The high refraction-index contrast between silicon and the surrounding cladding makes silicon-on-insulator devices highly polarization-dependent. However, it is greatly desirable for many applications to address the issue of polarization dependence in silicon photonics. Here, a novel ultra-compact polarization splitter and rotator (PSR), constructed with an asymmetrical directional coupler consisting of a rib silicon waveguide and a graphene-embedded rib silicon waveguide (GERSW), on a silicon-on-insulator platform is proposed and investigated. By taking advantage of the large modulation of the effective refractive index of the TE mode for the GERSW by tuning the chemical potential of graphene, the phase matching condition can be well satisfied over a wide spectral band. The presented result demonstrates that for a 7-layer-graphene-embedded PSR with a coupling length of 11.1 μm, a high TM-to-TE conversion efficiency (>−0.5 dB) can be achieved over a broad bandwidth from 1516 to 1602 nm. While the high-refractive index contrast between the silicon and surrounding cladding has the advantage of constructing photonic devices with a compact footprint, it inevitably poses a significant challenge to handling the polarization dependence. To address this issue, a polarization diversity circuit that consists of polarization beam splitters and rotators is highly desirable6. A polarization beam splitter can efficiently split one input beam with two orthogonal polarization states into two output beams with different polarization states5, while a polarization beam rotator can rotate the polarization of input beam by 90°4. For many applications, in the rest of an optical circuit only a certain polarization state can be guided. Therefore, it is highly desirable to develop polarization management devices that are capable of rotating one of the two polarization states to the orthogonal state so that merely one polarization state must be processed in the rest of the optical circuit. Recently, a type of polarization splitter and rotator (PSR) technology has been intensively proposed to achieve this purpose. With this technology, one polarization state of input beam is converted to the orthogonal polarization state using a directional coupler. Meanwhile, the orthogonal polarization state of input beam is much unaffected and output through the input waveguide because no coupling occurs in the region of the directional coupler12. Therein, a PSR using an asymmetrical directional coupler (ADC) composed of two waveguides with broken symmetry for the waveguide cross section has attracted much research attention12. However, the most apparent drawback of the scheme is that it can merely operate within a limited bandwidth, as the phase-matching condition should be precisely satisfied for an ADC. While promising steps have been taken to broaden the operation bandwidth by utilization of tapered10 and taper-etched11 ADCs, it is generally accompanied with a relatively large footprint, which is against the high-density integration on the SOI platform.Integrated photonic devices built on silicon-on-insulator (SOI) have been attractive for their compatibility with mature, complementary metal-oxide-semiconductor-compatible technologies16 and electro-refractive modulators17. A recent study has demonstrated that graphene will have different influences on the modal characteristics of silicon waveguides for different polarization states when graphene is involved in a dielectric waveguide, thus offering huge capability to address the issue of strong polarization dependence20.Graphene, a two-dimensional single layer of carbon atoms arranged in a hexagonal lattice, has attracted tremendous attention in recent years due to its unique optical and electrical properties. Owing to the large and flexible tunability of graphene by bias voltage and chemical doping, graphene is emerging as an attractive material for the development of highly efficient optoelectronics devices including electro-absorption modulatorsIn this article, we propose and numerically demonstrate a novel PSR using a graphene-embedded silicon rib ADC. When graphene is horizontally embedded into a rib silicon waveguide (RSW), the effective refractive index (ERI) of the TE mode undergoes a significant variation by tuning the chemical potential of graphene via bias voltage. Consequently, the phase-matching condition for the ADC can be satisfied over a wide spectral band by properly choosing the variation range of the chemical potential of graphene, and hence the operation bandwidth of the PSR can be significantly enlarged.21, and the optical property of the graphene layer has been provided in the Methods section. The chemical potential of the graphene multilayer has a stronger effect on the modal characteristics of the TE mode than that of the TM mode [shown in Fig. n) of the TE mode at 1450 nm is 0.057, while that of the TM mode is only 0.024. In the wavelength range of interest, the modal characteristics for both the TE and TM modes presents a sharp peak due to the dramatic change of the graphene’s conductivity around μc = 0.4 eV22. It should be noted that the propagation losses of the TE modes at the peaks in Fig. n) denoted by the star marks in Fig. μc is chosen.A graphene-embedded rib silicon waveguide (GERSW) is shown in Fig. wgap) between the two waveguides is unchanged in the ADC region, and an S-bend waveguide is connected to the GERSW. Metal 1 and Metal 2 act as two electrodes. The modal field of the TE mode is strongest in the centre of the RSW. Thus, in order to increase the influence on the modal characteristic of the TE mode, the graphene layers must be inserted into the centre of the RSW to maximize the graphene-light interaction. Further, the modal characteristic of the TE mode objects to a larger influence if more graphene layers are used22. In our work, we have chosen an odd number of graphene layers to illustrate the performance of the PSR. We emphasize that an even number of graphene layers applies to the design of the PSR as well, and the resultant PSR is expected to present a similar performance. We have noted a series of studies on the utilization of an even number of graphene layers for the construction of various graphene-based optoelectronics devices on a silicon platform, including silicon waveguide modulator23 and polarizer18. The odd number of graphene layers are extended to contact the Metal 1, and the remaining layers contact the Metal 2. The utilization of undoped, amorphous silicon ensures the formation of capacitor effect between the graphene layers contacted to the metallic electrodes23. Thus, the chemical potential of the graphene layers could be controlled by the bias voltage applied on these two metal electrodes. The chemical potential of graphene, μc, can be tuned by the gate voltage applied on graphene, 22, where 6 m/s is the Fermi velocity, and 18. For the fabrication of the PSR, we can first prepare two shadow masks for the RSW and two metal electrodes. By properly arranging the order of the two masks, choosing the deposition methods, and transferring the graphene layers (etched in an “L” shape by a focused ion beam without the utilization of masks) from the copper foil, the presented PSR, at least in principle, might be fabricated5. In order to enable an efficient conversion between two orthogonal fundamental modes, the phase-matching condition is required. Considering that the modal characteristics for the TE mode in the GERSW can be changed with a broader range than that for the TM mode by tuning the chemical potential of graphene, the TE mode in the GERSW and the TM mode in the RSW are employed to satisfy the phase-matching condition. In this case, the largest operation bandwidth might be achieved because the phase-matching condition can be satisfied within a wide spectral band. In addition, we employed rib waveguides to increase the vertical asymmetry in the waveguide cross section, which is helpful for the mode conversion between the fundamental TE and TM modes. By optimizing the widths of two rib waveguides and the coupling length (L), the input TM mode from the RSW could be efficiently converted into the TE mode in the GERSW and output from the cross port, while the input TE mode is rather unaffected and directly output from the thru port.Bearing these points in mind, we propose to construct a PSR consisting of a GERSW and a RSW, as shown in Fig. λ = 1550 nm. In order to find the optimum width of the GERSW (wg) that satisfies the phase-matching condition, the ERI of the fundamental TE mode in the GERSW as a function of wg has been numerically calculated [shown in the Fig. n) for the TE mode is roughly located in the middle of the tuning range [see Fig. wgap, is chosen as 150 nm to build a compact directional coupler with acceptable fabrication difficulty. The coupling length, associated with the device size, can be further decreased by use of a smaller gap separation but at the cost of remarkably increased fabrication difficulty. In the design, the total thickness of the silicon layer in the GERSW is set at a constant value of 210 nm. The height of the GERSW, h2, is increased as the number of graphene increases. To make the two waveguides have the same height, the height of the RSW, h2, increases accordingly. The resultant ERI of the TE mode in the GERSW [Fig. wg0, wg1, wg3, wg5 and wg7) must be optimized to satisfy the phase-matching condition between the TE mode in the GERSW and the TM mode in the RSW [Fig. L0, L1, L3, L5 and L7 in Fig. wg7) and the coupling length (L7), the TM input from the RSW is converted to the TE mode in the GERSW and efficiently outputs from the cross port, while the launched TE mode is barely influenced by the GERSW and directly outputs from the thru port. Because the modal characteristics of the TM mode in the GERSW would be negligibly influenced by using different numbers of graphene layers around μc = 0.5 eV [Fig. Next, we consider the performance of the designed PSR at RSW Fig.  and the RSW Fig. . In orde eV Fig. , the wav eV Fig. . It shouμc on the performance of the PSR. First, a PSR with seven layers of embedded graphene is studied as an example. All the structural parameters are the same as the above cases where the launched TM mode in the RSW could be efficiently converted to the TE mode in the GERSW at λ = 1550 nm. As for the TE mode input, an apparent phase-mismatching between the TE mode in the RSW and the TE and TM modes in the GERSW prevents light transferring from the RSW to the GERSW [Fig. μc is different for the case of λ < 1550 nm [Fig. λ < 1550 nm (>1550 nm), the ERI of the TE mode in the GERSW can be increased (decreased) by tuning the chemical potential of graphene to make the phase-matching condition well satisfied; thus, the operation bandwidth of the PSR might be effectively extended. In the case of λ < 1550 nm, the ERI of the TE mode in the GERSW can be made closer to the ERI of the TM mode in the RSW by tuning μc from the triangle-mark positions to the star-mark positions. However, the significantly increased propagation loss [see Fig. λ > 1550 nm [Fig. μc < 0.8 eV for λ = 1570 and 1590 nm, and the maximum transmission for λ = 1610, 1630, and 1650 nm occurs at μc = 0.8 eV in our case. It can be highly expected that the transmission can be further enhanced if a much higher carrier density of graphene, associated with a larger chemical potential, is used. Consequently, to achieve the maximum transmission for the TE mode at the cross port, one must properly select the ERI and propagation loss for the TE mode in the GERSW.Considering that the chemical potential of graphene could be tuned by bias voltage, we further investigate the influence of RSW Fig. . In othe nm Fig.  and λ >  nm Fig. . In the see Fig.  would susee Fig. . In the  nm Fig. , the maxμc as a function of wavelength, and μc versus μc ranging from 0.4–0.8 eV, is below 11.7 V, which is comparable to that in graphene-based SOI modulators23. Figure μc the device performance of the PSR has significantly improved as compared with that at μc = 0.5 eV. Here, for the TE mode input, the TE-TE conversion efficiency (CETE-TE) at the through port A2 and the cross-talk (XTTE) at the cross port B2 are defined as CETM-TE) at the cross port B2 and the cross-talk at the thru port A2 are defined as CETM-TE (>−0.5 dB) has been extended from 58 nm (1533–1591 nm) to 86 nm (1516–1602 nm), while the cross-talks XTTE and XTTM have improved significantly in the wavelength range of interest. No matter what chemical potential of graphene is used, the CETE-TE is kept less than −0.02 dB over the whole studied spectral band, which can be attributed to the fact that the TE mode passes through the ADC with little influence due to the significant phase-mismatch.The optimized value of 18. It should be noted that the electric field of the TE mode is strongest at the centre of the waveguide. As a result, the graphene layer in the middle has the largest contribution to the variation of the ERI, while the graphene layers on the upper and lower sides have the smaller influence on the ERI. This is why the growth rate of the operation bandwidth is decreased with an increase in graphene number [Table We further study the impact of the graphene number on the bandwidth broadening of the designed PSR. As shown in Fig. er Table . For thr25. The tapered ADC-based PSR has been recently proposed to overcome this limitation7, and the bandwidth is broadened as five times as large as the ADC-based PSRs25 but which is at the cost of a relatively large footprint. For the present graphene-based PSR, on one hand, the operation bandwidth can be four times larger than that in Refs25, while its length is several times smaller than that based on a tapered ADC-based PSR7. Our results here might offer a promising approach to construct a broadband and efficient PSR with an ultra-compact footprint. We have noted a recent study on a hybrid (de) multiplexer by combining mode, polarization, and wavelength together to increase the transmission capacity on a silicon platform26, where the polarization diversity circuit, comprised of a polarization beam splitter (PBS) and polarization rotation (PR), was used to address the issue of polarization dependence so that merely the TE mode was left for the arrayed-waveguide gratings (AWGs). Such a functionality with the combination of a PBS and PR, at least in principle, can be fulfilled with the proposed PSR, which has a larger working bandwidth that is required for the AWGs of 9 channels with the bandwidth of 9 × 3.2 nm (28.8 nm). In addition, more details regarding the performance of the PSR embedded with seven layers of graphene at 1550 nm have been listed in Table Table Finally, it is worthy emphasizing, when the TM mode is launched, one can modulate the transmission of the TE mode at the cross port by tuning the chemical potential of graphene, while the transmission at the thru port almost stays unchanged [see the blue lines in Fig. An ultra-compact PSR has been proposed and numerically demonstrated by utilizing an ADC consisting of a RSW and a GERSW. The ERI of the GERSW for the TE mode can be significantly changed by tuning the chemical potential of graphene. As a result, the phase-matching condition between the TM mode in the RSW and the TE mode in the GERSW can be well satisfied over a broad spectral band. The PSR embedded with seven layers of graphene and with a 11.1-μm coupling length and a 150-nm gap separation enables a high TM-to-TE conversion efficiency (>−0.5 dB) within a broad bandwidth (1516–1602 nm). Overall, this designed PSR presents a strong ability to address the issue of polarization dependence at 1550 nm with the TE-to-TE thru loss (>−0.01 dB), TM-to-TE polarization conversion loss (>−0.26 dB), and extinction ratio at the thru port (>14 dB).xp(−iωt) time dependence, the optical response of graphene can be characterized by surface conductivity (σg), which is related to the chemical potential (μc), using the Kubo formula27:e is the electron charge, kB is the Boltzmann constant, T is temperature (=300 K), ω is the angular frequency, and τ is momentum relaxation (τ = 0.5 ps in this work). Because an electron mobility of 100000 cm2V−1s−1 has been experimentally verified in high-quality suspended graphene28, which leads to τ > 1.5 ps, the choice of τ = 0.5 ps here is rather conservative. Here, the graphene sheet is treated as an anisotropic material29. The out-of-plane permittivity of graphene, εg,⊥, is equal to graphite (2.5). The in-plane permittivity of graphene, εg,∥, can be retrieved as30η0 (≈377 Ω) and k0 are the impedance and wave vector in the air, respectively, and dg (=0.34 nm) is the thickness of graphene. This equation has been extensively employed to characterize the optical property of graphene and estimate the electro-optical properties of graphene-based photoelectric devices both theoretically and experimentally31.With the assumption of an eIn the FDTD simulation, sufficiently small mesh girds for the silicon and graphene have been used to ensure the simulation accuracy. Four mesh grids are used to represent the thickness of the silicon layer between the adjacent graphene layers and each sheet of graphene. To precisely retrieve the device performance, different numbers of mesh grids have been tested in our modelling, and the present result indicates that the simulation date does not change if finer mesh grids are used. The perfectly matched layer-absorbing boundary condition is used at the boundaries of the computational window.


Graphene possesses extraordinary properties that promise great potential in biomedicine. However, fully leveraging these properties requires close contact with the cell surface, raising the concern of unexpected biological consequences. Computational models have demonstrated that graphene preferentially interacts with cholesterol, a multifunctional lipid unique to eukaryotic membranes. Here we demonstrate an interaction between graphene and cholesterol. We find that graphene increases cell membrane cholesterol and potentiates neurotransmission, which is mediated by increases in the number, release probability, and recycling rate of synaptic vesicles. In fibroblasts grown on graphene, we also find an increase in cholesterol, which promotes the activation of P2Y receptors, a family of receptor regulated by cholesterol. In both cases, direct manipulation of cholesterol levels elucidates that a graphene-induced cholesterol increase underlies the observed potentiation of each cell signaling pathway. These findings identify cholesterol as a mediator of graphene’s cellular effects, providing insight into the biological impact of graphene. Understanding the biological role of graphene in eukaryotic cells is essential for future biomedicine applications. Here, the authors investigate the interaction of neurons and fibroblasts with graphene substrates, which increase cell membrane cholesterol and potentiate neurotransmitter release and receptor signaling. A consequence of its unique atomic structure, graphene possesses a myriad of attractive chemical and physical properties: exceptionally high electron mobility, thermal conductivity, optical transmittance, mechanical strength, chemical stability, and surface area-to-volume ratio3. This combination of features has made graphene a promising material for a broad range of biomedical applications, including drug delivery, tissue engineering, biosensing, and neuroprosthetics6. Furthermore, its superior carrier mobility enables graphene-based electrodes to detect electrochemical changes associated with a variety of cellular activities or to deliver optical or electrical stimuli. For example, graphene electrodes have been used as voltage sensors to measure membrane potential changes at the single-cell level and to record electrical activity in neuronal networks in vitro and in vivo8. However, a significant drawback to the use of graphene-based devices remains, in that detection efficiency exponentially decreases as the distance from the cells or tissue increases. Therefore, graphene needs to be close to cell or tissue surfaces8 in order to maximize its utility in bioapplications. This then raises a fundamental question: how graphene affects the plasma membrane and related cellular functions.Graphene is a two-dimensional material composed of a single-layer of hexagonal Escherichia coli through the disruption of plasma membrane integrity11. Computational modeling and electron microscopy have suggested that this is the result of nanoscale GFs penetrating the bacterial plasma membrane and dispersing phospholipids12, which leads to membrane disintegration and cell death13. Interestingly, no such cytotoxicity has been reported in eukaryotic cells. Instead, cell proliferation14, differentiation15, and morphogenesis16 were improved when graphene with traditional tissue culture coatings was used as a culture substrate. For mouse neurons grown on bare graphene, Veliev et al.17 demonstrated that the crystallinity of graphene promotes cell adhesion and neurite outgrowth, suggesting the significance of close contact between graphene and the cell surface in mediating graphene’s effects. Aggregated GFs used as a substrate or applied acutely to mature neuronal cultures resulted in few developmental or morphological changes20. Aggregated GFs, however, have very different surface characteristics and physicochemical properties from monolayer graphene substrates21. Given the variation in experimental outcomes and the increasing interest in bioapplications of graphene, it remains important to understand how graphene interacts with the eukaryotic plasma membrane and how its diverse cellular effects are realized.Prior studies have documented that graphene flakes (GFs) are destructive to Gram-negative bacteria such as 22. For example, cholesterol influences membrane fluidity23, facilitates exocytosis/endocytosis24, regulates integral membrane proteins like G protein-coupled receptors (GPCRs)25, and organizes cytoskeletal attachment as well as cell adhesion26. The distribution of cholesterol within the cell membrane is highly heterogeneous and dynamic22, with trafficking between surface and intracellular membranes helping to maintain overall cholesterol homeostasis. Intriguingly, it has been empirically demonstrated that the planar tetracyclic ring structure of cholesterol may permit it to stack on the graphene surface31. We thus sought to investigate if and how the cellular effects of monolayer graphene are related to membrane cholesterol.We reasoned that, since the plasma membrane is the initial point via which any downstream cellular responses to graphene would be realized, membrane-associated molecule(s) were responsible for the variation of cellular effects in response to graphene. We hypothesized that cholesterol was involved for two reasons: it is unique to and abundant in the eukaryotic plasma membrane but absent from prokaryotic membranes, and it is related to many cellular processes, realized through both direct and indirect interactions with other lipids and proteins35. Bidirectional manipulations of membrane cholesterol demonstrate that a graphene-induced membrane cholesterol increase is an underlying mechanism for the potentiated neurotransmitter release. Furthermore, we demonstrate that graphene substrates allosterically enhance the Ca2+ responses of P2Y receptors to ATP stimulation in a cholesterol-dependent manner. Our findings collectively reveal that cholesterol is a key mediator of graphene’s biological effects on eukaryotic cells.Here we use liquid-phase exfoliation (LPE) of graphene powder to produce suspended GFs, which enables solution-based measurements to investigate a graphene–cholesterol interaction. GFs extract cholesterol from cholesterol-containing culture media and effectively quench a fluorescent cholesterol analog, suggesting an interaction between graphene and cholesterol. For cell-based studies, we grew both neurons and fibroblast cells on large-area monolayer graphene sheets. In neurons on graphene, we find increased cell membrane cholesterol and significant increases in the number, the release probability, and the turnover rate of synaptic vesicles—all of which are modulated by membrane cholesterol36. GFs were suspended in 2 wt% polyvinylpyrrolidone (PVP) at 260 ng mL-1 to prevent aggregation. We mixed this suspension with serum-containing neuronal culture media and incubated for 24 h to mimic chronic exposure during culture. Serum in our culture media contains cholesterol (see result below and Methods), which enabled us to measure cholesterol adsorption onto the graphene surface. GFs were separated out by size-dependent filtration and subjected to an enzymatic cholesterol assay37. The remaining fraction, untreated fresh media, and media mixed with PVP (as a vehicle control) were assayed in the same manner. After calibration by a cholesterol standard, we determined that cholesterol concentrations in the PVP-treated media (12.4 ± 2.3 μM) and untreated fresh media (12.3 ± 0.9 μM) were similar. The GF fractions (graphene incubated with media) showed a significantly higher cholesterol concentration (18.0 ± 1.0 μM), while there was a correspondingly lower concentration (4.3 ± 0.1 μM) in the remaining fraction (graphene-treated media)  Fig. , suggestner Fig. , suggest43 suggest that the graphene films are monolayer , and plated dissociated neurons directly on pristine graphene. Conventional hippocampal cultures contain a considerable number of astrocytes44, which if allowed to proliferate over the length of the culture period would have covered the substrate surfaces, further limiting the exposure of neurons to graphene. To minimize potential effects from the astrocyte layer, we applied a mitotic inhibitor (Ara-C) immediately after cell plating, which blocks astrocyte proliferation without any effect on non-proliferative cells 44. However, astrocytes play important developmental and functional roles for neurons, so to compensate for the reduced number of astrocytes, we routinely supplied cultures with conditioned media (see Methods) harvested from conventional hippocampal cultures prepared in parallel. As a negative control, the same astrocyte-deprived hippocampal cultures were grown on bare glass coverslips and maintained in the same manner.Having demonstrated an interaction between GFs and cholesterol, we set out to test whether this interaction plays a role in mediating graphene’s cellular effects. We produced large films of planar graphene via chemical vapor deposition (CVD), which were solution transferred to bare glass coverslips to be used as culture substrates Fig. . The quayer Fig. . Spatialyer Fig. . BecauseIt is well established that nanomaterial surfaces will be covered by a variety of biomolecules  after introduction to any biological system. Although we omitted a much thicker artificial protein-coating layer (Matrigel) in plating our cultures, we cannot exclude the likelihood of the formation of a protein corona on the graphene surface. However, our cell-free assays demonstrate that cholesterol enrichment on graphene still occurs even in the likely presence of a protein corona after 24-h incubation Fig. . We perf30. To experimentally investigate possible changes in cholesterol after chronic growth on graphene, we first performed Filipin staining, a conventional method for labeling cholesterol in fixed cells45. We found a 27% increase in Filipin staining intensity in the neurites of neurons cultured on graphene /(Iblue + G × Igreen), in which G is a correction factor and Iblue and Igreen are fluorescence intensity values46. We found that C-laurdan fluorescence exhibited a blue shift on graphene, which translated to an overall shift toward increased GP values, indicating increased plasma membrane cholesterol on graphene substrates  and lower concentration (1 μM) of TFC; this should largely result in TFC incorporation into the plasma membrane. However, we cannot exclude the possibility that membrane turnover will also result in some intracellular membrane labeling. We found that TFC is distributed throughout neurites  , which have distinct activation and decay kinetics , to determine whether the increased sEPSC frequency on graphene was driven by postsynaptic mechanisms, we measured INMDAR/IAMPAR. Here we observed a small but non-significant decrease in this ratio  Fig. . The amptio Fig. . Taken t52. It is generally established that increased neurite complexity is associated with more synaptic connections between neurons, and more synaptic connections in turn lead to more synaptic inputs 53. We performed Sholl analysis to quantify arborization, which counts the number of neurite intersections at concentric circles of increasing radius originating at the neuronal soma54. Although there was an overall increase in neurite complexity on graphene between 12 and 18 DIV , and a neurite-specific marker, β-III Tubulin Tuj1). Recorded neurons were retrospectively identified by biocytin infused through the patch electrodes Fig. . We firs. RecordeDIV Fig.  legend aDIV Fig. . The latDIV Fig. , in line55, thus differences in Syp staining intensity are positively correlated to differences in the number of vesicles within presynaptic terminals. We found that the mean fluorescence of Syp-positive puncta was ~21% greater in the graphene group ) using a combination of a high concentration of Qdots (100 nM) and strong stimulation (2-min 90 mM K+)59 , an approach which provides a more precise estimate of releasable synaptic vesicle amounts and their release probability)59 Fig. . Based o)59 Fig. . We next)59 Fig. . Quantal60. Neurons were treated with 1 μM TFC for 1 h, which was sufficient to allow incorporation into the plasma membrane. We used two independent measurements to assess cholesterol levels: Filipin staining (again measured in neurites)  of MβCD for a short time (10 min). For all four conditions, 10 µM D-AP5 and 5 µM NBQX were co-applied to prevent the activity-induced exposure of intracellular membrane cholesterol. Again, independent assessment of cholesterol levels by both Filipin staining in fixed neurites  Fig.  of fixedes) Fig.  of live es) Fig. . Notablyes) Fig. , similartes Fig.  and GP ites Fig.  confirmeity Fig.  of neuro62, we asked if and how cholesterol enrichment on graphene substrates could affect transmembrane proteins and the signaling pathways they mediate. Using the same approach that was used for our neuronal culture configuration, mouse fibroblast cells (NIH 3T3) were plated directly on graphene or glass. Filipin staining demonstrated a ~49% increase of fluorescence intensity in 3T3 cells on graphene  inhibited Ca2+ responses, confirming P2YRs as the ATP receptor  and Ca2+-free bath solution  reduced the second Ca2+ response, collectively confirming internal stores as the Ca2+ source  Matrigel coating. Because it gels upon incubation at physiological temperatures, Matrigel acts as an effective interlayer between the substrate and the cell membrane in traditional culture configurations. We reasoned that removal of this layer would better resemble scenarios where graphene is used as a biosensor. However, this approach does not prevent biomolecule deposition from culture media onto the graphene surface. A protein corona is likely formed over the length of our culture period. Hu et al.68 recently demonstrated that within minutes of exposure to serum-containing media a stable protein corona will form on graphene oxide, which has a more favorable surface for protein adsorption than graphene. We have found a similar time course for dissociated neurons to attach to bare glass surfaces  lie between the shifts we observed for weak (~0.089) and strong (~0.191) MβCD treatment, we conclude that the membrane cholesterol changes were moderate. Broadly, both GP and Filipin were qualitatively consistent with the observation that cells on graphene had increased TFC labeling . Consequently, cholesterol synthesis and/or uptake is upregulated, resulting in a total cholesterol increase over time , which are believed to act as the nexus for transmembrane protein complexes, mediating signal transduction across the plasma membrane, carrying out receptor-mediated endocytosis and more72. Therefore, further study is warranted for a comprehensive view of graphene’s effect on the organization and trafficking of eukaryotic cell membranes as well as membrane protein distribution and mobility.Cholesterol modulates many membrane-associated proteins and other cellular functions in addition to its role in the structural integrity of the eukaryotic plasma membrane. Thus it would be interesting to further study graphene’s impact on other signaling pathways regulated by cholesterol. For example, it helps to define the fluidity of the lipid bilayer, which in turn regulates the dynamics and subcellular distribution of many transmembrane proteinsFor the majority of graphene-based bioapplications, especially those seeking to harness its unique electrical properties, cells or tissue need to be directly interfaced with the surface to maximize detection efficiency. Given our findings, these applications should further consider the involvement of the cell membrane when evaluating the effect of the material on any biological system. The effects we observe may also occur in other carbon allotropes, for example, carbon nanotubes. It is possible that the variation of atomic structure among different allotropes may uniquely influence their cholesterol affinity; thus investigating the shared or unique effects of other carbon allotropes on the plasma membrane and whether cholesterol is involved in these effects will be highly informative. New advances in the ability to modify carbon nanomaterials without compromising their electrical properties may help to tailor interactions with cholesterol or other biomolecules. Since cholesterol is a precursor for many steroids, which also contain the same tetracyclic ring, graphene could potentially be utilized to detect, deliver, or manipulate steroids in vitro and in vivo. This opens future directions for graphene in biomedicine but also demands further structural and mechanistic investigation of the membrane interaction between graphene and diverse biomolecules.-1, from Sigma) water solution and sonicated for 9 h in a bath sonicator. The uniform GF suspension was then centrifuged with a Thermo Scientific Fiberlite F15-6 X 100y rotor at 4000 r.p.m. and at room temperature for 1 h to sediment large graphite aggregates. The upper 50% of supernatant was carefully decanted, resulting in PVP-functionalized GF suspension36. The transmission characterization of GF suspension was carried out on a Varian Cary 5000 UV-VIS-NIR spectrophotometer. The concentration of GF was estimated with an absorption coefficient of 2460 L/g/m at 660 nm36, which is typically 26 mg L-1 for freshly made GF suspension. The suspension is stored at 4 °C and remains stable for >3 years. Single-layer graphene sheets were synthesized by CVD on a copper foil with 100 sccm (standard cubic centimeter per minute) hydrogen and 10 sccm methane as the feed gases42. A layer of poly(methyl methacrylate) (PMMA) was then spin-coated onto the graphene film that was grown on the copper foil, which was later removed through wet etching in ferric chloride solution. Subsequently, the graphene film was transferred onto glass coverslips, and the PMMA support was dissolved in acetone.To produce GFs, we used liquid exfoliation of graphite powder  in the presence of 2 wt% PVP  and symmetric shape of the 2D peak indicate that the graphene is monolayer. For Raman mapping, the samples were moved by a motorized microscope stage at a step of 1 μm for both x and y axis. A Raman spectrum was recorded at each position, and the intensities of the G and 2D modes were plotted to form spatially resolved images.The quality of graphene sheets was examined by a DXR Raman Microscope (Thermo Scientific). A 532-nm laser (~5 mW power) was expanded and focused to a diffraction-limited laser spot (<1 μm) through a 50× Olympus objective. The intensity features of graphene Raman spectra could be found at ~1590 and ~2680 cm37. Briefly, a serial dilution of cholesterol standard  was used to generate a calibration curve. One-milliliter aliquots of media were incubated as prepared or with the addition of 0.002 wt% PVP in phosphate-buffered saline (PBS), or 260 ng mL-1 graphene nanoflakes (GFs) with 0.002 wt% PVP in PBS at 37 °C and 5% CO2 for 24 h. Graphene fractions were separated from media using Amicon centrifugal filters  and resuspended in 1 mL of fresh media. The flow-through media was also collected for the graphene fraction. Media alone, media incubated with PVP, GNFs separated from media, and flow-through media were assayed. The fluorescence intensities of all enzymatic reaction products were measured using a spectrofluorometer . Excitation and emission slit widths were set at 5 nm for all measurements. Samples were excited at 570 nm, and emission was measured at 590 nm. For each group, four different batches of media were used, and four independent measurements were performed for each media sample. Cholesterol concentrations were then calculated using the standard curve.An enzymatic assay  was used to quantify free cholesterol concentrations according to the manufacturer’s instructionsTFC (Avanti) or BODIPY (Thermo Fisher) were diluted in water at a final concentration of 1.3 µM and incubated at room temperature for all time points. All fluorescence emission measurements were performed using a FluoroMax-4 spectrofluorometer (Horiba). Excitation and emission slit widths were set at 5 nm for all measurements. Samples were excited at 400 nm to avoid spectral bleed-through and non-specific excitation in mixed solutions. Emission spectra were collected from 500 to 650 nm. Three replicates were scanned three times each for every sample and the result averaged. Subsequently, averaged intensities were corrected for the broadband absorbance of either graphene or PVP across the emission spectra, as defined by:F is the fluorescence emission value at a wavelength for TFC and T is the transmittance value at that same wavelength for graphene or PVP calculated from the Beer–Lambert relationship. Data smoothing was performed using a nine-point Savitzky–Golay filter in Matlab.44. Hippocampal neurons (CA1–CA3) derived from postnatal (P0/P1) Sprague–Dawley rats of both sexes were used. Neurons were dissociated to a single-cell suspension, recovered by centrifugation, and resuspended in plating media composed of Minimal Essential Medium  containing (in mM) 27 glucose, 2.4 NaHCO3, 0.00125 transferrin, 2 L-glutamine, 0.0043 insulin, and 10%/vol fetal bovine serum . Because of the addition of FBS, all neuronal media contained cholesterol. Cell resuspension was deposited on round 12 mm coverslips at a density of ~200,000 cells/mL for two different surface conditions: bare glass or graphene-coated glass. After 2 h, 1 mL of culture media was added, a 1:1 mixture of plating and 4-Arac-containing media: MEM containing (in mM) 27 glucose, 2.4 NaHCO3, 0.00125 transferrin, 1.25 L-glutamine, 0.0022 insulin, 2 Ara-C, 1 %/vol B27 supplement (Life Technologies), and 7.5 %/vol FBS. Ara-C mimimized astroglia proliferation. For cell attachment studies, coverslips were washed three times with Hank’s solution and cells remaining in randomly selected fields of view were counted. Sister cultures supplying conditioned media were prepared in the same manner, except that 1 mL of plating media was added 4 h after plating to allow glial cell proliferation. After the confluence of glial cells (~2 DIV), 1 mL of 4-Arac media was added. For cells growing on graphene and glass, after 2 DIV, 1 mL of culture media was replaced with an equal volume of conditioned media from sister cultures. Experiments were performed on cultures at 3 and 7 DIV for developmental studies and between 13 and 17 DIV for all other studies using neurons.All animal procedures and all experimental procedures were approved by the Vanderbilt University Animal Care and Use Committee  and were performed in accordance with the VUACUC approved guidelines and regulations. Rat hippocampal cultures were prepared as previously described2 in Dulbecco’s modified Eagle’s medium containing 4.5 g L-1 glucose and l-glutamine supplemented with 10% FBS, 100 units/mL penicillin, and 100 μg mL-1 streptomycin. Because of the addition of FBS, this media also contains cholesterol. Cells were regularly passaged to maintain adequate growth and were passaged at least five times before trypsinization and plating on either graphene-coated or bare glass coverslips (25 μL or ~2 × 106 cells per coverslip). Cells were grown to 50–80% confluence for 24 h on coverslips prior to imaging.NIH-3T3 cells were grown at 37 °C with 5% COn > 9 different coverslips). The total number of neurites or cells analyzed are reported (see figure legends). For the analysis of neurons, we manually selected ROIs covering neurites  for 2 h at room temperature. Fluorescence imaging was performed on an Olympus IX-81 inverted microscope using a Nikon Intensilight illuminator, a Nikon Plan Apo VC 20× objective (N.A. 0.75) and a fluorescence filter set . Images were acquired with a CoolSnap K4 CCD camera (Photometrics) via Micro-manager with the same acquisition settings (exposure time = 300 ms and gain = 1) across each experimental group. For analysis, three independent batches of cultures were analyzed . Secondary antibodies with distinct fluorophores  were then incubated at room temperature for 2 h. Fluorescence imaging was performed on an Olympus IX-51 inverted microscope with a 60× UPlanFL (N.A. = 1.25) objective and a Flash 4.0 sCMOS camera (Hamamatsu). The optical filter sets (Chroma and Semrock) for Alexa 488, 568, and 647 fluorescence were, respectively: Ex 470/20 DiC 510LP 535/25, Ex 565/25 DiC 585LP Em 630/90, and Ex 630/60 DiC 660LP Em 695/100. For each fluorescence channel, images were taken with the same acquisition settings . Biocytin-positive neurons were positioned approximately in the center of the fields of view for imaging. Not all electrophysiologically recorded neurons were identified and imaged, as some were damaged during Biocytin infusion.For Syp intensity analysis, a set of overlapping masks were used to restrict analysis to synapses on the processes of cells that were recorded (biocytin+). Binary masks of biocytin+ cells were generated by intensity-based global thresholding in ImageJ using a common threshold setting for the minimum value. Syp images corresponding to the same FOV were also thresholded to generate a second binary mask. Using the Boolean logic function in ImageJ, a new mask was generated from the intersection of the Syn+ and biocytin+ masks (AND function). This mask was then subjected to minimum particle size restrictions and ROI sets were generated for each FOV. These ROI sets were applied to the original Syp image and mean intensities were measured. For cluster analysis, the standard deviation of the values obtained from the Syp intensity measurement were used. Background subtraction was performed on intensity values measured from each FOV by subtraction of the average of at least three different manually selected ROIs from cell-free areas of the Syp channel. Data were pooled for analysis, and the total number of synapses analyzed for each condition are reported in the figure legends.73 to exclude possible artifacts from image thresholding and further to ensure that all counted projections originated from the cell of interest. Error bars represent the S.E.M. of the pooled intersections from all cells in a treatment group at each distance.Using the immunolabeled streptavidin-filled neurons from above, Sholl analysis was performed on images taken on an Olympus IX-51 inverted microscope with a 60× UPlanFL (N.A. = 1.25) objective and a Flash 4.0 sCMOS camera (Hamamatsu). The center of the filled soma was marked as the starting point and concentric circles spaced equidistantly were drawn. At each distance, the number of intersections was manually counted. Manual counting was taken as the best ground truth2, 2 CaCl2, 10 N-2 hydroxyethyl piperazine-n-2 ethanesulphonic acid (HEPES), 10 glucose, pH 7.35. The pipette solution contained (in mM): 120 Cesium Methanesulfonate, 8 CsCl, 1 MgCl2, 10 HEPES, 0.4 ethylene glycol-bis-(aminoethyl ethane)-N,N,N’,N’-tetraacetic acid (EGTA), 2 MgATP, 0.3 GTP-Tris, 10 phosphocreatine, QX-314 (50 μM), 5 biocytin (Tocris), pH 7.2. For the recordings of mEPSCs, bath solution was supplied with 1 µM tetrodotoxin . The last 50 mEPSCs at the end of 5 min recordings in the presence of TTX were collected and analyzed using template-based event detection. The template was generated from our own representative data. To measure AMPA receptor currents, 20 µM D-(-)-2-Amino-5-phosphonopentanoic acid , an NMDA receptor antagonist, was added to the bath solution. NMDA receptor currents were recorded in the presence of 10 µM 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione , an AMPA receptor antagonist, in 0 mM [Mg2+]/3 mM [Ca2+] bath solution at −70 mV holding potential. Isolated AMPA and NMDA EPSCs were recorded from the same neurons sequentially by first applying D-AP5 then completely replacing it with NBQX. The NMDA/AMPA ratio for every neuron was calculated from the average amplitudes of the last 10 NMDA and AMPA events during 5 min D-AP5 or subsequent NBQX application. No postsynaptic currents were detected if D-AP5 and NBQX were applied together. All signals were digitized at 20 kHz, filtered at 2 kHz, and analyzed offline in Clampfit (Molecular Devices).At least three coverslips from five batches of neuronal culture were selected for recording, using at least one neuron per coverslip. Whole-cell voltage clamp recordings were performed on 13–17-DIV neurons using a Multi-Clamp 700B amplifier, digitized through a Digidata 1440 A, and interfaced via pCLAMP 10 . All recordings were performed at room temperature. Cells were voltage clamped at −70 mV for all experiments. Patch pipettes were pulled from borosilicate glass capillaries with resistances ranging from 3 to 6 MΩ when filled with pipette solution. The bath solution  contained (in mM): 150 NaCl, 4 KCl, 2 MgCl2+ imaging, cells growing on bare or graphene-covered glass coverslips were pre-incubated with TFC , C-laurdan , or Rhod-2 AM ester  at 37 °C with 5% CO2. After dye loading, coverslips were washed and mounted in an RC-26G imaging chamber (Warner Instruments) bottom-sealed with a 24 × 40 mm2 size 0 cover glass (Fisher Scientific). The chamber was fixed in a PH-1 platform (Warner Instruments) fixed on the MP-78 stage and bath solutions were applied via gravity perfusion with a constant rate of ~50 μL/s. All perfusion lines were merged into an SHM-6 in-line solution heater (Warner Instruments). The temperatures of both the imaging chamber and the perfusion solution were maintained at 34 °C by a temperature controller . For FM dye or Quantum dot (Qdot) loading of the evoked pool of synaptic vesicles, mounted coverslips were incubated with 10 µM FM1-43, 10 µM FM4-64, or 100 or 0.8 nM Qdots  for 2 min in high K+ bath solution containing (in mM): 64 NaCl, 90 KCl, 2 MgCl2, 2 CaCl2, 10 N-2 hydroxyethyl piperazine-n-2 ethanesulphonic acid (HEPES), 10 glucose, 1 μM TTX, pH 7.35. For Qdot loading of the spontaneous pool of synaptic vesicles, cells were incubated with 100 nM Qdots for 15 min in normal Tyrode’s solution. The Qdots used here had a hydrodynamic diameter of ~15 nm, limiting loading to one per synaptic vesicle 59. After FM or Qdot loading, cells were washed with normal Tyrode’s solution containing 10 µM NBQX and 20 µM D-AP5 for 5 or 10 min, respectively, to remove surface dye/Qdots. Electric field stimulation  was triggered by a 5-V 2-ms TTL pulse generated by the Clampex software 30 s after imaging began and delivered via a pair of platinum wires attached to both sides of the imaging chamber by a Grass SD9 stimulator. Synchronization of perfusion with image acquisition was via a VC-6 valve system (Warner Instruments) and controlled in Clampex.Live cell imaging was performed with 13–17 DIV cells using an Olympus BX-51WI microscope equipped with a 60× LUMPPlanFl water-immersion objective (N.A. 0.9), a Sutter Instrument MP-78 xyz motorized stage, a Solamere laser combiner and launcher , a Yokogawa CSU-X1 spinning disk confocal head, and an Evolve 512 EMCCD (Photometrics). For TFC, GP, and Ca2+ imaging, a 100 mW 561-nm laser (20% power) and a filter combination of DiC 580LPXR and Em 605/52 were used. The exposure time was 150 ms, the EM gain was 500 for all images, and the acquisition rate was 1 Hz. For Qdot imaging, a 480-nm laser (80% power) and a filter combination of DiC 510LX and Em 605/10 were used. The exposure time was 200 ms, the EM gain was 250 for all images, and the acquisition rate was 5 Hz. For FM4-64 imaging, a 50-mW 640-nm laser (30% power) and a filter combination of DiC 660LX and Em 710/50 were used. The exposure time was 50 ms, the EM gain was 500 for all images, and the acquisition rate was 1 Hz. For static images, 10-frame stacks were averaged. All images were taken with the same acquisition settings between sample groups .For TFC imaging, a 100 mW 480-nm laser (20% power) and a filter combination of DiC 500LX and Em 520/20 were used. Exposure time was 100 ms and the EM gain was 250 for all images. For every FOV, 10 repeated images were taken and averaged. For FM1-43 imaging, laser and filter sets were the same as those for TFC. Exposure time was 50 ms, the EM gain was 300, and the acquisition rate was 1 Hz. For GP imaging using C-laurdan, a 405-nm laser (40% power) and a filter combination of a DiC 409LP and an Em 440/40 or 483/32  were usually used. For TFC pretreated cells, we used an arc lamp  and a D350x excitation filter (Chroma) to avoid spectral cross-excitation of TFC. The exposure time was 50 ms with an EM gain of 900 for all images. For every FOV and each fluorescence channel, 10 repeated images were taken and averaged. For CaImage analyses were performed in ImageJ. Four rectangular ROIs were drawn in cell-free regions in every FOV and their intensities averaged. For fluorescence imaging of TFC, C-laurdan (blue channel), FM1-43 (first 10 frames), FM4-64 (first 10 frames), and Rhod-2 AM, we pooled all background ROIs regardless of treatment differences in order to calculate the mean and standard deviation of the background intensity. Again, a masked threshold approach was applied in ImageJ, and the mean intensity plus two standard deviations was used as the common threshold for all images or image stacks. For every FOV, ROIs were generated by particle analysis based on a binary threshold mask. For TFC loading, watershed segmentation was used to generate ROIs. For FM1-43 and FM4-64, watershed segmentation and particle size limits (0.3–3 μm) were applied in ImageJ to isolate ROIs for synaptic boutons (~1 μm). ∆FM4-64 is defined here as the difference of FM4-64 fluorescence intensity before and after four rounds of exhaustive stimulation.IGP = (Iblue − G × Igreen)/(Iblue + G × Igreen), in which G is the sensitivity correction factor between the two channels74. G was empirically determined by imaging 1 μM C-laurdan diluted in dimethyl sulfoxide (DMSO) using the prescribed protocol. Given GPDMSO = 0.006, the G value of our imaging setup was calculated using the following formula: G = (Iblue × (1 − GPDMSO))/(Igreen × (1 + GPDMSO)). For Qdot images, FM4-64-defined ROIs were applied and the mean Qdot photoluminescence intensity in each ROI was calculated. Quantal analysis for single Qdots was performed as described previously59. Briefly, maximum likelihood estimates were used to fit Qdot counts to a distribution of intensities. Qdot intensities were binned every 30 a.u. without background subtraction. The estimated threshold based on the mean background signal plus two standard deviations was near 3000 a.u. by which we set the cut-off for a single Qdot. To analyze the behavior of vesicles labeled by single Qdots, we selected ROIs having only one Qdot. Time-dependent Qdot photoluminescence traces were extracted with a five-frame moving window.For Rhod-2, FM1-43, and FM4-64 time-lapse data, the average intensity from four background ROIs was subtracted from the average intensity of each individual ROI in the same FOV. Normalization was performed using the average intensity of the first 10 frames. For C-laurdan images, ROIs generated from blue channel images were used to analyze both channels. GP value was calculated as N ≥ 3). All imaging experiments were repeated with at least three randomly selected coverslips per batch with one randomly chosen FOV per coverslip. No statistical methods were used to predetermine sample size. All values presented are mean ± S.E.M. All fluorescence intensity values are background corrected except in Figure t-tests or the Wilcoxin rank-sum test were used for two-group comparison of average values. Cumulative distribution functions (FM dye and Qdot imaging) or histograms (GP imaging) were used for two-group comparison of pooled values. Cumulative distributions are an accepted measure to provide an overview of intensity distributions from synapses77 and more clearly demonstrate the overall trend of the individual data points than an average measure. For GP imaging data, all individual pixels from each FOV were pooled from each treatment condition as the GP distributions for the generation of test statistics. Kolmogorov–Smirnov tests were used to compare distributions. For Sholl analysis datasets, statistics were calculated using two-way analysis of variance (ANOVA) with repeated measures followed by the Bonferroni multiple comparisons test. ω2 values were calculated as effect-size metrics for relevant ANOVA data. A one-way ANOVA and the Tukey–Kramer method as post-hoc analysis was used for three or more groups. Fisher z-tests were used to compare correlation coefficients. Because the sample sizes of several reported values are large and thus may overestimate true significance78, Cohen’s d-statistic79 was also reported for some datasets.All experiments were carried out blindly and repeated in at least three different batches of cultures (The data supporting the findings in this study are available from the corresponding author upon reasonable request.Supplementary InformationPeer Review File


Metal–graphene nanocomposite is a kind of potential radiation tolerant material. Graphene damage of the composite is inevitable within radiation environments. In this paper, two kinds of copper–graphene nanocomposite (CGNC) systems containing perfect graphene and prefabricated damage graphene, respectively, were adopted to expound the influences of graphene damage on the properties (radiation-resistance and configuration) of CGNC under irradiation by atomistic simulations. In the CGNC containing perfect graphene, the increasing graphene damage induced by the increase of the number of cascades, did not obviously impair the role of copper–graphene interface in keeping the properties of CGNC. In the CGNC containing prefabricated damage graphene, the properties of CGNC would significantly deteriorate once the radius of prefabricated damage exceeds 10 Å, and even stacking fault tetrahedral would occur in the CGNC. The results highlighted that prefabricated graphene damage have greater effects on the change of the properties of CGNC. Therefore, it is very necessary to maintain the structural integrity of graphene for improving the radiation-resistance and configuration of CGNC. Nanostructured metals and composites provide a path for the design of radiation tolerant materials because they contain interfaces that can attract, absorb and annihilate point and line defects1234568910111314+ irradiation experiments of Kim et al.101220However, to our knowledge, metal–graphene nanocomposite is still new, and even the most fundamental radiation damage studies, such as point-defect evolution processes, are lacking. In our previous work, a copper–graphene nanocomposite (CGNC) system was adopted and observed under collision cascades by atomistic simulations2317In this work, two kinds of CGNC systems containing perfect graphene and prefabricated damage graphene, respectively, were adopted to study the effects of GD on the radiation-resistance and configuration of CGNC by the method of molecular dynamics (MD). To better distinguish the damage size  of pristine graphene of CGNC, six CGNC models, defined as Cu–Gr/R0, Cu–Gr/R5, Cu–Gr/R10, Cu–Gr/R15, Cu–Gr/R20, and Cu–Gr/R25, were used. As a control, pure copper with a prefabricated copper damage (CD), namely, Cu/R0, Cu/R5, Cu/R10, Cu/R15, Cu/R20, or Cu/R25, was also studied. Full details of these models are discussed in the methods section. For the CGNC system containing perfect graphene (Cu–Gr/R0), the evolution of GD induced by cascade overlaps was investigated firstly. Then, the relationship between the defect production of CGNC and the corresponding GD was revealed. Furthermore, the way that irradiated GD affected the structural change of CGNC, was also discussed. For the CGNC system containing prefabricated damage graphene , the transformation of prefabricated GD of different sizes before and after irradiation, the corresponding relationship between the defect number of CGNC and the size of prefabricated GD, and the structural change induced by prefabricated GD and irradiation were clarified, respectively. Our results highlighted that the GD of CGNC, introduced by either irradiation or artificiality, played an important role in the radiation-resistance and configuration of CGNC.The graphene of Cu–Gr/R0 obtained after the first cascade, and fourth, eighth, twelfth, fourteenth, and fifteenth overlapped cascades is presented in In order to clarify the role of Cu–C_gr interface in the defect formation or annealing stage, it is appropriate to adopt the results of first cascade because the first cascade is the starting point of fifteen overlapped cascades and can be better to distinguish the evolution of defects. The time evolution of the number of interstitials and vacancies produced in the bulk region during the first cascade was shown in With the increase of cascade overlaps, the number of defects in the bulk region slightly increases, and then may lead to the structural change near the Cu–C_gr interface. The atoms near the interface after the first cascade, and fifth, tenth, and fifteenth overlapped cascades are exhibited in The carbon atoms near the prefabricated GD of six CGNC models are shown in The number of defects in the bulk region versus R value of prefabricated GD after cascades is given in Combined with Prefabricated GD have great influences on the formation of defects which may disturb the structure of CGNC around the interface. To illustrate the pure effect of prefabricated GD on structural change before cascades, the Cu–Gr/R25 is as an example for analysis. As shown in The six CGNC structures obtained after cascades are shown in Classical molecular dynamics simulations were performed to study the influences of the GD derived from irradiation or artificiality on the radiation-resistance and configuration of CGNC. The influences are summarized as follows.The results of the CGNC containing perfect graphene by cascade overlaps showed that with the increase of the number of cascades, GD would gradually increase and the number of surviving defects in the bulk region fluctuated slightly, implying that the strict correspondence between the radiation damage of graphene and the radiation-resistance of Cu_Gr/R0 does not exist. Moreover, the defects in the bulk region were always less than that of Cu/R0, demonstrating that Cu_Gr/R0 has better radiation tolerance compared to its bulk counterpart in the early stage of irradiation. Furthermore, mutual infiltration would increasingly happen between the copper atoms at the top and the bottom of graphene, but had little effect on the configuration, indicating the role of perfect graphene in the configuration stability of CGNC.The results of the CGNC containing prefabricated GD by cascades showed that the radiation-resistance of CGNC can be maintained and the configuration of CGNC was seldom changed when R was less than 10 Å. The defects in bulk region would surge and SFTs emerged once R exceeded 10 Å. The above phenomena highlighted the role of prefabricated GD, which can induce the formation of SFTs and weaken the radiation-resistance and configuration of CGNC. Therefore, the root of SFTs, namely prefabricated GD, should be removed as much as possible.The CGNC containing perfect graphene may also generate SFTs near the Cu–C_gr interfaces after irradiation. However, within typical MD time span, no SFTs was observed in the CGNC model. The most likely reason may be that deformation stress did not occur in the copper along Cu–C_gr interface. Moreover, the profile of GD is also a key factor for the formation of SFTs and may affect the probability of the occurrence of SFTs. After a long-term irradiation, the perfect graphene of CGNC will be subjected to serious damage and plentiful SFTs may occur. Due to SFTs corresponding to the bad radiation-resistance of the nanocomposite, the observation may be important for evaluating the service lifetime of the nanocomposite.et al.σCu-C) used in this work were derived from Density Functional Theory calculationsThe interactions among carbon atoms (C–C) in graphene were described by the adaptive intermolecular reactive empirical bond order (AIREBO) potential which has been widely used in the radiation damage of graphene232423d = 15.4 Å) from the graphene and directed toward Cu–C_gr interface. The simulation time for each cascade run was 23 ps, which was similar to that of the Fe–Cr alloy2929To study the relationships between the radiation damage of perfect graphene and the property transitions of CGNC (the transitions of radiation-resistance and configuration) during long-term irradiation, the relaxed Cu–Gr/R0 model was used to simulate cascade overlaps. The simulations were carried out in the following three steps. In Step 1, each cascade was initiated by a PKA with 3.0 keV introduced at a certain distance , six relaxed CGNC models were used to simulate collision cascades. The simulation conditions were similar to that of Cu–Gr/R0. At the beginning, a PKA with 3.0 keV in each model was introduced at How to cite this article: Huang, H. et al. Graphene damage effects on radiation-resistance and configuration of copper-graphene nanocomposite under irradiation: A molecular dynamics study. Sci. Rep.6, 39391; doi: 10.1038/srep39391 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


We discovered an inherent trade-off in this mechanism between transcriptional redundancy, which maintains the robustness of TDP-43 metabolism, and vulnerability to specific interfering factors. These factors include an increased tendency of TDP-43 to aggregate, impaired nuclear-cytoplasmic TDP-43 transport, and a decreased efficiency of degrading abnormal proteins, all of which are functional abnormalities related to the gene that causes familial ALS/FTD. When these conditions continue at a certain intensity, the vulnerability of the autoregulatory machinery becomes apparent over time, and transcriptional redundancy enters a vicious cycle that ultimately results in TDP-43 pathology. The results obtained using this in silico model reveal the difference in TDP-43 metabolism between normal and disease states. Furthermore, using this model, we simulated the effect of a decrease in TDP-43 transcription and found that this decrease improved TDP-43 pathology and suppressed the abnormal propagation of TDP-43. Therefore, we propose a potential therapeutic strategy to suppress transcriptional redundancy, which is the driving force of the pathological condition caused by the specific factors described above, in patients with ALS presenting with TDP-43 pathology. An ALS animal model exhibiting TDP-43 pathology without overexpression of exogenous TDP-43 should be developed to investigate the effect of alleviating the transcriptional redundancy of TARDBP.Abnormal accumulation of TAR DNA-binding protein 43 (TDP-43) in the cytoplasm and its disappearance from the nucleus are pathological features of amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) and are directly involved in the pathogenesis of these conditions. TDP-43 is an essential nuclear protein that readily aggregates in a concentration-dependent manner. Therefore, cells must strictly maintain an appropriate amount of nuclear TDP-43. In one relevant maintenance mechanism, TDP-43 binds to its pre-mRNA and promotes alternative splicing, resulting in mRNA degradation via nonsense-mediated mRNA decay. The level of nuclear TDP-43 is tightly regulated by these mechanisms, which control the amount of mRNA that may be translated. Based on the results of previous experiments, we developed an  TARDBP gene, which encodes TDP-43, is present in 1–5% of patients with familial ALS, and these patients exhibit TDP-43 pathology similar to individuals with sporadic ALS. The same TDP-43 pathology has also been identified in patients with mutations in many ALS-causative genes, including hexanucleotide repeat expansions in C9ORF72, a major genetic cause of ALS/FTD  is a devastating neurological disease characterized by the degeneration of upper and lower motor neurons. ALS leads to death within 2–5 years as a result of muscle weakness, including respiratory dysfunction. Up to 50% of patients with ALS develop cognitive and behavioral abnormalities, and approximately 13% of patients with ALS present with concomitant behaviorally variant frontotemporal dementia (FTD) , a structured diagram editor used to draw gene-regulatory and biochemical networks  cells expressing human TDP-43 cDNA under the control of the endogenous mouse TARDBP promoter, the amount of TDP-43 protein increases approximately 3-fold in the absence of the TARDBP 3′-UTR, which is essential for autoregulation . The g Figure . A decreTARDBP pre-mRNA that undergoes alternative splicing in vivo. The percentage of alternatively spliced isoforms among all isoforms of the TARDBP mRNA was examined using the Droplet Digital PCR method  using NucleoSpin RNA II  to confirm the percentage of the d Figure . Since Dy Figure . For mosy Figure . The perA Figure ; Y axis 8 Figure ; R axis.8 Figure .TARDBP RNA is degraded by nonsense-mediated mRNA decay. This transcriptional redundancy may be influenced by several conditions, including conditions within the tissue or cell  model. Therefore, in a state in which no disturbance was applied, the levels of the TARDBP mRNA, nuclear TDP-43, cytoplasmic TDP-43, aggregates, and fragments were equal between these two models. We recorded the amount of each element that stabilized when each parameter was varied from half to twice its initial level.We subsequently examined the fragility of this autoregulatory mechanism by investigating how each element behaves when each parameter was varied. For this analysis, we compared a model with autoregulation  to a model without autoregulation [NAR (–)]. In the NAR (–) model, only the level of transcription was adjusted, which indicates that the initial expression level of the TARDBP heterozygous knockout mouse model  model, the level of nuclear TDP-43 decreased as its transcription level decreased, reaching approximately half of the initial value under conditions that mimic the l Figure . When thl Figure . The levl Figure . Howeverl Figure . Similarl Figure , 3A. ThuTARDBP mRNA  model, changes in the degree of nuclear import altered the nuclear TDP-43 level; however, these changes did not increase the fragmentation of TDP-43. In the NAR (+) model, the amount of nuclear TDP-43 remained robust until nuclear import was reduced to approximately three-quarters of the initial setting Figure . When nuA Figure . In the A Figure . As the A Figure . SimilarA Figure , 3B.TARDBP mRNA and subsequently exacerbate TDP-43-related pathology  are related to disturbances in the factors that lead to the vicious cycle of the autoregulatory system. Therefore, examinations of the TDP-43 metabolism and specifically the expression of TARDBP mRNA in model animals with mutations in these causative genes are necessary.At this stage, we propose that TDP-43 pathology involves a vicious cycle in which excessive TDP-43 is continuously produced as a result of an autoregulatory mechanism. As shown in a previous study, motor neurons from subjects with ALS displaying abnormal TDP-43 accumulation exhibit an increase in the amount of TARDBP mRNA was suppressed (Figure TARDBP mRNA levels under the pathological condition of persistent TARDBP mRNA expression driven by transcriptional redundancy. An animal model that maintains the autoregulatory mechanism that controls TDP-43 levels is necessary to provide supporting evidence for this treatment strategy. In this type of animal model, the endogenous TARDBP mRNA, but not exogenous TARDBP, should be overexpressed, as observed in motor neurons from patients with ALS (Koyama et al., TARDBP mRNA in ALS. Specifically, a strategy that employs an antisense oligonucleotide or CRISPR-dCas9 system to decrease the TARDBP mRNA level would conceivably be successful. A decrease in TDP-43 levels induces a loss in TDP-43 function, which may be detrimental to cell survival and function. However, because TARDBP expression is transcriptionally redundant, a 50% reduction in its transcription does not affect protein levels (Kraemer et al., TARDBP heterozygous knockout mice have normal survival times (Ricketts et al., TARDBP mRNA levels might be effective.Using this model, TDP-43 pathology was reversed and abnormal TDP-43 propagation was suppressed when the transcription of the d Figure . Similard Figure . Based oThe pathophysiology of neurodegenerative diseases is particularly difficult to understand due to the complexity, multifactorial, and dynamic aspects of the central nervous system. Approaches using systems biology may be useful for integrating all aspects of a given phenomenon and may constitute promising strategies. However, models of any scale have advantages and limitations (Vujasinovic et al., TARDBP transcription that is intended to maintain nuclear TDP-43 levels becomes the driving force that leads to TDP-43 pathology. This hypothesis answers several questions: why do ALS motor neurons enter a pathological state from which they cannot escape; why do many different ALS causative genes result in TDP-43 pathology; and why does TDP-43 pathology propagate between cells, thereby resulting in progression? Furthermore, based on our model, therapies for ALS targeting specific molecules are a possibility. In the future, an ideal animal model of this disease should be established to explore this hypothesis.Why does TDP-43 accumulate abnormally in the cytoplasm in patients with ALS/FTD when a mechanism that precisely maintains its expression exists? Studies aiming to resolve this issue are important to enhance our understanding of the pathology of sporadic ALS, which accounts for most ALS cases, and to promote the development of methods to treat this disease. Therefore, in the present study, we modeled and simulated TDP-43 dynamics within the cell. The trade-off relationship between the vulnerability and robustness of the mechanism that maintains nuclear TDP-43 levels might underlie TDP-43 pathology. Once this vulnerability is triggered, the redundancy of AS and OO designed the study. AS, TKA, and AK performed the biological experiments using the mouse cerebrum and investigated the results of previous experiments to create our model. AS analyzed the results. YK, SK, TKO, and TI contributed to the interpretation of the findings. AS and OO drafted the manuscript. All authors critically revised the draft and approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


PDAC) is considered a non‐immunogenic tumor, and immune checkpoint inhibitor monotherapy lacks efficacy in this disease. Radiotherapy (RT) can stimulate the immune system. Here, we show that treatment of KPC and Pan02 murine PDAC cells with RT and gemcitabine upregulated PD‐L1 expression in a JAK/Stat1‐dependent manner. In vitro, PD‐L1 inhibition did not alter radio‐ and chemosensitivity. In vivo, addition of anti‐PD‐L1 to high  but not low  RT doses significantly improved tumor response in KPC and Pan02 allografts. Radiosensitization after PD‐L1 blockade was associated with reduced CD11b+Gr1+ myeloid cell infiltration and enhanced CD45+CD8+ T‐cell infiltration with concomitant upregulation of T‐cell activation markers including CD69, CD44, and FasL, and increased CD8:Treg ratio. Depletion of CD8+ T cells abrogated radiosensitization by anti‐PD‐L1. Blockade of PD‐L1 further augmented the effect of high RT doses (12 Gy) in preventing development of liver metastases. Exploring multiple mathematical models reveals a mechanism able to explain the observed synergy between RT and anti‐PD‐L1 therapy. Our findings provide a rationale for testing the use of immune checkpoint inhibitors with RT in PDAC.Pancreatic ductal adenocarcinoma ( Surgeryet al, et al, et al, et al, et al, et al, et al, et al, + T‐cell infiltration  receptor and its ligand PD‐L1  is an important mediator of PD‐L1 signaling , anti‐PD‐L1 alone, or their combination . Additionally, we assessed the activation status of the CD45+CD8+ T cells based on IFNγ expression and found increased activated CD8+ T cells following combination of high RT doses and PD‐L1 blockade  KPC cells, then treated with anti‐PD‐L1 every 3 days. After 14 days, livers were harvested and weighted. Compared to control, prior treatment with 12 Gy reduced metastatic tumor burden that was further enhanced by anti‐PD‐L1 treatment Fig A. Combinrols Fig . Hence, et al, et al, Finally, we used mathematical modeling  RT induces a large population of tumor cells to become susceptible to immune cell killing and (ii) anti‐PD‐L1 increases the recruitment and killing ability of CD8 T cells and, importantly, prevents the establishment of suppressive microenvironment factors .et al, et al, et al, et al, et al, et al, et al, et al, et al, et al, et al, et al, et al, et al, et al, PD‐L1 plays a central role in permitting cancer evasion, mainly by interfering with T‐cell functions, and inhibitors of the PD‐1/PD‐L1 axis have changed the paradigm in the management of melanoma patients  that made tumor rechallenge impossible as the experiment had to be terminated.Although our observations with higher RT doses are in agreement with the majority of reports showing strong radiosensitization with PD‐1/PD‐L1  was sufficient to delay tumor growth in colorectal and triple‐negative breast cancers  or inflammatory processes via IFNγ (adaptive resistance)  are known to suppress antigen‐specific T cells  PDAC cells and assessed the impact of systemic PD‐L1 blockade (i.p.) in the development of liver metastasis. Both RT alone and anti‐PD‐L1 alone reduced metastatic colonization compared to untreated controls, but addition of anti‐PD‐L1 significantly enhanced the anti‐metastatic effect of RT. Recruitment of immune cells to the liver is known to precede development of liver metastases  tumors. Murine Pan02 pancreatic adenocarcinoma cells were obtained from the NCI–DCTD Tumor Repository, Maryland, USA. Gemcitabine was purchased from Eli Lilly and Company Ltd, UK. Murine anti‐PD‐L1 antibody (Clone 10F.9G2) was purchased from BioXCell.The murine PDAC cell lines KPC and Pan02 were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. KPC cells were derived from KrasLSL. Anti‐PD‐L1 antibody was added to the culture 1 h before irradiation with single dose of 2, 4, and 6 Gy. Similarly, cells were also treated with gemcitabine . Gemcitabine was diluted in DMSO (vehicle), and hence, we used DMSO for the untreated control group. Twenty‐four hours after treatment, cells were washed with PBS and replaced with fresh medium. The clonogenic survival of cells was assessed as described previously . Tumor size was measured with caliper using the formula Volume = (a × b2)/2, in which a and b are the largest and the smallest perpendicular diameters, respectively. Mice were randomized into different groups when tumors reached 100 mm3 and treatment was initiated (day 0) as described previously , 3, 6, and 9. We assessed the time it took for the tumors to grow to 400 mm3, starting from the beginning of treatment (day 0) to ensure that our experiments were according to the UK Home Office guidelines for animal welfare.C57BL/6 mice were injected subcutaneously (s.c.) with KPC and Pan02 cells (5 × 105 cells/100 μl in PBS) were injected intrasplenically into C57BL/6 mice that are either naïve or pre‐irradiated with 12 Gy 1 h before intrasplenic injection, as previously described  or long term  after completion of treatment with gemcitabine. Tumors were harvested and analyzed for PD‐L1 expression by flow cytometry as described below.The KPC transgenic mouse model has been described previously . All protocols were approved by the Committee on the Ethics of Animal Experiments of the University of Oxford.KPC and Pan02 cells were seeded in 6‐well plate. After 24 h, cells were treated with either 6, 4 × 2 Gy fractionated radiation given daily using cesium‐137 irradiators, or 20 nM gemcitabine. Gemcitabine was diluted in DMSO (vehicle), and hence, we used DMSO for the untreated control group and the radiation groups as well. Cells were harvested 24 h post‐treatment and washed with cold PBS. Cells were stained with mouse PE‐PD‐L1 and APC‐IFNγ. All the antibodies were purchased from eBiosciences.We also examined expression of PD‐L1 following blockade of either Stat1 or Stat3 using siRNA. For that purpose, cells were transfected with 50 nM negative control siRNA , 50 and 100 nM Stat1 , or 50 nM Stat3 siRNA  using Lipofectamine 2000 (Invitrogen) reagent. After 48 h, proteins were isolated and knockdown was confirmed by Western blotting. For flow cytometry analysis of PD‐L1 and IFNγ, siRNA‐transfected cells were either irradiated with 6 Gy or treated with gemcitabine and PD‐L1 expression examined as above. PD‐L1 expression was also assessed following addition of recombinant mouse IL‐6  for 1 h and subsequent treatments with 6 Gy and gemcitabine for 24 h.For flow cytometry analysis of mouse tissue samples, tumor, liver, and spleen were cut into small pieces and only liver samples were digested with 0.05% collagenase/dispase (Roche) for 15 min at 37°C in the presence of 0.01% trypsin inhibitor. Bone marrow was collected by flushing both femur and tibia with PBS using a 25‐G syringe. All cells were washed with cold PBS. Cells were then filtered through 70‐μm nylon strainer followed by red blood cell (RBC) lysis for 3 min at room temperature. Single‐cell suspensions were then stained with the following antibodies: FITC‐CD45, FITC‐CD8, PeCy7‐CD4, PeCy7‐CD11b, PE‐Gr1, PE‐Ly6G and APC‐IFNγ, PE‐CD25, APC‐FOXP3, CD69‐PE, APC‐CD44, and PeCy7‐FasL.g for 20 min at RT to isolate leukocyte population. The cells were washed with cold PBS and stained with FITC‐CD8, PeCy7‐CD4, and PE‐PD‐1. Flow cytometry was performed using a FACSCalibur (Becton Dickinson) and analyzed using FlowJo software . All the antibodies were purchased from eBiosciences, UK.Blood samples were collected in 40 mM EDTA and mixed with equal volume of PBS. Diluted blood was layered onto an equal volume of Histopaque (Sigma) and centrifuged at 400 Cells were first treated with 20 μM of JAK kinase inhibitor  1 h before RT (6 Gy) or addition of 20 nM gemcitabine. Following 24 h post‐RT and gemcitabine, cell lysates were prepared in RIPA buffer containing protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (Roche). In total, 30 μg of proteins were separated on SDS–PAGE (Invitrogen) and transferred to Hybond‐C membranes (Amersham Biosciences). Standard procedures were applied for Western blotting. The following antibodies were used: Stat1, phospho‐Stat1 (Y701), Stat3, mouse PD‐L1 , actin .et al, Serum was obtained from the KPC allograft‐bearing mice 5 days after treatment with either vehicle (control), 12 Gy, anti‐PD‐L1, or 12 Gy + anti‐PD‐L1. Serum was assessed for expression of cytokines using a standard Cytokine Profiler kit and SDF‐1 ELISA kit (both from R&D Systems) according to the manufacturer instructions. Array spot intensities were quantified using a Matlab software as reported : ε1 = 0.02, φ = −0.2, T1 day 0 = 0.4, T2 day 0 = 99.6; radiation + anti‐PD‐L1 (day 6): ε1 day 0 = 0.002, ε1 day 6 = 0.02, φ day 0 = 3, φ day 6 = −0.2, T1 day 0 = 0.4, T2 day 0 = 99.6. The ODEs were integrated in Matlab  using ode45.t‐test and one‐way ANOVA (Bonferroni test) with the GraphPad Prism version 5 . P‐values lower than 0.05 were considered statistically significant. Error bars represent standard deviation.Differences between groups were determined using Student's paired in vivo studies. SL helped with the manuscript preparation. All authors discussed and approved the manuscript.AA, SYL, and EF designed the experimental strategy. AA, SYL, ZD'C, and EF organized the experiments and collected and analyzed the data. AD and SYL provided assistance with the experimental methodology. AA, SYL, and EF prepared the protocols for mouse radiotherapy. OJS, WGM, and RJM provided experimental advice. PK and OD generated the mathematical modeling. AA, SYL, and EF wrote the manuscript with advice from OJS, WGM, OD, and RJM. KJ assisted with the Part of this work was presented at the ESTRO 35 and the NCRI 2015 conference. The authors declare that they have no conflict of interest.Pancreatic ductal adenocarcinoma is characterized by a dismal prognosis and it is refractory to conventional treatment, such as radiotherapy and chemotherapy, and immune checkpoint inhibitor monotherapy. In contrast to the notion that all patients with PDAC die of metastatic disease, at least 20% suffer and die of local recurrence. Hence, there is a need to improve radiotherapy efficacy to enhance local control and clinical outcome. Evidence indicates potentially complementary roles for immunotherapy and radiotherapy, but this combination has not been explored in PDAC.in vitro and in vivo. Inhibition of PD‐L1 strongly enhanced tumor response to high  but not low  RT doses, and to gemcitabine‐based chemoradiotherapy in different mouse models of PDAC. The increased tumor response to radiation observed upon PD‐L1 blockade correlated with increased intratumoral T‐cell numbers and activation and reduced myeloid cell infiltration, whereas depletion of CD8+ T cells abrogated the radiosensitization by anti‐PD‐L1. Additionally, inhibition of PD‐L1 enhanced the impact of radiotherapy in preventing development of liver metastases. We generated a novel mathematical model that explains the synergistic effect of radiation and PD‐L1.Here, we show that radiation and gemcitabine chemotherapy have substantial immunomodulatory effects, including upregulation of PD‐L1 In the present work, we provide a strong rationale for testing the use of PD‐L1 immune checkpoint inhibition with radiotherapy in PDAC in the clinical setting that could lead to improved outcome in patients with this disease.https://www.ncbi.nlm.nih.gov/pubmed/26433823https://www.ncbi.nlm.nih.gov/pubmed/26270858AppendixClick here for additional data file.Source Data for AppendixClick here for additional data file.Review Process FileClick here for additional data file.Source Data for Figure 1Click here for additional data file.Source Data for Figure 4Click here for additional data file.


In the last four decades, tumor immunology has shed light on identity and functions of cells and molecules involved in tumor rejection through the involvement of the immune system . SeveralIn response to the aim of the special issue, four original research papers and one review article are presented below.The study reported by B. Cerbelli et al. from Sapienza University of Rome, Italy, showed that immunohistochemical PD-L1 expression in ≥25% of triple negative breast cancer (TNBC) chemo-naïve cells, derived from core biopsies, is an independent predictor for pathological complete response (pCR) after neoadjuvant chemotherapy, thus discussing potentials and limits of PD-L1 future applications as a predictive biomarker for neoadjuvant treatment response in this subset of patients affected by such a clinically aggressive disease.M. Moschetta et al., at Sarah Cannon Research Institute of London, UK, presented a study assessing the impact of “neutrophil-to-lymphocyte ratio” (NLR) in predicting PFS among 55 advanced patients enrolled into PD-1/PD-L1 inhibitors phase 1 clinical trials. Results showed a significant longer PFS in patients with a reduction of NLR after two treatment cycles compared to the median baseline NLR, thus advancing the hypothesis that NLR may be a helpful predicting tool in cancer patients treated with anti-PD-1/PD-L1 agents.An international collaboration between USA  and Japan , coordinated by K. Suda et al., obtained evidence concerning molecular mechanism behind the low expression of PD-1/PD-L1 in NSCLC, associated with reduced efficacy of checkpoint inhibitors (CI) treatments. The study highlighted that EGFR-mutated lung cancer cell lines do not show high PD-L1 expression and, furthermore, after acquisition of resistance to EGFR-TKIs, EGFR phosphorylation affects PD-L1 expression, thus identifying a molecular event able to influence the expression of biomarkers, which regulate patients' access to CI agents.Apart from its immunomodulatory function, F. Zheng et al., from Huazhong University of Science and Technology, China, identify PD-L1 molecule as a potential biomarker of melanoma cancer stem-like cells, since blocking PD-L1 in melanoma cell lines expressing PD-L1 and ALDH1 impaired tumorsphere formation and induced the apoptosis of tumorsphere cells. These findings raise the need to elucidate the relationship between tumor response to checkpoint inhibitors and clonal evolution of cancer stem cells in the future.Finally, the review paper by M. Wei et al., Southeast University of China, discusses the role of gastric cancer patients' T cells immunity and disease prognosis, providing a critical synthesis of recent evidence on this still controversial topic.In conclusion, we find this special issue to be a good opportunity for improving knowledge in the field of cancer immunobiology and immunotherapy, which is a pivotal step to respond adequately to the questions of our time in the battle against cancer.Ilary RuscitoElena Ioana BraicuMaria Luisa GasparriIlaria Grazia Zizzari