BDNF)‐tyrosine kinase B (TrkB) (BDNF‐TrkB) signalling pathway plays a crucial role in regulating learning and memory. Synaptophysin provides the structural basis for synaptic plasticity and depends on BDNF processing and subsequent TrkB signalling. Our previous studies demonstrated that maternal exposure to propofol during late stages of pregnancy impaired learning and memory in rat offspring. The purpose of this study is to investigate whether the BDNF‐TrkB signalling pathway is involved in propofol‐induced learning and memory impairments. Propofol was intravenously infused into pregnant rats for 4 hrs on gestational day 18 (E18). Thirty days after birth, learning and memory of offspring was assessed by the Morris water maze (MWM) test. After the MWM test, BDNF and TrkB transcript and protein levels were measured in rat offspring hippocampus tissues using real‐time PCR (RT‐PCR) and immunohistochemistry (IHC), respectively. The levels of phosphorylated‐TrkB (phospho‐TrkB) and synaptophysin were measured by western blot. It was discovered that maternal exposure to propofol on day E18 impaired spatial learning and memory of rat offspring, decreased mRNA and protein levels of BDNF and TrkB, and decreased the levels of both phospho‐TrkB and synaptophysin in the hippocampus. Furthermore, the TrkB agonist 7,8‐dihydroxyflavone  reversed all of the observed changes. Treatment with 7,8‐DHF had no significant effects on the offspring that were not exposed to propofol. The results herein indicate that maternal exposure to propofol during the late stages of pregnancy impairs spatial learning and memory of offspring by disturbing the BDNF‐TrkB signalling pathway. The TrkB agonist 7,8‐DHF might be a potential therapy for learning and memory impairments induced by maternal propofol exposure.The brain‐derived neurotrophic factor ( Science publication by Ikonomidou et al. N‐methyl‐d‐aspartic acid (NMDA) antagonist neurotoxicity in developing neurons, the occurrence of long‐term damage to the central nervous system (CNS) has gained much attention and has become a new research focus in the anaesthesiology and neuroscience fields. Increasing evidence indicates that most general anaesthetics can induce neuronal apoptosis and long‐term neurobehavioural impairments in developing brains and in young animals It was traditionally thought that the effects of general anaesthetics were reversible. However, as a result of the Propofol is one of the most commonly used intravenous general anaesthetics and is used for paediatric anaesthesia. Increasing clinical evidence has shown that patients can exhibit symptoms of cognitive dysfunction several weeks or even several months after exposure to propofol anaesthesia et al. The exact mechanism for the long‐term effects of propofol on cognitive function still needs further clarification. Synaptic morphology and function is the basis of learning and memory. There are two different forms of synaptic plasticity: long‐term potentiation (LTP) and long‐term depression (LTD). Balancing the expression and biological function of LTP and LTD is critical for the correct formation and maintenance of learning and memory. Formation of LTP requires the involvement of brain‐derived neurotrophic factor (BDNF), which regulates neuron development in the CNS and is closely related to learning and memory Pregnancy is the key period for prenatal brain development, during which, the foetus is vulnerable to external noxious stimuli The following main reagents were used in this study: propofol ; 20% intralipid ; 7,8‐Dihydroxyflavone ; dimethyl sulphoxide ; TRIzol total RNA extraction kit ; RNAstore ; diethylpyrocarbonate (DEPC) ; MD101‐02 DNA Maker I ; Prime ScriptTM RT Reagent Kit with gDNA Eraser  ; SYBR Premix Ex TaqTM II (Tli RNaseH Plus) ; BDNF, TrkB and β‐actin RT‐PCR primers ; and DAB Colour Developing Reagent Kit for immunohistochemistry . Brain‐derived neurotrophic factor primary antibody (50 ug), rabbit polyclonal anti‐TrkB antibody (ab18987), rabbit polyclonal anti‐TrkB (phospho Y515) antibody (ab109684), rabbit polyclonal anti‐synaptophysin antibody (ab23754) and rabbit polyclonal anti‐β‐Actin antibody (ab8227) were purchased from Abcam Company.ad libitum. To exclude the possible contribution of genetic factors on the offspring, parental rats were assessed for learning and memory function using the Morris water maze (MWM) to ensure that there were no significant cognitive differences. Based on the learning and memory results, parental rats were then assigned to propofol (n = 10), control (n = 20) or intralipid (n = 5) treatment groups  rats were supplied by the animal science research department of the Jiangxi Traditional Chinese Medicine College (JZDWNO: 2011‐0030). Animals were maintained under the standard conditions of temperature between 22°C and 25°C, humidity at 55 ± 5%, and daily 12 hrs light/12 hrs dark cycle (with lights on at 7:00–19:00). Animals received food and water via the caudal vein and was followed by 20 mg/kg/hr of continuous infusion for 4 hrs. Equal volumes of saline were given to rats in the control group, while 20% intralipid was given to the intralipid group. The propofol infusion time was selected based on the following information: (i) It is proven that neuronal damage is highest when general anaesthetic exposure time is prolonged to between 6 and 8 hrs, but a 2 hrs exposure time has no significant effects; (ii) Most surgeries require 2–4 hrs of anaesthesia; and (iii) Our previous studies have shown that maternal exposure to propofol (at the same dosage used in this study) for 2 hrs during gestation had no significant effects on learning and memory in offspring, but exposure for 4 hrs caused significant impairments without affecting the levels of arterial blood gases of pregnant rats On day E18, 20 mg/kg propofol were injected into gestating rats in the propofol exposure group 2) and tail non‐invasive blood pressure were monitored during the time of propofol exposure. Tail arterial blood was collected for blood gas analysis after propofol infusion. If the cumulative time of SpO2 was <95% and/or the systolic blood pressure was <80% of baseline for more than 5 min., the maternal rat was removed from the study, and another rat was selected to supplement the sample size, thereby preventing toxicity from maternal ischaemia or hypoxia in rat offspring.Electrocardiograms, saturation of pulse oximetry  of the MWM system. Each rat was put into the pool and allowed to search for the platform one time per day for six consecutive days. The time for the rats to find the platform was recorded as the escape latency (which indicated learning ability). The animals were allowed to stay on the platform for 30 sec. if they were able to find the platform within 120 sec. However, if an animal did not find the platform within 120 sec., the animal was guided to the platform, allowed to remain there for 30 sec., and the escape latency was recorded as 120 sec. On the seventh day, the platform was removed. The rats were then allowed to perform the spatial probe test (a memory test) for 120 sec. The number of times that the rat swam across the site where the platform was hidden (number of platform‐crossing times) and the time spent in the target quadrant (target quadrant time) were recorded with a video connected to a computer. The mean value of the latencies, platform‐crossing times or target quadrant times of the offspring born to the same mother were calculated as the final results.Spatial memory and learning of rat offspring were tested by the MWM test at postnatal day 30 (P30). The trials began at 9:00 am in a MWM system that was filled with water at 24 ± 1°C. A platform was hidden 1 cm below the water surface in the second quadrant  and vehicle group  Figs  and 2. TThe day after the MWM test, rats were anaesthetized and killed by cervical dislocation. Hippocampus tissues were harvested and stored in Eppendorf tubes that had been treated with 1% DEPC and were stored at −80°C  or immersed in 4% paraformaldehyde (for IHC experiments).−[(Ct of target gene)−(Ct of β‐actin)], where Ct is the threshold cycle for each transcript).Total RNA was extracted with the TRIzol kit (Takara). cDNA synthesis was performed according to the manufacturer's instructions. Real‐time PCR was performed using the SYBR Green PCR Kit. The following primers were used: BDNF‐ forward primer (5′‐CCAACGAAGAAAACCATAG‐3′), reverse primer (5′‐AATACTGTCACACAGTCA‐3′); TrkB‐ forward primer (5′‐AGCATGAGCACATCGTCAAG‐3′), reverse primer (5′‐ATATGCAGCATCTGCGACTG‐3′); and β‐actin‐ forward primer (5′‐ACCACAGTCCAGCCATCAC‐3′), reverse primer (5′‐TCCACCACCCTGTTGCTGTA‐3′). The products of the PCR were analysed by the ABI 7500 system , and β‐actin was used as an endogenous control. The results were calculated by the ddCt method  and rabbit polyclonal anti‐TrkB antibody (1:500). After incubation with the appropriate secondary antibody, the protein complex was labelled with DAB chromogenic reagent, coverslipped with permount and observed by an optical microscope. The HPIAS‐1000 high definition colour pathological image analysis system was used to analyse the images. Three sections were obtained from each rat. Three visual fields were randomly selected and observed for each tissue section. The mean grey value of the protein complex of the offspring born to the same mother was regarded as the protein expression level.n = 10 in each group) in lysis buffer  containing a protease inhibitor cocktail (Sigma‐Aldrich) and phosphatase inhibitors . Protein concentrations of samples were determined using the BCA protein assay . Twenty micrograms of each protein sample were analysed by western blot using the following primary antibodies: rabbit polyclonal antiphospho‐TrkB at 1:1000, rabbit monoclonal anti‐synaptophysin at 1:1000 and rabbit polyclonal anti‐β‐Actin at 1:5000. Images were scanned by an Image Master II scanner  and were analysed using ImageQuant TL software v2003.03 . The signals for the protein bands of interest were normalized to those of β‐actin and were then expressed as fractions of the control samples from the same gel.Total protein lysates were prepared by homogenizing hippocampal tissues  version 17.0 software  was used to analyse the data. Escape latency data were analysed by two‐way P > 0.5) between the propofol exposure and control groups  or gender ratio. The litter number in the control and propofol groups was 276 and 139, respectively. The offspring survival rate of the control and propofol exposure groups was 94.6% (261/276) and 92.1% (128/139), respectively. The ratio of males to females in the control and propofol groups was 1.1 and 1.4, respectively. Maternal propofol exposure also had no obvious influence on offspring physical development. On day P30, the average weight of offspring in the control group (130.3 ± 10.9 g) was not significantly different from that in the propofol exposure group (132.7 ± 9.8 g). Dyskinesia was not observed in either of the two groups. These results indicate that propofol exposure on day E18 has no obvious effects on the offspring survival, sex ratio or basic physical development and suggests that the differences in learning and memory observed in the current study are induced by maternal propofol exposure instead of physical differences.Spatial learning and memory of the offspring was tested with MWM analysis on day P30. Preliminary results revealed no obvious gender differences in learning and memory; therefore, the learning and memory results of female and male offspring were combined. Compared to the control group, the offspring in the propofol exposure group required additional time (escape latency) to find the hidden platform but had shorter target travelling time and shorter platform‐crossing times (Fig. TrkB agonist 7,8‐DHF binds with high affinity to TrkB receptors and can activate the downstream signalling cascade To determine whether BDNF and TrkB are involved in the learning and memory impairments observed following maternal propofol exposure, the protein levels of BDNF and TrkB in hippocampus tissues of the offspring were tested. Because 20% intralipid exposure did not alter learning and memory in the offspring Fig. B–D, we dTo further verify whether the BDNF‐TrkB signalling pathway is involved in learning and memory impairments, a TrkB agonist was injected in rat offspring 2 hrs before each MWM trial. It was discovered that the protein levels of BDNF and TrkB in the PD group were higher than those in the P group but were still lower than those in the control group Fig. . FurtherTo explore whether the observed down‐regulation of BDNF and TrkB proteins is caused by inhibition of gene expression, the mRNA levels of BDNF and TrkB was measured by real‐time PCR in hippocampus tissues. We found that the levels of both BDNF and TrkB mRNA were significantly lower in the P group than those in the control group Fig. D and sugTo further verify whether the decreased BDNF and TrkB protein levels are caused by inhibition of gene expression, the mRNA levels of BDNF and TrkB in the PD group were compared to those in the P group and control group. It was discovered that that the mRNA levels of both BDNF and TrkB were significant higher in the PD group than those in the P group but lower than those in the control group Fig. D. 7,8‐DHTyrosine kinase B is autophosphorylated after binding to its ligand and the phosphorylation of TrkB facilitates long‐lasting synapse formation Synaptophysin hippocampal protein levels were detected by western blot. It was discovered that maternal propofol exposure during late pregnancy markedly down‐regulated synaptophysin expression in the hippocampus; however, this synaptophysin down‐regulation was significantly reduced in animals treated with DHF. Synaptophysin protein levels in the propofol exposed group were significantly lower than those in the control group and DHF‐treated group. Significant differences in synaptophysin levels were not observed when comparing between the C and CD groups Fig. B. SynaptThe results in present study showed that maternal exposure to propofol on day E18 impair learning and memory of rat offspring, inhibit expression of BDNF, TrkB and synaptophysin, and inhibit phosphorylation of TrkB in the hippocampus. Tyrosine kinase B agonist 7,8‐DHF significantly improved learning and memory, blocked down‐regulation of BDNF, TrkB and synaptophysin and facilitated phosphorylation of TrkB. Therefore, these results indicate that maternal exposure to propofol during late stages of pregnancy impair learning and memory by inactivating the BDNF‐TrkB signalling pathway in the hippocampus of offspring, and TrkB agonists may ameliorate these effects.2 of the maternal rats were monitored during propofol infusion. No significant changes were observed for any of these indicators; however, two maternal rats died during anaesthesia induction and 1 died during propofol infusion. The levels of various artery blood gases were analysed at the end of propofol infusion. The blood gas levels of maternal rats in the propofol exposure group were not significantly different from those in the control group (Table The body temperature, heart rate, respiratory rate, blood pressure and SPOvia TrkB receptor activation Studies have indicated that propofol may impair learning and memory by preventing synaptic plasticity, especially LTP, and facilitates the conception and maintenance of LTD in the CA1 areas of the hippocampus To verify the role of the BDNF‐TrkB signalling pathway in learning and memory, the TrkB agonist 7,8‐DHF was used to treat rat offspring. 7,8‐DHF is a recently discovered selective agonist of the TrkB receptor. 7,8‐DHF is a flavonoid compound that displays neuroprotective effects in neurological disorders such as Alzheimer disease Synaptophysin is a synaptic protein marker and provides a structural basis for synaptic plasticity The current results show that 7,8‐DHF can improve learning and memory impairments in offspring exposed to propofol, but the impairments were not completely eliminated. The possible reasons for the incomplete rescue are that first, the dosage of 7,8‐DHF may not be sufficient, although 5 m/kg of 7,8‐DHF has been shown to reverse memory impairments caused by immobilization stress Brain‐derived neurotrophic factor and TrkB can facilitate the dimerization and phosphorylation of TrkB and can thereby activate MAPK, PIK3 and phospholipase C‐γ1 signalling pathways to protect hippocampal neurons from damage induced by glutamate In the current study, we use the MWM to evaluate learning and memory. To provide a more comprehensive assessment of learning and memory effects in future studies, multiple behavioural test systems should be used, such as the Barnes maze test, the eight‐arm radial maze task and the fear conditioning test.In conclusion, the current results indicate that maternal propofol exposure during late pregnancy stages impairs spatial learning and memory in rat offspring by disturbing the BDNF‐TrkB signalling pathway. Additionally, the TrkB agonist 7,8‐DHF can ameliorate learning and memory impairments by facilitating the expression of BDNF and TrkB.The authors confirm that there are no conflicts of interest.Conceived and designed the experiments: Foquan Luo, Weilu Zhao. Performed the experiments: Liang Zhong, Liuqin Wu, Yunlin Feng, Jiamei Lin, Shengqiang Wang, Tianyin Liu, Xuexxue You, Wei Zhang. Analysed the data: Foquan Luo, Weilu Zhao. Contributed reagents/materials/analysis tools: Liang Zhong. Wrote the article: Foquan Luo, Yunlin Feng, Liang Zhong.


A new environmentally friendly one-step method for producing multilayer (preferably 7–9 layers) nitrogen-doped graphene (N-MLG) with a slight amount of oxygen-containing defects was developed. The approach is based on the electrochemical exfoliation of graphite electrode in the presence of azide ions under the conditions of electrolysis with pulse changing of the electrode polarization potential. It was found that usage of azide anions lead not only to the exfoliation of graphite but also to the simultaneous functionalization of graphene sheets by nitrogen atoms . Composition, morphology, structure, and electrochemical properties of N-MLG were characterized by C,H,N analysis, transmission electron microscopy, atomic force microscopy, FTIR, UV–Vis, and Raman spectroscopy, as well as cyclic voltammetry. The perspective of using N-MLG as oxygen reduction reaction electrocatalyst and for the electrochemical analysis of biomarkers  in their mixtures was shown. Functtability . It openNitrogen-doped graphene (N-graphene) can be prepared via direct incorporation of nitrogen atoms into graphene by means of, for example, chemical vapor deposition, arc discharge, and solvothermal processes –7 or by 4)2SO4 and NH4OH [The electrochemical exfoliation of graphite is a promising approach to produce graphene and graphene-related materials due to its easy, fast, and environmentally friendly nature . Recentlnd NH4OH  were repnd NH4OH , high cond NH4OH –14, enviPreviously, we have shown the possibility of the formation of multilayer graphene (MLG) with slight amounts of oxygen-containing defects by means of exfoliation of graphite electrode in presence of benzoate anions in a pulse mode of electrolysis . It is sConsidering the above, the purpose of this study was to establish the possibility of electrochemical one-step production of N-graphene with slight amounts of oxygen-containing defects via exfoliation of graphite in an aqueous solution of sodium azide in a pulse mode of electrolysis without using concentrated ammonia solution, expensive ionic liquids, and high potentials, as well as clarification of its electrocatalytic activity in the oxygen reduction and oxidation of such biomarkers as ascorbic acid (AA), dopamine (DA), and uric acid (UA).2), and commercially available chemicals —NaN3, KCl, H2SO4, ascorbic acid, dopamine, and uric acid—were used as supplied without additional purification. The distilled water was used for electrolyte preparation. The graphene oxide (GO), used in the study for comparison, was obtained via liquid phase exfoliation of graphite oxide, synthesized by the modified Hummers method [High-purity graphite rods , gasses . In order to modify electrode, 2 μL of aqueous or alcoholic dispersion (1 mg/mL) of the corresponding graphene material was dropped onto its surface, followed by drying on air. TEM images were recorded using a transmission electron microscope TEM125K (Selmi) with an accelerating voltage 100 kV (samples were deposited onto copper grids coated with amorphous carbon film). Atomic force microscopy (AFM) of thin film graphene samples on the surface of silicon wafers coated with silicon nitride (Agar Scientific) was performed on a Nanoscope IIIa Dimension 3000TM  instrument. FTIR spectra were taken on Fourier transform infrared spectroscope SPECTRUM ONE (PerkinElmer); samples were prepared as tablets with KBr. UV–Vis spectra of dispersions were registered via UV–visible spectrometer 4802 (Unico). Raman spectra were obtained with a triple spectrometer ; samples were deposited onto silicon templates. C,H,N-elemental analysis was performed on Carlo Erba 1106 elemental analyzer  based on modification of the classical Pregl and Dumas method  using 0.5–1.0 mg of sample per analysis. The oxygen content in the samples was evaluated by difference between the total weight of the samples and content C,H,N in them .Electrochemical studies were carried out via computer complex based on potentiostat PI-50-1.1 using a three-electrode undivided cell (working electrode—glassy carbon (GC) disk with visible surface area of 0.03 cm3 was used as electrolyte. Dispersion of obtained graphene material was filtered through a membrane filter with a pore diameter 0.2 μm (SUPELCO°), rinsed with water, and dried in oven at 60 °C. If there was necessity, the dried precipitate was transferred to appropriate solvent , where regeneration of material dispersion was occurred by using ultrasound treatment for 2 min in the ultrasonic washing bath (Selmi). For comparison, the synthesis was carried out using other electrolyte concentrations: 0.1 and 2 M.Electrochemical exfoliation of graphite was carried out in undivided cell  Fig.  using a 3−) into graphite interlayer space followed by its partial anodic decomposition, N3− → 3/2 N2 + e− [3− occurs and also its partial decomposition: N3− + 3H2O + 2e− → N2 + NH3 + 3OH− [2, NH3, and O2 evolution during electrolysis provide separation of graphene layers, forming multilayer packages of graphene, doped with nitrogen, that are passing to electrolyte volume. At the same time, ammonia, evolved as a result of cathodic decomposition of N3−, acts as a nitrogen source for the in situ graphene doping, while using of low potentials and nitrogen evolution as a result of electrochemical process promotes low number of oxygen-containing defects in N-graphene. Furthermore, over time pH of the electrolyte was changed from 7 to 11–12 . It should be noted the appearance of so-called Moire contrast on some TEM micrographs of the N-MLG particles , C–O in carboxyl (1460 cm−1), and C–O in epoxy and alkoxy (1100 cm−1), are observed in both spectra. It should be noted much smaller intensity of bands caused by vibrations in oxygen-containing groups in N-MLG spectrum if compare with spectrum of GO, which indicates on significantly lower content of such groups in obtained MLG doped with nitrogen. The unambiguous interpretation of intense band at 1629 cm−1, which present in spectra on Fig. 2-C clusters of graphene [−1 in FTIR spectrum of N-MLG, unlike GO, related to C–N stretching vibrations [Figure graphene –23. It ibrations , which cPartially oxidized state of obtained material and functionalization of it by nitrogen were also confirmed by C,H,N-analysis data. Thus, calculated nitrogen content in N-MLG was about 0.6% and atomic ratio C/O ~17. Such C/O ratio indicates that oxygen-containing groups although are present in obtained N-MLG, but their content is much less than for example in chemically or thermally reduced GO, where C/O ~8–11 . It is inπ*-junction with the participation of unshared electron pairs of the oxygen atoms in oxygen-containing groups [The presence of band with maximum absorption at 267 nm in UV–Vis spectrum of N-MLG dispersion in ethanol Fig. , which c−1 along with the impossibility of its approximation via only one Lorentz line [Raman spectrum of obtained N-MLG differs from spectrum of starting graphite, and it is characterized by typical for carbon materials D, G, and 2D bands Fig. . The posntz line  as well ntz line  indicatentz line .Fig. 5Ra2SO4 in presence and absence of oxygen were investigated by means of cyclic voltammetry. For comparison, the same features were measured for graphene materials, which did not contain nitrogen in their composition—electrochemically obtained multilayer graphene (MLG) [onset) and catalytic current maxima potential. Improving of electrocatalytic properties of N-MLG in comparison with MLG and ERGO can also be an additional indirect proof of functionalization of obtained material by nitrogen atoms. It should also be noted that the electrocatalytic activity in ORR of obtained N-MLG is comparable with the established in the literature for the N-modified graphenes obtained by other methods [As it is known, N-doping of graphene leads to significant improving of its functional characteristic in particular electrocatalytic activity in oxygen reduction reaction (ORR), which is current-forming process in fuel cells . In ordene (MLG)  and elec methods .Fig. 6CVOne important application of N-MLG may be its use as an electrode material for electrochemical analysis of biomarkers—dopamine (DA), ascorbic (AA), uric (UA) acids, etc., because doping graphene with heteroatoms may improve their sensitivity and selectivity at electrochemical determination of these substances. As a result of electrochemical studies, it was found that the biomarkers on glassy carbon (GC) electrode modified with N-MLG are oxidized at different potentials—AA 285 mV, DA 415 mV, and UA 535 mV Fig. . It shouIn conclusion, the possibility of one-step electrochemical preparation of multilayer graphene functionalized with nitrogen (N-MLG) via exfoliation of graphite electrode in presence of azide anions in a pulse mode of electrolysis was presented. Sodium azide provides not only exfoliation of graphite via multiple repetition of anion intercalation/deintercalation cycles into graphite interlayer space but also simultaneous functionalization of graphene sheets by nitrogen atoms as a result of partial electrochemical decomposition of azide anions with ammonia evolution. Particles of N-MLG preferably consist of 7–9 individual graphene layers with a low amount of oxygen-containing defects (C/O ratio ~17), which was proved by means of C,H,N-analysis, TEM, AFM, FTIR, UV–Vis, and Raman spectroscopy. It was shown that increase of electrolyte concentration during electrochemical synthesis (from 0.1 to 2.0 M) allows change nitrogen content from 0.2 to 0.9% in resulting material.It was found that N-MLG is a promising electrode material. By means of cyclic voltammetry, it was shown higher electrocatalytic activity of N-MLG in the oxygen reduction reaction, which is realized in fuel cells, compared to electrochemically prepared multilayer graphene or electrochemically reduced graphene oxide, that do not contain nitrogen atoms in their structure. Also, N-MLG was shown to be electrochemically active toward oxidation of such biomarkers as dopamine (DA), ascorbic (AA), and uric (UA) acids. Significant difference of oxidation potentials of DA, AA, and UA  suggests the possibility of using N-MLG in electroanalysis of mentioned above biomarkers in biological liquids.


Originating from the investigation of condensed matter states, the concept of quantum Hall effect and quantum spin Hall effect (QSHE) has recently been expanded to other field of physics and engineering, e.g., photonics and phononics, giving rise to strikingly unconventional edge modes immune to scattering. Here, we present the plasmonic analog of QSHE in graphene plasmonic crystal (GPC) in mid-infrared frequencies. The band inversion occurs when deforming the honeycomb lattice GPCs, which further leads to the topological band gaps and pseudospin features of the edge states. By overlapping the band gaps with different topologies, we numerically simulated the pseudospin-dependent one-way propagation of edge states. The designed GPC may find potential applications in the fields of topological plasmonics and trigger the exploration of the technique of the pseudospin multiplexing in high-density nanophotonic integrated circuits. Photonic topological insulators –4, opticSurface plasmon polaritons (SPPs) , elementIn this work, we theoretically explore the topological properties of two-dimensional (2D) graphene plasmonic crystals (GPCs) constructed by periodically arranged graphene nanodisks. Dirac cones at the Brillouin zone (BZ) corners are folded to a double Dirac cone at the BZ center by utilizing the zone folding mechanism. In order to obtain topological band gaps, we take further deformations on honeycomb lattice. By shrinking or expanding the graphene nanodisks, the double Dirac cone is opened and the band inversion occurs between pseudospin dipole and quadrupole modes, which further leads to topological phase transition between nontrivial and trivial states. Furthermore, one-way propagation of edge states is numerically simulated along an interface constructed by the trivial and nontrivial GPCs, which further demonstrates the pseudospin characteristics and topological robustness of our designed plasmonic crystals.a = 40 nm, μc1, and r are the chemical potential and radii of the graphene nanodisks; μc2 denotes the chemical potential of the surrounding graphene. By solving the Maxwells equations with boundary conditions, we obtain the dispersion relation for transverse magnetic (TM)-polarized SPP modes supported on the graphene layer surrounding by air and silica  can be expressed as [k± = kx ± iky, and A comes from off-diagonal elements of the first-order perturbation term α = 1, 2 and β = 3, 4. The effective Hamiltonian Heff(k) takes a similar form as the Bernevig-Hughes-Zhang (BHZ) model for the CdTe/HgTe/CdTe quantum well system [To further explore the topological property of the band gaps shown in Fig. essed as , 35.5\dol system , implyinl system .6\documeM = (Ep – Ed)/2 is the frequency difference between E2 and E1 representations at the Γ point. B is determined by the diagonal elements of the second-order perturbation term and is typically negative [C± = 0 is obtained when having a normal band order as displayed in Fig. M becomes positive when band inversion occurs. Therefore, C± = ±1 is simply obtained, and the gap in Fig. Here, negative . Thus, CΓK direction for such a ribbon, where a bulk band gap is spanned by additional topological edge states as indicated by the double degenerated red curves. Fig. kx = − 0.05π/a) and B (with kx = 0.05π/a) marked in Fig. By overlapping the band gaps with different topologies , one can create edge states that are spatially confined around the interface between two plasmonic crystals. Here, we consider a ribbon of topologically nontrivial plasmonic crystal  of anticlockwise (clockwise) circular polarization of in-plane magnetic field. One of the most distinguishing features of topological edge states is that they are robust against perturbations/imperfections. To verify this robustness, we construct sharp bends as displayed in Fig. S−. The SPP wave vanished eventually after a long traveling distance along the sharp bends due to the intrinsic loss of graphene material. To further confirm this topological transmission, we also exhibit the electric field intensity distribution by ignoring the intrinsic loss of graphene for comparison. As can be seen from Fig. The pseudospin-dependent unidirectional transmission of edge states are also demonstrated in a finite 20In summary, we have systematically investigated the band topologies of the GPCs constructed by periodically patterned graphene nanodisks. By employing zone folding mechanism, the Dirac cones at the BZ corner are folded to a double Dirac cone at the BZ center. Further, topological band gaps are realized by deforming the honeycomb lattice GPCs. Based on the effective Hamiltonian derived by the


Many human introns carry out a function, in the sense that they are critical to maintain normal cellular activity. Their identification is fundamental to understanding cellular processes and disease. However, being noncoding elements, such functional introns are poorly predicted based on traditional approaches of sequence and structure conservation. Here, we generated a dataset of human functional introns that carry out different types of functions. We showed that functional introns share common characteristics, such as higher positional conservation along the coding sequence and reduced loss rates, regardless of their specific function. A unique property of the data is that if an intron is unknown to be functional, it still does not mean that it is indeed non-functional. We developed a probabilistic framework that explicitly accounts for this unique property, and predicts which specific human introns are functional. We show that we successfully predict function even when the algorithm is trained on introns with a different type of function. This ability has many implications in studying regulatory networks, gene regulation, the effect of mutations outside exons on human disease, and on our general understanding of intron evolution and their functional exaptation in mammals. While many introns behave as neutral elements, many others has been shown to carry out a wide variety of cellular functions, and are involved in virtually every step of mRNA processing2. Here, we define intron function in the “causal role” sense, meaning that it contributes to cellular processes, and it need not necessarily adhere to the definition of “selected effect” functions, which are selected at the organism level3. For instance, introns participate in regulating transcription initiation, elongation, termination and timing7, in exporting mRNAs to the cytoplasm8, and in the quality control of mRNAs9.Spliceosomal introns are one of the hallmarks of eukaryotic species, and inhabit genomes from the full spectrum of the eukaryotic domain. Many species, such as mammals, are intron-rich, harboring on average multiple introns per geneD. melanogaster. The pre-mRNA of the predominant transcript of this gene is 60 kb long, but the mature mRNA is only 6 kb long. Thummel et al. measured the RNA polymerase II elongation rate on this gene and suggested that the time it takes to transcribe the introns generate delays that are crucial to the function of this gene during metamorphosis10. Other intronic functions depend on the position of the intron along the mRNA rather than on its sequence. This is the case, for example, in nonsense-mediated decay in mammals, where the splicing of introns sufficiently downstream of a termination codon marks the transcript for degradation12. Still more intronic functions depend on the fact that splicing occurred along the transcript, regardless of which specific intron had been excised. This is the case, for example, in functions related to mRNA export and quality control13.This makes the identification of functional introns and the estimation of their abundance fundamental questions in functional genomics. However, it is not possible to detect functional introns by using traditional strategies such as looking for high sequence conservation. Intronic sequences – even of functional introns – rarely show elevated sequence conservation. One reason is that many intronic functions come about from short cis-regulatory elements that are hard to detect against a background of a very long non-functional intronic sequence, particularly when their location within the intron varies. A second reason is that not all intronic functions are sequence-dependent. Some intronic functions depend on the intron length rather than on its sequence. This is demonstrated by the E74 gene in 15. We conjectured that introns that harbor elements that are under purifying selection would experience a different selection regime than other introns, and therefore display distinct evolutionary characteristics that are reflected in lower loss rates and higher positional conservation. In a previous paper, we provided support to this conjecture. We identified 503 human introns that harbor miRNAs and snoRNAs. We used several features to quantify their level of positional conservation, compared them to 5,603 human introns that are not known to harbor functional elements, and demonstrated that indeed introns harboring functional elements show unique evolutionary patterns16. Specifically, they tend to be of ancient origin (otherwise their function would not have impacted their evolutionary pattern) and to show elevated levels of positional conservation.As opposed to their generally poor sequence conservation, some introns show high conservation of their position with respect to the coding sequence, which is sometimes stable across billions of yearsHere, we developed a statistical framework to predict which introns harbor functional elements. First, we examined the characteristics of functional introns whose function originates from an entirely different class of functional elements. To this end, we used introns that harbor transcription factor binding sites (TFBS) for which we have functional evidence. We found that these introns show almost identical characteristics to introns that harbor small RNAs, thus demonstrating that high positional conservation is a universal property of such functional introns, regardless of the particular element embedded within their sequence. Based on these universal characteristics, we have devised a statistical model that computes the probability of each human intron to be functional in the sense that it harbors a functional element. Our statistical framework is unique in that it accounts for the inherent uncertainty in labeling an intron as non-functional. Whereas there is a high certainty that an intron tagged as functional is indeed so, many introns whose function is not known and are thus tagged as non-functional, may in fact be functional. Our approach explicitly models this uncertainty, and provides estimates to its magnitude.16.In a previous work we identified 391 sets of orthologous transcripts across 28 species, in which the human transcript harbors at least one functional intron, and where the function stems from embedded miRNA or snoRNA elementsHomo sapiens, Pan troglodytes, Pongo abelii, Mus musculus, Monodelphis domestica, Ornithorhynchus anatinus); a bird ; a lizard (Anolis carolinensis); an amphibian ; two fish ; and one member of Cionidae (Ciona savignyi), which together make the representatives of Chordates in this data set. In addition, our data set includes three protostomes , which, together with the chordates, are all bilaterians. Furthermore, our data set contains four fungi . Other Eukaryotic subgroup include Viridiplantae ; two representative of Stramenopiles ; a single Alveolate ; a single Amoebozoan (Dictyostelium discoideum); and a single Euglenozoan (Leishmania major). This list of species may not be ideal, as it includes lineages that had gone through massive intron loss (like S. cerevisiae and U. maydis) and lineages that harbor a small number of introns . In addition, some lineages are connected to the rest through long branches in the phylogenetic tree, presenting some challenges to the interpretation of the results. On the other hand, these species were selected based on the fact that their genes have known lists of orthologs, allowing for large-scale comparative genomics. Indeed, the species included in this study were shown to provide meaningful information on evolutionary trends of intron evolution16.The 28 species comprise of: Six mammals . The mere presence of a TFBS within an intron is not an indication of functionalityFor each set of orthologs we generated multiple sequence alignment of the orthologous transcripts at the mRNA level. Upon this multiple sequence alignment we had mapped the gene architecture using a ternary representation, where 1 marks the last nucleotide of an exon, 0 marks all other nucleotides, and 2 marks a lack of ortholog or a gap. Each column in the ternary representation is called a pattern because it shows the intron presence-absence configuration at that position across the 28 species Fig. . We lookAs long as introns are assumed to evolve independently of each other, two positions with the same pattern hold the same information on the underlying evolutionary processes. Therefore, pattern information in a data set can be compactly summarized by a list of all unique patterns, together with the number of times each unique pattern appears in the data set Fig. . We have16. Notably, these features do not include any gene-specific properties like expression level, as the same pattern can be present in multiple genes.In a previous work, we examined the effect of many pattern-characterizing features. Our initial list consisted of 48 features, but features that were highly associated with other features were removed. This process led to a final list of 13 features that represent diverse properties of the intron such as its antiquity, how its evolutionary pattern resembles that of other introns, the level of its positional conservation, and its position along the coding sequence Table 16. Notab16.Analysis of the 13 features for introns that harbor miRNAs and snoRNAs revealed some characteristic properties of these introns: First, their associated patterns have low likelihood, suggesting that their evolutionary history is different than that of the bulk of introns. Second, they show high positional conservation across metazoans. In particular, they have low intron loss rate even compared to the generally low loss rate in metazoans. Third, their origin is ancient, and usually predates the origin of metazoans. Indeed, as the identification of functional introns relies on their unique evolutionary histories, we do not expect to be able to identify introns which have recently gained their function−6, t-test); greater antiquity, reflected by high values of LCA_AGE  that measures the age of the last common ancestor of all species that harbor the intron; and a strong inclination to reside closer to the 5′ UTR .We started the analysis by examining these 13 features on Dataset 1. First, we compared the values of each feature across miRNA/snoRNA-bearing and -lacking patterns using Bonferroni-corrected t-test Table . Indeed,−22, t-test). Moreover, introns that harbor TFBS in humans tend to be found also outside of metazoans: in fungi , in protists , and in plants . This suggests the plausible evolutionary scenario by which these introns became functional much earlier than the birth of metazoans. It should be emphasized that it does not mean that the same TFBS populated these intron for such a long period, as TFBS turnover rate is known to be high24. Rather, different elements from within the same intron may have similar functions, keeping the intron functional. This is further supported by the values of the feature LCA_AGE. The average LCA_AGE of TFBS-bearing introns is significantly higher than that of TFBS–lacking patterns , but also than that of miRNA- and snoRNA-bearing patterns . Also in common to all functional introns is that both SANKOFF_G3L1 and SANKOFF_G1L3 are higher, which is again a result of their antiquity, providing them with more time to accumulate evolutionary events.To test whether introns with a completely different function have similar characteristics, we repeated the above comparison, contrasting TFBS-bearing patterns with patterns associated with a human intron not known to be functional Table . We foun25. In a previous work we showed that this tendency for introns harboring small RNAs is fully explained by the fact that first introns tend to be longer26. In order to test whether this effect also explains the proximity of TBFS-bearing introns to the CDS start, we conducted a permutation test. To this end, we assumed as a null hypothesis that a functional element has an equal probability to reside at any position inside an intron, regardless of the distance of the intron from the CDS start. Then, we ran 1,000 simulations in which we uniformly repositioned the functional elements within the introns. Compatible with our previous observations, in more than 95% of the cases the randomly positioned miRNAs and snoRNAs resided in their actual hosting intron, or in an intron closer to the CDS start. In contrast, in less than 1% of the cases a TFBS resided in its actual hosting intron or in an intron closer to the CDS start. We conclude that the tendency of TFBS-bearing introns to reside in introns that are close to the CDS start is not a simple outcome of the fact that 5′-most introns are longer. Rather, this is likely to be a result of the fact that many of the TFBS affect transcription initiation and are located in the vicinity of the transcription start site.All types of functional patterns have a higher tendency to reside closer to the 5′-side of the gene Table . This teIn order to further demonstrate that functional introns share common characteristics, regardless of their specific function, we performed Fisher discriminant analysis to separate miRNA/snoRNA-bearing patterns from miRNA/snoRAN-lacking ones Fig. . On thisFisher discriminant analysis shows that functional introns tend to have high values of the Fisher discriminant vector, but that they are mixed with numerous non-functional introns Fig. . This mai be represented by the 13-dimensional feature vector xi, and let its functionality be labeled by i to represent a functional intron is given by the logistic function0 and ω are parameters of the model (which we hereinafter group together as ω).Let a unique pattern zi and the true label yi, and treat zi as a noisy version of yi. The relationship between the true and the noisy labels is described by θij’s may be determined from the particular nature of our problem. If a unique pattern is labeled as functional, we assume that it is indeed functional, which is formally expressed as θ value is θ11 (as θ10 = 1−θ11).To incorporate the uncertainty in the labeling of unique patterns, we make a distinction between the actual label  is Fig. :2\documeθ11, and then used (2) to compute the probability of each unique pattern to be functional. To determine which unique pattern corresponds to a functional intron we used a threshold T = 0.5, such that if We used the Expectation-Maximization (EM) algorithm to learn the parameters of the model, −11, hypergeometric-test).We used three different strategies to validate our model. First, we trained the model on Dataset 1, treating only the 134 small RNA-bearing unique patterns as functional, and tested the predictions of the model on the 88 TFBS-bearing unique patterns. We found that 82% of the TFBS-bearing patterns (72 out of 88) were correctly predicted as functional. Under our unique setup that the labeling of non-functional introns is uncertain, we cannot directly estimate the rate of false positives. To show that our success rate is not a result of a general tendency of our classifier to inflate the number of predicted functional patterns, we showed that the fraction of unique patterns predicted to be functional is higher among unique patterns labeled as TFBS-bearing than among those that are labeled as non-functional . We have already discussed the fact that our approach is unable to detect recent acquisition of function, as the intron do not show unique evolutionary patterns. This is further reflected by the fact that the LOGLIKE values of all these patterns is significantly higher than the average value for TFBS-bearing patterns, and similarly their median distance from the CDS start is significantly greater than the value for TFBS-bearing patterns.−104).As a second validation, we have trained the model over Dataset 1 and tested it over Dataset 2. We correctly labeled 77% of the TFBS-bearing introns (581 out of 754). Again, to verify that our model does not inflate the number of predicted functional introns we performed a hypergeometric test , and coefficient of variation (mean/STD). The results show the robustness of the training procedure, with the values of the estimated parameters insensitive to the initialization of the algorithm , followed by LCA_AGE and IN_PLANT , preceded by LCA_AGE and SANKOFF_G1L3 , those that are predicted to be functional by our model (PRED-FUNC), and those that are predicted to be non-functional (PRED-NON-FUNC). We wanted to compare these groups in terms of the density of functional regulatory elements they harbor. To this end, we used chromatic states as inferred by the ENCODE project. These states were determined based on the combination of two techniques (ChromHMM and Segway) which identify functional genomic segments based on experimental data that marks open chromatin, certain histone modifications, and transcription factor binding. Their data are available for six human cell types , and is used to predict seven states: promoter region including TSS; promoter flanking region; enhancer; weak enhancer or open chromatin cis regulatory element; CTCF-rich element; transcribed region; and repressed or low activity regionχ2-test to check whether the proportion of ‘active’ introns is different across the three groups of introns  bins  are enriched with certain biological functions. To this end, we generated three list of genes. The first list contains all genes hosting at least one known functional intron. The second list contains all genes that are not present in the first list and that host at least one intron predicted to be functional by our model. The third list contains all other genes.27, we tested each list for enrichment in biological processes against a background of all human genes  is significantly above the average (P = 0.003).In this work we developed a model that computes the probability of a human intron to be functional. Based on a comparative study of gene intron-exon architecture, we characterized introns by 13 features that mostly describe their evolutionary histories. Importantly, we do not look at intronic properties that are considered more evolutionary volatile, such as their sequence or length. Nor do we examine properties that characterize genes (like expression level), as the same pattern may be found in multiple genes. We showed that functional introns are characterized by a similar set of features, confirming the hypothesis that functional introns display lower loss rates and higher positional conservation, reflected in lower log-likelihood of the pattern, and in dense presence within certain clades ), the level of positional conservation  and the antiquity of the intron (LCA_AGE).We build a statistical model that accounts for the asymmetrical state of knowledge regarding intron function. While an intron that harbors a functional element is highly likely to be functional, an intron that lacks such an element is not necessarily non-functional. It may well be functional by harboring a yet undetected functional element, by harboring a functional element not looked for in the current study, or by having a function that does not depend on an embedded functional element. Therefore, there is high certainty in labeling an intron as functional, but no certainty when labeling an intron as non-functional. We estimated the parameters of the model, and estimated that roughly 20% of the functional introns are labeled as such, and roughly 38% of the introns are functional. This number is expected to be an overestimation, because we had only taken highly conserved transcripts into the analysis. We expect that many introns within less conserved transcripts would lack function, and will thus reduce the above estimate.We provide further validation of our predictions by showing that functional introns, as well as introns predicted to be functional, host more active genomic segments. In addition, genes that harbor functional introns, whether known to be functional or predicted to be one, tend to be enriched in GO terms of the same biological processes.The model was trained on introns that are functional in humans, and therefore we expect our predictions to be most accurate for mammals. The ability to predict functional introns has obvious implications in fields such as studying the effect of mutations on human diseases and designing DNA constructs.28, that was updated to include new annotations from the Ensembl Genomes release 2129.Gene exon-intron architecture data was downloaded from the JuncDB databasePan troglodytes, Mus musculus, Pongo abelii, Ornithorhynchus anatinus, Monodelphis domestica), a bird , two fish , two amphibians , an insect (Drosophila melanogaster), three other invertebrates , four fungi , four plants , and five protists . The phylogenetic tree that spans these 28 species was taken from our previous work16, with the exception that it has been modified to reflect the Ecdysozoa topology31, which is now the commonly accepted alternative to the Coelomata topology33 . The modified topology was taken from The Tree of Life Web Project (http://tolweb.org/) and from NCBI36, and the updated divergence times were estimated using Time Tree37.Sets of orthologous genes were defined over 28 eukaryotic species. In addition to human, the sets include five mammals , and crossing the data with the JuncDB database, we obtained 391 sets of orthologous transcripts over 28 species, in which the human transcript harbors at least one functional intron. In order to identify additional human functional introns, where the function stems from factors other than embedded small RNAs, we have extended our data set in two ways.The starting point of our previous work was a list of 450 human transcripts that host functional introns, where the intron function comes about from embedded miRNA or snoRNA elements38. Out of the 450 transcripts, 306 were found to harbor TFBS-bearing introns within their CDS. Combining this with our previous data set of small RNA-bearing introns, we ended up with a set of 404 orthologous groups whose human transcript harbors at least one functional intron. To avoid a bias towards functional introns, we screened the Ensembl Genomes database (release 21) for transcripts whose introns are not known to contain small RNAs or TFBS, or to be otherwise functional. This added 56,957 human transcripts. Ensembl Compara API39 was used to search for orthologs of these transcripts in the other 27 species, resulting in 2,017 sets of orthologs. Obviously, not all 27 species have representatives in all the sets, and to avoid excessive amounts of missing data we removed from the data sets of orthologs with representatives from less than 13 species. This left 367 sets of orthologs. Altogether, Dataset 1 consist of 771 sets of orthologs: in 404 of the sets the human transcript is known to harbor a functional intron, and in 367 sets the human transcript lacks a known functional intron.First, we looked at all the introns in the above 450 human transcripts, and queried for the presence of intronic transcription factor binding sites (TFBS), using TFBS positions from the MotEvo databaseIn order to build a larger data set, we queried the MotEvo database again, this time against the complete Ensembl human transcripts data set, omitting only the transcripts who partake in Dataset 1. This resulted in 65,064 human transcripts harboring TFBS-bearing introns. Using Compara API, we have searched orthologs for these transcripts in the remaining 27 species, and formed a set of 8,661 orthologous groups in which the human transcript harbors a TFBS-bearing intron within the CDS, and in which there are at least five orthologous transcripts in other species. Among transcript variants that share introns and are members in different set of orthologs, we left in the analysis only the set with the highest number of representatives from other species.40 to align the protein products of the transcripts in each group, and then used Matlab’s seqinsertgaps function to properly align the mRNA. The aligned mRNA was then translated to a ternary representation; positions just before exon-exon junctions were denoted by 1, gaps and missing data were denoted by 2, and all other positions were denoted by 0. Each position across the alignment is an absence-presence pattern of introns. Patterns consisting of 45% or more missing values were filtered out, as well as patterns lacking 1’s. In addition, as the analysis is centered on humans, we filtered out patterns with 0’s or 2’s in human. TFBS that were not located in active ENCODE segments23 were filtered out as well.For both data sets, we generated the multiple sequence alignments of the orthologous groups at the mRNA level. First, we used MUSCLE41.Mann–Whitney U-test, and t-test with unknown and equal variance were used to test whether a distribution of a feature is different in function-bearing patterns. Tukey’s multiple comparison test for proportions was used to test whether there were significant dissimilarities between the proportions of introns bearing ENCODE active states across the three groups: functional introns, introns predicted to be functional, and introns predicted to be non-functionalIn order to avoid the use of excessively dependent features, we first applied principal component analysis to the data, and then used Fisher discriminant analysis on the data as represented by the first seven principal components.n unique patterns x1,x2,…,xn and their corresponding binary labels z1,z2,…zn, which are a noisy version of the hidden true labels y1,y2,…yn, the log likelihood of the model iscti to be an estimation of the hidden label yt given the features vector xt and the noisy label zt, based on the current values of the parameters, ω0 and θ0,Given Then, the EM auxiliary function is:t = 1,…,n and For θ can be reduced to θ11, therefore:ω, it is sufficient to maximizeω, meaning this optimization problem is concave and therefore gradient ascent can be used in order to find the global maximum of L2(ω). Therefore, for iteration n and learning rate τ,Based on the auxiliary function above, in order to find the updated value of Xi is the i’th feature, and Y is the predicted binary label.In order to evaluate the contribution of each feature to the prediction of functional introns, we computed for each feature the mutual information, defined as42. In each iteration, the pair of features that affects the performance the least is removed (added to the “rejected” set). Then, the rejected feature that contributes most to the performance is added back. For each set of features Pr0(p) denotes the probability of pattern p to be functional based on the full set of features, and PrF(p) denotes the probability of pattern p to be functional based on the subset F.In order to find the smallest set of features needed for classification with minimal effect on performance, we have implemented a sequential ‘remove two add one’ features selection algorithmSupplementary Figures and Tables


A set of high-affinity, high-specificity posttranslational modification (PTM) enrichment tools was developed to generate an unbiased snapshot of four key PTM profiles  for the clinically important protein programmed cell death ligand 1 (PD-L1). The results showed that epidermal growth factor (EGF) treatment induced tyrosine phosphorylation, acetylation, and ubiquitination of PD-L1. Further characterization of EGF-induced PD-L1 ubiquitination revealed a significant increase in mono- and multiubiquitination of PD-L1 that occurred on glycosylated PD-L1. EGF induced mono- and multiubiquitination of PD-L1 preceded EGF-induced increases in PD-L1 protein levels. Chemical inhibitors of the EGFR pathway, gefitnib and SCH772984, suppressed PD-L1 mono- and multiubiquitination, and inhibition of the ubiquitin E1 activating enzyme, with the chemical inhibitor PYR41, was sufficient to block EGF-stimulated increases in PD-L1 protein levels. This study highlights the significance of identifying novel PTMs for PD-L1 and reveals potentially critical regulatory mechanisms that may be valuable therapeutic targets. In a broader context, this report validates an approach whereby one can gain insight into novel mechanisms of action by a simple and unbiased analysis of a PTM profile of potentially any endogenous protein of interest. Programmed cell death ligand 1 (PD-L1) is a 33-kDa type I transmembrane protein that is comprised of an extracellular, N-terminal domain that interacts with receptor PD-1 expressed on Tcells to inhibit the immune response. PD-L1 is expressed in a wide range of cell types and tissues and has been shown to be upregulated in many of these cell and tissue types under inflammatory conditions Host immune suppression by cancer cells through cell surface expression of checkpoint inhibitors like PD-L1 is a key mechanism for cancer progression Nature Communications describing the regulation of PD-L1 via both polyubiquitination and glycosylation PTMs A large body of work exists describing the transcriptional and posttranscriptional regulation of PD-L1 expression Several PTMs have been studied in great detail; of these, serine/threonine phosphorylation, tyrosine-phosphorylation (pY), acetylation (Ac), ubiquitination (Ub), and SUMOylation (SUMO 2/3) have been shown to be key regulators in almost all cellular processes, including signal transduction, protein expression, stability and localization, and cellular immunity A431 cells were grown in DMEM media  supplemented with 10% FBS  and penicillin/streptomycin . Trypsin/EDTA was obtained from Gibco . Unless otherwise noted, chemicals were obtained from Sigma . Human epidermal growth factor (EGF) was obtained from Cytoskeleton, Inc. . For EGF stimulation experiments, A431 cells were serum restricted for 24 hours with serum-free DMEM to synchronize the cells. Cells were then treated with 33 ng/ml of EGF for 15 minutes or 1, 2, and 4 hours in individual 15-cm dishes  followed by subsequent lysis with BlastR lysis buffer . Gefitnib, SCH772984, and PYR-41 were obtained from Selleckchem . For chemical inhibition experiments, synchronized A431 cells were pretreated with 0.5 μM gefitnib, 2 μM SCH772984, or 100 μM PYR-41 for 30 minutes at 37°C prior to EGF treatment for 1 hour followed by lysis with BlastR lysis buffer.3VO4, and protease inhibitors . DNA was removed by passing the lysate through the BlastR filter system . BlastR lysis buffer is a complete cell lysis reagent that is comprised of a proprietary mixture of detergents, salts, and other buffer additives. After dilution with BlastR dilution buffer, protein concentrations were determined with Precision Red Advanced protein reagent  and measured at 600 nm OD. Protein lysate samples were separated using Tris-glycine SDS-polyacrylamide gel electrophoresis  and transferred to Immobilon-P membranes . Membranes were blocked for 30 minutes at room temperature in Tris-buffered saline  containing 0.05% Tween-20 (TTBS) and 5% milk  and then incubated with 0% to 2.5% milk in TTBS solution containing primary antibodies for 1 to 3 hours at room temperature (RT). Membranes were washed in TTBS 3 ×10 minutes prior to secondary antibody for 1 hour at RT. Bound antibodies were visualized with horseradish peroxidase–coupled secondary antibodies and chemiluminescent reagent  according to the manufacturer's directions. Antibodies used were as follows: P-EGFR ab40815 , PD-L1 ab213524  or PD-L1 13,684 , anti-ubiquitin-HRP AUB01-HRP , tubulin ATN02 , HRP-anti-sheep secondary , and HRP-anti-rabbit secondary . Changes were quantitated by densitometry using Image J software (rsb.info.nih.gov).A431 cells were lysed with ice-cold BlastR lysis buffer containing a cocktail of NEM, TSA, NaAfter obtaining A431 lysate as described above in the western immunoblotting section, samples were immunoprecipitated using pY, Ub, Ac, and SUMO 2/3 PTM identification, Signal-Seeker kits, with equal protein concentration and IP volumes according to the manufacturer's protocol . The appropriate amount of pY (APY03), ub (UBA01), SUMO 2/3 (ASM24), Ac (AAC01), IgG beads (CIG01), or control beads (CUB01) was added to the respective samples for 1 to 2 hours at 4°C on an end-over-end tumbler. After incubation, the affinity beads from each sample were pelleted and washed 3× with BlastR wash buffer. Bound proteins were eluted using the elution buffer and spin columns in the Signal-Seeker kits, and PTM modified target proteins were detected by western immunoblotting.3VO4, and protease inhibitors. IP was performed on lysate using ubiquitin affinity beads (UBA01) or control beads (CUB02). Ubiquitinated proteins were eluted with an alternative elution buffer  for 15 minutes at 25°C. Immunoprecipitated and WCL samples were then deglycosylated using PNGase F from NEB according to the manufacturer's instructions  with the following modifications: PNGase F was used at 0.2 μl per reaction with an incubation time of 30 minutes at 37°C. Additionally, during the SDS neutralization step, additional NP-40 was used to account for the additional 0.2% SDS in the elution step. Finally, samples were run on SDS-PAGE, transferred to PVDF, and analyzed by western immunoblotting.A431 cells were lysed with ice-cold BlastR lysis buffer containing a cocktail of NEM, TSA, NaA, lanes 1 and 2, recapitulated previously published results that PD-L1 protein levels increased in response to EGF stimulation in the A431 cell line Figure 1A, lanes 5,6,9,and10); conversely, neither untreated nor EGF-treated A431 cells showed any evidence of SUMO 2/3 modification of PD-L1 . Most interestingly, a marked increase in PD-L1 Ub was detected in response to EGF stimulation .Several studies have linked increased epidermal growth factor receptor (EGFR) signaling, either through mutation or ligand-induced activation, to increased PD-L1 expression, which has been shown to contribute to the immune evasion of EGFR-driven cancers Figure 1A, densitometry). This is a minimal estimate of the fractional stoichiometry as the absolute efficiency of ubiquitin enrichment for PD-L1 Ub has not been determined. In this regard, it is known that the vast majority of endogenous PTMs is substoichiometric and require an enrichment step for detection Densitometric quantitation of ubiquitinated PD-L1 normalized to input:immunoprecipitation (IP) ratio suggests that an estimated .41% of the total PD-L1 pool is ubiquitinated in response to EGF . The presence of NEM protects ubiquitinated species from rapid de-ubiquitination by DUBs during lysate processing. The results in Figure 1B show that cell lysates lacking DUB inhibitor have more than an 80% decrease in ubiquitinated PD-L1 signal and demonstrates that the ubiquitinated PD-L1 signal is dependent on protein Ub and is not likely a false-positive signal. The detection of endogenous levels of PD-L1 Ub that is dynamically regulated by EGF suggested physiological relevance of PD-L1 Ub and warranted further investigation.To confirm that the ubiquitinated PD-L1 signal was truly ubiquitinated, cell lysates were processed from untreated and EGF-treated A431 cells in the absence of the pan de-ubiquitinase (DUB) inhibitor A and B), which are representative of a molecular weight shift of 1 to 2 ubiquitin modifications. To gain further insight into the type of Ub occurring on PD-L1, IP of Ub was performed with affinity reagents that are known to bind monoubiquitinated proteins (FK2 antibody) The EGF-induced Ub signal for PD-L1 appears to represent a pattern of mono- and multi-/di- (here forward termed multi-) Ub as indicated by the discreet bands of 55 to >64 kDa , A and Bin vitro deglycosylation with PNGase F, which resulted in a single unmodified PD-L1 protein band at 33 kDa. The in vitro deglycosylation method was utilized in this study and resulted in a collapse of the WCL bands between 45 and 55 kDa to a single species right below the 36-kDa molecular weight marker , which was very similar to the previously reported findings. The in vitro deglycosylation step was also performed on ubiquitin immunoprecipitated samples from EGF-treated cell lysate, which resulted in a collapse of the ubiquitinated PD-L1, 55- to >64-kDa smear, to three discrete bands that migrated below the 36-kDa maker, between the 36-kDa and 50-kDa marker, and right at the 50-kDa marker . These size changes of roughly 8 kDa between bands correlate with the molecular weight of ubiquitin, providing further evidence that EGF stimulation induces a mono- and multi-Ub of PD-L1. The exact PD-L1 Ub site has not been defined; however, the UbPred prediction software The smeared banding of PD-L1 in both the ubiquitin IP and the whole cell lysate (WCL) made it difficult to decipher which band corresponded to the monoubiquitinated PD-L1. A previous study reported that the smeared PD-L1 signal observed in the WCL was due to extensive glycosylation of PD-L1 A). The increase in PD-L1 protein levels in response to EGF corroborates previously published findings by several groups Figure 2A). Surprisingly, a rapid increase in PD-L1 mono-Ub at 15 minutes consistently preceded the increase in PD-L1 levels . Densitometric analysis highlighted the significant difference between the change in mono-Ub versus total Ub at 15 minutes with a measured three-fold increase in PD-L1 mono-Ub but no measureable increase in total PD-L1 protein levels . The mono- and multi-Ub (50->64 kDa) bands of PD-L1 are the predominant species throughout this time course, particularly at the 15-minute time point . These findings were clarified even further by deglycosylating the WCL and ubiquitinated PD-L1 samples, which resulted in two distinct bands corresponding to a mono- and multi-Ub molecular weight shift at the 15-minute time point without any significant increase in total PD-L1 . The fact that mono-Ub precedes the increase in PD-L1 levels suggests that PD-L1 mono-Ub is not simply a consequence of increased PD-L1 expression and raises the intriguing possibility that mono-Ub is required for the EGF-driven enhancement of PD-L1 protein levels. Later time points, 1 to 4 hours, also show clear evidence of polyubiquitinated species which are indicated by the nondiscreet, smeary signal from 80 to 200 kDa (A and C). Whether or not the same PD-L1 molecule is both mono- and polyubiquitinated is not known, but based on the information that glycosylation and poly-Ub are mutually exclusive, one may hypothesize that the mono-Ub and poly-Ub are occurring on different PD-L1 molecules. Why EGF would activate both mono- and poly-Ub mechanisms is also unknown, but it may suggest that there is a constant flux or turnover of PD-L1 molecules that may be important for EGF-regulated cellular function.Previously published data on Ub of PD-L1 identified poly-Ub as a mechanism for proteasomal-dependent PD-L1 downregulation Figure 1B). Effective inhibition of EGFR activation and signaling significantly decreased PD-L1 mono- and multi-Ub  and prevented the accumulation of PD-L1 protein . Similar results on PD-L1 protein levels and mono- and multi-ubiquitination were observed with the specific Erk inhibitor SCH772984 while having no effect on upstream activation of EGFR (C and D), thus suggesting that EGF-regulated Ub of PD-L1 is mediated at least in part through ERK signaling. Together, these results indicated that the inhibition of total PD-L1 accumulation in response to EGF stimulation is proportional to the reduction in PD-L1 mono- and multi-Ub and support the hypothesis that EGF-induced PD-L1 Ub is intricately intertwined with EGF-mediated increase in PD-L1 protein levels. Although this current study focused specifically on EGF regulation of PD-L1 Ub, it will be interesting to determine if alternative growth factors and cytokines  that are known to enhance PD-L1 expression also regulate PD-L1 Ub. Conversely, utilization of non-EGF pathway inhibitors may shed light on the specificity of the regulation of PD-L1 Ub by EGF.To gain further insight into the relationship between PD-L1 mono- and multi-Ub and PD-L1 protein levels, inhibitors of the EGF signaling pathway that are known to block EGF-induced PD-L1 expression Figure 3A and C) while having no effect on the relative activation of the EGFR as determined by phosphorylation of EGFR (B). Quantitation of total PD-L1 revealed that the EGF-mediated increase of PD-L1 was abolished in PYR-41–treated cells (B and C). Although obvious caveats exist with interpretation of these data against a backdrop of total inhibition of the global ubiquitin machinery, taken as a whole, the data are consistent with the hypothesis that mono-Ub of PD-L1 can operate as a mechanism to regulate EGF/EGFR-enhanced PD-L1 protein levels. Although utilizing an E1 chemical inhibitor to block Ub may not provide the desired therapeutic specificity, identification of the E3 ligase that regulates mono-Ub of PD-L1 may have promise as a therapeutic target.To further explore the possibility that PD-L1 mono-Ub and multi-Ub regulate total PD-L1 protein levels, we wanted to inhibit the Ub of PD-L1 through direct interference with the ubiquitin machinery. The E3 ligase that regulates PD-L1 mono- and multi-Ub is unknown, but since there is only one E1 ubiquitin activating enzyme in humans that regulates the entire Ub machinery, inhibiting E1 function is a valid tool to examine ubiquitination. The E1 specific inhibitor PYR-41 has been shown to effectively block protein Ub  of EGFR B. Quantied cells , B and CIn this study, PTMs of PD-L1 in response to EGF stimulation were analyzed through a nonbiased snapshot, and three novel PTMs of PD-L1 were identified, specifically, tyrosine phosphorylation, acetylation, and mono- and multi-Ub. An important role for PD-L1 mono- and multi-Ub was elucidated, which was to facilitate EGF-induced PD-L1 protein levels. Our study combined with the work by Li et al. As the focus of these studies was PD-L1 Ub, no additional information regarding the role for PD-L1 tyrosine phosphorylation or acetylation was uncovered. For example, whether they may regulate PD-L1 expression, function, or interaction with target proteins like the PD-1 receptor was not investigated. The signaling mechanisms that regulate the dynamics of these newly identified PD-L1 PTMs are still unknown, and it will be interesting to further dissect why EGF signaling activates all three PTMs as well as whether cross-talk or cross-regulatory mechanisms exist between these three PTMs and other PTMs like poly-Ub. These findings not only have implications for basic PD-L1 biology but may also aid in the design of more efficacious PD-L1/PD-1 drugs and/or diagnostic markers.N ≥ 3 independent experiments.Characterization of PD-L1 mono- and multiubiquitination. (A). Serum-restricted A431 cells were either unstimulated or stimulated with EGF 1 hour prior to lysis with BlastR lysis buffer. Samples were immunoprecipitated with ubiquitin binding beads (UBA01), UbiQ poly-ubiquitin preferential binding beads, or FK2 antibody-based mono- and polyubiquitin binding beads. Samples were separated by SDS-PAGE and analyzed by Western blot for PD-L1. Shown is a representative Western blot from Supplemental Figure S1The following are the supplementary data related to this article.H.H. conceived and performed experiments, and wrote the manuscript. A.L. and S.H. provided reagents, expertise, and feedback. K.M. conceived experiments, wrote the manuscript, provided expertise and feedback, and secured funding.


In EBV+ GC, we observed PD-L1 staining in tumor cells in 50% (16/32) and immune cells in 94% (30/32) of cases. Among EBV-negative GCs, PD-L1 expression within tumors cells was observed only in cases with microsatellite instability (MSI), although 35% of EBV-/MSS GCs possessed PD-L1 expression of inflammatory cells. Moreover, distinct classes of GC showed different patterns of PD-L1+ immune cell infiltrations. In both EBV+ and MSI tumors, PD-L1+ inflammatory cells were observed to infiltrate the tumor. By contrast, such cells remained at the tumor border of EBV-/MSS GCs. Consistent with these findings, we utilized gene expression profiling of GCs from The Cancer Genome Atlas study to demonstrate that an interferon-γ driven gene signature, an additional proposed marker of sensitivity to PD-1 therapy, were enriched in EBV+ and MSI GC. These data suggest that patients with EBV+ and MSI GC may have greater likelihood of response to PD-1 blockade and that EBV and MSI status should be evaluated as variables in clinical trials of these emerging inhibitors.Gastric cancer (GC) is a deadly disease with limited treatment options. Recent studies with PD-1 inhibition have shown promising results in GC, but key questions remain regarding which GC subclass may respond best. In other cancers, expression of the PD-1 ligand PD-L1 has been shown to identify cancers with greater likelihood of response to PD-1 blockade. We here show with immunohistochemistry that Epstein-Barr Virus (EBV)+ GCs ( Gastric cancer (GC) is a deadly disease that, despite its molecular heterogeneity, has been largely treated through uniform approaches. As new profiling approaches and molecular characterization efforts enable better sub-classification of GC, one hope is that these subclasses may help guide optimal selection of therapy, both cytotoxic and biologic therapies. Among biologic agents, there is great enthusiasm regarding use of novel immune checkpoint inhibitors in cancer therapy, most notably those targeting the programmed cell death protein 1 (PD-1) pathway. Together with its ligands PD-L1 and PD-L2, PD-1 participates in regulating the balance between T cell activation and tolerance in response to antigenic stimulation . HoweverThrough a recent comprehensive molecular analysis of 295 gastric adenocarcinomas as part of The Cancer Genome Atlas (TCGA) , we and EBV is a human herpes virus that contributes to the development of a diversity of malignancies such as B and T cell lymphomas, nasopharyngeal carcinomas and ~9% of gastric adenocarcinomas , resultsBesides amplification of 9p24.1, additional features of EBV+ gastric cancers may possess alternative mechanisms able to induce PD-L1 expression. EBV positive classical Hodgkin's lymphoma, for instance, have increased PD-L1 expression levels as a result of EBV induced AP-1 expression and JAK/STAT signaling . AdditioIn this study, we investigate the status of candidate markers to guide the use of PD-1 therapy, immunohistochemical analysis of PD-L1 status and gene expression based analysis of IFN-γ immune signatures, in GCs with and without EBV-positivity. The primary question we hope to address is if markers that suggest greater likelihood of potential response to PD-1 blockade are a broad feature of EBV+ GCs or are more restricted to the smaller subset of EBV+ tumors with 9p24.1 amplification.in situ hybridization using probes against Epstein-Barr encoded RNA 1 (EBER1) as previously described [In order to determine whether PD-L1 expression is restricted to 9p24.1 amplified EBV+ GCs we collected a sample series of 12 EBV+ and 10 EBV negative GCs (2 microsatellite instable (MSI), 3 genomic instable (GS), 5 chromosomal instable (CIN)) from the TCGA study, using cases in which we were able to retrieve tissue slides [escribed . We evalescribed , only onin situ hybridization [P < 0.001), predisposition to a proximal tumor location (P < 0.001) and a lower TNM classification (P < 0.015) compared to EBV negative GCs. As single only a single tissue slide was available for analysis, we restricted our testing to PD-L1 . In the EBV+ samples, we identified PD-L1+ tumor cells in 50% (16/32) and PD-L1+ immune cells in 94% (30/32) of tumors. By contrast, EBV negative GCs had significantly less PD-L1 staining in tumor cells (10% (5/49), P < 0.001) and immune cells . When we broke out the EBV negative GC by MSI status we found that MSI GCs had PD-L1+ tumor and immune cells in 33% (5/15) and 46% (7/15) of cases respectively, which was higher than EBV negative microsatellite stable (MSS) GCs that had no PD-L1 positive tumor cells (0% (0/34), P < 0.001) and PD-L1 positive immune cells in only 35% of cases . Again, PD-L1+ immune cells had a tumor infiltrating patterns in 57% of PD-L1+/EBV+ cases and 43% in MSI GCs which was 0% in PD-L1+/EBV- /MSS gastric cancers (P < 0.001)  Figure .n = 12) and EBV negative GCs (n = 10) from the TCGA study. Supervised clustering of genes that discriminate the EBV+ tumors identified IFN-γ signaling genes CXCL9, CXCL10, CXCL11, IDO and GZMB among the top 40 most enriched genes  MSI GCs (n = 47) have high IFN-γ response gene expression compared to GS (n = 42) and CIN (n = 106) GCs (P < 0.001). As expected, IFN-γ GSE was strongly associated with PD-L1 mRNA expression within all GC subgroups (P < 0.001). Furthermore, within these subgroups there was no association between IFN-γ signature and total number of mutations . These results demonstrated that besides EBV+ GCs  GCs that did not have PD-L1+ tumor cells and PD-L1+ immune cells in only 35% of GCs. Remarkably, beyond the differential presence of PD-L1+ cells in EBV+ and EBV negative GCs, we observed a difference in infiltration pattern of PD-L1 positive immune cells; while PD-L1+ immune cells were able to infiltrate the center of EBV+ and MSI GCs, in EBV negative MSS GCs PD-L1+ immune cells stayed mainly at the invasive margin.Although molecular predictors of clinical benefit from PD-1 inhibitors remain uncertain, PD-1 blockade has been found to be most effective in PD-L1+ tumors or tumors whose PD-L1+ immune cells are able to infiltrate the tumor center rather than remain at the invasive margin –8. FurthWith mRNA sequencing analyses we here showed that compared to EBV negative GCs, EBV+ GCs indeed have strong enrichment of IFN-γ response genes CXCL9, CXCL10, CXCL11, IDO and GZMB. The combination of PD-L1 positivity and enrichment for an IFN-γ signature in EBV+ GCs suggests the potential for PD-L1 expression and activation of the PD-1 pathway to be a critical mechanism in these tumors to control an antecedent cytotoxic anti-tumor immune response, which increases the likelihood of response to PD-1 blockade in this GC subtype.Interestingly, besides EBV+ GCs, we also observed that MSI GCs have high IFN-γ response gene expression, perhaps reflecting the large lymphocyte infiltrate that is typical for mismatch-repair deficient cancers with a high mutational load. Whether MSI GC will respond as well to anti-PD-1 directed therapy as MSI colorectal cancers  needs fuFurthermore, although IFN-γ response gene expression occurs less frequent in genomic stable (GS) and chromosomal instable (CIN) GCs, we also noted the presence of PD-L1 expression in inflammatory cells in a significant minority of these tumors. Moreover, PD-L1 mRNA expression was associated with an IFN-γ signature. This finding is in agreement with a recent study of 34 GCs  that alsIn summary, we here show that PD-L1 expression by tumor or tumor infiltrating immune cells is a general phenomenon in EBV+ GCs and MSI GCs. Enrichment of an IFN-γ gene expression signature indicates a interferon-mediated adaptive immune response in EBV+ and MSI GCs, which provides a strong rationale for testing of PD-1 blockade in this patient population and for evaluating EBV status along with MSI status as key variables in immunotherapy trials in gastric cancer.In order to study PD-L1 protein expression in EBV+ gastric cancers in relation to 9p24.1 amplification, formalin-fixed, paraffin-embedded (FFPE) tissue sections were retrieved from tumor block of 12 EBV+ and 10 EBV negative gastric cancers (2 microsatellite instable (MSI), 3 genomic instable (GS), 5 chromosomal instable (CIN)) that were part of a recent comprehensive molecular analysis of 295 gastric adenocarcinomas as part of The Cancer Genome Atlas (TCGA) [in situ hybridization using probes against Epstein-Barr encoded RNA 1 (EBER1) as previously described [In this series EBV status was determined by whole-genome, whole exome, mRNA and miRNA sequencing and confirmed by escribed . 9p24.1 in situ hybridization for EBER1 and MSI status was assed at Bat 25, Bat 26, BAT40, D5S346 and D2S123 loci [An independent series of FFPE tissue sections to validate PD-L1 expression results was composed of 32 EBV+ and 49 EBV negative, among which 15/49 EBV negative GCs were microsatellite instable (MSI) . EBV+ ca123 loci . Tumors PD-L1 IHC was performed using mouse anti-human PD-L1 (clone 405.9A11) as previously described , 17. 405http://gdac.broadinstitute.org/) [http://software.broadinstitute.org/gsea/msigdb). Enrichment scores of the IFN-γ gene set for each case were obtained by single-sample GSEA using the R code from Genepattern (http://www.broadinstitute.org/cancer/software/genepattern/). Enrichment scores of the IFN-γ gene set were correlated to GC subgroup status, PD-L1 mRNA expression status and total number of mutated genes. The total number of mutated genes were obtained from whole exome sequencing data (http://gdac.broadinstitute.org/) [Differentially expressed genes between EBV+ and EBV negative GCs were identified using TCGA mRNA-sequencing data of 214 GCs of different GC subgroups; 19 EBV+ GCs, 42 MSI GCs, 42 GS GCs and 106 CIN GCs (t-test and Pearson's chi-2 or Fisher's Exact test where appropriate. Associations between IFN-γ signatures and GC subgroups, PD-L1 mRNA expression status and mutational load were analyzed using Spearman's rank order correlation and one-way Anova. All p-values are two sided, and a p-value < 0.05 was considered statistically significant.Associations between clinical, pathological or molecular characteristics were analyzed using two-tailed Student


Metasurfaces utilizing engineered metallic nanostructures have recently emerged as an important means to manipulate the propagation of light waves in a prescribed manner. However, conventional metallic metasurfaces mainly efficiently work in the visible and near-infrared regime, and lack sufficient tunability. In this work, combining the pronounced plasmonic resonance of patterned graphene structures with a subwavelength-thick optical cavity, we propose and demonstrate novel graphene metasurfaces that manifest the potential to dynamically control the phase and amplitude of infrared light with very high efficiency. It is shown that the phase of the infrared light reflected from a simple graphene ribbon metasurface can span over almost the entire 2π range by changing the width of the graphene ribbons, while the amplitude of the reflection can be maintained at high values without significant variations. We successfully realize anomalous reflection, reflective focusing lenses, and non-diffracting Airy beams based on graphene metasurfaces. Our results open up a new paradigm of highly integrated photonic platforms for dynamic beam shaping and adaptive optics in the crucial infrared wavelength range. Metamaterials are rationally engineered composite materials with exotic properties not easily obtained or altogether unavailable in nature123568101213151718195So far, most metasurfaces rely on judiciously designed metallic nanostructures. At optical frequencies, metallic nanostructures with varied geometries support strong surface plasmon resonances, acting as local antennas in frequency selective surfaces (FSS)222325272829303132333335363839414243444546313238394048Although fairly extensive effort has been devoted to graphene-based metamaterials and plasmonics, previous work mainly focuses on the distinct resonance features and their spectral tuning. In this letter, we propose and demonstrate graphene plasmonic metasurfaces to steer infrared light with high efficiency, which drastically expand the functionalities and performance of graphene devices towards complete wave shaping. This is made possible by combining the plasmonic resonance of patterned graphene nanostructures with a subwavelength-thick optical cavity to enhance the intrinsically weak interaction between light and single-layer graphene. Furthermore, the graphene metasurfaces can be dynamically tuned by changing the Fermi energy of graphene via simple chemical or electrostatic tuning that is unachievable in conventional metasurfaces made of metals. As a proof of principle, we numerically study graphene metasurfaces consisting of rationally designed graphene ribbons. It is shown that the phase of the reflected infrared light can range almost from –π to π by tailoring the dimensions of the graphene ribbons as well as the optical cavity, while the amplitude of the reflectivity is sufficiently high without substantial variations. This allows us to design graphene metasurfaces to implement anomalous reflection, focusing and non-diffracting Airy beam generation. We envision that such transformative graphene metasurfaces would open up a new paradigm of integrated photonic systems with multifunctional applications, including dynamic beam shaping, hyperspectral imaging, adaptive optics, biomedical sensing, and energy harvesting in the critical infrared region.s of the dielectric spacing layer is smaller than the wavelength. Consequently, we can achieve strong and even perfect absorption with monolayer graphene structures as theoretically proposed and recently demonstrated in experiments525354Graphene can interact with light strongly via plasmonic resonance27383950Graphene metasurfaces coupled with an optical cavity exhibit much more striking properties and applications beyond strong absorption. Our numerical simulations show that the phase of the reflected light can vary from almost –π to π by selecting the width of the graphene ribbons as well as the thickness of the dielectric spacing layer, while the amplitude of reflectivity remains high and only shows moderate variations. These two properties allow us to design a variety of planar graphene photonic devices in the reflective configuration that are easy to fabricate and integrate. It should be emphasized that the full 2π phase coverage is crucial for designing functional metasurfaces, which necessitates the bottom metallic mirror. The bare graphene ribbons on a dielectric layer only provide a phase modulation range of about π 56, becau8913s) of the dielectric spacing layer and the width (w) of the graphene ribbons. TM polarized light can excite the plasmonic resonance of graphene ribbons. In contrast, for transverse electric (TE) polarization, the electric field is parallel to the graphene ribbons, and no plasmonic resonance can be excited to modulate the phase and amplitude of reflected light (p) of the graphene ribbon is fixed at 3 μm. In the finite element simulation utilizing commercial software COMSOL Multiphysics, the optical conductivity of graphene is described by Kubo model with the Fermi energy Ef = 0.64 eV. The refractive index of the dielectric spacing layer is taken as 1.4, and Drude model is applied to fit the experimental data of permittivity of gold for the back reflective mirrorIn this paper, we focus on the far-infrared region, while the operation wavelength of the proposed graphene metasurfaces can be readily extended to mid-infrared by scaling the dimension of the graphene patterns. ed light . The fres = 4.37 μm, the reflectivity is larger than 48.6% and the phase smoothly spans over from almost –π to π with different graphene ribbon widths as shown in From 52dφ/dx at the interface between two media. Anomalous reflection and refraction phenomena have been reported in this regime, which are in sharp contrast to the well-known refraction and reflection law8The results in ni is the refractive index of the material at the incidence side, and θi and θr are the incident and reflected angle, respectively. There is a nonlinear relation between θi and θr, distinctly different from conventional specular reflection. For normal incidence and ni = 1 (air), the reflection angle can be calculated aswhere Hy) of the reflected light. According to the field distribution, we can retrieve the reflection angle, which is about 13.1° and in excellent agreement with the predicted value. The slight deformation of the phase front arises from the finite periodicity and non-uniform reflectivity of the metasurface. If we set the incident angle as 15.0°, the reflected angle should be about 29.0°. The simulation result is shown in In our simulation, we set 36 graphene ribbons with periodicity of 3 μm. The width of each ribbon is properly selected  so that F, the phase shift distribution φ(x) along the interface should satisfyThe second graphene metasurface device to be demonstrated is a reflective focusing lens. To design such a lens, we must ensure that the light reflected from every graphene ribbon has the same phase or with 2π multiplied by an arbitrary integer when propagating to the focal point. In other words, the metasurface should carry a phase profile to compensate the propagation phase between graphene ribbons and the focal point, so that the radiated waves from the metasurface can constructively interfere with each other at the focal point. For a certain focal length F = 100 μm, and x = mp with p equal to the periodicity of the graphene ribbons and m = 0, ±1, ±2… From the results presented in x axis  and w2 = 1058 nm  to match the simplified profile  planar systems59 profile . From FiWe have introduced graphene metasurfaces based on simple ribbon geometries to efficiently steer infrared light in the reflective configuration. The graphene ribbons coupled with a subwavelength-thick optical cavity substantially enhance the light-graphene interaction, allowing us to achieve almost 2π phase modulation and high reflectivity amplitude. Anomalous reflection, reflective focusing lenses and non-diffracting beam generation with capability of dynamic tuning have been successfully demonstrated. Although we have focused on the far-infrared frequency, the design strategy can be readily applied to mid-infrared region by varying the dimension of graphene ribbons and thus their plasmonic resonance. Moreover, extending ribbons to other 2D patterns provides additional degrees of freedom in the design, promising other important advantages for light steering such as 3D focusing, polarization-independent functionalities, and strong polarization conversion. Leveraging the remarkable plasmonic properties of graphene at infrared wavelengths, the graphene metasurfaces demonstrated here manifest unprecedented control over the electromagnetic properties of materials on a completely 2D platform, which enable disruptive light-weight, ultra-compact, and high-speed optical architectures for diverse applications.In the terahertz and infrared region, the optical conductivity of graphene is dominated by the intraband transition. It can be described by a semi-classical Drude modelkB is the Boltzmann constant, Τ is the temperature, Ef is the absolute value of the Fermi energy, and τ is the electron relaxation time. When the Fermi energy Ef is much larger than the thermal energy kBT, the conductivity of graphene can be further simplified to τ is related to the carrier mobility μ, the Fermi energy Ef and Fermi velocity vf as τ = μEf/evf2, which indicates that increasing carrier mobility of graphene will reduce the loss and improve the device efficiency. The carrier mobility μ of graphene film ranges from ∼1000 cm2/Vs in chemical vapor deposition (CVD)-grown graphene to 230,000 cm2/Vs in suspended exfoliated graphene. In our simulations, we assume the Fermi energy of graphene Ef around 0.64 eV, a moderate mobility μ = 10,000 cm2/Vs, Fermi velocity vf = 106 m/s and scattering time τ = 6.4 × 10−13 s27Ef = 1–2 eV has been reported652/Vs has been achieved by using a hexagonal boron nitride substrate even with thickness as small as 14 nm67σv = σ/t assuming that t is a very small value2), Calcium Fluoride (CaF2), or Magnesium Fluoride (MgF2), is employed in the design as the dielectric spacing layer, which is assumed to have a refractive index of 1.4.where How to cite this article: Li, Z. et al. Graphene Plasmonic Metasurfaces to Steer Infrared Light. Sci. Rep.5, 12423; doi: 10.1038/srep12423 (2015).