvant chemotherapy before radical resection; 3). with enough paired tumor tissue samples around NACT for IHC staining of PD-L1.This retrospective study was approved by the Institutional Review Board of Sun Yat-sen University Cancer Center. All the patients had provided written informed consent before tissue samples were collected. All procedures performed in studies involving human participants were in accordance with the ethical standards of the national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The studies conducted in laboratory were operated under exploratory research principles.2 IV, days 1) plus carboplatin  or cisplatin , every 3 weeks. 2. Pemetrexed-based NACT, including pemetrexed  plus cisplatin , every 3 weeks. 3. TKI-based NACT, means treatment including erlotinib , gefitinib  or icotinib  for a period of at least 4 weeks, stopped 72 hours before surgery. All patients were re-evaluated by radiographic method after two cycles of treatment (four weeks for TKI-based NACT) for resectability. Adjuvant chemotherapy was administrated rouinely after resection, according to the sensitivity to neoadjuvant regimen and pathological type of cancer.Patient diagnosed as stage IIIA (N2)/IIIB (T4N0-1) by invasive mediastinoscope were transferred to medical oncology department for neoadjuvant chemotherapy. According to the regimens given to patients, the neoadjuvant chemotherapy (NACT) were classified as three types: 1. Paclitaxel-based NACT, which refers to paclitaxel , EGFR mutation status and classification of neoadjuvant regimens.The expression of PD-L1 in human NSCLC specimens was performed with IHC staining using rabbit monoclonal anti-human antibody . Five-μm-thick Sections were cut from the formalin-fixed, paraffin-embedded (FFPE) tumor block and then routinely deparaffined and rehydrated. For antigen retrieval, slides were heated in a microwave oven for 30 minutes in citrate buffer solution (pH = 7.4) and cooled slowly at room temperature for 20 minutes. Then, we blocked the activity of endogenous peroxidase with 3% hydrogen peroxide for 8 minutes. Thereafter, the sections were treated with primary antibodies and incubated for overnight (more than 12 hours). Subsequently, the slides were rinsed in PBS three times and incubated in HRR-linked secondary antibodies. After incubation, slides were washed again with PBS and then visualized using diaminobenzidine. Finally, Mayer’s hematoxylin was used to counterstain the sections and dehydrated and mounted. This mature method was applied in our previous work and detailed describedSemiquantitative H score  was defined as multiplying the percentage of stained cells by an intensity score 19p-value of <0.05 was considered statistically significant at all situation.All the statistical analysis was performed using SPSS 20.0 for Windows . Non-parametric quantitative data were presented as median value and interquartile range (IQR). Wilcoxon or Mann-Whitney method was used for comparision of non-parametric data. Pearson’s chi-squared test or Fisher exact test was used to assess the correlation between changes of PD-L1 expression and clinicopathologic variables. DFS was defined as the time from data of surgery to recurrence. Survival analyses were performed by Kaplan-Meier method and Cox proportional hazard regression model. A two sided How to cite this article: Sheng, J. et al. Expression of programmed death ligand-1 on tumor cells varies pre and post chemotherapy in non-small cell lung cancer. Sci. Rep.6, 20090; doi: 10.1038/srep20090 (2016).


E(k), with a slight p-doping for graphene/Au(111) and a larger n-doping for graphene/Ag(111). The obtained experimental data  are very well reproduced within DFT-D2/D3 approaches, which provide a detailed insight into the site-specific interaction between graphene and the underlying substrate.We investigate the electronic properties of graphene nanoflakes on Ag(111) and Au(111) surfaces by means of scanning tunneling microscopy and spectroscopy as well as density functional theory calculations. Quasiparticle interference mapping allows for the clear distinction of substrate-derived contributions in scattering and those originating from graphene nanoflakes. Our analysis shows that the parabolic dispersion of Au(111) and Ag(111) surface states remains unchanged with the band minimum shifted to higher energies for the regions of the metal surface covered by graphene, reflecting a rather weak interaction between graphene and the metal surface. The analysis of graphene-related scattering on single nanoflakes yields a linear dispersion relation  EF) and the Dirac point (ED). However, theoretical and experimental investigations allow a distinction between graphene which is weakly and strongly interacting with metals. In the first case 1213141516n- or p-doped with a linear dispersion around ED. In the latter case of graphene interacting strongly with a metal 1418192021n-doping of the graphene layer as well as a strong band bending and hybridization, leading to a complete destruction of the linear dispersion of graphene at ED, are observed.Graphene, a flat monolayer of carbon atoms arranged in a honeycomb lattice, is of particular interest as a possible material for many electron- and spin-transport devices135679ene on Au1, Ag2325262829In the present work, the structural and electronic structure of graphene nanoflakes on noble metal surfaces, Au(111) and Ag(111), was studied by means of scanning tunneling microscopy and spectroscopy (STM and STS). These systems were formed via intercalation of a thick layer of noble metal in GNFs/Ir(111). We show that STS measurements and the corresponding Fast-Fourier-Transform (FFT) analysis allow to unambiguously identify the scattering features arising from the metallic substrate and from graphene. The experimentally obtained energy dispersions of the charge carriers for the metal surface state electrons and for charge carriers in graphene are analyzed relying on state-of-the-art density functional theory (DFT) calculations, providing detailed information about the graphene-metal interaction.R0 structures are considered when the metal inverted contrast for the low bias voltages used for the atomically-resolved imaging. In the latter case, the ATOP positions of the moiré structure are imaged as dark spots and other sites are brighter in the STM images. A similar effect was found also for the graphene/Ir(111) system and here this effect was assigned to the moiré-structure modulated interaction that leads to the formation of sites in the graphene moiré structure, where interface states are formed that are responsible for the observed STM imaging contrast32Structural properties of graphene/Au(111) and graphene/Ag(111) were studied by means of STM at the atomic scale and compared with the results of DFT calculations. zmax,C − zmin,C), are 9.9 pm and 10.8 pm for graphene/Au(111) and graphene/Ag(111), respectively. These values are very close to the ones of the moiré corrugation of 4 ± 1 pm and 6 ± 1pm, respectively, obtained from STM experiments. In order to take into account the presence of the herringbone reconstruction, two absorption configurations - graphene/Au fcc and graphene/Au hcp were considered in the DFT calculations [DFT calculations yield an almost flat graphene layer in both systems. The corresponding binding energies (in meV/atom) and graphene-metal distances (in Å) calculated within DFT-D2 or DFT-D3 approaches are presented in ulations . Both coρ(r) = ρGr/M(r) − ρM(r) − ρGr(r) and plotted in units of e/Å3, where M stands for metal. As can be deduced from these data, the charge distribution at these graphene/metal interfaces is different reflecting the respective interaction strength between graphene and metal surface. For the graphene/Au(111) system, the Δρ distribution is very similar to the one for gr/Ir(111)17p-doped with the Dirac point located at ED = 0.05(0.17) eV above EF as obtained from DFT-D2 (DFT-D3) calculations [n-doped after its adsorption on Ag(111) with ED = −0.53(−0.41) eV, from DFT-D2 (DFT-D3) [bond-like states are formed at the HCP and FCC positions of the graphene/Ag(111) moiré structure similar to the graphene/Cu interface. The comparison of the carbon site projected partial density of states (PDOS) shows that the difference between the different high-symmetry positions for both systems is negligible [d and Ag 4d states are localized and overlap in the energy space with graphene π states. However, this effect does not influence the observed energy dispersion of graphene π states around the Fermi energy studied in this paper.ulations . The sit(DFT-D3) . In thisgligible . The maiI/dV images [I/dV maps can be used for the FFT analysis34E(k) can be obtained upon measuring wave vectors at different energies. In the case of the studied noble metals, the backscattering process within the ring-like constant energy contour of the parabolic surface state centered at the Γ-point leads to a circle around k of the surface state electrons can further be obtained by using the relation q = 2k, with q being the radius of the scattering circle.Quasiparticle interference in the proximity of defects leads to standing wave patterns in the topography  and evenV images  on both V images . Such dIE(k) for clean metal surfaces are parabolic as expected for the surface states and are in good agreement with previously published ARPES and STS data for Au(111) and Ag(111)3637383940424446h-BN29d and graphene π states discussed before may, however, lead to a slightly shorter distance between graphene and metal, compared to the distance if only van der Waals interaction is considered. Such a reduction of the distance between graphene and noble metal will then lead to an even stronger localization of the surface state wave function, giving a small correction to the position of the band minimum. A quadratic fit of the obtained data points allows to obtain the position of the band minimum as well as the effective mass of charge carriers for the pure Au(111) and Ag(111) surfaces and for graphene covered Au(111) and Ag(111) as summarized in Along with the scattering circles of the Shockley surface state, additional features arising from scattering solely within the graphene flake are observed within the FFTs. These features can be assigned to two specific backscattering processes: scattering between two neighbouring Dirac cones  and scattering within a single Dirac cone 253435EF.In the case of graphene/Au(111), the Au surface state scattering circle is still rather pronounced in the FT-LDOS within our measurement range of the electronic dispersion relation of graphene, whereas for graphene/Ag(111) the surface state is shifted towards the unoccupied states, therefore not being visible in the mapping energy range. The opening of a band gap in graphene at the Dirac point as observed in ARPES measurementsvF depending on the direction in ED and vF extracted from the plotted dispersions are compiled in While the scattering features in the FFTs of graphene on Au(111) appear to be almost circular, the corresponding features on Ag(111) show a trigonal warping indicating a larger energy shift of the Dirac point with respect to the Fermi energy. This effect leads to an enlargement of the structures visible within the FFT, thus increasing evaluation precision, but also yielding variation of the Fermi velocity d states . The expp-doping of 0.24 ± 0.07 eV, whereas graphene/Ag(111) shows a much larger n-doping of −0.56 ± 0.08 eV, hence already displaying trigonal warping at the Fermi energy. Despite the doping, the interaction between graphene and the chosen noble metal substrates is weak, since no additional band bending occurs within the measured energy range and the flakes appear to be quasi-freestanding. The obtained experimental data on the electronic structure of the flakes close to EF are compared with the results obtained within DFT-D2/D3 approaches and good agreement between all data is found. However, for graphene/Au(111) the experimental data fits better to the DFT-D3 method, whereas DFT-D2 delivers a better result for the graphene/Ag(111) system.Graphene nanoflakes have been produced and investigated on Au(111) and Ag(111) in order to obtain information about their structural and electronic properties. Quasiparticle interference mappings on both pure and graphene covered Au and Ag have revealed scattering due to the metals’ surface state, which underneath graphene has been shifted towards lower binding energies. For both substrates, we find that the presence of graphene does not influence the behaviour of the surface state electrons. The possibility to observe metal-related and graphene-related scattering features allows us to trace each sample’s electronic dispersion relation separately for graphene and metal. Graphene/Au(111) exhibits a GNFs were fabricated on Au(111) and Ag(111) using the method described elsewhereOmicron Cryogenic STM under ultra-high vacuum (UHV) conditions (<5 ⋅ 10−11 mbar). Polycrystalline tungsten tips flash-annealed in UHV were used for all STM/STS measurements. The sign of the bias voltage corresponds to the potential applied to the sample. Differential conductance (dI/dV) maps were recorded by means of standard lock-in technique, using the modulation voltages  and frequencies given in the figure captions.STM and STS measurements were performed at low temperatures in an et al.56k-space sampling of (4 × 4 × 1) (model-i) and (28 × 28 × 1) (model-ii) was used, where the Γ-point was explicitly included. After structure relaxation successive single-point calculations using denser (7 × 7 × 1) (model-i) and (39 × 39 × 1) (model-ii) k-grids were carried out in order to determine the density of states as well as binding energies and simulate STM images via the Tersoff-Hamann approximationDFT calculations were performed using the Vienna Ab initio Simulation Package (VASP)How to cite this article: Tesch, J. et al. Structural and electronic properties of graphene nanoflakes on Au(111) and Ag(111). Sci. Rep.6, 23439; doi: 10.1038/srep23439 (2016).


The world is facing an energy crisis due to exponential population growth and limited availability of fossil fuels. Over the last 20 years, carbon, one of the most abundant materials found on earth, and its allotrope forms such as fullerenes, carbon nanotubes and graphene have been proposed as sources of energy generation and storage because of their extraordinary properties and ease of production. Various approaches for the synthesis and incorporation of carbon nanomaterials in organic photovoltaics and supercapacitors have been reviewed and discussed in this work, highlighting their benefits as compared to other materials commonly used in these devices. The use of fullerenes, carbon nanotubes and graphene in organic photovoltaics and supercapacitors is described in detail, explaining how their remarkable properties can enhance the efficiency of solar cells and energy storage in supercapacitors. Fullerenes, carbon nanotubes and graphene have all been included in solar cells with interesting results, although a number of problems are still to be overcome in order to achieve high efficiency and stability. However, the flexibility and the low cost of these materials provide the opportunity for many applications such as wearable and disposable electronics or mobile charging. The application of carbon nanotubes and graphene to supercapacitors is also discussed and reviewed in this work. Carbon nanotubes, in combination with graphene, can create a more porous film with extraordinary capacitive performance, paving the way to many practical applications from mobile phones to electric cars. In conclusion, we show that carbon nanomaterials, developed by inexpensive synthesis and process methods such as printing and roll-to-roll techniques, are ideal for the development of flexible devices for energy generation and storage – the key to the portable electronics of the future. The demand for energy in the 21st century is increasing due to the increase in the world’s population and rapid technological advancement .Solving this energy demand using more efficient or clean alternative energy sources will not only save the planet from harmful effects caused by pollution but could also reduce disparity and create a more peaceful world . Energy EnergyWaterFoodEnvironmentPovertyTerrorism and warDiseaseEducationDemocracyPopulationThe energy problem is on the top of the list because, according to Smalley, it directly influences the other problems and thus should be prioritized accordingly by governments worldwide. The first immediate solution to this problem would be to work on energy efficiency programs. With the implementation of such programs, it has been demonstrated that developed nations could already reduce energy consumption by 25% .The majority about 87%) of energy produced in 2013 was composed of fossil fuels such as oil, gas and coal, which represented the best choice of energy production at competitive costs in the 20th and 21st century  [7]. Unf% of ener2 per year per watt  and intermittent. This is why concentration and storage become two critical issues to solve in order to make this energy source cost competitive with fossil fuels. The challenge over the next few years will not just be to produce electricity in a safe and clean way but also how to store the energy produced using technologies more efficient and more environmentally friendly than chemical batteries fulleroid ester and subsequent isomerization to the methanofullerene by refluxing it with o-dichlorobenzene solution or with trifluoroacetic acid , wCarbon nanotubes (CNTs), discovered by Ijima in 1991 , are anoTensile strength of at least 37 GPa and strain to failure of at least 6% –39Young’s modulus, ≈0.62–1.25 TPa Electrical resistivity, ≈1 μΩ cm Thermal conductivity, ≈3000 W/mK 3 ..43].n, m). If m = 0 the SWNTs are called zigzag; if n = m, they are armchair, otherwise, they are chiral if n ≠ m . In, m), is very important because it strongly affects the electronic properties of the SWNTs. For a given  nanotube, if n = m, the nanotube is metallic; in all other cases, the nanotube is semiconducting with the remarkable situation that when (n − m) is a multiple of 3, the nanotube has a very small band gap . It. It61]. 2 V−1 s−1) . T. T154]. The two major polytypes are α-SiC and β-SiC. The α-SiC exhibits a hexagonal crystal structure (similar to wurtzite) and is usually formed at temperatures above 1700 °C. β-SiC exhibits a cubic crystal structure with a stacking sequence of ABCABC along the (111) direction, which is typical of a zinc blende crystal structure (similar to diamond) and is formed at temperatures below 1700 °C. The α-SiC and the β-SiC can also be catalogued with the Ramsdell classification scheme where a number indicates the layer and a letter indicates the Bravais lattice type, such as cubic (C), hexagonal (H) or rhombohedral (R) .For example the α-SiC can also be called 2H-, 4H- or 6H-SiC, depending on the unit cell, while β-SiC can also be called 3C-SiC because of the ABC stacking  Figure .The formation process of graphene on SiC is also called graphitization. It consists of the sublimation of Si atoms from the SiC surface caused by high temperatures with a consequent rearrangement of the carbon atoms on the surface to form a graphene lattice –159. GraHexagonal polytypes of SiC (such as 6H and 4H) with orientation (0001) are preferred because of the lattice structure that matches the graphene lattice. However, 3C polytype structures have also been used on (111)-oriented surfaces in order to maintain a good match with the hexagonal lattice of graphene –162.The SiC wafer is usually precleaned in ultrahigh vacuum (UHV) or in other environments with different techniques in order to increase the graphene quality during graphitization. The three most common techniques are: heating the sample ex situ while hydrogen etching for 30 min at 950 °C; preparing a silicon rich phase (3 × 3) in a Si flux and then heating the sample for 30 min at 1000 °C; or heating the sample at around 1000 °C under Si flux to remove the native oxide and to avoid silicon depletion of the surface .Graphene growth occurs at temperatures of 1200–1350 °C in UHV –165 evenThe graphene can also be grown on a C face. The advantages are the absence of a buffer layer and the possibility to easily grow multilayer graphene (MLG) in all directions on the SiC surface  171]. T. T171]. 2 V−1 s−1) of graphene grown on SiC. An IBM research group recently reached the 300 GHz cut-off frequency for a graphene-based FET grown on SiC. They showed the stable operation of an integrated circuit containing graphene  , the devTypically, an organic solar cell consists of two electrodes (one of which is transparent) and an active layer between them where the generation of free charge carriers occurs. A buffer layer is usually included between each electrode and the active layer in order to prevent charge recombination, which reduces the efficiency of the device. The active layer can be a double layer (one has a stronger affinity for electrons (donor) and the other has a stronger affinity for holes (acceptor)) or a bulk heterojunction . The bulk heterojunction (BHJ) provides a larger volume of paths for the transport of free carriers and more efficient charge separation in comparison to the other structures –186. TheThe generation of a photocurrent due to light incident upon an organic solar cell device consists of three steps  189–190190:Photon absorption in the conducting polymer .Creation of an exciton. An exciton is a bound state of an electron and a hole that are attracted to each other by the electrostatic Coulomb force. Its diffusion length is about 10 nm.Exciton separation at the interface between the donor and the acceptor. Because of the built-in electric field at the interface, the electron is transferred to the acceptor and the hole to the donor (creation of the photocurrent).One problem is the short lifetime of excitons generated by light. Considering that their recombination distance is between 4 and 20 nm –192, the61-butyric acid methyl ester (PC61BM or PCBM) as the acceptor material and the conducting polymer poly (P3HT) as the donor material -phenyl-Cmaterial –198. Botmaterial –200.61BM/P3HT as the active layer, PEDOT/PSS as the electron blocking layer and gold (Au) as the anode methanofullerene or the PC61BM derivative is more soluble in organic solvents than pristine C60 ..242].71BM, semiconducting SWNTs, and reduced GO, achieving a PCE of 1.3%. They also used ab initio calculations to demonstrate efficiency limits of up to 13% for this device, which is comparable to those predicted for polymer solar cells 2NH) ..253].EDLCs typically store the energy at the electrode/electrolyte interface as shown in Positive and negative ions are accumulated at this interface, typically on the order of 5–10 Å . The con0 is the permittivity of the vacuum, εr is the permittivity of the electrolyte and d is the thickness of the double layer with surface area A. The thickness (d) of the interface is very small (on the order of Å), as discussed previously, while the surface area (A) of the electrode is usually very due to the choice of porous structures with a large internal surface area usually chosen for supercapacitor applications. In this way, the capacitance can reach a high value (>10 μF/cm2).where εCH) and diffusion region capacitance (Cdiff):The model described to store the charge at the electrode/electrolyte interface was first derived by Helmholtz  in the 1The ideal total capacitance in an EDLC is given by the sum of the capacitances established at the two electrode/electrolyte interfaces :R1, R2), the electrode resistances  and the interelectrode resistance (RS) ..C) of thIn summary, several important characteristics of an EDLC have to be considered to maximize the performance of the device :The specific surface area of the electrodes to increase the capacitanceThe conductivity of the electrodes to reduce the power density lossesThe resistance to any oxidation/reduction on the surface of the electrode to maintain good stability and performanceControlled size distribution of the pores that should match the size of the electrolyte ionsElectrochemical stability of the electrolyte in the voltage operating window of the deviceLow interconnected resistance of the electrolyteGood wettability of the electrolyte on the electrode.2/g) but with a wide pore size distribution consisting of macropores (>50 nm), mesopores (2–50 nm) and micropores (<2 nm) [2) when compared to the theoretical calculations [Activated carbons (ACs) are the most commonly used materials for commercial electrodes in supercapacitors because of their stable electrical properties, large surface area and low cost. ACs can be produced by thermal and/or chemical activation of various types of materials containing carbon such as wood, coal, etc. For thermal activation, high temperatures are usually employed (from 700 to 1200 °C) in the presence of oxidizing gases. For chemical activation, the process temperatures are lower (from 400 to 700 °C) and require activating agents such as phosphoric acid, sodium hydroxide and others . These t (<2 nm) –272. Eveulations . This diulations . Pore shulations .Carbon nanomaterials such as CNTs and graphene are excellent candidates to replace ACs as electrode materials in supercapacitors because of their remarkable chemical stability, large specific surface area, and high electric conductivity –275. Com2 g−1) caused by the entangled arrangement of the CNTs with only the outermost tubes of the bundles exposed to the electrolyte [CNTs in the form of either arrays grown on a substrate –278 or nctrolyte   [Recently, graphene and graphene derivatives have been considered for supercapacitor electrodes, not only for their exceptional electrical, thermal and mechanical properties previously described, but also for two other main reasons: the theoretical high surface area (2630 m2/g)  and the 2/g) . In fact2/g) .2/g  by employing inexpensive synthesis and process methods based on printing and roll-to-roll techniques.In this paper, different approaches to synthesize and employ carbon nanomaterials for energy generation and storage applications have been explored. Carbon nanomaterials used for organic solar cells have been thoroughly reviewed. Graphene produced by electrochemical exfoliation was discussed as a viable solution to produce a large quantity of transparent electrodes for organic solar cells. Achieving a high quality material on a large scale still remains an issue in order to compete with conventional conducting transparent electrodes such as ITO. However, its potential for production on flexible substrates makes it very appealing for the organic solar cell field where roll-to-roll techniques have been recently employed to increase the production volume. Additionally, fullerene derivatives, CNTs and graphene oxide could help to boost the performance of organic solar cell devices if employed in the active or buffer layers. In fact, their semiconducting properties can be tuned by doping with other materials or by changing their physical structure in order to absorb a broader range of solar spectrum wavelengths.Carbon nanomaterials, in particular carbon nanotubes and graphene, have also been proven to be very efficient and reliable materials for energy storage. The high specific surface area and conductivity of graphene are two key features for employing this material in supercapacitors. The ability to use a solid-state electrolyte composed of graphene oxide or a gel polymer electrolyte allow for the possibility of printable, flexible devices that do not require encapsulation. Although carbon nanotubes generally have a relatively low specific surface area, they can still be employed in combination with graphene to increase the conductivity of the electrode or the surface roughness of the film, resulting in an increase in the number of ions stored at the electrode/electrolyte interface.Carbon continues to surprise researchers with its extraordinary properties. Completely new carbon structures have been synthesized over the last 20 years, from 0D fullerenes to 1D nanotubes and 2D graphene. The low cost of this element, the sixth most abundant element on earth, makes it an attractive choice to replace conventional materials for energy generation and storage applications.