O,O’-acac)(γ-acac)(DMS)] ([Pt(acac)2(DMS)]) exerted substantial cytotoxic effects in SH-SY5Y neuroblastoma cells, and decreased metalloproteases (MMPs) production and cells migration in MCF-7 breast cancer cells. The ubiquitously distributed sodium-hydrogen antiporter 1 (NHE1) is involved in motility and invasion of many solid tumours. The present study focuses on the effects of [Pt(acac)2(DMS)] in SH-SY5Y cell migration and also on the possibility that NHE1 may be involved in such effect. After sublethal [Pt(acac)2(DMS)] treatment cell migration was examined by wounding assay and cell invasion by transwell assay. NHE1 activity was measured in BCECF-loaded SH-SY5Y as the rate of Na+-dependent intracellular pH recovery in response to an acute acid pulse. Gelatin zymography for MMP-2/9 activities, Western blottings of MMPs, MAPKs, mTOR, S6 and PKCs and small interfering RNAs to PKC-ε/-δ mRNA were performed. Sublethal concentrations of [Pt(acac)2(DMS)] decreases NHE1 activity, inhibites cell migration and invasion and decreases expression and activity of MMP-2 and -9. [Pt(acac)2(DMS)] administered to SH-SY5Y cells provokes the increment of ROS, generated by NADPH oxidase, responsible for the PKC-ε and PKC-δ activation. Whilst PKC-δ activates p38/MAPK, responsible for the inhibition of MMP-2 and -9 secretion, PKC-ε activates a pathway made of ERK1/2, mTOR and S6K responsible for the inhibition of NHE1 activity and cell migration. In conclusion, we have shown a drastic impairment in tumour cell metastatization in response to inhibition of NHE1 and MMPs activities by [Pt(acac)2(DMS)] occurring through a novel mechanism mediated by PKC-δ/-ε activation.We previously showed that [Pt( Among these exchangers, NHE1 is considered a main regulator of intracellular pH (pHi) in cancer cells; it is regulated by pHi and oncogenic transformation + across the plasma membrane Neuroblastoma is the most common tumor among children under 1 year of age and is therefore prone to early metastasis; in 60–70% of neuroblastoma cases, systemic organ or bone marrow metastasis is detected at presentation and is the leading cause of death in these patients. It is of great significance to explore the metastatic mechanisms of neuroblastoma, particularly the mechanisms underlying its systemic dissemination. Cell migration is a complex dynamic process that involves multiple biological features. Highly proliferating cancer cells generate increased levels of lactic acid by Warburg 2(DMS)]), a Pt(II) complex specifically designed and synthesized by some of us cisplatin. [Pt(acac)2(DMS)] is very stable under physiological conditions 2(DMS)] on various cell cultures invitro  indicates that it does not require extracellular organismic activation. The possibility that it may act as an intracellularly activated prodrug is also very low. This is because being DMS (dimethylsulphide) the only labile ligand in the metal coordination sphere of this complex, covalent binding to a biological target via DMS substitution or host guest interaction with a specific cellular receptor are the expected drug action mechanisms. Recent work also demonstrated that in these complexes substitution of the sulphur ligand with the much more inert ammonia gave almost the same in vitro cytotoxicity results on cisplatin resistant MCF-7 cells, therefore an activity based on some molecular recognition mechanism may be involved cisplatin. [Pt(acac)2(DMS)] exerts higher and selective cytotoxicity towards cancer, as observed in immortalized cell lines and confirmed in breast cancer cells in primary cultures and in mouse xenograft model of breast cancer 2(DMS)] is able to exert cytotoxic effects also on the human neuroblastoma SH-SY5Y cells, which are immature neuroblasts 2(DMS)] to exert specific antimetastatic responses invitro. In this paper, we provide evidence that sublethal doses of [Pt(acac)2(DMS)] altered some basic biological features of the SH-SY5Y cells, such as the NHE1-mediated regulation of pHi, migration and invasion.In the present study we use [Pt(γ-acac)(DMS)] ( administration.Several protein kinases have been proposed to regulate NHE1 activity, including ERK1/2, NIK, ROCK1, mTOR, p902(DMS)] exerts antimetastatic responses in vitro, decreasing metalloproteases production in MCF-7 cells. Thus, to further investigate the molecular mechanism affecting invasion of SH-SY5Y cells, we studied the [Pt(acac)2(DMS)]-mediated decrease of MMP 2 and 9 activities.Regarding invasion, migrating cells must break through the basement membrane by secreting enzymes, including metalloproteinases 2(DMS)] inhibits Na+/H+ exchange.Our results provide new insights into the relationship between NHE1 and metastatic process, and also demonstrate that activation of ERK is a critical component of the signaling pathway through which  treatment, the culture medium was collected and SH-SY5Y cells were washed with PBS  and harvested by scrapping in 0.1 M Tris–HCl containing 0.2% Triton X-100. Both conditioned medium and cell lysates were centrifuged at 14,000 rpm for 5 min at 4°C to remove cells and debris as described previously After  was prepared according to previously reported procedures  at concentrations ranging from 1 to 200 µM resulted in a dose-dependent inhibition of cell survival 2(DMS)] had effects on SH-SY5Y cell invasion and migration without affecting cell viability, we here used low drug concentrations  and assessed that were not able to induce apoptosis nor assayable cytotoxicity 2(DMS)] on the invasive potency of neuroblastoma cells. As illustrated in 2(DMS)]. [Pt(acac)2(DMS)] reduced the migration ability of these cells by 80% (P = 0.026 by Anova) after 24 h (2(DMS)] inhibited the invasion of SH-SY5Y cells by 83% (2(DMS)] prevents invasion and migration of SH-SY5Y human neuroblastoma cell line.We showed previously that exposure of the SH-SY5Y cells to [Pt(acac)toxicity . In vitrter 24 h . Similars by 83% . These r2(DMS)] effect. Both the migration ability and the transwell invasion potential were strongly inhibited by the specific NHE1 inhibitor, Zoniporide niporide , leading4Cl in the absence of extracellular Na+, after which the NH4Cl was removed from the superfusate (0 Na+ solution), resulting in an intracellular acidification of ∼0.9 pH units to ∼6.3. As seen in +. Reintroduction of extracellular Na+ resulted in a Na+-dependent intracellular alkalinization back to a pHi of ∼6.75, at a rate of 0.03 pH U/min. The rate (slope) of the Na+-dependent intracellular alkalinization, calculated from the point at which recovery starts, as indicated by the dashed line in +-dependent pHi recovery suggesting that NHE1 is responsible for the Na+-dependent pHi recovery in these cells.SH-SY5Y cells constitutively express amiloride-sensitive NHE1 +) and its activities in the presence of increasing [Pt(acac)2(DMS)] concentrations: such activity decreased, in a dose-dependent manner, by 41%, 78% and 96% for 0.10, 0.25, 0.50 µM [Pt(acac)2(DMS)], respectively (p<0.001). In SH-SY5Y cells, the inhibition of Na/H exchanger activity was restricted to [Pt(acac)2(DMS)] since cisplatin , did not affect it (new t it new .2(DMS)] activated ERK1/2 2(DMS)] and analyzed for activated ERK1/2. Results show that [Pt(acac)2(DMS)] provoked a time dependent activation of ERK1/2. To determine whether [Pt(acac)2(DMS)] block NHE1 activity by ERK1/2 activation, cells were preincubated with PD98056, a MEK inhibitor that prevented ERK1/2 activation (2(DMS)] inhibited the recovery response by 96% , whereas PD98056 restored the response to control (from 0.18±0.02 to 0.19±0.04). These results are consistent with [Pt(acac)2(DMS)]-induced ERK1/2 activation playing an inhibitory role on NHE1 activity. To confirm this, we blocked the MEK-induced ERK1/2 activation by preincubating with PD98059 (0–25 µM) and then determined whether [Pt(acac)2(DMS)] would still effect cell migration. The effects of [Pt(acac)2(DMS)] on wound-healing assays were reversed by PD98056 (We previously showed that cytotoxic doses of [Pt(acac)tivation . As show PD98056 .2(DMS)] treatment reaching a maximum at 10 min (2(DMS)] had effects also on additional signaling components in the p70S6K pathways, i.e. increased mTOR phosphorylation, with the same kinetic of S6 phosphorylation, without changing the total mTOR level (2(DMS)] (The role of p70S6K in cell migration t 10 min . [Pt(acaOR level . PD98059(2(DMS)] .2(DMS)] effects; thus, two commonly used PI3K inhibitors, LY294002 and wortmannin and the mTOR inhibitor rapamycin (γ-acac)(DMS)]-induced mTOR and S6 phosphorylations and also abolished the inhibition of NHE1. Rapamycin did not affect the [Pt(acac)2(DMS)]-induced ERK1/2 phosphorylation (2(DMS)] (2(DMS)] effects on NHE1 inhibition (2(DMS)]-induced inhibition of SH-SY5Y cells Matrigel invasion were observed (2(DMS)], restored cell invasion capacity (We investigated the role and mTOR and PI3K in [Pt(acac)rylation . LY29400(2(DMS)] . These rhibition  and on whibition , but notobserved . Finallycapacity .2(DMS)] on cells viability (data not shown). These data implied that the effect of MMP-2/MMP-9 inhibition on Matrigel invasion cannot be simply attributed to the inhibition of cell proliferation. [Pt(acac)2(DMS)] had effects on both the levels and activities in of MMP-2 and -9 (It is known that MMP-2 and MMP-9 expression may be related to metastasis and progression of neuroblastoma. The role of MMP-2 and MMP-9 in SH-SY5Y cell invasion was assessed using the MMP-2/MMP-9 inhibitor II. Pretreatment of cells with MMP-2/MMP-9 inhibitor II blocked cell invasion in a concentration-dependent manner  but did 2 and -9 .2(DMS)] provoked the phosphorylation of p38MAPK and JNK1/2 2(DMS)] induced a time-dependent phosphorylation of p38MAPK, without affecting the overall level (2(DMS)]-induced MMP 2 and 9 inhibition were examined. When SH-SY5Y cells were pretreated for 30 min with SB203580 (1 and 10 mM), and then with 0.50 µM [Pt(acac)2(DMS)] for 24 h, the inhibition of MMP-2 and MMP-9 expression levels was blocked (2(DMS)] on Matrigel invasion were unchanged (2(DMS)] on Matrigel invasion were reversed when SH-SY5Y cells were pretreated with SB203580 plus PD98059 or SB203580 plus rapamycin (We showed previously that exposure of SH-SY5Y cells to 1–100 µM -mediated PKC-δ and -ε activation and consequential phosphorylation of p38MAPK and ERK1/2 2(DMS)] (2(DMS)] on both NHE1 activity (2(DMS)] was blocked when PKC-ε and PKC-δ were both silenced (We previously showed that ROS (reactive oxygen species) generated by NADPH oxidase was responsible for the [Pt(acac) 2(DMS)] . PKC-ε-sactivity  and cellactivity . The inhsilenced .2(DMS)], led to inhibition of migration of mammary tumour cells 2(DMS)] inhibition of MMP-2 and MMP-9. In addition, the effects of [Pt(acac)2(DMS)] on NHE1 activity activity , wound-hactivity  and tranactivity  were revThe tumor acidic microenvironment exerts profound influence on cancer cell invasion and metastasis and these characteristics are considered as important targets for the development of new anti-cancer therapies. Despite numerous studies in this field, the different relationship between pHi and cell invasion is still poorly understood. Nine mammalian NHE isoforms (NHE1–NHE9) are known and several are upregulated and/or activated in tumours 2(DMS)]. We previously showed that besides its potent anti-cancer activity invitro and invivo2(DMS)] also prevented the events leading to metastasis via alterations of MMP-2 and -9 production and activity in MCF-7 breast cancer cells 2(DMS)] activated PKC-ε/PKC-δ and ERK1/2 via the ROS generated by NADPH oxidase. The inhibition of ERK1/2 blocks the [Pt(acac)2(DMS)]-induced NHE1 inhibition and restores SH-SY5Y cell motility. Similarly, ERK1/2 activation by Troglitazone decreased NHE activity and pHi in MCF-7 cells and in proximal tubule cells 2(DMS)] increases the phosphorylation of mTOR and also of the downstream component in rapamycin-dependent mTOR signaling pathway, S6. Little is known regarding the mechanism by which p70S6K regulate acid extrusion; however, it has been reported that, in cultured human renal proximal tubule cells, gastrin-induced S6 activation increased the phosphorylation and internalization of NHE3, therefore decreasing its expression at the cell surface In this paper we show that the inhibition of NHE1 constitutively expressed by SH-SY5Y cells 2(DMS)] activates Akt, we here show that the inhibition of PI3K does not affect mTOR phosphorylation. Thus, regulation of p70S6K in response to [Pt(acac)2(DMS)] must therefore involve other signaling events. Although the p70S6K and ERK cascades have been believed to lie on distinct pathways 2(DMS)] to NHE1 inhibition through ERK, mTOR, and S6, as shown in 2(DMS)] as anticancer and immunostimulatory agent for a vast array of malignancies would appear to be worthwhile. In addition to its well-known ion translocation function, the C-terminal cytoplasmic tail of NHE1 interacts with actin-binding proteins resulting in promotion of cytoskeletal reorganization and cell migration. Indeed, the inhibition of critical component in these pathway restored SH-SY5Y cell motility in wound-healing assays but not in transwell invasion assays Akt is a downstream target of PI3K that links PI3K to mTOR 2(DMS)] inhibited expression and secretion of MMP2 and MMP9 in SH-SY5Y cells.Cell invasion typically characterize cancer cells with actin remodeling being one of the crucial elements of a deregulated actin system in malignant transformation. Degradation of ECM is another of the first steps in tumor invasion and metastasis and metalloproteases (MMPs) have been strongly implicated in this step 2(DMS)] on tumor cell invasion is associated with p38/MAPK. In fact, the p38/MAPK inhibitor SB203580 reversed the [Pt(acac)2(DMS)]-provoked inhibition of MMPs secretion (2(DMS)]-provoked inhibition of cell migration through trans-wells (2(DMS)] was blocked by the inhibition of the NADPH oxidase (2(DMS)] may be through a down-regulation of MMP2, MMP9 levels and NHE activity of SH-SY5Y cancer cells.The effects of [Pt(acac)ecretion . We detens-wells . The act oxidase . Taken t2(DMS)] occurring through a novel mechanism mediated by PKC-ε/ERK/mTOR pathway. Since altered proton gradients established through upregulated NHE activity sensitise tumour cells much more than normal cells to intracellular acid damage when NHEs are inhibited 2(DMS)] of the molecular mechanisms leading to NHE1 activation may therefore represent an alternative approach to decrease cancer invasion and motility.In conclusion, we have shown a drastic impairment of cell motility in response to the inhibition of the NHE1 by [Pt(acac)


We show that these points are responsible for the development of the superstructure, while charge from the Pt substrate is injected into the graphene, inducing a local n-doping, mostly localized at these specific pinning-point positions.The intrinsic atomic mechanisms responsible for electronic doping of epitaxial graphene Moirés on transition metal surfaces is still an open issue. To better understand this process we have carried out a first-principles full characterization of the most representative Moiré superstructures observed on the Gr/Pt(111) system and confronted the results with atomically resolved scanning tunneling microscopy experiments. We find that for all reported Moirés the system relaxes inducing a non-negligible atomic corrugation both, at the graphene and at the outermost platinum layer. Interestingly, a mirror “anti-Moiré” reconstruction appears at the substrate, giving rise to the appearance of  Several substrates are routinely used in the search for high-quality free-standing graphene single layers, among which transition metal (TM) surfaces represent a good model system where to rationalize their basic properties223Graphene growth on metal surfaces is one of the most promising routes towards scalable production of high-quality grapheneThe interaction between high-symmetry metal substrates and graphene varies from weak physisorption to strong chemisorption6910111216171819et al.Although there is a large amount of works devoted to the study of the Moiré structures6789101112131415161718192021pinning-points for the Moiré superlattice, whilst the rest of the graphene layer interacts weakly with the metallic substrate, and can be considered unaffected by the metal underneathIt is important to remark that the aforementioned phenomenological model only takes into account very simple geometrical considerations. The outcome rises from the numerical search of the best matching pair of C—Pt atoms for any given rotational angle, considering any lateral relaxation within the system negligible. Surprisingly, the determination of the minimum mismatch exclusively for a pair of atoms describes the stable domain orientations in the Gr/Pt(111) system. The main consequences of this experimental observation are noteworthy. The fact that minimization of the mismatch exclusively at the coincidence point (rather than minimization of the strain over the whole unit-cell) yields the best predictions introduces a novel scenario where most of the interaction must be ruled by the coincidence of some particular atoms within the Moiré unit-cell. These regions would act as sort of To better understand all these effects, a characterization by means of first-principles density functional theory (DFT) of the structural and electronic properties of the different Moirés becomes necessary. However this turns into a very challenging task due to large size of the unit cells involved. To this aim, in the present work we analyze the nature of the graphene—substrate interaction for several Moiré superstructures appearing for Gr/Pt(111) by using an adequate combination of local ultra-high vacuum (UHV) STM experiments and first-principles calculations, accounting for an accurate – given the high amount of atoms involved in some of the calculations – van der Waals (vdW) interaction. We show hereafter that the Pt surface atoms tend to relax out of plane, approaching towards the graphene layer and originating a sort of low-dimensional draining points where charge can efficiently flow between the two materials.−10 mbar equipped with low energy electron diffraction (LEED) optics and STM at room temperature (RT). The temperature of the samples was monitored by an IR pyrometer with an emissivity of 0.25. The samples were cleaned by the conventional procedure of repeated cycles of argon sputtering and annealing (at 1200 K for 20 min). The first annealing cycle was performed in an oxygen atmosphere (P = 1 × 10−6 mbar) to remove major carbide impurities. The sample surface cleanliness was checked by STM and LEED prior to the graphene growth. Commercial C60  was deposited at a rate of 0.4 monolayers per hour during 30 min, keeping the sample at room temperature. The samples were then annealed by electron bombardment at temperatures up to 1200 K for 5–10 min intervals. Importantly, during this procedure the pressure did not exceed 5 × 10−10 mbar. Submonolayer coverage of flat and homogeneous graphene islands was observed on top of clean Pt regions and, thus, modifying the deposition time the total coverage can be controlled. A long deposition time leads to complete monolayer coverage. STM images were recorded using topographic and current modes with typical biases of −250 to 250 mV and currents ranging between 0.1 and 4 nA. WSxM software was used for data acquisition and image analysisExperiments were carried out in an ultra-high vacuum (UHV) chamber with base pressure of 1 × 10We have used two different atomistic simulation packages: i) the plane-wave code PWSCF25−6 correction to add dispersive forces to conventional density functionals – see details in previous literature27303167316673133Thus, as starting point we use the atomic configurations predicted by the phenomenological model. Commensurate unit cells have been used to perform the calculations; while the geometrical model neglects relaxations and does not account for the commensurability of the system. However, the mismatches between commensurate and the incommensurate superstructures (predicted models) are smallThe ion–electron interaction is modeled by ultrasoft pseudopotentialsSTM simulations have been performed for all the Gr/Pt(111) Moiré configurations considered in this study, to be compared with the experimental UHV-STM images. In order to obtain STM images and tunneling currents, we used an STM theoretical simulation technique that includes – by construction – a detailed description of the electronic properties of both the tip and the sample. Using this technique, based on an effective combination of a Keldysh—Green formalism and local orbital density functional theory3132We have performed a quantitative theoretical characterization of five of the most stable and representative Moiré superstructures predicted by our aforementioned geometrical modelpinning-point just at the perpendicular coincidence between a C atom “on top” a Pt atom. The calculated Pt surface relaxation, induced by the interaction with the graphene, manifests the high malleability of Pt under these a-priori soft interactions. The region affected by the structural relaxation involves 2 Pt and 4 C atoms for the structure d, takes its highest value of 3.23 Å for the configuration 66pinning-points, this quantity does increase for increasing rotation angles. At 30°, the maximum possible angle, our group has reported the existence of an atomic vacancy network in the By inspecting the structural ripple heights see  observed642s from −0.5 to 0.5 eV. As an illustration, s up to 0.2 V. It can be seen that the periodic Moiré pattern in the STM image is well reproduced by theory, and not only the relative intensity but also the apparent size of the different STM features as valleys  and brighter zones (corresponding to topmost regions of the graphene Moirés).The simulated constant-current STM topographic images are in rather good agreement with the experimental ones in the range of VVs in the interval  V along the same selected scanning paths. The experimental results were obtained at room temperature in different experimental STM sessions, involving different tips (W-etched) and measuring conditions. The criteria for tuning the bias, current and feedback parameters are to maximize the resolution within the graphene superstructures. The overall variation with the tunnel bias is fairly well reproduced by theory for all the Gr/Pt structures. In particular, for the case of the A more stringent test is to compare STM profiles of apparent height (corrugation) along specific directions as the tunnel voltage varies. Vs = −0.15 and 0.15 V in Itunnel = 0.1 nA) for all the Gr/Pt structures considered in this study  interface has been previously reported− per C atom for the n-doped character of the graphene layer in all the superstructures, as reported previouslyOn one side, the charge transfer occurring in each Gr/Pt(111) Moiré has been calculated by means of a Bader analysisleft-shift  by effect of the formation of the different Moirés, we have designed a theoretical procedure consisting in obtaining the equilibrium geometries of flat graphene on Pt(111)  for each periodicity analyzed in this study. In this way, one can just integrate downwards the PDOS profile from the Fermi energy up to a point in the density of states profiles fulfilling the condition of integrating just up to the charge transfer obtained by the Bader analysis for all the cases. This theoretical strategy permits a direct comparison between the projected DOS on flat graphene on Pt for every domain with the projected DOS on graphene on Pt once the Moirés have been formed. By construction, this direct comparison yields the Dirac-cone shift by effect of just the formation of the different corrugated Moirés, eliminating in the shift values so obtained any possible contribution coming from the tendency of Gr and Pt chemical potentials to align.On the other hand, in order to estimate the Dirac-cone left-shifts for all the different configurations , may be explained considering that the DOS moves towards lower energies to make available some of the empty states in the non-deformed graphene to accommodate the local excess of electronic charge coming from the Pt substrate in each Moiré with the respect the Fermi level of the interacting systems, in agreement with previous resultsleft-shift is more pronounced, of 0.26, 0.31 and 0.33 eV for pinning-point. The pinning-points in the side of the Pt substrate for the pinning-points for the a priori one could think, and demonstrates that many structural and electronic factors participate in such particular interaction.ions see . This obe Pt see  showing ions see . This fathe pinning-points extends to 3 near-neighbourset al.44pinning-points.The concept of pinning points for explaining epitaxial graphene overlayers on Pt(111) can be extended to other transition metal surfaces, although the interaction area can be different. On Ru(0001) a small relaxation of the outermost metal layer has been found, and the interaction area around pinning-points are still the closer to each other, and that they will be the point from where to inject charge until the separation between C and Pt atoms stop the coherence of the Moiré.Moreover, it is important to have in mind that our DFT-based model imposes commensurability, which may be absent in the real system. Nevertheless, the low mismatch between substrate and graphene makes that the atoms at the pinning-points in all the Moiré superstructures reveals the origin of the non-negligible charge transfer in all the configurations. The mechanism underlying these interface interactions can be understood in terms of the increase of the chemical overlapping between the Pt substrate and graphene, which is caused by a migration of electronic charge from the s orbitals in the Pt atoms at the pinning-points towards their own pinning-point as compared with a Pt atom of a clean Pt(111) surface. This enhances the orbital perpendicular orientation in the Pt atoms, favoring the hybridization with the pz orbitals from the Gr C atoms at the pinning-points. Those pz orbitals arise from the emerging sp3 character of the C atoms at the pinning-points as a result of the pristine graphene sp2 hybridization rupture by the Gr buckling just at those pinning-points. It is important to mention that, very recently, hybridization between pz orbitals in the Gr has been proposed by Vita et al. as origin of band-gap formation in the Gr/Cu/Ir(111) interfacen-doping mechanism in Gr/Pt(111) proposed here.An additional analysis of the projected density of states carried out on the substrate Pt atoms forming the pinning-points, which can be understood as the best coincident atoms between substrate and overlayer for a given rotational angle. At these pinning-points there is a significant atomic relaxation approaching the C and Pt atoms. As the distance is shorten, migrated electronic charge from the s towards the pz orbitals from the buckled Gr C atoms at the pinning-points. Thus, every one of the Moiré domain superstructures manifests a different n-doping and, therefore, the multidomain graphene/Pt(111) can be regarded as true graphene-laboratory for testing fundamental properties of doped graphene.Summarizing, we have shown that the structural and electronic properties of graphene superstructures on Pt(111) surfaces are dictated by the existence of How to cite this article: Martínez, J. I. et al. Role of the Pinning Points in epitaxial Graphene Moiré Superstructures on the Pt(111) Surface. Sci. Rep.6, 20354; doi: 10.1038/srep20354 (2016).


Under perpendicular external magnetic fields, two-dimensional carriers exhibit Landau levels (LLs). However, it has recently been reported that LLs have been observed on graphene and graphite surfaces without external magnetic fields being applied. These anomalous LLs have been ascribed primarily to a strain of graphene sheets, leading to in-plane hopping modulation of electrons. Here, we report the observation of the LLs of massive Dirac fermions on atomically flat areas of a nitrogen-doped graphite surface in the absence of external magnetic fields. The corresponding magnetic fields were estimated to be as much as approximately 100 T. The generation of the LLs at the area with negligible strain can be explained by inequivalent hopping of π electrons that takes place at the perimeter of high-potential domains surrounded by positively charged substituted graphitic-nitrogen atoms. In the presence of an external magnetic field, charge carriers of conductive material circulate in cyclotron orbits perpendicular to the field, with quantised energies called Landau levels (LLs). In the case of a two-dimensional electron gas, the energy differences between adjacent LLs are equal, and the quantised energy levels are described by the following equation:n is the Landau index, En is the energy of the nth LL, e is the electron charge, B is the external magnetic field and m* is the effective mass of the carrier arising from interactions with the lattice. The energy differences of adjacent LLs are, however, not the same if the carriers are massless Dirac fermions (DFs)123where vF represents the Fermi velocity. When two graphene layers form an AB-stacking bilayer, the carriers are no longer massless but rather massive DFs, i.e., they form a unique electronic structure5156Here, Note that the field-independent zero-energy state is still preserved as in equation 5The formation of LLs in graphene even without external magnetic fields being applied was theoretically predicted to be possible if a specific structural symmetry break, such as non-uniform shear strain in the lattice, occurred and a pseudo-magnetic field was generatedβ-site carbon of Bernal-stacked graphite, 0.245 ± 0.01 nm, which is typically observed at the flat region of the pristine graphite surface by STMz orbitals of carbon atomsThe nitrogen-doped graphite used in this study was prepared by nitrogen ion bombardment of an HOPG surface at room temperature, followed by annealing at 900 K in an ultra high-vacuum chamber. Scanning tunnelling microscopy (STM) images of this surface are shown in r was greater than 0.998 for the assignment of the STS peak at ~260 meV as the n = 0 energy level of the LLs of the bilayer graphene (Bs could thus be estimated from the slope of the fitted line as Bs = 63 ± 3–105 ± 5 T using equation m* of the bilayer graphene is 0.03–0.05 meme is the mass of an electron). The corresponding magnetic length (cyclotron radii) l can be derived as 2.5–3.2 ± 0.1 nm by calculating The discrete peaks in the STS spectra were found to correspond to the LL peaks of the bilayer graphene. We used equations graphene . The psegraphene . On the graphene .Although strain-induced LLs under no external magnetic field have been reported in several studies on graphene with corrugation, as described above, the region in which LLs were observed in our current study was indeed atomically flat, and the atomic periodicity was the same as that of pristine graphite, as shown in 1819+ in pyridinium ion, C5H5NH+, is 401.2 eV1821et al., the 1% strain has been calculated to produce a pseudo-magnetic field of 7 T9, which is much smaller than the estimated pseudo-magnetic field of 60–100 T for the nitrogen-doped graphite.The significant chemical shift in N1s BE for graphitic N (401.2 eV) compared to that for pyridinic N (398.5 eV) indicates positive charge of the graphitic N, given that the N1s BE for NHere, we discuss the origin of the LLs observed on the flat nitrogen-doped graphite surface using the domain model proposed previously24z orbitals of C and N change the layer–layer interaction, compared to that of pristine graphite. Enhancement of the coupling between the first and second graphene layers can also be realized if the charge transfer occurs not only from nitrogen atom to the π* states but also to the interlayer bandAs for the reason why primarily bilayer graphene LLs appear rather than graphene LLs or graphite LLs, the decoupling of the bilayer graphene from the bulk HOPG should be considered. Because our sample was prepared by nitrogen ion bombardment, decoupling of a few layers of the HOPG surface may occur as a result of the bombardment. Subsequent annealing to 900 K most likely causes re-stacking of the decoupled layers to form the nitrogen-doped bilayer graphene at the HOPG surface. The other possible cause of the decoupling is coupling between the first and second graphene layers of the HOPG due to the presence of nitrogen. For example, if graphitic N is located at the α site, the interactions of the p2+ ions of 200 eV at the normal incidence using a commercial ion-gun  at room temperature. The total ion dose applied to the sample was set to be 1.9 × 1012 ions/cm2 for the samples used for STM and STS, as measured independently using a Faraday cup. After the bombardment, the samples were annealed at 900 K for 300 sec . The sample temperature during heating was measured using an infrared thermometer.The fresh HOPG  samples were cleaved in air using adhesive tape, put into an ultra-high-vacuum (UHV) chamber, and annealed at 900 ± 50 K. The HOPG samples were then bombarded by N−10 Torr. An SPM 1000 from RHK Technologies was used as the STM controller. The nitrogen doping of graphite samples were conducted in the preparation chamber of the STM apparatus. The samples were cooled down to 5 K using liquid He. STM images were recorded in constant-current mode with a PtIr tip (Pt:Ir = 8:2). STS measurements were carried out by measuring the differential conductance (dI/dV) with a lock-in technique using a 1.0-kHz alternating-current (AC) modulation voltage with an amplitude of 20–100 mV. Each STS data point in the figure represents an averaged spectrum of ten spectra measured at the same position. Measurements were conducted for three different nitrogen-doped graphite surfaces to check the reproducibility of the experimental results.STM/STS measurements were performed in a UHV chamber  with a base pressure of 1 × 105/2 peak width of a clean Ag sample, uncertainty of binding energy ±0.05 eV). The XPS peak energy was calibrated using the peak position of C1s (284.6 eV) of the clean HOPG surface. The nitrogen-doped graphite samples used for the XPS measurements were prepared in the preparation chamber of the XPS apparatus. The nitrogen concentration (at %) was calculated as follows: {(peak area of N1s)/(sensitivity factor of N1s for Al Kα:7.5129)}/{(peak area of C1s)/(sensitivity factor of C1s for Al Kα:4.2586) + (peak area of N1s)/(sensitivity factor of N1s for Al Kα:7.5129)} × 100, where the Shirley background was subtracted from each peak prior to estimation of the area of the peak, using the XPSPeak4.1 software. The fitting analysis was conducted using the Gaussian–Lorentz function (Gaussian:Lorentz ratio = 2:8) using the XPSPeak4.1 software. We set the full width at half maximum of each peak as 2.0 eV to fit the results well.The XPS measurements were carried out using JEOL, JPS 9010 TR  using the Simulation Tool for Atom Technology (STATE). We used a local density approximation to treat the exchange-correlation interaction among the interacting electrons, using the Perdew–Zunger functional form to fit the Ceperley–Alder numerical results. Ultrasoft pseudopotentials were adopted to describe the electron–ion interaction generated by using the Vanderbilt scheme. The valence wave function and charge density were expanded in terms of the plane wave basis set with cutoff energies of 25 and 225 Ry, respectively. We considered the bilayer graphene  and one of the carbon atoms at the α site to be substituted by a nitrogen atom to simulate nitrogen-doped graphene. For this unit cell, the N concentration in the doped graphene was 1.6 at %. Integration over the two-dimensional Brillouin zone (BZ) is carried out using equidistant k-points sampling with 2 × 2 k point in the rectangular BZ. The atomic structures were fully optimised under a zero electric field until the force acting on each atom was less than 5 mRy/Å.How to cite this article: Kondo, T. et al. Observation of Landau levels on nitrogen-doped flat graphite surfaces without external magnetic fields. Sci. Rep.5, 16412; doi: 10.1038/srep16412 (2015).


Different doses of luteolin were added to VSMC cultures, and the proliferation and migration rate were observed by MTT and Transwell chamber assays, respectively. In addition, the expressions of p-Akt (308), p-Akt (473), and proliferative cell nuclear antigen (PCNA) in VSMCs were monitored by Western blotting. This study demonstrated that luteolin has an inhibitory effect on Ang II-induced VSMC proliferation and migration. Further, the levels of p-Akt (308), p-Akt (473), and PCNA were reduced in VSMCs treated with both Ang II and luteolin compared to VSMCs treated with only Ang II. These findings strongly suggest that luteolin inhibits Ang II-stimulated proliferation and migration of VSMCs, which is partially due to downregulation of the Akt signaling pathway.Luteolin is a naturally occurring flavonoid found in many plants that possesses cardioprotective properties. The purpose of this study was to elucidate the effect of luteolin on vascular smooth muscle cells (VSMCs) proliferation and migration induced by Angiotensin II (Ang II) and to investigate the mechanism(s) of action of this compound. Rat VSMCs were cultured  Atherosclerosis (AS) is a chronic inflammatory disease of arteries , and theAbnormal VSMCs proliferation and migration have been implicated in a number of cardiovascular pathologies, including the pathogenesis of hypertension, the development of restenosis after percutaneous coronary intervention (PCI), and the progression of AS , 5. In tAkt is a serine/threonine protein kinase and the Akt signaling pathway is involved in the regulation of multiple biological processes, including cell survival, proliferation, migration, and glycogen metabolism . Akt is Many studies have reported that dietary flavonoids provide protection against VSMCs disorders –15. LuteIn the present study, we carried out a preliminary study on the effects of luteolin on Ang II-induced VSMCs proliferation and migration. To further evaluate the effects of luteolin on the regulation of Ang II-induced VSMCs at a nuclear level, PCNA expression is examined. Furthermore, we also explored the hypothesis of the antiproliferative and antimigration effects of luteolin by inhibition of the Akt/PCNA signaling pathway, one of the many interrelated underlying mechanisms.Luteolin was obtained from Sigma , dissolved in dehydrated alcohol, and stored at 4°C until further use. Ang II was purchased from Beijing Union Pharmaceutical Factory , dissolved in sterile deionized water, and stored at 4°C until later use. Fetal bovine serum (FBS) was acquired from Gibco/Invitrogen . The assay reagent 3--2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma . Transwell chambers were manufactured by Millipore (Germany). An anti-PCNA polyclonal antibody and phosphospecific polyclonal antibodies against p-Akt (308) and p-Akt (473) were purchased from Cell Signaling Technology . The anti-Akt polyclonal and anti-PCNA polyclonal antibody was obtained from Zhongshan Golden Bridge.2 atmosphere, and maintained in Dulbecco's modified Eagle's medium  containing FBS (20% FBS for primary culture and 10% FBS for subculture), penicillin (100 IU/mL), and streptomycin (100 μg/mL). The purity of VSMCs cultures was confirmed with an antibody against smooth muscle α-actin . For all experiments, only cell passages 3–5 were used. Before each experiment, cells were serum starved for 24 h in DMEM containing 5.5 mM glucose and 1% antibiotics. The experimental VSMCs were given 12.5 μM (low-dose group), 25 μM (middle-dose group), and 50 μM (high-dose group) of luteolin. VSMCs were divided into groups and treated with Ang II for different lengths of times, with luteolin for different periods of time, or with luteolin at different concentrations.Thoracic aortas were quickly excised from sacrificed Sprague-Dawley rats . Rat aortic VSMCs were harvested using the explants culture technique, incubated at 37°C in a 5% COμL of VSMCs suspension, with 1 × 104 cells per well. After being cultured for 24 h, VSMCs were treated with serum-free media for about 24 h to achieve synchronization at G0/G1 phase. After pretreatment with different concentrations of luteolin for 12 h, VSMCs were stimulated with Ang II. MTT solution (5 mg/mL) was added to each well, after incubation for 4 h at 37°C. The cell culture medium was removed and 100 μL of DMSO was added to each well. Absorbance at 570 nm was measured for each well with an ELx800 Universal Microplate Reader . The proliferation of the control VSMCs was considered to be 100%; results were expressed as relative proliferation rates.MTT was used to measure the viability and proliferation of VSMCs. VSMCs at logarithmic growth phase were digested, centrifuged, and inoculated into 96-well flat-bottomed plates. Each well contained 100 2 atmosphere for 8 h. Depending on the experimental group to which they were assigned, VSMCs were cultured with Ang II (1 μM) and different concentrations of luteolin for 12 h. The VSMCs migrated through the micropores and stayed on the bottom side of the Transwell chamber. The upper chamber was removed out from the 24 pore plates and the residual cells on the inner side of the Transwell filters were wiped with cotton swab. The Transwell filters were flushed with 0.01 M PBS, fixed with 4% paraformaldehyde, and washed for 2 min with distilled water. After HE staining, 5 visual fields were chosen at random from each of the Transwell filters, and the average number of cells that migrated through the Transwell filters was counted under the microscope.The migration of the cultured cells was examined by Transwell chamber assay. The VSMCs were cultured in the upper chamber while only 10% FBS was placed in the lower chamber without cells. The cells were incubated at 37°C in a 5% COThe cell culture medium was poured off and the culture plates were washed twice with PBS at 4°C. Cells were lysed with lysis buffer on ice for 30 min. During this procedure the culture plate was agitated from time to time to ensure that the lysis buffer fully covered the cells. The cell lysates were centrifuged at 12,000 g for 15 min at 4°C. Equal amounts of soluble protein were separated by electrophoresis on a 12% SDS-PAGE gel and proteins were then electrotransferred to a polyvinylidene fluoride membrane. The membranes were immunoblotted with the appropriate primary and secondary antibodies. The membranes were photographed with 5-bromo-4-chloro-3-indolyl phosphate/nitroblue-tetrazolium (BCIP/NBT) coloration and grey scale values were determined with the Image J 3.0 system .P < 0.05.All data were expressed as mean ± SEM. Statistical analysis was performed with GraphPad Prism 4.0. The statistical significance of differences was determined using ANOVA followed by Bonferroni correction for post hoc analysis. Differences were considered statistically significant when μM (P > 0.05). As seen in μM Ang II significantly increases proliferation compared to the control group (P < 0.05).To test for cytotoxic effects of luteolin on VSMCs viability or cell proliferation, VSMCs were pretreated with luteolin or vehicle for 12 h. The MTT assay was then used to measure the viability and proliferation of VSMCs. As shown in μM Ang II clearly promoted the migration of VSMCs from the upper chamber to the lower chamber as compared to the control group (P < 0.05). Pretreatment with luteolin for 12 h significantly suppressed the migration stimulated by Ang II in a dose-dependent manner (P < 0.05). Thus, as the concentration of luteolin increased, the number of cells migrating decreased.The migration of VSMCs following Ang II stimulation was examined by Transwell chamber assay. As shown in μM Ang II for different lengths of time. The stimulatory effect of Ang II on the Akt pathway of VSMCs was apparent after only 5 min, and the effect lasted up to 40 min (see P < 0.05); phosphorylation then went back down to background levels. Our results demonstrated that Akt phosphorylation is increased by Ang II stimulation and is abolished by pretreatment with luteolin (P < 0.05).To examine whether luteolin inhibited Ang II-induced VSMCs proliferation or migration by affecting Akt activation, VSMCs were pretreated by luteolin for 12 h and then stimulated with 1  min see . After 5μM Ang II. As shown in P < 0.05). As shown in P < 0.05). LY294002  was used as a positive control in these experiments.VSMCs were pretreated with various concentrations of luteolin for different lengths of time before stimulating for 12 h with 1 μM luteolin was 29.16%, 38.58%, and 66.86%, respectively.To estimate the inhibitory effect of luteolin on VSMCs proliferation at the nuclear level, the expression of PCNA was detected. As shown in Due to various changes in social and living conditions, the morbidity and mortality of cardiovascular diseases have increased, with AS being one of the leading causes. Consequently, elucidation of the molecular and cellular mechanisms that drive the development and progression of AS is necessary for formulating strategies to treat and prevent AS. However, due to its complicated causes and pathologies, there are relatively few safe and effective drugs for treating AS. Therefore, the search for natural, inexpensive, and effective anti-AS drugs with little or no adverse reactions has become an intense area of research.There are many cells that play a role in the formation of AS, and the activities of VSMCs are particularly important. AS and arterial injury-induced neointimal hyperplasia involve medial VSMCs proliferation and migration into the arterial intima. The media of vessels chiefly consist of VSMCs, which have the ability to shrink and differentiate in normal culture conditions. However, VSMCs are prone to hypertrophy and lose their differentiation capacity due to the action of many pathological factors. VSMCs can synthesize high levels of extracellular matrix and inflammatory factors, proliferate, and migrate to the intima in excess. A high restenosis rate and poor long-term therapeutic outcomes following PCI have become the important factors driving the development of PCI procedures.2O2-induced proliferation and migration in VSMCs by attenuating the phosphorylation of Akt, published in journal of Pharmacy and Pharmacology (JPP) by Lang et al. [2O2 is one of the most important molecules of signal transduction in ROS [Many published studies support anti-AS and cardioprotective roles for dietary flavonoids that are ubiquitous in fruits and vegetables . The resg et al. . In the n in ROS , which c2O2-induced oxidative stress [Some documents have shown that the mechanisms for apoptosis, inflammatory response, oxidative stress, and so forth maybe be involved in proliferation and migration of VSMCs. Ang II can be more extensively involved in the production for inflammatory factor, oxygen free radical, and apoptosis related protein than that of He stress , 26. So μM Ang II, while pretreatment with 50 μM luteolin significantly suppressed this proliferation and migration without any cytotoxic effects.VSMC proliferation and migration are crucial events underlying the complications of vascular injury. The purpose of the present study was to determine which effect was played on VSMC proliferation and migration by luteolin pretreatment. The present study demonstrated that VSMC proliferation and migration can be significantly induced by 1 Acting as a proinflammatory factor, Ang II plays a crucial role in the pathogenesis of cardiovascular diseases , 28. AngThe Akt pathway is crucial for mediating changes in gene expression that regulate cell survival, proliferation, migration, and growth. The phosphorylation of both its activation sites, Thr308 and Ser473, is essential for the full activation of Akt. It has been suggested that different signaling pathways regulate the proliferation and migration response to Ang II, and our findings indicate that the effects of luteolin on VSMCs may be mediated by a coordinated inhibition of the Akt signaling pathway.1 and S phases of the cell cycle and to behave as a marker of cell proliferation in both normal and disease states, usually assayed in tumor cell not for VSMCs [To further evaluate the effects of luteolin on the regulation of VSMCs at a nuclear level, we examined PCNA expression. PCNA is known to be synthesized during the early Gor VSMCs , 30. In In conclusion, with the ultimate goal of preventing AS and restenosis following angioplasty, the identification and characterization of effective compounds that can inhibit VSMCs proliferation and migration have become one hot spot of investigation. The results obtained from this study suggest that luteolin can inhibit Ang II-stimulated VSMC proliferation and migration. The effect of luteolin is partially associated with inhibition of the PI3K/Akt pathway. Additionally, this study shows that luteolin inhibits the Ang II-induced expression of PCNA.Further studies centered on the detailed molecular mechanisms of luteolin are now in progress. We hope that our study lays a solid foundation for the clinical application of luteolin for treatment of AS. As the proliferation of VSMCs is an important determinant of atherosclerotic plaque development and stability, luteolin may be a valuable therapeutic compound for the treatment of cardiovascular diseases in the near future.


Then, antibody of PSA was immobilized on the surface of modified electrode surface through the adsorption of Au/Ag toward proteins, leading to the decrease of the ECL intensity. As proven by ECL spectra test and electrochemical impedance spectroscopy (EIS) analysis, the fabrication process of the immunosensor is successful. Under the optimal conditions, the ECL intensity decreased linearly with the logarithm of PSA concentration in the range of 1 pg/mL ~ 10 ng/mL. The detection limit achieved is 0.29 pg/mL. The immunosensor results were validated through the detection of PSA in serum samples with satisfactory results. Due to excellent stability, high sensitivity, acceptable repeatability and selectivity, the immunosensor has promising applications in disease and drug analysis.Prostate-specific antigen (PSA) was used as the model, an ultrasensitive label-free electrochemiluminescent immunosensor was developed based on graphene quantum dots. Au/Ag-rGO was sythsized and used as electrode material to load a great deal of graphene quantum dots due to the large surface area and excellent electron conductivity. After aminated graphene quantum dots and acarboxyl graphene quantum dots were modified onto the electrode, the ECL intensity was much high using K Immunosensors based on antibody-antigen binding are one of the most widely used to detect disease related substances, which are known as biomarkers, in clinical diagnosticsThere are two types of immunosensors. One is sandwich-type immunosensors and the other is label-free immunosensors. Compared with sandwich-type immunosensor, label–free immunosensors have some other advantages, for instance, they could be directly used to monitor the binding process of antibody–antigen reaction and overcome the condition limitation of enzymes. Therefore, the label–free immunosensors has become a remarkable analytical tool for detection of biomolecules. Many immunosensors for the determination of PSA are continuously being put forward based on electrochemistry, fluorescence, electrochemiluminescence (ECL) and so on45678Graphene quantum dot (GQD), a class of carbon nanomaterials containing one or few layered sheets with lateral dimension less than 100 nm, have several unique properties1112131415163, HAuCl4 and bovine serum albumin  were purchased from Sigma-Aldrich . 1-ethyl-3-(3dimethylamino-propyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), sodium citrate and K2S2O8 were obtained from Shanghai Aladdin Chemistry Co., Ltd, China. All other chemicals were of analytical grade and used without further purification.Aminated graphene, aminated graphene quantum dots and carboxyl graphene quantum dots were purchased from Nanjing XFNANO Materials Tech Co., Ltd. (China). The PSA and corresponding antibody were purchased from Beijing Dingguo Changsheng Biotechnology Co. Ltd. (China). AgNOECL measurements were carried out by using the MPI-F flow-injection chemiluminescence detector . Electrochemical measurements were operated by employing a CHI760D electrochemical workstation . A conventional three-electrode system was used including a platinum wire electrode as the auxiliary electrode, a saturated calomel electrode (SCE) as the reference electrode and a glassy carbon electrode  as the working electrode. The scanning electron microscope (SEM) images were obtained by the field emission SEM .3, 5 mL 1% HAuCl4 and 5 mg PVP were added into the above solution. 4 mL 50 mM sodium citrate was added dropwise to reduce AgNO3 and HAuCl4 after 6 h of stirring. After 24 h of stirring, the Au/Ag-rGO suspension was filtered and then dried at 35 °C in vacuum.Au/Ag-rGO was prepared using the following procedure. 80 mg aminated graphene powder was dispersed in 50 mL ultrapure water and under continuous ultrasound for 10 h. Then, 40 mg AgNOAminated graphene quantum dots , carboxyl graphene quantum dots , 40 mg EDC and 20 mg NHS were mixed into a centrifuge tube and stirred for 5 h at 4 °C. Au/Ag-rGO/Aminated-GQDs/Carboxyl-GQDs were prepared by adding 5 mg of Au/Ag-rGO into the above Aminated-GQDs/Carboxyl-GQDs solution and incubating for 24 h at 4 °C. After that, the solution was centrifugated and the products were redispersed in 1 mL of PBS (pH 7.4) and stored at 4 °C for use. The final concentration of Au/Ag-rGO is 5 mg/mL.The illustration of ECL immunosensor fabrication process is depicted in 6]3−/4− solution containing 0.1 mol/L KCl are displayed in Electrochemical impedance spectroscopy (EIS), a tool for evaluating electron transfer resistance, was used to characterize the stepwise modification of the electrodes18In order to further prove that the electrode was modified successfully, the stepwise fabrication process of the immunosensor was also characterized by ECL, as shown in As shown in To obtain an optimal ECL signal, pH value of substrate solution was investigated. Keeping the concentrations of TB constant, the effect of pH on the ECL intensity was studied over a pH range from 6.0 to 8.0, as shown in 2O82−. However, the intermediate SO4•− from S2O82− reduction would be scavenged by OH•− leading to a decrease in the ECL intensity in basic solution2S2O8 concentration in the substrate solution on ECL intensity was also tested. As shown in 2S2O8 concentration increased from 40 to 100 mmol/L because more GQDs* is produced from oxidation of GQDs•− by the electrogenerated SO4•− and remained constant in the range 100 ~ 140 mmol/L. Thus, 100 mmol/L K2S2O8 was chosen for further study in order to ensure an adequate sensitivity and save the dosage.In acidic solution, the reduction of proton to hydrogen would take place at the applied negative potential, which might inhibit the reduction of SIECL = 1294.0 − 756.63 lgc. Compared with the other immunosensors previously reported for the detection of PSA . As shown in The amount of PSA was measured 5 times in human serum sample and the relative standard deviation (RSD) was calculated to obtain the precision. The accuracy was also studied through a recovery experiment using standard addition method. An appropriate amount of PSA standard solution was added to corresponding samples. With the same experiments measured for five times, the average recovery was calculated. It can be found from This work demonstrated an ultrasensitive electrochemiluminescent immunoassay based on graphene quantum dots. The success of this work relied on the use of Au/Ag-rGO to assemble quantities of aminated graphene quantum dots and carboxyl graphene quantum dots, thus making ECL signal amplification. Great amplification together with the specificity of immunoreaction made sensitive detection of PSA possible. The proposed immunosensor achieved excellent performance with high sensitivity, good selectivity and stability. On one hand, this type of electrochemiluminescent immunosensor should be feasible for the field of cancer biomarker diagnosis and other life science fields. And on the other hand, this principle could be also applied to many other bioassays.How to cite this article: Wu, D. et al. Label-free Electrochemiluminescent Immunosensor for Detection of Prostate Specific Antigen based on Aminated Graphene Quantum Dots and Carboxyl Graphene Quantum Dots. Sci. Rep.6, 20511; doi: 10.1038/srep20511 (2016).


An obvious reduction of the metallic state of the MoS2 nanolayer is noticed as electrons are transferred to form a strong contact with the reduced graphene support. The missing metallic state associated with the unsaturated atoms at the peripheral sites in turn modifies the hydrogen evolution activity. The easiest evolution path is from the Mo edge sites, with the presence of the graphene resulting in a decrease in the energy barrier from 0.17 to 0.11 eV. Evolution of H2 from the S edge becomes more difficult due to an increase in the energy barrier from 0.43 to 0.84 eV. The clarification of the chemical bonding and catalytic mechanisms for hydrogen evolution using this strongly coupled MoS2/graphene nanocatalyst provide a valuable source of reference and motivation for further investigation for improved hydrogen evolution using chemically active nanocoupled systems.Inorganic nano-graphene hybrid materials that are strongly coupled via chemical bonding usually present superior electrochemical performance. However, how the chemical bond forms and the synergistic catalytic mechanism remain fundamental questions. In this study, the chemical bonding of the MoS To date, the reaction mechanisms on MoS2 were inconclusive because of the wide range of HER Tafel slopes reported182 catalyst supported on graphene sheet is therefore of fundamental importance for understanding the reaction that takes place during HER. The two types of exposed edges which both have predominantly unsaturated atoms, may provide different bonding geometries with hydrogen, resulting in a dual functionality for the hydrogen evolution reaction. Besides edges, the kink sites that exist as joints between the Mo and S edges may also have some effect on the overall catalytic activity. The presence of corners and kink sites was revealed by experimental studies, but the role of kink sites has not previously been considered2 catalysts without support also remained inconclusive. Detailed information about the peripheral structures of MoS2 of both free-standing nanolayers and when supported on a graphene sheet is thus essential in order to understand the nature of their catalytic activities.A complete picture of the kinetics of the hydrogen evolution reaction (HER) involves, firstly, a primary discharge step (Volmer reaction) where bonding of hydrogen to the catalyst H2 nanolayer and vacancy mediated graphene support. In addition, the nature of the active sites of the MoS2/graphene nanocontact involved in the essential step of the hydrogen evolution reaction (HER), that is, hydrogen bonding and recombination, was studied. The functionality of the MoS2 nanolayer when a graphene base is present turns out to be different for the two types of edges, wherein the Mo edge becomes more active in facilitating the hydrogen evolution reaction with a lower hydrogen binding energy |ΔEbond| and hydrogen recombination barrier compared with the free-standing MoS2 nanolayer, but the catalytic effect of support on the S edge is the opposite. The calculated results provide a motivation for further investigations on improved hydrogen evolution using supported MoS2 and provide a valuable source of reference of the use of the chemically active nanocoupled systems as well.In this paper, we analyze from a computational point of view the chemical and electronic coupling mechanism between the MoS2 consists of layers of S-Mo-S sandwiches. The stoichiometry and coordination numbers of the edge atoms can differ from the bulk. The most active sites are Mo edges passivated by S atoms that have low coordination numbers, providing a sulfide rich environment2/graphene nanocontact and free-standing MoS2 nanolayer were compared, and as shown in 2 nanolayer which is deficient in S. Therefore, in this manuscript we focus on the case where a MoS2 nanolayer with pristine edges is allowed to relax to its minimum energy structure since this is what occurs under standard reaction conditions and is therefore an environment that often occurs experimentally. 2 nanolayer, the sulphur-terminated 2 using high-resolution TEM telescope2 nanolayer, and the structure was optimised. This leads to the presence of a number of dangling bonds on the neighboring carbon sites, as denoted with numbers in 2 nanolayer. 2 on the vacancy mediated graphene surface showing the covalent bond that is formed between them, i.e. between the dangling bond of the graphene surface (C2 and C4 carbon atoms) and the edge sulphur atom of the MoS2 nanolayer. These S-C bonding distances are ~(1.76–1.78) Å, consistent with typical bond lengths in most sulphur compounds. The real applications of nanocatalysts supported on graphene are limited as weak bonding is usually found, involving the π orbital perpendicular to the graphene plane2 dangling bonds on the graphene surface as a result of the formation of carbon vacancies, resulting in vacancy-mediated graphene23MoS2 is quite similar whether or not the support is present, and therefore its optimised geometric shape in isolated form is not displayed. The optimized structure in the presence of the support is shown in 2 nanolayer undergoes considerable deformation from the bulk structure on structural optimization. As is clear from 2 edge configuration in sulfide rich environments based on thermodynamic calculation and TEM observation, i.e.We found that the optimized structure of the MoS2 nanolayer and graphene support was carried out. As shown in 2/graphene, and significant overlap  was found between 3p orbitals of S and 2p orbitals of C in the wave functions, also indicating a strong covalent bond is formed between the nanolayer and the support. The character of the charge redistribution between the MoS2 nanolayer and graphene sheet can be also evaluated as the difference between the electron charges of the MoS2 nanolayer and graphene sheet alone and the one formed when they are in contact, and is shown in the inset of 2 nanolayer and graphene support, originating from the vacancy-mediated hybridization between the C 2p and S 3p orbitals.Characterization of the electronic bonding between the MoS2 nanolayer and the one in nanocontact with the graphene support, shown in 2 layer as a whole. In both cases, the periphery-induced relaxation breaks the symmetry of semiconducting bulk MoS2, leading to the presence of metallic states at the Fermi energy level. Note that the states of the attached MoS2 nanolayer are similar to that of the free-standing one, except for two distinctive changes as pointed out by the arrows in the top part of 2 is bound to the graphene substrate. Based on the analysis of the projected density of states to the atomic sites as shown in Next, we compare the total and projected density of states for two cases: the free-standing MoS2 formation on MoS2 catalysts directly correlates with the mean MoS2 particle diameter2 particle, which would correspond to atoms in corner or edge positions in nanolayers above the support interface. To find out whether the decreased metallic nature of the states associated with the active periphery sites of the MoS2 nanolayer in the supported case affects their catalytic activity in hydrogen evolution, we first probed the chemical bonding of hydrogen atoms in the free-standing nanoparticle using the supercell model. In the peripheral sites (edges and corners) the cationic Mo atoms and anionic S atoms usually have low coordination numbers. Both of these are able to coordinate H, and therefore, they are active for initial bonding with H atoms. Our calculations identified the S and Mo atoms in the supercell which have low coordination numbers as potential active sites on which H atoms can be bonded and recombine (which were located at the periphery of the MoS2 layer). Binding energies of single or two H atoms on a free-standing or supported MoS2 nanolayer were calculated as EnH-MoS2[/Graphene] being the total energy of the hydrogen bonded to the complex with or without the graphene support, EMoS2[/Graphene] being the total energy of the corresponding catalyst model, and EH2, the energy of the H2 molecule placed in a 10 × 10 × 10 Å3 cubic box.It has been unequivocally demonstrated that the rate of HGbond, and this has been found to serve as a reasonable descriptor of hydrogen evolution activity for a wide variety of metals and alloys2627Gbond should be around 0. The phonon contribution to the free energy is typically quite small, <5%, which is negligible in the supercell model used. Ignoring entropy terms we can set |ΔGbond| approximately equal to enthalpy |ΔEbond| and we therefore use calculated value |ΔEbond| to judge the catalytic activity for a variety of peripheral sites of MoS2 based catalysts, and we will assume in the following that the closer |ΔEbond| is to zero, the better the catalytic activity of specified periphery sites. The free energy of hydrogen bonded states ΔGbond was also estimated using the approximated method proposed by Nørskov by simplifying the entropy energy difference to 2 in the gas phase at standard condition, for isolated MoS2 nanolayer and MoS2@graphene as well  with the reactive intermediates as well 27. HowevEbond when hydrogen is bound to the peripheral atoms of the isolated MoS2 nanolayer and MoS2 in contact with the graphene substrate and show structural configurations for the latter (H bonded isolated MoS2 layers show similar structures to the attached one so are not displayed). We found that for the free-standing MoS2 nanolayer, H can bind to the S- or Mo-edge and Mo-corner, resulting in negative ΔEbond, as shown in 2 distorted sandwich structure. The S-edge less strongly binds the H atoms with a |ΔEbond| which is closer to zero, suggesting for isolated MoS2 particles, the S edge could play a major role on its catalytic performance for hydrogen evolution. A stable complex of H atoms bound on the Mo corner is formed in which the two H atoms are bound to the same corner Mo atom. The main difference between the isolated and attached MoS2 nanolayers is that the atomic sites at the Mo-edge have a positive ΔEbond in the graphene attached MoS2, with a much lower magnitude than that obtained for the isolated MoS2 nanolayer. For free-standing MoS2, the S-edge, with its low H binding energy, is the predominant factor which would lead to a high activity for the HER of the whole layer. The reactivity of the graphene supported MoS2 nanolayer might be a better catalyst for the HER because the Mo-edge can contribute to its catalytic activity with a much reduced |ΔEbond|. Although in our study the support does not play any direct role in bonding or activating the H atoms, except for the role of stabilizing and modifying the MoS2 nanolayer, the S atoms on the Mo-edge which have low coordination numbers will be less trapping of the H atoms. This could be the result of the fact that some metallic states associated with the unsaturated peripheral sites are missing after the MoS2 is bound to the reduced graphene support, and therefore the MoS2 will more easily release H in the next step of electrochemical hydrogen evolution.In 2/graphene nanocontact and free-standing MoS2 nanolayer. As shown in The effect of inclusion of a van der Waals correction on the conclusions was considered for the two-hydrogen-atom binding energy at the Mo and S edges of MoS2/graphene structures with two hydrogens, we also selected the edge sites that weakly bind hydrogen and the corner sites that strongly trap hydrogen and have calculated the complete reaction path for H recombination and release after two atoms are attached to the substrate. The initial state was selected to be those shown in Eact were then obtained as the energy difference between the maximum energy transition state and the initial adsorption complex along the reaction pathway, and reaction energies ΔE were obtained as the energy difference between initial and the final state, and these results are shown in Among the different possible MoS2 nanolayer, relative to the energy of the initial structure in each case. The detailed atomic configurations of the initial state, a number of intermediate states, and the final state are also plotted. For two H atoms on the Mo-edge, the global process is exothermic with an activation barrier around 0.17 eV for the free-standing MoS2 nanolayer and 0.11 eV for the attached nanolayer. During the reaction path the first step is a rotation of one H atom on the S site followed by a later migration to a high coordination number inner Mo atom of the nanolayer. After overcoming an energy barrier in the subsequent step, we found both H atoms are situated above the nanolayer and come closer to each other. The distance between them decreases to 0.75 Å in the final state, indicating that an H-H bond is formed. The lower barrier of H recombination from the Mo edge on attached MoS2 could be due to the nanocontact-induced diminishment of metallic states on filling the antibonding 2π* orbital of formed H2. We note that the final energy of the H2 on the substrate is lower than the separated substrate and H2, which might suggest that it remains bound at low temperatures.In 2 from the S edge might be qualitatively considered to be similar to that on the Mo edge but the energetics differ. The energy barrier was determined to be 0.43 and 0.84 eV, respectively, as the H atom is released from the S of the free-standing and attached MoS2 nanolayers. For the isolated MoS2 nanolayer, rearrangement to form H2 results in an even more stable structure, indicating that this site will not be useful for the HER at moderate temperatures. For the MoS2/graphene, there is only a small difference in energy between the initial and final structures as the H2 is formed, however the activation energy is significant, making this process difficult to proceed. Therefore, the evolution of H2 from the S edge becomes more difficult due to the increase in the energy barrier. Nevertheless in the presence of the graphene, H2 evolution is enhanced due to the easier evolution path on the Mo edge sites, resulting from a decrease in that energy barrier. The excellent HER activity of the MoS2/RGO hybrid catalyst reported by Li et al.The first stage of the recombination and release of H2/graphene substrate. The hydrogen atoms at the S corner stay bound to different S atomic sites initially. As shown in 2 molecule is assumed to form, but has slightly higher energy than the initial structure. From the Mo-corner site, the two H atoms are initially bound to the same corner Mo atom. The meta-stable product on the potential energy surface, that is 0.22 eV more stable than the physically bonded H2, consists of two H atoms located on bridge positions near the Mo corner site, with H-H optimized distances around 0.78 Å. Along the further reaction pathway, the H–H bond length starts to decrease to a final equilibrium value of 0.75 Å.The activation energies for the recombination and release of hydrogen were determined to be 1.40 and 2.10 eV, respectively, for highly trapped S and Mo corners in the case of MoS2 evolution process on a MoS2 nanolayer without reducing its high activity, we have investigated by means of DFT calculations the nature of the active periphery sites involved in the H bonding and later recombination and release of hydrogen on a series of models in which a graphene support is in effect. We found that the coupling with the reduced graphene support causes noticeable changes in the shape of the Mo-edge in MoS2 nanolayer and also a certain degree of charge transfer. The optimized Mo-terminated 2 nanolayer turns out to be similar to the S-terminated 2 nanolayer and graphene support, originating from the vacancy-mediated hybridization between the C 2p and S 3p orbitals. When MoS2 is attached to graphene, the metallic states around the Fermi energy level in association with the unsaturated S atoms at the periphery sites in MoS2 also decrease as electron transferring to form contact path with reduced graphene support.In order to understand and improve the H2/graphene nanocontact using supercell methods that have been shown to be suitable for this type of system. Considering the favourable peripheral sites, the hydrogen binding energies were used to evaluate the catalytic activity, and the activation energy for the essential hydrogen evolution step of the recombination process was determined. The Mo edge of MoS2 nanolayer in the presence of graphene support clearly bound the H atoms less strongly, as was revealed by the small |ΔEbond|, which is enough to modify the energy profile so that the following H recombination and release process becomes relatively easier with a decreasing activation energy to 0.11 eV. In contrast, the same H evolution on S edge becomes more difficult due to a higher energy barrier of 0.84 eV for the MoS2/graphene. The functionality of MoS2 nanolayer when a graphene base is present is different depending on the types of edges, wherein an improved synergistic catalytic effect was obtained on Mo edges characterized by lower hydrogen binding energies |ΔEbond| and hydrogen recombination barrier, but an opposite catalytic effect of graphene support was found on S edges.We also checked all of the possible active sites on the MoS2 having a 3 × 3 in-plane periodicity and a single layer on the z axis of the unit cell, see 2 model system was studied as an isolated structure and also when supported on a 7 × 7 × 1 graphene basal plane repeated in a supercell geometry and with two carbon atoms removed  facet exposed for both the Mo-terminated 2 nanolayers was examined.Here we consider a supercell model of MoS2/graphene is smaller than 0.002 eV, which validates the suitability of our current computational methodology. In optimizing the atomic structure, a conjugate-gradient algorithm was employed and the force convergence criterion was set to 0.01 eV/Å.All calculations were based on density functional theory (DFT) as implemented in the PWSCF codeAfter the evaluation of the active bonding sites for H atoms and its probable recombination path, the next step was to find the activation energy along that path, so that kinetics of the recombination reaction could be predicted. The climbing-image nudged elastic band (CI-NEB) method, widely used for locating transition states, was adopted to find the minimum energy path (MEP) between a given initial and final state of the recombination reactionT.L. performed the calculations, analyzed the results and wrote the manuscript. Z.Q.S. conceived the idea and reviewed the manuscript. C.H.S. and S.X.D. took part in discussion. D.J.S. discussed and reviewed this manuscript.Dataset 1


The role of epithelial growth factor (EGF) as a mediator of enterocyte proliferation and the innate immune response has been described. In this paper, we review the most recent literature on the mechanisms responsible for triggering the up-regulation of these mediators in CD by gliadin peptides. We will discuss the role of P31–43 in enterocyte proliferation, structural changes and the innate immune response in CD mucosa in cooperation with EGF and IL-15, and the mechanism of up-regulation of these mediators related to vesicular trafficking. We will also review the literature that focuses on constitutive alterations of the structure, signalling/proliferation and stress/innate immunity pathways of CD cells. Finally, we will discuss how these pathways can be triggered by gliadin peptide P31–43 in controls, mimicking the celiac cellular phenotype.Celiac disease (CD) is a frequent inflammatory intestinal disease, with a genetic background, caused by gliadin-containing food. Undigested gliadin peptides induce innate and adaptive T cell-mediated immune responses. The major mediator of the stress and innate immune response to gliadin peptides ( Ingested food can cause tissue inflammation through different mechanisms. In the intestine, and particularly in the enterocyte, nutrients are modulators of various cellular functions and may be involved in tissue immune response and inflammation . An examGliadin, the major protein component of wheat and other cereals, is a peculiar protein very rich in glutamine and proline. Several gliadin peptides are recognized by T-cells (TC) of the celiac intestine, and can induce the adaptive immune response, but most of them are digested by gastric, pancreatic and intestinal proteases. Only two main peptides remain undigested ,6,7,8: tin vitro and in vivo [The inflammation of the intestinal mucosa is due not only to the adaptive but also to the innate immune responses to wheat gliadin. The A-gliadin 33-mer that is deamidated by tissue transglutaminase (tTG), binds to human leukocyte antigen (HLA) DQ2 and/or DQ8 and induces an adaptive Th1 pro-inflammatory response . The P31 in vivo ,11,12. M in vivo ,14 and a in vivo ,16 with Although the structural changes of the celiac mucosa are considered a consequence of sustained mucosal inflammation due to the Th1-TC response, recent data have shown that gliadin peptides, in particular P31–43, induce proliferation of celiac enterocytes. This process is epithelial growth factor (EGF) and IL-15 dependent, and has profound upstream effects in inducing the crypt hyperplasia, which is characteristic of the remodeling of the celiac mucosa ,18,19. Mi.e., P31–43) can have several different non-T cell mediated effects, both in cell lines and cells and in biopsies from CD patients, that can be grouped into three sets: cell structural changes, including apoptosis, signaling/proliferative effects and stress/innate immunity activation  and intestinal biopsies, highlighting their relationship with IL-15 and EGF/EGFR deregulation in CD.IL-15 exerts pleiotropic activity that ultimately results in immunoregulatory cross-talk between cells of the innate and adaptive branches of the immune response. Moreover, IL-15 can induce the proliferation of intestinal epithelial cells ,28.The increase of IL-15 and IL-15R-α is a major event in the initial phases of CD ,30,31. IIL-15 inhibits regulatory T-cells (TREG) ,35 and tIL-15 stimulates the effector functions of intestinal epithelial lymphocytes (IEL) in active celiac disease with the production of γ-INF and perforin-granzyme-dependent cytotoxicity against the enterocytes ,33.In fact, the IL-15 in the intestinal mucosa of celiac disease up-regulates the expression in IEL of two receptors (CD94 and NKG2D) and in the enterocytes of their ligands (MICA and HLAE), resulting in apoptosis ,29,39,40Transgenic mice that express human IL-15 specifically in the enterocytes develop villous atrophy, crypt proliferation and severe duodenal-jejunal inflammation with massive accumulation of NKG2D-expressing CD8+ intraepithelial lymphocytes in the affected mucosa, reproducing the intraepithelial inflammation and damage typical of CD in a gluten-independent way. The blockage of IL-15 signaling with antibodies leads to a reversal of the intestinal damage . These mIL-15 is therefore a major mediator of the stress and innate immune response to gliadin peptides  and a central regulator of celiac disease immunopathology. For a recent review on this subject see the publication of Abadie and Jabrì [Although in the past it was suggested that gliadin peptides could promote IL-15 expression by IEC  . More reDamage to the intestinal mucosa in CD is mediated both by inflammation due to the adaptive and innate immune response to gliadin and by proliferation of crypt enterocytes as an early alteration of CD mucosa causing crypt hyperplasia ,3,4. TheGliadin peptides and P31–43 induce cell proliferation and actin rearrangements ,17 in vaP31–43 is also able to enhance proliferation of the celiac enterocytes in an EGF-dependent way and to delay the trafficking and degradation of EGFR at the epithelial level, suggesting a role for EGFR activation in CD, particularly in determining crypt hyperplasia and the tissue remodeling of the celiac intestine. Persistent epithelial cell proliferation leads to inhibited maturation and differentiation of epithelial cells and loss of the normal villous structure . GliadinIn particular, in Caco2 cells, proliferation can be induced by both IL-15 and EGF and is dependent on interplay between EGFR and IL-15R-α. The cooperation is mediated by a complex between IL-15R-α and EGFR which is increased and activated by each ligand. The signaling, beginning with the complex of the two receptors can be activated by EGF and IL-15, with each of them able to stimulate its own and the other receptor. Moreover, both EGF and IL-15 can induce transcriptional activation of each other. P31–43, which induces enterocyte proliferation and the activation of IL-15 in CD, increases the complex, the activation and the downstream signaling of both receptors together with the transcripts of both ligands. These data show that the proliferation of enterocytes can be regulated by interplay between growth factors (EGF) and cytokines (IL-15) and that P31–43 can stimulate growth and the innate immune response by employing such cooperation .The mechanisms through which P31–43 might induce the celiac intestine innate immune response and EGF- and IL-15-mediated enterocyte proliferation have recently been investigated. Recent evidence ,47,48 poEndocytosis has many effects on signaling; in fact, signaling pathways and endocytic pathways are regulated in a reciprocal manner. It is now widely accepted that the “endocytic matrix” is a master organizer of signaling, governing the resolution of signaling in space and time. Consequently, endocytosis affects several cell functions, ranging from proliferation to actin organization, cell motility and stress/innate immunity activation ,50 Figua,b.P31–43 is strikingly similar to a region of hepatocyte growth factor regulated substrate (HRS) kinase, a key molecule regulating endocytic maturation, which is localized on the membranes of early endocytic vesicles  Figure b.The sequence similarity between gliadin peptide P31–43 and HRS is in a small area of the proline/glutamine rich domain of HRS. The COOH- terminal of HRS contains a clathrin-binding domain that binds clathrin to clathrin-coated vesicles  and is oin vitro in an IL-15-dependent way, demonstrating that the effects of P31–43 on enterocytes can activate signaling in lymphocytes. Interestingly, more IL-15R-α is expressed in CD enterocytes and in patients on a gluten-free diet [In Caco2 cells, P31–43 interferes with the correct localization of HRS at the level of the early endosomes, interfering with HRS-mediated maturation of early endosomes. By interfering with the localization to the endocytic membrane of HRS, P31–43 induces two important effects: (a) it delays endocytic maturation and (b) it alters the recycling pathway. By delaying the maturation of endocytic vesicles, P31–43 reduces the degradation of EGFR and other receptor tyrosine kinases (RTKs) that are endocytosed in these vesicles, and prolongs their activation, resulting in increased proliferation, actin remodeling and other biological effects. The alteration of the recycling pathway is able to direct more transferrin receptor  and IL-1The production of IL-15 is tightly controlled at multiple levels, not only at the level of intracellular trafficking but also of transcription and translation . P31–43 By increasing the synthesis of IL-15 and the amount of the cytokine that is trans-presented to the neighboring cells, P31–43 affects both enterocyte proliferation, which is EGFR-IL-15 dependent, and the activation of innate immunity .We would like now to review the hypothesis that the effects of gliadin and P31–43 on endocytosis, a pathway nodal to many cell functions, could explain the sensitivity to gluten of CD cells.Recent observations suggest an effect of P31–43 on the maturation and function of early endocytic vesicles and consequently on EGFR signaling, enterocyte proliferation and IL-15 trans-presentation and synthesis ,47,48,51PARD3 and MAGI2 have been associated with disease susceptibility in a Dutch cohort [Different studies have identified the importance of the disruption of the integrity of the epithelial layer in CD. One of the first structural alterations identified is the alteration of the cell-to-cell junctional complexes that regulate intestinal permeability. Patients show enhanced intestinal permeability and altered tight junction (TJ) morphology, and these disruptions persist in patients who are on a gluten-free diet (GFD) with a normalized intestine, suggesting that permeability may play a driving role in the development of CD ,59,60,61h cohort . Interesh cohort . These oLPP gene presents the strongest non-HLA association signal, mapped in intron 2 [Recent genetic studies point to the importance of polymorphisms of CD genes that are involved in actin remodeling and cell adhesion. Among these, the intron 2 . More reintron 2 . The LPPintron 2 . A constintron 2 . MoreoveTaken together, these data indicate that structural alterations are present in CD cells independent of gluten.Alterations in signaling pathways and cell proliferation have been demonstrated in CD biopsies and cells  of patients on a GFD.The NF-κB pathway is constitutively altered in CD, with more than 20 components of the pathway increased in GFD-CD biopsies. Most of the mRNA over expressed in GFD-CD was central to the regulation of the pathway . InteresConstitutive activation of the EGF receptor/ligand system is also present in CD enterocytes . IncreasCellular stress has been implicated in the early events of the disease, in particular in the epithelium ,14. HeatIL-15 is elevated in intestinal biopsies from CD patients on GFD . In dendIn conclusion, the data in the literature point to several constitutive alterations of cell structure, signaling, proliferation and stress/innate immunity in CD cells . These pSeveral reports in the literature introduced the concept that gliadin may not be safe for non-celiac individuals .In the 1980s, it was reported that gluten could induce small intestinal mucosal structural alteration and symptoms in normal subjects, suggesting that gliadin may not be safe for non-celiac subjects ,79.More recently, we have demonstrated that the peptide P31–43 reproduces the structural alterations of the celiac cells in several cell types. These include actin modifications and cell shape alterations ,80. RemaExposure of the intestine of normal subjects to gliadin may cause the overall deregulation of NF-κB-related gene expression similar to the alteration of the pathway augmented in the CD intestine with and without gliadin treatment, although these alterations are more evident in the CD intestine .Moreover, treatment with P31–43 can transiently increase EGFR staining of normal intestinal biopsies . After tIL-15, the main mediator of the innate immunity response to gliadin in the celiac intestine, can be induced in intestinal biopsies of normal subjects by gliadin , confirmThus, gliadin is an activator of various signals at the cellular level mimicking the constitutive alterations found in celiac cells and intestinal biopsies. These pathways, constitutively altered in celiac cells, render them more susceptible to the effects of the gliadin peptides that, acting on the same pathways but in the celiac background, are able to produce long-term damage, including structural alterations, over-proliferation of crypt enterocytes and stress/innate immune response activation.In the general issue of food and tissue inflammation, gliadin and its undigested peptides plays a leading role. In this paper, we have reviewed most of the effects of the gliadin peptide P31–43 in intestinal biopsies and cells from normal subjects and CD patients at different stages of the disease. We have highlighted the effects of an alimentary peptide, contained in very common foods, that is biologically active on cell structure, signaling/proliferation pathways and stress/innate immune activation. These are also constitutively altered in celiac cells and biopsies, rendering them more sensitive to the effects of gliadin.What remains to be understood is the molecular defect explaining the alterations of the celiac cells, most likely due to a particular genetic make-up.i.e., a particular fraction of wheat albumin, able to inhibit amylase and trypsin, can cause Toll-like receptor 4 (TLR4)-mediated intestinal inflammation [Gluten and other wheat proteins can induce inflammation in the intestine and outside. This also may occur in non-celiac subjects; ammation .in vivo [in vitro [Gluten itself could play a role in the pathogenesis of diseases different from CD, such as type 1 diabetes. In children with insulin-dependent diabetes but not celiac disease, intestinal inflammation ,88 is trin vivo , and thein vitro .In conclusion, gliadin and its undigested peptides have biological effects not only in cells and the intestinal mucosa of patients with CD but also in normal subjects or in different diseases. How these effects can affect the health of non-celiac subjects will be the object of future research.


Regulation of gene expression by histone-modifying enzymes is essential to control cell fate decisions and developmental processes. Two histone-modifying enzymes, RPD3, a deacetylase, and dKDM5/LID, a demethylase, are present in a single complex, coordinated through the SIN3 scaffold protein. While the SIN3 complex has been demonstrated to have functional histone deacetylase activity, the role of the demethylase dKDM5/LID as part of the complex has not been investigated.Sin3A or lid resulted in decreased cell proliferation in S2 cells and wing imaginal discs. Conditional knockdown of either Sin3A or lid resulted in flies that displayed wing developmental defects. Interestingly, overexpression of dKDM5/LID rescued the wing developmental defect due to reduced levels of SIN3 in female flies, indicating a major role for dKDM5/LID in cooperation with SIN3 during development. Together, these observed phenotypes strongly suggest that dKDM5/LID as part of the SIN3 complex can impact previously uncharacterized transcriptional networks. Transcriptome analysis revealed that SIN3 and dKDM5/LID regulate many common genes. While several genes implicated in cell cycle and wing developmental pathways were affected upon altering the level of these chromatin factors, a significant affect was also observed on genes required to mount an effective stress response. Further, under conditions of induced oxidative stress, reduction of SIN3 and/or dKDM5/LID altered the expression of a greater number of genes involved in cell cycle-related processes relative to normal conditions. This highlights an important role for SIN3 and dKDM5/LID proteins to maintain proper progression through the cell cycle in environments of cellular stress. Further, we find that target genes are bound by both SIN3 and dKDM5/LID, however, histone acetylation, not methylation, plays a predominant role in gene regulation by the SIN3 complex.Here, we analyzed the developmental and transcriptional activities of dKDM5/LID in relation to SIN3. Knockdown of either We have provided genetic evidence to demonstrate functional cooperation between the histone demethylase dKDM5/LID and SIN3. Biochemical and transcriptome data further support functional links between these proteins. Together, the data provide a solid framework for analyzing the gene regulatory pathways through which SIN3 and dKDM5/LID control diverse biological processes in the organism.The online version of this article (doi:10.1186/s13072-016-0053-9) contains supplementary material, which is available to authorized users. Chromatin, composed of DNA wrapped around histone proteins, acts as the template for gene transcription in eukaryotes. The activity of nucleosome remodeling factors and histone-modifying enzymes, as well as the incorporation of histone variants, regulates dynamics of chromatin packaging . Dense pDrosophila, and HDAC1 and 2, in mammals, render catalytic activity to the complex. Investigations using different model organisms have identified the interaction of SIN3 with a KDM, dKDM5/LID, in Drosophila, and the homolog KDM5A in mammals  sonicator. Sonicated samples were subjected to centrifugation at 15,000g for 15 min at 4 °C and the pellet was discarded. 75 μg of prepared chromatin was diluted to a final volume of 500 μl with NaCl buffer and used for immunoprecipitation. For immunoprecipitation of HA-tagged proteins 30 μl of anti-HA beads ) were added to 500 μl of prepared chromatin samples and placed on a nutator at 4 °C for 4 h. For immunoprecipitation of native protein or modified histones antibody specific to SIN3  at 45 °C for 1.5 h and subjected to phenol chloroform extraction and ethanol precipitation. Precipitated DNA was resuspended in 50 μl of ddH20. Input DNA was prepared from 75 μg of chromatin samples directly after RNase treatment and reversal of crosslinks as described above. Input DNA (diluted 1:100) and immunoprecipitated samples (diluted 1:4) were subjected to real-time quantitative PCR with Absolute SYBR Green ROX master mix (Fisher Scientific) using a Stratagene MX3005P real-time thermocycler. The primer sets used in the PCR reactions are listed in Additional file 4 × 10 to SIN3  (5 μg), http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE68775.The datasets supporting the results of this article are available in NCBI Gene Expression Omnibus (GEO) database under accession number GSE68775 at