Age-friendly communities promote active, healthy, socially connected aging. Opportunities for social connections are key for older residents to enjoy the best possible health and well-being. Communities that join the AARP Network of Age-Friendly States and Communities (AARP NAFSC) include an aging lens in eight areas of community life—social participation, respect and social inclusion, civic participation and employment, communication and information, housing, transportation, community support and health services, and outdoor spaces and buildings. By addressing factors in these eight areas, communities encourage residents to enjoy formal participation in activities and groups and informal contacts with friends, neighbors and other residents. The purpose of this exploratory study was to find out if communities that join the AARP NAFSC plan and implement changes to enhance social connectedness. A review of 62 AARP-approved action plans nationwide, showed that social connectedness was included in 74% of the mission statements and was a goal in 92% of the plans. The lack of resources in rural communities creates special challenges; many age-friendly initiatives depend on community volunteers to implement changes on a shoe-string budget. To learn how rural age-friendly communities promote social connections, an email survey was distributed to 46 AARP NAFSC communities in rural Maine. All the communities responded. Fostering social connectedness was an explicit goal for 88% of the communities. Areas of implementation included services and activities (83%), communication (61%), transportation (30%), programming to include isolated residents (26%), accessible public spaces (22%), and intergenerational volunteering (17%). Implications will be discussed.


The transplantation of neural stem cells (NSCs) has become an emerging treatment for neural degeneration. A key factor in such treatments is to manipulate NSC behaviors such as proliferation and differentiation, resulting in the eventual regulation of NSC fate. Novel bionanomaterials have shown usefulness in guiding the proliferation and differentiation of NSCs due to the materials' unique morphological and topological properties. Among the nanomaterials, graphene has drawn increasing attention for neural regeneration applications based on the material's excellent physicochemical properties, surface modifications, and biocompatibility. In this review, we summarize recent works on the use of graphene-based biomaterials for regulating NSC behaviors and the potential use of these materials in clinical treatment. We also discuss the limitations of graphene-based nanomaterials for use in clinical practice. Finally, we provide some future prospects for graphene-based biomaterial applications in neural regeneration. Neural stem cell (NSC) transplantation has become an emerging technology over the past decade for application in tissue engineering and regenerative medicine . More anAdhesion, proliferation, and differentiation of NSCs are regulated by numerous growth factors, environmental stimuli, culturing topographies, extracellular matrix (ECM), cell-material interactions, and cell-cell interconnections. Among these factors, cell-material interactions have drawn increasing attention as an important factor for the manipulation of NSC behaviors. The properties of biomaterials such as their topography, stiffness, and composition have been shown to be effective at influencing NSC behavior, mainly through the stimulation of mechanosensors and spatiotemporal dynamics in the cells .With the development of nanotechnology, novel bionanomaterials have become candidates for manipulating NSC behavior both in vivo and vitro. Through nanomanufacturing, the bionanomaterials can precisely simulate the in vivo microenvironment of NSCs and thus regulate their behaviors in the direction of a desired cell fate. As reported in recent studies, porous silicon nanoparticles loaded with growth factors promote the extension of neurites , multilaThe novel nanomaterial graphene, a monolayer of sp2-hybridized carbon atoms arranged in a hexagonal honeycomb lattice , was firBased on the development of graphene-based biomaterials and the study of NSC transplantation, novel graphene-based NSCs have emerged as possible clinical treatments for neural degeneration. The integration of graphene-based nanomaterials and NSCs provides a microenvironment that can be manipulated to suit the transplanted cells and can directionally regulate the adhesion, proliferation, and differentiation of the NSCs. Different kinds of graphene-based biomaterials have undergone significant improvements for use in NSC transplantation and have proven promising for use in neural regeneration in clinical trials . In thisGraphene-based nanoparticles can be internalized by NSCs. Different from the lamellar structure of graphene, the internalized graphene-based nanoparticles can interact with the subcellular organelles, regulate the expression of relevant genes and proteins, and ultimately influence the proliferation and differentiation of NSCs. The study by Kim et al. focused on the self-renewal and differentiation abilities of NSCs after graphene oxide nanoparticle (GO-NP) treatment. Based on immunofluorescence staining, the GO-NP-treated NSC spheres exhibited enhanced cell-cell and cell-matrix interactions, leading to enhanced self-renewal ability. The rate of differentiation of NSC spheres was also accelerated at the same time  Figures. In addico-glycolic acid)). In recent years, nanofibers manufactured from graphene-hybridized nanomaterials have been heavily studied and used in NSC transplantation. Shah et al. reported that electrospun PCL nanofibers covered with a thin layer of GO could promote NSC differentiation into oligodendrocytes [Antheraea pernyi silk fibroin (ApF)/(poly(L-lactic acid-co-caprolactone)) (PLCL) nanofibers by electrospinning, and GO was coated onto the surface of the nanofibers. The Schwann cells cultured on the nanofibers showed enhanced migration, proliferation, and myelination compared to the controls, and the molecules secreted by the cultured Schwann cells also promoted the differentiation and focal adhesion kinase expression of PC12 cells. Animal experiments showed the successful repair of a 10 mm sciatic nerve defect using the nanofibers [Graphene-based fibers have proven to be very useful in neural injury repair. With the integration of the neural interfacing effect provided by graphene and the “contact-guidance” topography offered by fibers, graphene-based fibers can enhance the neural differentiation of NSCs and direct the oriented extension of neural axons to bridge gaps in injured and severed nerves. The fibers could also be used in the construction of a 3D culturing system for NSCs because of excellent spatial assembly. The fibers could be classified as nano- and microfibers based on diameter size. Nanofibers have a linear morphology within the diameter size range of 1 nm to 100 nm. Nanofibers are usually manufactured from biocompatible materials like PCL (polycaprolactone), PLL (poly-L-lysine), and PLGA  and seeded NSCs onto the surface of the hybrid material. Immunofluorescence staining for nestin indicated that the NSCs on the GONR grid exhibited enhanced attachment and proliferation compared to quartz, TiO2-nanoparticle/SiO2, and rGONR grid substrates, and the NSCs preferred to align along the grid patterns. After flash photostimulation, there were significantly more neurons on the rGONR grid and TiO2-NPs/SiO2 substrates compared to the samples that were kept in the dark, and the neurons preferred to form two-dimensional neural networks around the grid patterns [Graphene-based composite nanomaterials have become an emerging area of focus with the development of modern nanomanufacturing methods. Through hybridization with biocompatible and biodegradable materials, graphene-based composite nanomaterials integrate electrical, cytofriendly, and biodegradation properties, providing potential nanomaterials for clinical use in neural regeneration. Solanki et al. introduced a hybrid nanomaterial manufactured by coating 300 nm SiOed axons   maintain the cells' natural morphology in vivo, (2) enhance proliferation depending on the specific materials and cell types, (3) direct differentiation under the guidance of specific materials and topographies, and (4) upregulate the expression of differentiation-relevant genes and proteins. Thus, it is necessary to set up in vitro 3D culturing systems for simulating the ECM microenvironment of NSCs. During the process of developing 3D culturing systems for NSCs, graphene-based biomaterials have been studied and developed further. Several new kinds of novel three-dimensional graphene-based nanomaterials for the proliferation and differentiation of NSCs have been reported in the past decade, including porous materials, fibers, and scaffolds.Graphene foam is constructed as a porous 3D structure and has been proven in the past few years to be an excellent 3D culturing system for NSCs in tissue engineering applications. Graphene foam can effectively enhance NSC adhesion, proliferation, differentiation, and neural network formation  in vitroGraphene-based scaffolds are another key approach to mimicking the NSC microenvironment for clinical applications in neural regeneration. Different from the 3D spatially aligned graphene-based fibers, scaffolds show more complicated 3D structures, including fibers, surfaces, and interconnections. Thus, such scaffolds are more similar to the in vivo ECM microenvironment of NSCs compared to other materials. In addition to more precisely simulating the microenvironment of NSCs, the scaffolds also integrate physiological cues in the 3D geometry like surface wrinkles and ripples of graphene and the contact-guidance of fibers. NSCs seeded in the 3D geometry of graphene-based scaffolds receive regulatory signals from the surrounding environment, and this allows their behaviors to more closely mimic those of cells in vivo. Using nanomanufacturing technology, regulatory cues can be integrated into the scaffolds during the manufacturing process in order to improve the neuronal differentiation of NSCs.μm to ensure sufficient water and oxygen exchange between the external environment and the tubular scaffold lumen. The immunofluorescence staining indicated that the expression of Tuj-1 was highest in differentiated cells on the PDA/RGD-SG/PCL scaffold, and western blotting also showed increased expression of neurotrophic factors like nerve growth factor on a PDA/RGD-SG/PCL scaffold compared to control scaffolds. After implantation, the walking track analysis indicated that the recovery of the sciatic nerve was faster on the PDA/RGD-SG/PCL and PDA/RGD-MG/PCL scaffolds than on other materials at 6 and 12 weeks after the surgery and was similar to the autograft group at 18 weeks after the surgery [A study by Ma et al. established two kinds of 3D scaffolds made of graphene foams with stiff and soft properties, respectively. The stiff scaffold significantly enhanced the attachment and proliferation of NSCs, and the expression of proliferation markers, including BrdU and Ki67, was much higher on the stiff scaffold compared to the soft scaffold. However, the NSCs cultured on the stiff scaffold mainly differentiated into astrocytes. The expression of the neuron marker Tuj-1 was significantly lower than that on the soft scaffold, and the expression of the axonal marker GAP-43 was much higher on the stiff than the soft scaffold. Thus, the stiff scaffold enhanced axon genesis and suppressed the differentiation of neurons  Figure . Further surgery . So to sOver the past decade, graphene-based materials have shown significant advantages for the guided manipulation of NSC fate by directly regulating the attachment, proliferation, and differentiation of NSCs. Novel graphene-based nanomaterials with the potential for clinical use have been tested in trials for further application in the treatment of neural regeneration. However, for long-term implantation, the mechanisms behind the biodistribution, biocompatibility, and biodegradation of graphene-based materials must be better understood .Similar to the general properties of all nanomaterials, the biocompatibility of graphene-based nanomaterials depends on the morphology, size, and surface modifications of the material. The morphology and size of graphene-based materials are key factors for their uptake by NSCs. Flat GO sheets with a thickness of hundreds of micrometers have been shown to be an excellent neural interfacing substrate for NSC culture . In cont125I-labeled PEGylated graphene nanosheets accumulate in the reticuloendothelial system (RES), especially in the spleen and liver, after the injection of the material and that the accumulated graphene nanosheets are metabolized through excretion [The biodistribution of graphene-based nanomaterials is mainly due to nanoparticles or sheets originating from the implanted biomaterials. The biological effect of distributed nanoparticle materials has been studied systematically, and the introduction of radioisotope tracking has led to significant advances in the study of biodistribution. It has been shown that xcretion . HoweverFor long-term in vivo implantation, biodegradation is an important factor to consider regarding clinical treatment. Combined with the metabolization routes, the hybridization of graphene-based and biodegradable nanomaterials offers an ideal strategy. Guo et al. reported a composite material fabricated by assembling rGO nanosheets onto the surface of a 3D bioactive porcine acellular dermal matrix. The hybridized materials showed good enhancement of mesenchymal stem cell differentiation and newborn neurite extension, while at the same time exhibiting good biodegradation . The comThe study of graphene-based nanomaterials for the application in neural regeneration is an emerging area and has shown the potential for such materials in clinical use. Graphene-based nanomaterials combine biocompatibility with the excellent mechanical, electrical, and optical properties of graphene for manipulating NSC fate. However, clinical applications are still at an early stage, and there are still a few issues that need to be addressed, such as the simulation of the in vivo NSC microenvironment, the toxicity of long-term implantation, and the degradation of implanted materials. Significant progress has been made in the use of hybridized nanomaterials and surface-modified graphene, and we anticipate that implanted graphene-NSC systems will provide a solid foundation for further studies of clinical neural regeneration.


Generation of large areas of graphene possessing high quality and uniformity will be a critical factor if graphene-based devices/sensors are to be commercialized. In this work, epitaxial graphene on a 2" SiC wafer was used to fabricate sensors for the detection of illicit drugs (amphetamine or cocaine). The main target application is on-site forensic detection where there is a high demand for reliable and cost-efficient tools. The sensors were designed and processed with specially configured metal electrodes on the graphene surface by utilizing a series of anchors where the metal contacts are directly connected on the SiC substrate. This has been shown to improve adhesion of the electrodes and decrease the contact resistance. A microfluidic system was constructed to pump solutions over the defined graphene surface that could then act as a sensor area and react with the target drugs. Several prototypic systems were tested where non-covalent interactions were used to localize the sensing components (antibodies) within the measurement cell. The serendipitous discovery of a wavelength-dependent photoactivity for amphetamine and a range of its chemical analogs, however, limited the general application of these prototypic systems. The experimental results reveal that the drug molecules interact with the graphene in a molecule dependent manner based upon a balance of  Ever since its discovery and isolation by Novoselev and Geim , graphen[π-bands . Whilst [π-bands ,5,6,7, t[π-bands  on silic[π-bands ,10. The [π-bands .Within the field of forensics, there is a constant demand for the development of new and more cost-efficient tools for detection of narcotics in the field at both the bulk and trace level. Whilst many different techniques based on presumptive testing, such as chemical ‘spot’ tests , colorimIn the present work, we describe and demonstrate chemical sensors for illicit drug detection based on epitaxial graphene synthesized on the silicon face of a silicon carbide (SiC) wafer with an application as a forensic field testing apparatus in mind. The devices were characterized in terms of their structural and electrical properties by the transmission line measurements (TLM) method, shear testing and measurements of the current-voltage characteristics. The electrodes of the sensors were processed on the graphene surface with a series of anchors where the metal contacts were in direct contact to the SiC substrate, which increases adhesion of the contacts and decreases the contact resistance due to an increased carrier injection through edge effects . In ordeGraphene is not expected to display significant differences in sensitivity between closely related analytes and thus in this study antibodies were utilized to establish selectivity. Two different prototypic systems were investigated. In the first, antibodies were immobilized onto magnetic beads to enable their localization upon the graphene surface using an external neodymium magnet positioned directly under the cell. This non-covalent interaction allows magnetic beads immobilized with different antibodies to be easily exchanged within the sensor cell. The second prototype involved the in-situ derivatization of antibodies with 1-pyrene butyric acid-N-hydroxysuccinimide ester (pyrene-NHS) already bound to the graphene surface through non-covalent interactions .The 1x phosphate buffered saline (PBS) pH 7.4 (97062-732) tablets were purchased from VWR International, Stockholm, Sweden. PBS-Tween , bovine serum albumin  and Dynabeads MyOne tosylactivated 1 In short, the device fabrication process consisted of isolating individual active graphene areas by dry etching a 2-inch graphene-on-SiC wafer, forming contacts/electrodes, passivating non-active areas and finally dicing the wafer to chips. The graphene wafer was produced by means of Si sublimation from semi-insulating, Si-face, on-axis, 4H-silicon carbide (SiC) substrates, forming epitaxial graphene on SiC (EG-SiC) ,20. The The TLM method  was used15–655 Ω ,23,24.The adhesion quality of the metal contacts was evaluated by attaching bond wires in a loop within the openings using a West-Bond 7476E-79 manual wedge bonder. Destructive and non-destructive shear tests were performed with a DAGE 4000Plus bond tester fitted with a 100 g cartridge. Measurements were carried out by manually lowering a shear tool, inserting the tip into the bond wire loop and applying an upwards force to either a predetermined value of 2.4 g or until the wire broke.To maximize the coverage of the magnetic beads and overall affinity towards the antigen, a mixture of the three anti-amphetamine antibodies was used in a 1:10:10 (8.F.10.B: M9905225:BDI0) ratio. For those beads designed for the detection of cocaine, only a single strain of anti-cocaine antibody IP3G2 was used due to limited commercial availability. Antibody affinities for their respective antigens were tested using standard Dot blot methodology see . The schThe anchoring of antibodies onto the graphene surface using pyrene-NHS was performed directly in a polydimethylsiloxane (PDMS) chamber. Pyrene-NHS (375 mg/mL) in EtOH (99.5%) was flowed over the surface for two minutes, followed by EtOH for 10 minutes. Before immobilization of the antibodies, PBS solution (1x pH 7.4) was applied for three minutes. Anti-cocaine antibody solution (1:2000 from 1 mg/mL in PBS) was then passed over the system for 30 minutes and the surface was thereafter washed with PBS for an additional 30 minutes. Atomic force microscopy (AFM) was used to examine the graphene surface under the surface modification process see .Detection of the drugs was performed with the fabricated graphene-based device using electrical measurements and a flow dynamic system to pump analytes over the active sensing area of the device, as schematically shown in The general detection method consists of applying a bias to the graphene-based device and probing the openings on either side of the active sensing area. The current over time is further registered when analytes are pumped through the flow system. To detect the analytes pumped through the system, the scheme summarized in The adhesion of metal contacts is of great interest for graphene-based devices in general and is investigated due to it being a limiting factor in terms of the general quality and performance of the devices. Here, eight of the fabricated graphene-based devices were independently measured and characterized with the TLM method for the contact resistance to ensure reproducibility. Each measured graphene-based device yielded a data set with six resistance values, if there was an outlier in the data set, i.e., nonlinear resistance values, or showing non-ohmic characteristics, the whole data set was discarded from the reported results. Out of the measured eight devices, six showed pure ohmic contacts. In In total eight metal contacts were tested for mechanical stability, four at 2.4 g in a non-destructive fashion, and four at the maximum required force to break the wire. All contacts passed the set limit for the non-destructive test. The required force to break the respective bonding wires are listed in Current–potential (I–V) measurements between the electrodes on the fabricated-based graphene devices were always performed before any measurement with the flow dynamic system. A typical I–V measurement is shown in In order to characterize the functionalized surface of the magnetic beads, the device surface was thoroughly washed, and the solution phase allowed to evaporate in order to permit SEM experiments. This process was performed on both the native and the functionalized beads in order to examine the structural changes resulting from immobilization of the antibodies. The images revealed a homogeneous morphology and size for the beads, before and after immobilization of antibodies, as well as an increased diameter of approximately 10 nm. Even though the washing and evaporation processes can have disturbed the sensing element-graphene interface it became clear from the SEM images that the magnetic beads were clustered in isolated clumps and not spread as an ideal monolayer see . These rThe first prototypic system utilized antibody-immobilized magnetic beads localized onto the sensor surface using a neodymium magnet. During the measurement of resistivity changes, the flow across the sensor cell was alternated between antigen solutions (both amphetamine and cocaine) and pure PBS solution in order to better elucidate changes in resistivity arising as a result of sensor-antigen events from baseline drift. Despite repeated attempts, no clearly identified responses could be connected to the various flow sequences over the cell when a potential of +50 mV was placed over the graphene.Due to these findings, a fluidic approach with two steps was established, in which magnetic beads were used for the isolation/selection of antigen on a secondary surface which could later be released to the sensor cell. This demonstrates a method to achieve specificity without competing interactions on the graphene surface see .N-ethyl amphetamine, phentermine, p-methyl amphetamine) were studied over time.As previously discussed in N-methylated analog, methamphetamine, only showed measureable photoactivity when the microscope lamp was set to 3000 K, no response was seen for 2600 K, see The N-Ethylamphetamine displayed similar results to methamphetamine, with a clear increase in the current when exposed to illumination at 3000 K, but with a barely visible increase in current at 2600 K. Compared to methamphetamine, there was however no need for prolonged flow of the amphetamine analog solution through the system before this response became measurable, see Phentermine on graphene rendered a decrease in current upon light exposure. With the light set to 3000 K a larger decrease, compared to 2600 K, could be seen as well as a change in slope after the initial response, see p-methylamphetamine, but setting the light to 3000 K for an extended time (minutes instead of seconds) gave a rapid increase following the initial decrease, see p-methylamphetamine can therefore be seen as an increase in conductivity after prolonged light exposure.The current also decreased upon light exposure for Electrical measurements were performed for a solution containing cocaine diluted in PBS and investigated for the conductivity and photoactivity following the scheme in Pyrene-NHS was allowed to interact with the graphene surface followed by the linkage of anti-cocaine antibodies as described in Our findings in this study show that the EG-SiC was formed with low contact resistance and processed with metal contacts of high adhesion capabilities. The specific processing of the metal contacts anchored to the substrate layer could prove to be vital for future prospects, especially for applications where a low contact resistance is required as for instance in high-frequency applications ,26.Two prototypic systems incorporating the non-covalent anchoring of antibodies (specific against amphetamine and cocaine respectively) to the graphene surface were tested. Both systems possessed design elements that could be utilized in future prototypes. Whilst difficulties were encountered in achieving a monolayer of magnetic beads (and thus a reproducible device), the use of immobilized antibodies to capture and release antigens in a preselection step has been demonstrated. The attachment of antibodies using a planar hydrophobic pyrene anchor in the second prototype provided promising results for cocaine, demonstrating that binding events between antigen–antibody can be relayed to the graphene and monitored electrically. The mechanism resulting in resistivity changes for this prototype is currently under study to examine if the observed effects are due to changes in strain, conformation, electrical changes, solvation etc. However, due to the later-detected photoactivity of amphetamine analogs, the generality of this second prototype is limited since the small molecular size of these antigens allows their ready access to exposed graphene surfaces and thereby a competing process of resistivity-changing events.Since the detection scheme involved alternated pumping of the different solutions with and without analyte and with and without light exposure in an otherwise unchanged environment, possible spurious effects on the measured current by e.g., temperature fluctuations could be excluded.p-methyl-amphetamine possessed an unsubstituted phenyl group All amphetamine analogs demonstrated a wavelength-dependent photoactivity with a higher activity when irradiated with a microscope lamp set at 3000 K compared to 2600 K. Adsorption of organic molecules onto graphene has been studied both theoretically and experimentally, resulting in the conclusion that the photoinduced charge carriers such as electrons or holes depending upon the adsorbates can interact actively with graphene ,30,31,32 surface ,29,32.N-ethylamphetamine gave similar responses to amphetamine, although considerably weaker than for the parent compound. The overall response for these three analogs was an increase in conductivity when a solution of the analog was flowed over the graphene surface most likely as a result of the amine nitrogen acting as an electron donor.Both methamphetamine and Irradiation of the sensor when a solution of phentermine was flowed across the graphene surface resulted in a decrease in conductivity for the graphene surface. Phentermine possesses a crowded primary amine functionality with p-methylamphetamine displays an initial reduction in conductivity  before interaction with the graphene surface, thereby creating high substance-specificity even for complex mixtures of active substances and cutting agents.The use of immobilized antibodies on the graphene surface via a hydrophobic pyrene anchor proved promising as long as the antigen itself showed no photoactivity with the surface. Antigen–antibody binding events relayed from the antibody through the butyric amide linker to the pyrene anchor and ultimately to the graphene surface resulted in resistivity changes.The serendipitous discovery of photoactivity between amphetamine analogs and graphene rendered the viability of the two initial prototypic systems conjectural. The intensity of these photo-responses was found to be wavelength-dependent. The nature of the overall response is conjectured to arise from a balance between The combination of preselection of analyte using immobilized antibodies with detection using a native EG surface shows promise as the technology behind hand-held devices utilizing batch-fabricated graphene-based sensors for rapid front-line detection of illicit drugs.


We also discuss topics related to the creation and configuration of disorders in graphene, such as corrugations, topological defects, vacancies, adatoms and sp3-defects. The effects of these disorders on the electrical, thermal, chemical and mechanical properties of graphene are analyzed subsequently. Finally, we review previous work on the modulation of structural defects in graphene for specific applications.Monolayer graphene exhibits extraordinary properties owing to the unique, regular arrangement of atoms in it. However, graphene is usually modified for specific applications, which introduces disorder. This article presents details of graphene structure, including sp  Due to Graphene film consisting of few layers was employed to fabricate transistors, due to its strong ambipolar electric field effect . However60 molecules, the lithium storage capacity of graphene nanosheets could be increased up to above 700 mAh/g, making them suitable for use in rechargeable lithium ion batteries , , AB stacAB-stacked bilayer, the vertical hopping can be very complex: besides the hopping between the dimer sites Bilayer graphene has not only in-plane hopping (e.g. nearest neighboring hopping and next nearest neighboring) in the same layer, just like that in monolayer graphene, but also vertical hopping which is dependent on the stacking order. In the case of AA-stacked bilayer graphene, vertical hopping mainly occurs between The single spin Hamiltonians for AA-stacked and AB-stacked bilayer graphene and their corresponding matrix representations are given in Refs ,158. By The stacking orders can become more complicated for multi-layer graphene. Theoretically, there are two stable stacking arrangements, namely Bernal (in ABA stacking order) and rhombohedral (in ABC stacking sequence), as seen in The controlled growth of FLG with specified stacking sequences can be performed in the chamber of an environmental scanning electron microscope (ESEM) which enables real-time imaging of the shape and size evolution of graphene islands . Infrare3.3-defects. Therefore, this part will first introduce the configurations of diverse types of disorders contained in the crystal structure of graphene in detail, followed by a brief discussion about the formation energy and immigration energy of these disorders. After that, the influence of different types of disorders on properties of graphene are analyzed. Lastly, we will introduce the generation of various disorders during graphene preparation procedures, and provide approaches for reducing these disorders, so that near-defect-free samples of monolayer graphene can be obtained.An ideal graphene with highly ordered structures exhibits zero band gap , high te3.1.In the standard harmonic approximation , the lonL is the distance between two ripples, where Apart from the intrinsic disorder of ripples, wrinkles and crumples are other two types of corrugations in graphene sheets  see Fig. Unlike g strain  can be dg strain (31)λ=tLL are the thickness and width of a graphene thin film respectively, and where graphene ,200. Howgraphene :(32)λ=tAs the values of d stress . When uswhere ectively .Crumples are dense deformations that are generated by rapid evaporation, usually occurring isotropically in two or three dimensions ,201. Foring form  as:(34)where 3.2.The graphenes produced via the CVD method are usually polycrystalline, due to the presence of topological defects: disclinations, dislocations and GBs, which are able to alter the lattice orientations. An intriguing feature of these topological defects is that they can exist in graphene without introducing local disorder into crystalline lattice. As seen in Å), and the semi-infinite strip is inserted along the zigzag direction. The core of dislocation with a Burgers vector the same as that of dislocation  can be also constructed from two dislocations, such as + dislocation pair.where L + θR (0° < θ < 60°), describing the mutual orientation of the two crystalline domains, is a critical topological invariant of GBs. GBs can be viewed as a type of line defects formed by one-dimensional chains of aligned dislocations , which fequation , the misHigher densities of dislocations correspond to smaller separations between neighboring dislocations, and consequently are expected to lead to larger misorientation angles. Further, the small-angle GBs in suspended graphene tend to form the out-of-plane buckling for further reducing their formation energies, while large-angle GBs are almost flat .10.1080/Both STM and SEM are suitable for the identification of GBs in graphene lattice. It is evidenced from the atomically resolved STM image see ) of a reocations . As seenocations . The proocations ,210. It ocations , by specocations ,212, andocations .3.3.3-defects, and their configurations can be seen in Other three types of common defects are vacancies, adatoms and spTransition-metal (TM) atoms adsorbed on perfect graphene sheets exhibit low migration barriers, in the range of 0.2–0.8 eV, indicating the high-mobility of adatoms at room temperature . Hence, 3 defects can be observed in hydrogenated [3 defects produced by the chlorination of graphene following the method described in a previous study [3 defects usually appear in form of dimers or clusters rather than being isolated [The spogenated , fluorinogenated ,219, chlogenated ,221 and ogenated . Figure us study . Cl atomisolated .3.4.Isolated disclinations require out-of-plane deformations of the graphene sheet and therefore have high formation energies . This maÅ the length of Burgers vector for  dislocation, and misorientation angle where ocations .The θ = 21.8° and the θ = 32.2° symmetric large-angle linear GBs have particularly low formation energies of 0.338 and 0.284 eV/Å, respectively see . Among tIt is noted that sometimes the defects in the crystal structure of graphene are migratory rather than stationary, and their migration may influence the properties of a defective crystal, especially the annealing and reconstruction behaviors. Each defect shows a certain mobility parallel to the graphene plane. For instance, the mobility for SW defects and divacancies are immeasurably low, while the mobility for adatoms is fairly high. The migration of each defect is governed by an activation barrier which is indicated by the migration energy. Therefore, the migration is expected to increase exponentially as temperature increases. For a SV in graphene, the calculated migration energy ranges from 1.2 to 1.4 eV , which a3.5.3.5.1.In an idealistic planar graphene model without disorders, the Fermi energy level lies at the Dirac point, where the valence band and conduction band intersect, and the dispersion relation around the Dirac point is isotropic and linear . Howeverectories ,238. As ectories , creatinectories  and induectories . Whereasectories ,242, carectories ,243, banectories , suppresectories  and quanectories .Disclinations and dislocations induce distortions in the graphene lattice which probably alter the carbon–carbon bond length, and consequently the band structure is changed. Due to the strong scattering of charge carriers, GBs can impede electronic transport, thus degrading the electrical properties (e.g. decreasing mobility and increasing resistance) of polycrystalline graphene ,247–250.Point defects such as SW defects, vacancies, and adatoms can serve as scattering centers for electron waves, and thus reducing the conductivity of graphene –258. The3.5.2.2 [The room-temperature in-plane thermal conductivity of suspended monolayer graphene is among the highest of any known materials, 4840–5300 W/(m K), as determined by micro-Raman thermometry . However2  or confi2 , due to 2 . Numeric2  indicate2  also obs2 . Besides2 –274. A h2 .Due to the scattering effect induced by GBs, the thermal conductivity of CVD-grown polycrystalline graphene is generally lower than that of exfoliated graphene . In non-3.5.3.2 hybridized surfaces enables the resulting graphene materials to be hydrophilic and more reactive [Defect-free graphene surfaces appear to be chemically inert, and these surfaces usually interact with other molecules via physical adsorption . Besidestrength –296 were2 to sp3 bonding but also the easy-rotation of unsupported sp3 bonds. On the other hand, it is reported [3-type defects, in contrast to significant degradation of mechanical properties in the vacancy-defect regime.In addition, MD simulations  suggest reported  that theIn polycrystalline graphene, the Young’s modulus and fracture strength are more sensitive to the variations of temperature and strain rate than that in monocrystalline graphene ,300. Bes3.6.Disorders can be deliberately introduced into the graphene lattice for tailoring properties by additional treatment, which will be discussed in the next part. Besides, it is possible to bring defects into the crystal structure of pristine graphene during preparation process. Up to now, a large number of methods have been developed for fabrication of graphene see . These mFabrication of graphene by CVD comprises two steps: CVD-growing graphene on metal substrates and transferring graphene to a desired substrate. In the growing step, high-temperature growth facilitates the rapid annealing of defects, and thus generates less defects than low-temperature growth. Due to the high formation energy of vacancies and fast migration of adatoms, isolated vacancies are uncommonly seen in graphene sheets after growth. However, graphene spontaneously grows at different positions on the substrate surface, and consequently grain boundaries are formed at the amalgamation areas, leading to polycrystallinity. These GBs or other small topological defects generate in-plane stress, which is responsible for the out-of-plane deformations (ripples and wrinkles) . Normall2 and trapped H2O also significantly influence the mobility of the transferred graphene [2 for practical applications (e.g. nanoelectronic devices). The graphene corrugations observed by a novel combined SEM/AFM/STM technique are suggested to be attributed to the partial conformation of the graphene to the SiO2, not to the intrinsic corrugations of graphene [2O may be trapped at the graphene/SiO2 interface [The graphene transfer is commonly executed by spin-coating a polymer layer (e.g. polymethylmethacrylate (PMMA)) to support the graphene while etching the metal catalyst away in an etchant . Polymergraphene . The CVDgraphene . Besidesnterface ,318. Watnterface .2/N2 or H2/Ar [Most of these introduced defects are undesirable, and somewhat exert negative impacts on the properties of graphene. Therefore, there is a need to provide approaches for removal of these disorders. Typically, the intrinsic ripples may be suppressed by depositing graphene on atomically smooth substrates . Wrinkleor H2/Ar ,324,325.4.Disorders are predominantly brought in during the production process, and unavoidably occur due to the interaction with substrates and environment. As discussed in Section 3.5, the properties of graphene are sensitive to these intrinsic or extrinsic disorders. Sometimes these defects need to be removed for maintaining remarkable properties of pristine graphene. However, in most cases the properties of graphene are supposed to be tailored for satisfying different requirements in industries. Tuning properties of graphene can be realized by several defects modulations approaches, including particle irradiation –328, the4.1.Irradiation usually introduces disorders and leads to the self-organization or self-assembly in nanostructured carbon materials . Typical2+) irradiation, a sufficient number of vacancies may result in the hole doping of graphene due to the charge interaction between broken carbon bonds and ambient O2 molecules, and the increase of work function due to hole doping showed a logarithmic behavior with respect to the irradiation dose [+ at energies (10–30 keV) allows the passage of more than 97% He+ particles without creating vacancies on the hexagonal lattice of freestanding graphene, which was corroborated by Raman spectroscopy. In contrast, large Ga+ ions at energies (5–30 keV) collide more often with the graphene lattice, and impart a notable higher sputter yield of ~ 50%.Ion irradiation can be employed to selectively create defects as well as to pattern or mill graphene sheet by utilizing a focused ion beam (FIB) . As seenion dose . Buchheiion dose  found thin situ monitoring. Alternatively, it is possible to in situ monitor the defects fabrication at atomic level by using an aberration-corrected TEM [5 e−1 nm−2 s−1, a focused beam with high current density (108 e−1 nm−2 s−1) employed to create defects, and a broad beam with low current density adopted to observe the formed disorders on graphene. It is evidenced from the electron irradiation experiments that both the location and average complexity of defect formation in graphene could be controlled by tailoring the electron beam current density and the exposure time. Some of the created defects (i.e. closed-loop defects) were stable, whereas others (i.e. SVs and divacancies) relaxed to simpler structures through bond rotations and surface adatom incorporation. Kotakoski et al. [However, defects created by ion beams are sporadically formed over a wide area of graphene, which lacks nanoscale spatial control and cted TEM  followini et al.  investig4.2.3-hybridization of carbons when annealing over a long period or at a high-temperature of 200 °C, due to the random scission of polymer chains. The rehybridization alters graphene’s band structure near the Fermi level , and thevel see ). Furtheevel see  that sust of rGO , a smallt of rGO . Conductt of rGO .10.1080/4.3.3 defects. Though the room-temperature reaction is limited by the high-inertness of pristine graphene, strong oxidizing acids (e.g. HNO3 and H2SO4) used in Hummers method can easily react with graphene and attach chemical groups (e.g. oxygen and hydroxyl and carboxyl) to the graphene surface. Due to the chemical inhomogeneity and irreversibility of the resulting GO, an alternative approach using atomic oxygen in ultrahigh vacuum is presented for reversible and uniform oxidation of epitaxial graphene on SiC(0001) [The reaction of carbon atoms in the graphene sheet with other species can lead to the formation of vacancies or spiC(0001) . SpecifiPlasma treatment in ammonia gas are commonly used in efforts to dope graphene by nitrogen ,346. The2 into sp3 orbitals. Daukiya et al. [2 conjugation of carbon atoms and the generation of local sp3 bonds. Moreover, the grafted molecules perturb the graphene lattice, and consequently an anisotropic standing-wave pattern is generated due to the  cycloaddition. In their experiments, the formation of covalent bonds was ascertained by the increased sp3 components in the XPS spectrum, while the standing wave patterns were visualized by STM.Diels−Alder or other cycloaddition reactions are effective approaches for surface modification of graphene by converting spa et al.  successf2. In addition, sequential creation of vacancies (e.g. irradiation of energetic particles) and sequential exposure to NO molecules make it is possible to achieve fine control of N-doping.On the other hand, disorders in graphene lattice can be reconstructed via chemical reactions between defective graphene sheet and foreign species. Wang and Pantelides  carried 4.4.Atomic thickness of graphene makes it amenable to external influences, including mechanical deformation. It is intuitive that strain can cause distortion or other defects to the hexagonal lattice, thus changing the electronic band structure of graphene. Pereira and Castro Neto  demonstrMoreover, strain can be applied to a defective lattice for tailoring the properties of graphene. Kumar and Guo  used ato5.3 defects) are usually deliberately introduced into the structure of graphene. The configuration, formation of these disorders and their influences are systematically introduced. Various approaches  for defects modulation in graphene are discussed at the end of this article. This review is expected to facilitate the understanding of the structure of graphene and its contained disorders, and consequently assist in investigations into graphene modification.This article describes the structure of graphene from a fundamental perspective, explaining in detail the formation of the honeycombed structure and studying the electronic band structure of graphene via tight-binding approximation. The characterization methods for edge orientations, number and stacking arrangements of graphene layers, and their effects on the properties of graphene are also discussed. To satisfy various demands in practical applications, disorders (i.e. corrugations, topological defects, vacancies, adatoms and sp


To depict the genomic landscape of Chinese early-stage lung squamous cell carcinoma (LUSC) and investigate its correlation with tumor mutation burden (TMB), PD-L1 expression, and immune infiltrates.+ tumor-infiltrating lymphocyte (TIL) density and PD-L1 expression were evaluated by immunohistochemistry. Six immune infiltrates were estimated using an online database.Whole-exome sequencing was performed on 189 surgically resected LUSC. TMB was defined as the sum of nonsynonymous single nucleotide and indel variants. CD8+ TILs density were identified in 24.3% and 78.8%. PIK3CA amplification was associated with significantly higher TMB (P = 0.036). Frequent genetic alterations had no impact on PD-L1 expression but PIK3CA amplification and KEAP1 mutation were independently associated with significantly lower CD8+ TIL density . Low TMB and high CD8+ TIL density were independently associated with longer disease-free survival (DFS) while none of them could individually predict the overall survival (OS). Combination of TMB and PD-L1 expression or TMB and CD8+ TIL density could stratify total populations into two groups with distinct prognosis. Classifying tumor-immune microenvironment based on PD-L1 expression and CD8+ TIL density showed discrepant genomic alterations but similar TMB, clinical features, and OS. Notably, patients with different smoking status had distinct prognostic factors.The median TMB was 9.43 mutations per megabase. Positive PD-L1 expression and CD8The combination of TMB, PD-L1 expression, immune infiltrates, and smoking status showed the feasibility to subgroup stratification in Chinese patients with early-stage LUSC, which might be helpful for future design of personalized immunotherapy trials in LUSC.The online version of this article (10.1186/s13045-019-0762-1) contains supplementary material, which is available to authorized users. Lung squamous cell carcinoma (LUSC) is one of the most common histological subtypes of non-small-cell lung cancer (NSCLC), the remaining leading cause of cancer-related death worldwide for a long period –3. UnlikFor patients with early-stage LUSC, curative surgery with adjuvant chemotherapy is the main therapeutic option even though adjuvant chemotherapy just brought limited survival benefit . RecentlHerein, we performed this study in 189 Chinese patients with early-stage LUSC to evaluate (1) the genomic landscape of LUSC and its correlation with PD-L1 expression, TMB, and six immune infiltrates and (2) their associations with clinical parameters, disease-free survival (DFS), and overall survival (OS). Additionally, we also compared our data to other ethnicities and tumor types such as adenocarcinoma.We retrospectively identified patients who underwent surgical resection of the lung (lobectomy or pulmonectomy) and histologically confirmed LUSC at Shanghai Pulmonary Hospital from 2012 to 2015. We firstly checked the histological types of each case using medical electronic records. Then all primary diagnoses were further independently evaluated by two experienced pathologists (Z.W.D and L.K.H) according to the WHO nomenclature for squamous carcinoma. The specimens of eligible case must have a confirmed diagnosis of LUSC and had at least 50% tumor cellularity. Major exclusion criteria were inadequate or poor quality samples, missing baseline clinicopathological features, mixed histology, and incomplete follow-up data. Corresponding formalin-fixed and paraffin-embedded (FFPE) tissues were used for immunohistochemistry (IHC) staining and whole-exome sequencing. The major baseline features including age, sex, smoking history, Eastern Cooperative Oncology Group performance status (ECOG PS), tumor size, node status and stage, vascular invasion, differentiation degree, tumor stage, DFS, and OS. A never-smoker was defined as a patient who had smoked less than 100 cigarettes during his/her lifetime. DFS was defined as the time from the initial surgical resection until recurrence. OS was calculated from the date of LUSC diagnosis to death from any cause or was censored at the last follow-up date. This study was conducted in accordance with the provisions of the Declaration of Helsinki and was approved by the ethics committee of Shanghai Pulmonary Hospital.+/−). PD-L1 > 50% was defined as PD-L1 strong positivity. For SP142 staining, positive cells were defined as cancer cells displaying membranous staining for PD-L1, and the proportion of PD-L1+ cells was evaluated as the percentage of total cancer cells in whole sections. The cutoff values of 1%, 5%, 10%, and 50% were set and 1% was determined for PD-L1+/− [PD-L1 expression was firstly tested by using anti-human PD-L1  according to the manufacturer’s recommendations and previous publications using 4–5 μm FFPE sections , 23. ThePD-L1+/− , 26. Sec+ TILs density was assessed by using a mouse anti-CD8 monoclonal antibody . Lymphocytes with immunostained CD8 infiltrating within a tumor region  were defined as CD8+ TILs. On the basis of the percentage of CD8+ TILs displayed within a tumor region, we determined high/low CD8+ TIL density (CD8+ TIL+/−) with a cutoff of 5%, which was analogous to the previous studies [CD8 studies , 27.The details of whole-exome sequencing and data processing were listed in Additional file Sn represents the absolute number of somatic mutations, and N represents the number of exonic bases coverage depth ≥ 100 ×.+ T cells, CD8+ T cells, macrophages, neutrophils, and dendritic cells (DC) in specific groups of LUSC were estimated by using online database, named Tumor Immune Estimation Resource (TIMER). TIMER is a comprehensive resource for systematical analysis of immune cell infiltrations across diverse cancer types, which is validated using pathological estimations. The details and statistical methods were listed in this website (https://cistrome.shinyapps.io/timer/) and their previous publications [The abundances of six immune cell infiltrations including B cells, CD4ications , 29.+ TIL density. Correlations between PD-L1 expression/CD8+ TIL density and clinical parameters were analyzed using the chi-squared or Fisher’s exact test for categorical variables. The continuous variable was analyzed by ANOVA and Tukey’s multiple comparison tests. Mann-Whitney U tests or Kruskal-Wallis rank sum tests were used for comparisons of continuous variables across multiple groups. The Kaplan-Meier curve was leveraged to assess the patients’ survival curves. The log-rank test was used to test the significance of differences between two or four groups. Cox proportional hazards model was utilized for uni- and multivariate survival analyses to calculate the hazard ratios (HR) and related 95% confidence intervals (CI). P < 0.05 was considered significant. All statistical analyses were performed using GraphPad PRISM 6.0 and the SPSS statistical software, version 22.0 .The Pearson correlation coefficient was used to determine the correlation of PD-L1 expression level between two antibody assays. Spearman’s rank correlation was utilized to assess the correlations among PD-L1 expression, TMB, and CD8TP53, KMT2C, NFE2L2, KEAP1, CDKN2A, PTEN, FBXW7, and PIK3CA. This would have a slight difference to TCGA which identified 10 genes  with a false discovery rate (FDR) Q value < 0.1 [TP53, MLL2, PIK3CA, NFE2L2, CDH8, KEAP1, PTEN, ADCY8, PTPRT, CALCR, GRM8, FBXW7, RB1, and CDKN2A [TP53 mutation was found in 67% of all cases . There were 10 genes with significant copy number variations  was the most frequent. PIK3CA and FGFR1 amplifications were found in 23% and 20% of all cases, respectively. Interestingly, we found a high frequency of EML4-ALK fusion (3.2%), one of the most common driver gene alterations in LUAD. There was no significant correlation between EGFR amplification or EML4-ALK fusion and clinicopathological features . There was no significant relationship between TMB and other clinical features , which was lower than it in Caucasian with LUSC [H-score ≥ 50, or 63.9% using > 1% tumor cell positive as a cutoff, which is consistent with those in the literature on the Western population [+  and representative IHC images of CD8+ TIL were showed in Additional file + TIL density and clinicopathological features. Patients with CNVs had similar CD8+ TIL density to those without CNVs .The mean TMB was 10.77 mutations per megabase (mut/Mb) and median TMB was 9.43 mut/Mb . Additionally, most of frequent CNVs including FGFR1, EGFR, and PIK3CA amplifications and loss of CDKN2A were associated with significantly lower six immune infiltrates .We then investigated the associations between frequent genomic alterations and TMB, PD-L1 expression, or CD8+ TIL density and lower TMB were independently associated with significantly longer DFS . However, patients with different PD-L1 expression, CD8+ TIL density, and TMB had comparable OS  and OS   while CD8+ TIL+ plus lower TMB was associated with longer DFS (P = 0.011). The combination of high TMB and PD-L1− or CD8+ TIL− did not reach the statistical significance in stratifying patients with different OS . Multivariate analysis revealed that stages II and III were associated with both markedly poor DFS  and OS . Only the combination of CD8+ TIL+ and lower TMB was associated with significantly longer DFS  . We found that type IV  TIME was dominant while type III  was the least common in Chinese early-stage LUSC  and 17.5% (n = 33), respectively . There was no any correlation between TMB, PD-L1 expression, or CD8+ TIL density and other clinical parameters in both groups. In never-smoking group, PD-L1+ was associated with marginally significantly longer DFS  in this cohort, we further conducted the subgroup analysis of the associations between TMB, PD-L1 expression, CD8.7% in thr P = 0.08. There r P = 0.08. There r P = 0.08. There r P = 0.08. There r P = 0.08. There r P = 0.08. There r P = 0.08. There SOX2 amplification was only found in 6.0% of all cases, which was significantly lower than other studies [EML4-ALK fusion (3.2%), one of the most common driver alterations in LUAD, which might mainly due to a high rate of never-smoker in our cohort, and infer the necessity to detect the common driver gene alterations in never-smokers with LUSC.Whole-exome sequencing of our cohort identified significant somatic mutations and CNVs that was consistent with previous publications on the genomic profile of early-stage LUSC , 34, 35. studies , 34. ImpFGFR1, EGFR, and PIK3CA amplifications and loss of CDKN2A, were associated with significantly lower six immune infiltrates. This finding could partly explain that the fraction of copy number altered genome was highest in NSCLC patients treated with anti-PD-1/PD-L1 therapy but lack of durable benefit due to the importance of these immune infiltrates in antitumor immune response [LUSC was previously found to have higher TMB than other solid tumors due to the close correlation to tobacco exposure , 36, 37.response . It also+ TIL density. The results showed that only KEAP1 mutation was significantly associated with lower CD8+ TIL density, and NFE2L2 mutation was associated with marginally higher TMB. Moreover, a recent study reported that there was a higher rate of KEAP1/NFE2L2 >mutations in Chinese LUSC than those in Western populations [KEAP1 or NFE2L2 would destroy this process and lead to oncogenesis and drug and radio resistance in different types of solid tumors. Considering these findings, we could infer that tumor with KEAP1 or NFE2L2 mutation would have a higher level of oxidative stress, which could lead to the destruction of immune cells including CD8+ TILs and increased DNA damage level, resulting in the increase of somatic mutations of tumor cells. We also used the online database to calculate the abundance of tumor-infiltrating immune cells [KEAP1 mutation had the negative effect on CD8+ T cell abundance. KEAP1 mutation was reported to be associated with poor response to adjuvant chemotherapy in both LUSC and LUAD [KEAP1 mutation and lower CD8+ T cell infiltration could explain the negative predictive value on adjuvant chemotherapy warrants further examinations.Understanding the interplay between molecular underpinnings and immune landscape may help improve strategies for precise immunotherapy , 39. Towulations . KEAP1-Nulations . Geneticne cells , 29. Conand LUAD , 34, 41.+ TIL density but a significant positive correlation was found between CD8+ TIL density and PD-L1 expression. Similar to previous reports [+ TIL density and lower TMB were independently associated with significantly longer DFS even though none of TMB, PD-L1 expression, or CD8+ TIL density were associated with OS. The combination of TMB and PD-L1 expression or CD8+ TIL density showed an improved yield in stratifying patients with discrepant DFS and OS, suggesting that the incorporation of these biomarkers into multivariable predictive and prognostic models worth further investigation in the future.Currently, TMB and PD-L1 expression are the two developed predictive biomarkers for anti-PD-1/PD-L1 therapy. We found no association between TMB and PD-L1 expression, which was consistent with previous findings. Yu et al. reported that PD-L1 protein expression was not correlated with TMB in both tumor cells and tumor-infiltrating immune cells of early-stage LUSC . Rizvi e reports , 43, pos+ TIL density. Our data indicated that type IV TIME was dominant while type III was the least common in Chinese early-stage LUSC, which is in contrast to the previous studies [Furthermore, we classified the TIME into four types based on PD-L1 expression and CD8 studies , 44. The studies –9. We al+ TIL+ was not associated with DFS in never-smoker but associated with markedly longer DFS in former/current smoker. Both TMB level and CD8+ TIL+ were not associated with OS. PD-L1+ was not correlated with both DFS and OS in two groups. NSCLC of never-smokers are entirely different from these of former/current smokers. In never-smoking group, high TMB in tumors with never-smoking would mainly come from the intrinsic mechanism, which means the high possibility to be recognized and eliminated by the immune system. In this process, more and more CD8+ TILs would be induced and translated into the exhausted phenotype. Hence, high TMB was correlated with significantly longer DFS but CD8+ TIL+ was not associated with DFS in never-smoker. Conversely, high TMB in smokers caused by tobacco exposure could not predict the clinical outcome. However, CD8+ TILs in smokers could recognize and eliminate the malignant cells after exposure to the different carcinogens. Hence, high CD8+ TIL density was found to associate with markedly longer DFS in the current study. We must mention that the relatively small sample size and multiple different subsets based on distinct cutoffs of TMB, PD-L1 expression, CD8+ TIL density, and/or smoking status for these exploratory analyses would also be the potential reason for these discrepancies. Future investigations with a larger number of patients with LUSC are still needed.Additionally, we observed that high TMB was correlated with significantly longer DFS in never-smoker but not associated with DFS in former/current smoker. CD8PIK3CA amplification was associated with higher TMB but lower CD8+ T cells density while the common genetic alterations had no impact on PD-L1 expression. Combination of TMB and PD-L1/CD8+ TIL could be helpful to stratify the whole population into distinct DFS and OS subgroups. TIME classifications according to PD-L1 expression and CD8+ TIL density showed distinct genomic alterations but similar TMB, clinical variables, and prognosis. Notably, TMB and CD8+ TIL+ might have a distinct role in patients with different smoking status. These findings shed a light and might be helpful to guide future design of personalized immunotherapy trials in LUSC.In summary, this large-scale study found Additional file 1:Figure S1. (A) the distribution of TMB; (B–D) the comparison of TMB (B), PD-L1 expression (C), and CD8+ TIL density (D) between patients with CNVs and those without CNVs. Figure S2. (A) The representative immunohistochemical images of PD-L1 expression, (B) the correlations of PD-L1 expression score between SP142 and E1L3N, and (C) the distribution of PD-L1 expression score in each antibody assay. Figure S3. The representative immunohistochemical images of CD8+ TIL expression. Figure S4. The distribution of different PD-L1 expression (A) and CD8+ TIL density (B); (C) correlation between TMB and PD-L1 expression, (F) TMB and CD8+ TIL density, and (E) PD-L1 expression and CD8+ TIL density. Figure S5. Association between frequent somatic mutations and six immune cells infiltration. Figure S6. Association between frequent CNVs and six immune cells infiltration. Figure S7. DFS at PD-L1 expression cutoff of 5% (A) and 50% (B); CD8+ TIL expression cutoff of 5% (C) and 50% (D); TMB cutoff of 25th (E), 50th (F), 75th (G) and 90th (H) percentile; TMB plus PD-L1 at TMB cutoff of 25th (I), 50th (J), 75th (K) and 90th (L) percentile; TMB plus CD8+ TIL expression at TMB cutoff of 25th (M), 50th (N), 75th (O) and 90th (P) percentile. Figure S8. OS at PD-L1 cutoff of 5% (A) and 50% (B); CD8+ TIL expression cutoff of 5% (C) and 50% (D); TMB cutoff of 25th (E), 50th (F), 75th (G) and 90th (H) percentile; TMB plus PD-L1 at TMB cutoff of 25th (I), 50th (J), 75th (K) and 90th (L) percentile; TMB plus CD8+ TIL expression at TMB cutoff of 25th (M), 50th (N), 75th (O) and 90th (P) percentile. Table S1. The relationship between EGFR amplification/EML4-ALK fusion and clinicopathological features. Table S2. Baseline characteristics of included patients according to smoking status. (DOCX 3849 kb)


Graphene is a good candidate for filling the market requirements for cheap, high sensitivity, robust towards contamination, low noise, and low power consumption gas sensors, thanks to its unique properties, i.e., large surface, high mobility, and long-term stability. Inkjet printing is a cheap additive manufacturing method allowing fast, relatively precise and contactless deposition of a wide range of materials; it can be considered therefore the ideal technique for fast deposition of graphene films on thin substrates. In this paper, the sensitivity of graphene-based chemiresistor gas sensors, fabricated through inkjet printing, is investigated using different concentrations of graphene in the inks. Samples have been produced and characterized in terms of response towards humidity, nitrogen dioxide, and ammonia. The presented results highlight the importance of tuning the layer thickness and achieving good film homogeneity in order to maximize the sensitivity of the sensor. Increasing consciousness within the population and institutions is translating into a substantial increase in the demand for cheap, low power, high sensitivity, and reliable gas sensors for monitoring the concentration of pollutants in indoor and outdoor environments. Gas sensors partially filling these market’s requirements are MOX  or polymer based chemiresistor gas sensors ,2,3. HowThis paper is structured as follows: 3, a viscosity between 8 and 14 cP (at 25 °C and using a shear rate of 500 s−1), and a total surface energy between 30 and 35 mN/m; it therefore showed compatible rheological characteristics for optimum jetting performance and printing results, according to the dimensionless figure of merit, used to characterized the drop behavior in the DOD (Drop On Demand) printing technology [The ink used in this work was developed by an external partner, to meet the requirements of the employed printing hardware. The exact composition of the ink was not disclosed but the ink uses terpineol as the main solvent and few-layers graphene flakes with very low oxygen content. Due to confidentially agreements, more information on the ink may not be disclosed. For more information on the ink you may contact caterina.travan@infineon.com. The ink was characterized in terms of density, viscosity, and surface tension at Joanneum Research in Weiz in order to assess its compatibility with the printhead used and to calculate its Ohnesorge numbers before testing its jetting performance. The density of the ink was measured using a volume measuring container and a scale. The rheological measurements were performed with a rotational viscometer Brookfield DV-III Ultra. The surface tension was measured with the pendant drop method using Krüss DSA 100, a drop shape analyzer produced by Krüss. The employed ink had a density of about 1 g/cmchnology ,18,26. TRaman spectroscopy was used to investigate the amount of defects in the graphene flakes present in the ink. Raman spectra were measured at CTR (Carinthian Tech Research) in Villach using a Renishaw inVia Reflex Raman spectroscope. The spectra were acquired using a laser excitation of 532 nm, an aperture of 50%, and an exposure time of 1 s. The obtained laser spot had a size of about 5 µm. The samples were kept at room temperature during the measurements. A Raman analysis was performed in combination with an optical investigation using SEM, which provided information about lateral flake size and flake distribution. SEM pictures were taken using an Ultra-High-Resolution HitachiSU-8010. After verifying the good droplet formation and jetting performance, the ink was printed on gold interdigitated electrodes (IDEs) on a silicon substrate with an isolating silicon nitride layer between the IDEs and the silicon. All the jetting and printing trials were carried out using a LP50 printer, an R&D (Research and Development) DOD piezoelectric printer produced by Meyer Burger. The printheads used in this work are SE128AA produced by FUJIFILM Dimatix, Inc. After printing, samples were thermally treated in IR vacuum reflow oven with a single process chamber SRO-700, produced by ATV, in order to remove residues of solvent and burn off stabilizers and surfactants present in the ink.2 and NH3, inks, the following graphene concentrations were used: 5 g/L (sensor S0 and S4), 2.5 g/L (sensor S1 and S5), 1 g/L (sensor S2 and S6), 0.5 g/L (sensor S3 and S7) as shown in 4 resulted in a strong coffee stain due to a small misalignment of the print which caused a very low spreading of the ink. This sensor was used to investigate the difference in the gas sensitivity between a homogeneous graphene film and a sensor with strong coffee stain, thus a highly inhomogeneous graphene layer.In order to investigate the influence of the graphene concentration on the sensitivity towards NO2 and NH3) and synthetic air (20% O2 and 80% N2) as dilution gas. Additionally, it included a bubbler using 250 sccm of synthetic air as input flow and generating an output flow of humid air capable of producing a relative humidity up to 90%. Both NO2 and NH3 flows were provided by gas bottles produced by Linde with fixed concentrations of 100 ppm and 1000 ppm respectively, while their flow was regulated by a precision MFC (mass flow controller) subject to periodic calibration. The gas flows were therefore diluted by the synthetic dry air and by the humid air from the humidity generator, to reach the target gas concentration. The mixture of gas flows and synthetic air flow was kept constant at 500 sccm in order to keep a constant humidity in the total flow.The setup, illustrated in The MEMS heater located underneath the sensor was used for setting different sensor temperatures. The sensor heaters were controlled by an FPGA (Field Programmable Gate Array) and the sensor resistance were read by 8 multimeters (Agilent HP 3458A). The data were recorded in real time by the measuring software (MultiMess7) installed on the computer. The MFCs (mass flow controllers), the bubbler, and the heater temperature were regulated using a Visual Studio Macro. A steel chamber was used for the measurements to reduce the adsorption of water or gases.The graphene layer was investigated after printing and drying, using Raman spectroscopy and SEM. SEM analysis shows that part of the graphene flakes crumbles on the surface while the other part is flat and adhering on the substrate with their whole area, as can be seen in G peak, which is a primary in-plane vibration mode and the 2D peak, which is a double phonon scattering (second-order overtone of a different in-plane vibration). Other three peaks can appear in the spectrum: D (at 1350 cm−1 from a 532 nm excitation laser), D’ (at 1620 cm−1 from a 532 nm excitation laser), and D + D’ (at 2940 cm−1 from a 532 nm excitation laser). The D peak is caused by intervalley phonons (scatters from K to K’) and defect scattering and it is not visible in pristine graphene due to crystal symmetries, since it needs a defect to cause a second elastic scattering. The D peak increases with the amount of defects but it is not dependent on their geometry. The D’ peak is due to intravalley phonons (scatters from K to K’) and defect scattering. Its level depends on the type of defect, i.e., is more sensitive to vacancies than sp3 sites. The sp3 sites introduce a different arrangement of the carbon atoms, but do not break the network [G peak [D + D’ is also a defect activated peak and is only visible in the presence of high amount of defects [Raman spectroscopy is a technique largely used to investigate the chemical and physical properties of graphene; it can be used for studying thickness, disorder, edge, and grain boundaries, doping and thermal conductivity ,28,29,30 network ,30,31,32 [G peak ,34. The  defects ,18.D peak indicates the presence of point and edge defects. The D peak is low and sharp and lower than the G peak indicating a relatively low amount of defects in the material. In the case of nanocrystallite graphite (low defect density regime), DI\GI will increase with the increasing amount of defects due to the higher elastic scattering. In the case of amorphous carbon (high defect density regime), the lower the DI\GI, the smaller the inter-defect distance, since the defect density in this structure is so high that all the Raman peaks are attenuated [DI\GI of the graphene flakes investigated is about 0.6, which indicates an average crystallite size of 13 nm [tenuated ,31,35. TD peak. In few-layer graphene, compared to monolayer graphene, the intensity of the 2D peak decreases and the peak broadens and shifts to lower frequency [D peak would suggest that the material consists of few-layer thickness graphene platelets. No D’ modes are visible due to the superposition of the G and D’ modes, but it is always present in graphene with defects and can be estimated as half of the 2D’ peak (at about 3160 cm−1 at 2.33 eV) [D’ peak superposed by the G peak. Since D’ is more sensitive to vacancies compared to sp3 sites, this means that the majority of the defects in this material are sp3 hybridizations rather than vacancies [The number of layers can be estimated from the shape and position of the 2requency ,35,36. T2.33 eV) . As can acancies .2, printed using a graphene concentration of 1 g/L towards humidity. The sensor was heated up at 250 °C directly before starting the measurements and kept at this temperature during the measurements; this procedure helps remove the water molecules from the surface. All produced sensors do not exhibit a strong change in the resistance with respect to relative humidity. This represents a significant advantage to MOX sensors. The motivation for this behavior is not yet fully understood. One hypothesis is that the resistance change due to adsorption of water molecules at the boundary defects is partially compensated by the edge defects, where molecules form conductive chains through the graphene flakes [e flakes . A secone flakes .2 and NH3 were measured keeping the relative humidity of the synthetic air and gases mixture to a value of 20%, which represent a dry ambient environment. In 0 has a thick graphene layer, homogeneously distributed over the electrode surface, and its response to the different concentrations of gases is represented by the black curve in 4 has the same amount of graphene flakes, but a strong coffee stain, which means that the sensing area is drastically reduced compared to S0. Its response is represented by the red curve. The response of sensor S0 is faster and shows a higher sensitivity compared to S4, as expected due to the smaller sensing area. The recovery of sensor S0 is slower due to the higher adsorption of the gas molecules. The fast response of the sensor can be explained by an initial physisorption mechanism. The long recovery time is caused by the presence of both physisorption and chemiadsorption phenomena; the defects in the graphene flakes indeed cause, most likely, chemiadsorpion on the defect sites [3 and S7, probably also due to an insufficient amount of material to form a continuous film, the sensor resistance is high and a high thermal noise level can be observed on the output signal. In this case, a deviation in the sensitivity of similar sensors is observed, caused by the noise and the smaller sensing area. In all the presented measurements of the 8 sensor fields, the signal during adsorption did not reach the saturation.The response of the different sensor fields towards NOct sites . It can ct sites . Neverth2 the sensors exhibit a first rapid response, which corresponds to the first steep part of the slope, followed by a slower response which corresponds to a shallower slope. According to Robinson et al., the rapid response comes from the adsorption of gas molecules into low energy binding sites, like 2sp bonded carbon, while the slow response is caused by the gas molecular interaction with higher energy binding sites, e.g., vacancies, structural defects, and oxygen functional groups. The adsorption of molecules into low energy binding sites occurs through physisorption and involves weak dispersive forces, while the interaction with higher energy binding sites involves a chemisorption mechanism and therefore energies of several hundred meV/molecule. The physisorption which occurs during the rapid response is therefore recoverable, while the chemisorption which occurs during the slow response needs some moderate heating to recover [During exposure to NO recover ,39.3 and S7 and the small reaction at low gas concentration, this analysis is presented just for 6 of the sensors and for NO2 concentration of 100 and 200 ppb. It can be observed in 2 and 100 s when at 200 ppb NO2, caused by a faster occupation of low energy binding sites when more gas molecules are present. The peak amplitude of the signal’s first derivative has been used to compare the reaction time of the different sensors, this provides a quantitative value for the maximum steepness of the sensor’s initial response. Results are visible in The response time of the sensors cannot be properly calculated since the signal did not reach saturation, however, the physisorption time has been estimated through an analysis of the first derivative, as shown in 0, S1, S2, S3, S4, S5, S6, and S7 after exposure to different concentrations of NO2. Sensors S0 and S4, which have higher amounts of graphene flakes show a very slow recovery, 30 min for desorbing 20 ppb NO2 and more than 40 min for desorbing 100 and 200 ppb NO2. Sensors S1 and S5 show three times faster recovery from 20 ppb NO2 exposure, while the recovery from higher gas concentration is very similar to S0 and S4. The other sensors show a noisier signal and therefore different response times even with similar amounts of graphene. However, the results indicate that thinner graphene layers have faster recovery time. The hypothesis is that the thick graphene layer presents a porous structure due to the stacking of crumbled graphene flakes which can slow down the desorption of the gas molecules from the film.The sensor’s recovery time has been calculated as the time for the sensor to reach the 90% of its stable recovered condition, as displayed in 4) has a very low sensitivity and almost no reaction at 0.5 and 1 ppm NH3. Sensor S0 shows response even to the lower concentration but also a drift in the baseline, due to an incomplete desorption. In 1 and S5 is plotted, showing the same response for the two sensors. It can be observed that at low NH3 concentration the slope of the response is shallow, indicating that the adsorption centers with high binding energy are occupied first, this also explains that almost no recovery happens at low gas concentration [ntration .3 concentration (10 ppm) a fast response occurs, followed by a slower one, which would suggest physisorption on the low binding energy adsorption centers and just after this phase the occupation of adsorption centers with high binding energy. In this case a fast recovery of the sensor is observed. As visible in 2 and S6 is noisy and a response towards NH3 can be detected just at high concentration. Sensors S3 and S7 (At the higher NH3 and S7 d show toThe sensitivity of these sensors towards ammonia is significantly lower than the one towards nitrogen dioxide due to the lower binding energy which causes a lower adsorption density ,41,42.0, S1, S4, and S5 and during exposure to 10 ppm NH3 due to the low signal to noise ratio and very low response of the other sensors at lower concentrations. 1 and S5 compared to the sensors with higher amount of graphene, indicating a stronger response of the sensors, which is consistent with the sensor’s sensitivity (displayed in The physisorption time and the first derivative’s peak amplitude have been calculated only for sensors S0, S1, S4, and S5 after exposure to 10 ppm NH3 is illustrated in The recovery time of sensors S0 towards 200 ppb NO2 and 10 ppm NH3 at two different temperatures of the sensor, 100 and 250 °C, is shown in 3 at higher temperature might be caused by thermal fluctuations of the NH3 molecules which decrease the probability of the molecules to attach on the surface [2, this would cause higher thermal fluctuations in the presence of ammonia rather than nitrogen dioxide. Therefore, the operation temperature of the sensor should be optimized if higher sensitivity towards ammonia is required.The response of sensor S surface . Since t surface  and ammo3 and different concentrations of NO2. As expected some cross sensitivity can be observed between NO2 and NH3 due to the reaction of the sensor to both gases. Since ammonia is donating electrons while nitrogen dioxide is accepting them and behaving graphene as a p-type material, the first gas will increase the resistance of the sensor and the second will decrease it. This would lead to an underestimation of the NO2 concentration compared to the real value and a non-detection of NH3. However, since the sensitivity of the sensor towards ammonia is significantly lower, if the ammonia concentration is below a few ppm this will not severely affect the sensitivity of the sensor.2 because of the lower adsorption density and the operation temperature of the sensor being optimized for nitrogen dioxide [3 concentration the adsorption seems to be dominated by chemisorption and this would explain the slow desorption, while at higher concentration physisorption also contributes and desorption occurs faster. Cross sensitivity measurements at high NH3 concentration have shown little influence compared to NO2, however, to be able to distinguish and predict the concentration of two different gases, employing more sensors with differently doped graphene becomes necessary. Some drift in the baseline has been observed but this is due to incomplete desorption and can be reduced by introducing higher temperature pulses to enhance the gas desorption or can be compensate by periodic calibration and data post processingThe presented sensors show a stable response when exposed to very different humidity levels, showing a great advantage compared to MOX. When the sensing layer is not homogeneous and does not cover all the electrode area, i.e., when coffee ring forms during ink drying, the reaction of the sensor towards gases is slower and so is the sensitivity due to the small sensing area and thus reduced number of adsorption sites. The right trade-off in the sensing film thickness is important: On one side when the layer is too thick the current flows through independent parallel paths, which brings to a lower sensitivity, on the other side when the film is too thin, thermal noise degrades the signal to noise ratio at the output. Moreover, thinner graphene films show higher sensitivity and faster recovery time. The sensitivity of the sensor towards ammonia is lower than the one towards NO dioxide ,42. At l


Protein S-acylation is a reversible post-translational modification involving the addition of fatty acids to cysteines and is catalyzed by transmembrane protein acyltransferases (PATs) mainly expressed at the Golgi complex. In case of soluble proteins, S-acylation confers stable membrane attachment. Myristoylation or farnesylation of many soluble proteins constitutes the initial transient membrane adsorption step prior to S-acylation. However, some S-acylated soluble proteins, such as the neuronal growth-associated protein Growth-associated protein-43 (GAP-43), lack the hydrophobic modifications required for this initial membrane interaction. The signals for GAP-43 S-acylation are confined to the first 13 amino acids, including the S-acylatable cysteines 3 and 4 embedded in a hydrophobic region, followed by a cluster of basic amino acids. We found that mutation of critical basic amino acids drastically reduced membrane interaction and hence S-acylation of GAP-43. Interestingly, acute depletion of phosphatidylinositol 4-phosphate (PtdIns4P) at the Golgi complex reduced GAP-43 membrane binding, highlighting a new, pivotal role for this anionic lipid and supporting the idea that basic amino acid residues are involved in the electrostatic interactions between GAP-43 and membranes of the Golgi complex where they are S-acylated. Protein S-acylation, also known as palmitoylation, consists of the addition of a lipid molecule to one or more cysteine residues through a thioester bond. This modification, which is widespread in eukaryotes, allows the reversible association of peripheral proteins with membranes or, in the case of integral membrane proteins, modulates their behavior within the plane of the membrane due to their partition to different micro- and nanodomains via interaction with other proteins and lipids .S-acylation is mediated by protein acyltransferases (PATs) belonging to the zinc finger DHHC (aspartate-histidine-histidine-cysteine) family ,5, whereFor many soluble proteins, myristoylation or farnesylation on specific amino acid residues provides sufficient lipophilicity for transient membrane adsorption prior to S-acylation, which strongly increases the binding to the lipid bilayer . Thus, fBiophysical and biochemical studies have demonstrated the active participation of domains rich in basic amino acids in the electrostatic interaction between acylated proteins and anionic lipids of artificial and biological membranes ,17–21. FN13GAP-43), comprising the two S-acylatable cysteines close to a cluster of positively charged amino acids  were kindly provided by Dr. Tamas Balla  and are described in  proteins. CHO-K1 cells transiently transfected to express N13GAP-43 or each of the mutants was analyzed by confocal fluorescence microscopy. As shown in N13GAP-43 localized in the perinuclear region (identified as TGN), plasma membrane and recycling endosomes, whereas the mutant N13GAP- 43 remained completely cytosolic  protein, which was found largely accumulated in the cytosol, with a minor fraction associated with the Golgi complex and completely absent from the plasma membrane   and N13GAP-43, whose net charge is +1. Interestingly, the mutation of arginine residues—but not that of lysine residues—caused a noticeable accumulation of the mutants N13GAP-43 and N13GAP-43 in the cytosol (N13GAP-43(R6A) and N13GAP-43(R7A), and the double mutant N13GAP-43. Imaging of single mutants revealed that either of these residues (R6 and R7) are critical for S-acylation, since both mutants distributed to the same compartments as N13GAP-43  also distributed to the same compartment as N13GAP-43, confirming that the physical properties of amino acids at positions 6 and 7 are more critical for the efficient S-acylation of the N13 motif than the amino acid itself. In order to quantify the amount of cytosolic, deacylated protein fraction, we analyzed the extent of membrane association and the S-acylation status of these mutants by centrifuging extracts from mechanically lysed cells  was completely associated with the soluble fraction. The single mutant N13GAP-43 mostly partitioned in the pellet fraction, in accordance with the subcellular localization reported above, while a significant fraction of the triple mutant N13GAP-43 was present in the soluble fraction after centrifugation. It should be taken into account that the soluble protein fraction could be underestimated due to the length of time required for expression in biochemical experiments. In agreement with the drastic change in the subcellular localization observed for N13GAP-43, we found that most of the protein was present in the soluble fraction and only a minimal amount remaining attached to membranes, indicating that most of the mutant lack the lipid modification  mutant, which could not be assessed because of the reduced expression of this mutant protein and/or low sensibility of the ABE assay. It should be mentioned that the ABE assay is an indirect and very useful method to demonstrate S-acylation of protein. However, it is not quantitative and complementary techniques are required to perform comparative and quantitative analysis.The fact that a basic cluster is highly conserved at the N-terminal (N13) motif of GAP-43 led us to hypothesize that electrostatic interactions could mediate the initial absorption of this protein to the membranes, to be later modified by PATs. To investigate the role of basic residues, we generated several alanine (A) substitutions C and comytosolic A. Imaginmembrane A,C. ThisK9A) see C and the cytosol A,C. It i13GAP-43 A. The doed cells B. N13GAPfication B. Using fication D except N13GAP-43, N13GAP-43 and N13GAP-43  present in the N13 motif by acidic glutamic residues  A. By bio/K9,13E) B,C, and fullGAP-43) protein. Our results indicate that the presence of arginines is the minimal requirement for efficient S-acylation of N13GAP-43, but mutation of the four basic residues  drastically affected this lipid modification. Thus, we generated the fullGAP-43 and fullGAP-43 mutants, fused to YFP, to further characterize their subcellular distribution in CHO-K1 cells. Confocal fluorescence imaging revealed that mutation of arginine residues caused an accumulation of the mutant in the cytosol , a minor fraction of fullGAP-43 mutant was still associated with membranes, while the fullGAP-43 mutant was found mainly in the cytosol. Overall, these results indicate that signals present in the first 13 amino acids of GAP-43 are essential for its proper S-acylation.We then sought to evaluate the role of basic residues in S-acylation in the context of the full-length GAP-43  or N13GAP-43 and their subcellular localization analyzed. A similar analysis was also carried out using the full-length versions of both GAP-43 and mutants  was drastically reduced and a minor fraction remains associated with perinuclear membranes. The full-length mutant fullGAP-43 was able to associate with membrane, although an evident pool was observed in the cytosol  polypeptide fused to a fluorescent tag, mCherry (mCh), followed by the PH domain of the PtdIns4P adaptor protein-1 (FAPP1) -mCh-PH FAPP1). The PH domain specifically interacts with PtdIns4P, the major phosphoinositide present in the TGN, forcing N13GAP-43, found mainly in the cytosol, to interact with membranes where the PATs reside. CHO-K1 cells were co-transfected to express N13GAP-43 and the PH chimera. Interestingly, N13GAP-43-mCh-PH FAPP1 was found completely anchored to membranes and localized at the same subcellular compartments as N13GAP-43  motif and probably depends on S-acylation. To explore this possibility in detail, CHO-K1 cell expressing both the PH chimera and N13GAP-43 proteins were incubated in the presence of the PAT inhibitor 2BP (50 μM) and the subcellular localization was analyzed. 2BP should be used carefully to avoid erroneous interpretations since it is a non-specific inhibitor of S-acylation. Thus, 50 μM 2BP was the optimal and recommended concentration used in our experiments to avoid an effect on protein S-deacylation  and phosphatidylinositol 3,4,5-trisphosphate [PtdInsP3] lipids to the regulated plasma membrane targeting of proteins containing clusters of polybasic amino acids ,19,39. MClick here for additional data file.Click here for additional data file.


Graphene-based nanomaterials (GBNs) have attracted increasing interests of the scientific community due to their unique physicochemical properties and their applications in biotechnology, biomedicine, bioengineering, disease diagnosis and therapy. Although a large amount of researches have been conducted on these novel nanomaterials, limited comprehensive reviews are published on their biomedical applications and potential environmental and human health effects. The present research aimed at addressing this knowledge gap by examining and discussing: (1) the history, synthesis, structural properties and recent developments of GBNs for biomedical applications; (2) GBNs uses as therapeutics, drug/gene delivery and antibacterial materials; (3) GBNs applications in tissue engineering and in research as biosensors and bioimaging materials; and (4) GBNs potential environmental effects and human health risks. It also discussed the perspectives and challenges associated with the biomedical applications of GBNs. Structures and properties of graphene-based nanomaterials (GBNs) including graphene, graphene oxide (GO), reduced graphene oxide (RGO) and modified graphene are introduced briefly.Recent advances in GBNs for the biomedical applications in drug delivery, biosensor, bioimaging and tissue engineering are summarized and analyzed.Potential risks resulted from the vast production and applications of GBNs to the environment and health are discussed to ensure sustainable development of GBNs.This review focuses on the recent advances in graphene-based nanomaterials (GBNs) in the field of biomedical applications and their potential environmental and health risks. Graphene, the mother of all carbon atoms, is a single atomic thick, nanosized, two-dimensional structure and provides high surface area with adjustable surface chemistry to form hybrids. It was synthesized from graphite. In this review, we addressed the current state of the science and identified the knowledge gap for the future research development. The broad family of GBNs listed in this review includes graphene, graphene oxide (GO), reduced graphene oxide (RGO) and chemically modified graphene  .Although carbon-based materials such as fullerene, graphite, graphene and carbon nanotubes have been widely used due to their unique properties and nanoscale dimensions –7, GBNs Even though graphene came into existence in the year 1859 by a British Chemist Benjamin Collins Brodie , it has GBNs have been classified based on the number of layers in the sheet, oxygen content and their chemical composition. There are many structural differences between GO and RGO which determine their physicochemical properties. Figure sp2-hybridized and has four bonds, one σ bond with each of its three neighbors and one π-bond that is oriented out of the plane. It has a hexagonal pattern, forming a honeycomb crystal lattice. It is produced by mechanical or chemical exfoliation of graphite via chemical vapor deposition. It has a large specific surface area, high intrinsic mobility and high thermal conductivity. Graphene is considered as hydrophobic because of the absence of oxygen groups.Graphene is a single carbon layer of the graphite structure. It is a two-dimensional planar and hexagonal array of carbon atoms. Each of these carbons is sp2) and aliphatic (sp3) domains which facilitate the interactions at the surface  is commonly used in various forms and shapes in bone and tissue engineering. However, due to their lack of mechanical strength, its usage is discouraged from replacing various parts of the bone system  fr fr151] f−1. The quality of the effluent was deteriorated by increasing the turbidity of water and the reduction in sludge dewaterability. They also confirmed the reactive oxygen species generation is responsible for the toxicity of GO on microbial communities. Deng et al. studied the characterization factors such as toxic effect factor, fate factor and exposure factor of GO in the environment to study the life cycle impact assessment of GO-based nanomaterials [As it is projected that the GBNs-based products market to reach millions of dollars by 2020, there will be a generation of GBNs-based wastes into the environment. It is essential to evaluate the potential toxic effects and fate of GBNs in the environment. Only a few researchers evaluated the impact of GBNs in environment. Ahmed et al. investigated the acute effects of GO on waste water microbial community . They coaterials . More reaterials . In addiaterials .It is evident that GBNs, because of their unique properties and functionalization, raise a great interest and provide more avenues for the research and development in their applications of translational medicine. The biomedical applications related to the unique physiochemical properties of GBNs focus on their thermal, mechanical and electrochemical features. Many reports have paid attention to GBNs as therapeutics in cancer therapy along with PTT and PDT, gene/drug delivery and as antibacterial agents. The intrinsic optical properties of GBN-based hybrids in the visible and NIR range along with their small size effects, low toxicity and low production costs make the hybrids attractive for bioimaging in clinical diagnostics and photo-thermal cancer therapy. This targeted therapy aids in their high therapeutic effects and fewer side effects. Among GBNs, GO and RGO are considered as the most potent antibacterial agents which can be used in nanohybrids to synthesize novel antibacterial agents. The use of GBNs in bioimaging and biosensing fields is an emerging biomedical application. As the GBNs are used in various fields for biomedical purposes the safety and efficacy of GBNs in clinical trials such as diagnostics and therapeutics require standardized parameters; mainly, biocompatibility, solubility and selectivity are the predominant factors that will further the biomedical applications of GBNs. More studies in computational simulations of GBNs need to be explored for the efficacy of GBNs in clinical trials.The applications of GBNs have expanded quickly into various fields, but still, there is a lack of systematic understanding of biological interactions of GBNs. The experimental data on the toxicity are limited more to in vitro rather than to in vivo. In addition, there should be more knowledge of the long-term toxicity effects of GBNs to further enhance their applications in the biomedical field to assure the human safety. The existing literature does not provide detailed information on the various synthesis procedures and characterization techniques before proceeding to the toxicological assays. More emphasis should be given on the comprehensive understanding of GBNs-based products on adsorption, dispersion and toxicity, and transformation is recommended. Currently, most of the GBNs are focused on lungs and liver. Studies on other organs including brain/central nervous system are very limited or remain unexplored. Moreover, the GBNs due to excellent physicochemical properties can specifically disrupt the neuroendocrine/reproductive organs. To our knowledge, the reports on GBNs as endocrine disruptor are very limited. Additional studies in these areas are also necessary. Furthermore, GBNs can interact with DNA and thus affect the genetics of human populations. Therefore, studies are needed to elucidate transgenerational effects or effects of GBNs on the epigenome. Reina et al. emphasized the guidelines and the development and evaluation of biocompatibility of GBNs . These g