The demand for high‐performance absorbers in the microwave frequencies, which can reduce undesirable radiation that interferes with electronic system operation, has attracted increasing interest in recent years. However, most devices implemented so far are opaque, limiting their use in optical applications that require high visible transparency. Here, a scheme is demonstrated for microwave absorbers featuring high transparency in the visible range, near‐unity absorption (≈99.5% absorption at 13.75 GHz with 3.6 GHz effective bandwidth) in the Ku‐band, and hence excellent electromagnetic interference shielding performance (≈26 dB). The device is based on an asymmetric Fabry–Pérot cavity, which incorporates a monolayer graphene and a transparent ultrathin (8 nm) doped silver layer as absorber and reflector, and fused silica as the middle dielectric layer. Guided by derived formulism, this asymmetric cavity is demonstrated with microwaves near‐perfectly and exclusively absorbs in the ultrathin graphene film. The peak absorption frequency of the cavity can be readily tuned by simply changing the thickness of the dielectric spacer. The approach provides a viable solution for a new type of microwave absorber with high visible transmittance, paving the way towards applications in the area of optics. For instance, carbon‐based materials including carbon nanotubes, carbon composites, and graphite nanosheets are widely utilized due to their lightweight and efficient microwave absorbing characteristics.In this work, we present a general strategy to design the new type of transparent perfect MA based on asymmetric Fabry–Pérot resonant cavity by employing a multilayer structure in the form of graphene‐dielectric‐ultrathin doped silver (GDS). Here the optically transparent graphene and an ultrathin doped silver (Ag) film function as the microwave absorber and reflector respectively, and fused silica as the dielectric spacer. In addition, by tuning the dielectric spacer thickness to achieve critical coupling in the cavity, we show that this device yields near‐unity (≈99.5%) microwave absorption and remains highly transparent (≈74%) in the visible range, and the relative visible transmittance of the cavity can reach ≈93.5%. Furthermore, by employing the simple building block, an ultrathin planar graphene film, we theoretically and experimentally demonstrate that nearly all the microwave is absorbed in the graphene layer by this transparent MA in contrast to other metamaterials and metasurface absorbers where microwave is mainly absorbed in the tailored metallic components (2D arrays of subwavelength elements).22.1FigureThe microwave absorption by a single graphene layer is very limited. However, with its partially microwave absorbing and reflecting properties, graphene can be an excellent candidate as the top absorbing medium in an asymmetric Fabry–Pérot cavity, where one of the mirrors has significantly higher loss than the other. A schematic view of the proposed visibly‐transparent MA based on cavity resonance is depicted in 2D bands. The 2D band exhibits a single Lorentzian line shape with a full width at half maximum (FWHM) of ≈26 cm−1 located at ≈2686 cm−1 and a higher intensity relative to the G band. In addition, the intensity of D band at ≈1350 cm−1 is below the Raman detection limit, which proves the absence of a significant number of defects. Afterwards, the Fermi energy level of graphene is determined by comparing the G and 2D peak positions we measured, with the characterized correlation from Ref. c = ≈0.3 eV.The designed GDS cavity can be easily fabricated. The synthesis process of the graphene by the typical chemical vapor deposition (CVD) method is shown in Figure S4 in the Supporting Information. Figure The doped Ag and ITO layers were deposited on polyethylene terephthalate (PET) substrate by sputtering. SEM and AFM images of the as‐deposited ITO/Cu‐doped Ag/ITO are shown in Figure 2.2Figuren1, n2, k2, n3, n4, k4, d2, d3), including complex refractive index and thickness of interested layers, are involved in designing the transparent MA in this configuration. The metal thickness is fixed at 8 nm to ensure both microwave reflection and optical transparency. It is convenient to describe the graphene's property using its conductivity (σ) since that this quantity can be modeled or measured directly ranging from radio wave to optical frequencies, and the corresponding refractive index (n) and extinction coefficients (k) can be derived from the conductivity.e is the electron charge, kB is the Boltzmann constant, T is the temperature, ℏ is the reduced Planck constant, ω is the frequency of the incident electromagnetic waves, and Γ is the relaxation time which is assumed to be independent of energy. It is worth nothing that graphene's conductivity strongly depends on the value of its Fermi energy level μc which can be controlled by chemical doping or electrostatic gating.As shown in E(z) is the electric field in the ultrathin graphene film, E0 is the incident electric field, c is the light speed in the vacuum, and f is the frequency of the incident electromagnetic wave. Therefore, the total absorption in the graphene film isWe consider the theoretical condition for perfect microwave absorption in the planar graphene layer by destructive interference through multiple reflections within the GDS cavity. As graphene is the only lossy medium and there is no restriction placed on the metallic substrate in the GDS cavity, so even metal approaching the perfect electric conductor limit can be used. For simplicity, we assume that all the power is consumed by the graphene layer and the bottom reflective layer can provide 100% reflectivity. Here, we adopt an analytical method for the absorber structure studied in the optical regime,d2 ≈ 0.34 nm) is much smaller than the wavelength of the incident microwave (d2 ≪ λ), a sheet of graphene layer minimally changes the electric field profile inside the cavity, and the electric field can maintain its intensity within the monolayer absorber layer, which is also validated by calculating the electric field distribution within the graphene layer at different frequencies . In view of the zero reflection in the critical coupling condition, we can assume thatSince the thickness of monolayer graphene (E(z) = E0 into Equation Atotal = 100%, the sufficient and necessary condition for perfect microwave absorption is obtainedSubstituting πn2κ2d2f/cn1 for normal incidence, revealing that perfect absorption at around 15 GHz can be achieved in the GDS cavity by the graphene film under the condition of μc = 0.3 eV and Γ = 20 ps.The dotted line in the Figure R) and absorption (A = 1−R) of the GDS cavity at interested frequencies. Due to the symmetry of the cavity in the X–Y plane, the device should be independent of polarization at normal incidence. In the following numerical calculations, we only take TE‐polarized incident microwaves into consideration. Figure d3 of 3 mm. It illustrates that the near‐unity absorption of the GDS cavity can be achieved with different Fermi levels in the Region A (black dotted circle), which also shows that the conditions for achieving strong absorption are not very narrow. In addition, the ideal perfect absorption is attained with μc = ≈0.3 eV at near 15 GHz, which is consistent with the predicted result based on Equation d3 varies from 1 to 15 mm, the absorption peak shifts continuously from ≈40 to ≈2.5 GHz.Furthermore, by employing TMM, we can calculate the reflection (d3) and resonant frequency (f0) can be understood by investigating the condition of partial reflected wave for the destructive interference in the Fabry–Pérot configuration, which can be roughly estimated as f0 = c/(4n3d3). However, due to the strong loss in the graphene layer, the reflection phase of partial reflected wave at the air–graphene interface is not equal to π, resulting in a slight resonant peak shifts under the destructive interference condition. Meanwhile, several higher‐order modes start to appear with thicker cavities and higher frequencies, forming several bands on the absorption map. Hence, it is obvious that the peak absorption frequencies can be readily tuned by changing the dielectric thickness of the cavity. Note that at a fixed dielectric thickness , the bandwidth of lower‐order resonances is slightly broader than that of higher‐order modes  and reflection (R) are measured at normal incidence using waveguide configuration, and then used to calculate absorption (A), which is defined as A = 1 − T − R, where R = |S11|2 and T = |S21|2 are obtained from the measured S‐parameters. First, by analyzing the microwave responses of the GDS cavity layers individually, we checked the respective role of each layer. The microwave transmission and reflection of the monolayer graphene on silica substrate and the ITO/Cu‐doped Ag/ITO on PET substrate in the Ku bands are plotted in Figure S6 in the Supporting Information. As predicted, the monolayer graphene is partially reflective and absorptive to the microwaves with averaged ≈25% reflection and ≈45% transmission. On the other hand, besides the high visible transmittance, the ITO/Cu‐doped Ag/ITO layer supports a broadband high microwave reflection, corresponding to only ≈0.3% transmission, which is nearly perfect as a microwave reflection mirror in the cavity.To confirm the microwave absorption performance of the GDS cavity, the microwave transmission  at different frequencies.d3 ranging from 1 to 4 mm. The overall shape, trend and position of the experimental features in Figure Then, we examined the microwave absorption performance of the GDS cavities. The measured microwave reflection and transmission for the GDS cavities with different dielectric thicknesses are shown in d3 = 3 mm) in the Figure d3 = 3 mm, broadband absorption is obtained with the measured absorption of >50% from 8 to 18 GHz, covering the entire X and Ku bands, while all maintaining ultralow microwave transmission through the cavities. In addition, the effective bandwidth is defined as the width of the band with microwave absorption intensity of over 90%. For the GDS cavity at d3 = 3 mm, the measured effective bandwidth is 3.6 GHz, from 12 to 15.6 GHz in Figure d3 = 2.5, 3.5, and 4 mm, the maximal absorption values all exceed 98% at their resonant frequencies, showing excellent microwave absorption performance of the GDS cavities.In addition, with the increase of the dielectric thickness, the location of the absorption peaks gradually red‐shifted to the low frequencies and was able to be tuned across the measured spectrum from 8 to 18 GHz. As characterized by the purple curves d = 3 mm i2.3d3 = 3 mm) at interested wavelengths, are presented in FigureIn order to get a better understanding of the extraordinary microwave absorption by an atomically thin graphene layer, numerical calculations using TMM of the electromagnetic responses,In simple physics, the GDS cavity resembles an asymmetric Fabry–Pérot (FP) cavity comprising of a lossless dielectric core with a partially reflective top layer and back reflector. At resonances, microwave beams reflected from the air/graphene and the silica/metal interfaces form destructive interference, resulting in zero reflection, which accounts for the period absorption peaks in the Figure 2.4FigureThe thickness of such GDS cavity is on the order of several millimeters, which is much larger than the visible wavelengths, so it is hard for the absorbing layer and reflective layer to form an optical cavity in this case due to the incoherent situation. Therefore, the optical transmittance of the GDS cavity can be improved by enhancing the transparency of individual layers or reducing dielectric layers. To characterize the optical property of the GDS cavity, we measured transmittance spectra in the range of 300–1000 nm of the individual layers and GDS cavities with different dielectric thicknesses, as shown in the d3 = 3 mm, we have achieved the near‐unity absorption in the microwave range, and an averaged visible transmittance of 74% (relative to PET substrate) can still be maintained. It is important to note that the transparency of the GDS cavity can be improved by using one single dielectric spacer instead of employing multilayer configuration at the designed thickness, and the visible transmittance of the GDS cavity will be enhanced by eliminating the interface reflection loss up to nearly 87%  could achieve an EMI SE of ≈26 dB, a microwave absorption of ≈99.5% with visible transparency of ≈74% (and can be improved up to >85% by using single layer of silica). With a lower microwave absorption, the GDS cavity with 1‐mm‐thick dielectric spacer shows a visible transmittance of ≈87%. All these values match or outperform most of the reported results shown in the table.3In summary, we have experimentally demonstrated a transparent and near‐unity absorption microwave absorber consisting of graphene, transparent spacer and ultrathin doped Ag based on asymmetric FP cavity. The experimental results are consistent with the theoretical calculations. The physical origin can be explained by the asymmetric FP cavity model. Experimental results show a maximal absorption up to ≈99.5% at 13.75 GHz, an EMI shielding effectiveness of 26 dB with an averaged visible transmittance of 74% (limited by the light reflection from the air/silica interface used in the current experiment). In addition, the GDS cavity is easy to be tuned either by chemical doping or electrostatic gating for the graphene layer or simply altering the cavity thickness. The devised configuration and physical insight opens up a new pathway for transparent microwave absorbing materials and for developing novel microwave optical components.4Graphene Synthesis: The graphene films were grown on 25 µm thick Cu foils  using CVD method at the center of a tube furnace. The foils were subsequently heated to 1000 °C under H2 at 60 Pa for half an hour. Then, the carbon source (CH4) was introduced into the quartz tube for 2 h. After following gas mixtures, we rapidly cooled down the samples to the room temperature (25 °C). The fast cooling rate is critical in suppressing the multiple layers formation of graphene.Graphene Transfer: As‐grown graphene on the Cu foils was transferred by spin‐coating 5 µm thick PMMA on the graphene. After PMMA dried, the sample was immersed into the Marble's etchant (HCl: H2SO4: CuSO4 = 50 mL: 50 mL: 10 g) solvent to etch the Cu foil. After removal, the graphene sample was washed in distilled water and transferred to the 1 mm silica substrate to be used.Film Deposition: Cu‐doped Ag films were fabricated by co‐sputtering of Cu and Ag. The concentration of Cu and Ag was controlled by adjusting the individual sputtering power of the Cu and Ag targets. The calibrated deposition rates for the Cu‐doped Ag film employed in this study are 1.109 and 0.019 nm s−1 for Ag and Cu, respectively, which leads to ≈2% atomic concentration of Cu in the obtained film. The film thickness was controlled by the deposition time. The chamber base pressure was pumped down to about 0.13 mPa before film deposition. During deposition, the Ar gas pressure was 0.6 Pa and the substrate holder was rotated at the speed of 10 rpm.Characterization and Measurements: ITO/Cu‐doped Ag/ITO film thickness of each layer was calculated by the calibrated deposition rates and confirmed by the spectroscopic ellipsometry measurement (J. A. Woollam M‐2000). The morphology of the graphene and ITO/Cu‐doped Ag/ITO films were investigated by SEM (FEI HELIOS Nanolab 600i) and tapping mode AFM (Bruker Dimension FastScan) on fused silica and PET substrates, respectively. The optical transmittance of the films and GDS cavities were measured by a UV‐vis–Infrared spectrophotometer (PerkinElmer Lambda 950) ranging from 300 to 1000 nm with normal incident radiation. The microwave transmission and reflection of the samples was calculated from the measured S‐parameters (S11 and S21) using waveguide methods in the X and Ku bands. Schematic illustration and microwave measurement setup and are shown in the Figure S7 in the Supporting Information.The authors declare no conflict of interest.SupplementaryClick here for additional data file.


Spinal sarcoidosis is a rare subgroup of neurosarcoidosis. Although most sarcoid lesions develop in the intramedullary compartment, intradural extramedullary (IDEM) spinal sarcoidosis is an extremely rare entity.  We herein report a case of IDEM spinal sarcoidosis mimicking a meningioma. A 32-year-old man presented to the hospital with clumsy hands and was unable to walk without assistance. Magnetic resonance imaging (MRI) of the cervical spine revealed a dural tail sign that is common in meningiomas. The patient underwent gross total resection. The pathological findings consisted with a sarcoid leision of the spinal cord. The patient's myelopathy recovered after surgery.  Physicians should be alert for the possibility of IDEM sarcoidosis mimicking a meningioma in the differential diagnosis of IDEM spinal cord tumors. Sarcoidosis is a systemic disease of unknown etiology characterized by noncaseating giant-cell granulomas affecting all organs. Several case series have detected the nervous system involvement of sarcoidosis in up to 5% of all sarcoidosis cases . FurtherWe herein report a case of IDEM spinal sarcoidosis mimicking a meningioma.A 32-year-old man presented to the hospital with progressive numbness and paresthesia of the right hand, trunk, and both lower limbs. He also complained of clumsy hands and was unable to walk without assistance. His history was significant for systemic sarcoidosis two years earlier. Steroid therapy had not been performed because the systemic sarcoidosis was not active, and he had stopped follow-up.Manual muscle testing was 2/5 in the deltoid, 3/5 in the biceps, 4/5 in the wrist flexors, 5/5 in the wrist extensors, 5/5 in the finger flexors, 4/5 in the finger extensors, 2/5 in the finger abductors, 5/5 in the iliopsoas, 5/5 in the quadriceps, 5/5 in the tibialis anterior, 4/3 in the extensor halluces longus, 5/5 in the gastrocnemius, and 4/5 in the flexor halluces longus. The patient's performance on the 10-second test was slow. Pin prick and temperature sensations were impaired from the T2 dermatome bilaterally. His Japanese Orthopaedic Association (JOA) score for cervical myelopathy, according to the criteria proposed by the association itself, was 3 of 17 points . Laboratory investigations showed normal values for all parameters. Cervical X-ray and computed tomography (CT) showed no apparent abnormalities. MRI of the cervical spine revealed isointensity in the intradural extramedullary on T1-weighted imaging and a low-intense mass at the C3-4 levels on T2-weighted imaging. Following the intravenous administration of gadolinium-diethylenetriaminepentaacetic acid (Gd-DTPA), an intradural extradural mass with a marked gadolinium enhancement and a dural tail sign were observed .The patient underwent laminotomy of the caudal side of the C2, right open door laminoplasty of the C3 to the C4, and laminotomy of the cranial side of the C5. On opening the dura, a firm intradural extramedullary mass was found. The mass was partly adhered to the spinal cord but was not attached to the dura. Of note, the appearance of the actual mass differed from that of a meningioma, as it had been preoperatively suspected to be based on the MRI findings . Gross tAfter the surgery, the patient's myelopathy recovered gradually and he returned to full-time work. Steroid therapy was not performed because the systemic sarcoidosis was not considered to be active. Four years later, his JOA score for cervical myelopathy was shown to be 14 of 17 points, giving a 78% recovery rate. No abnormalities on MRI were noted at that time .Spinal sarcoidosis can occur in intramedullary spaces, intraspinal epidural spaces, intradural extramedullary spaces, and vertebral bodies . Most caIDEM spinal sarcoidosis has been reported to occur usually in the cervical spine. Indeed, in previous reports, the anatomic location of IDEM spinal sarcoidosis was in the cervical spine in eight cases and thoracic spine in two –11. The Although MRI and CT are useful evaluation tools for diagnosing spinal sarcoidosis, the diagnosis of IDEM spinal sarcoidosis is often difficult because of its similarity to spinal cord tumors. In our case, a mass lesion was observed to be isointense on T1-weighted imaging and low intense on T2-weighted imaging. Although meningiomas usually show iso- or high-intensity findings on T2-weighted imaging, approximately 10% of meningiomas show a low intensity on T2-weighted imaging , 13. In The causes of spinal sarcoidosis remain unclear, although Schaller et al. hypothesized that IDEM spinal sarcoidosis represents a very early stage of intramedullary spinal sarcoidosis .Subtotal or total resection of the mass is required for the surgical treatment of IDEM spinal sarcoidosis. Of note, the risk with surgery in cases of IDEM is lower than that in cases of intramedullary lesions because the mass is extramedullary, so several cases of tumor resection have been reported .There is no consensus regarding the pharmacological treatment of neurosarcoidosis. In previous reports of IDEM spinal sarcoidosis, postoperative steroid treatment was performed in seven cases, but not in three others. Regardless of steroid treatment, recurrence was not observed during the follow-up period , 5. HighPhysicians should be alert for the possibility of IDEM sarcoidosis mimicking meningiomas in the differential diagnosis of IDEM spinal cord tumors.


Kainate receptors (KARs) are glutamate-gated ion channels that play fundamental roles in regulating neuronal excitability and network function in the brain. After being cloned in the 1990s, important progress has been made in understanding the mechanisms controlling the molecular and cellular properties of KARs, and the nature and extent of their regulation of wider neuronal activity. However, there have been significant recent advances towards understanding KAR trafficking through the secretory pathway, their precise synaptic positioning, and their roles in synaptic plasticity and disease. Here we provide an overview highlighting these new findings about the mechanisms controlling KARs and how KARs, in turn, regulate other proteins and pathways to influence synaptic function. There are three major subclasses of ionotropic glutamate receptors, kainate receptors (KARs), AMPA receptors and NMDA receptors. KARs are widely distributed throughout the brain and, depending on the cell type in question, they can be localised at pre-, post- and/or extrasynaptic sites. In general, presynaptic KARs modulate both excitatory and inhibitory neurotransmitter release, postsynaptic KARs contribute to excitatory neurotransmission and extrasynaptic KARs play a role in determining neuronal excitability (for reviews of KAR physiology see –4). For (EPSCs) –8. IntriR(EPSCs) , 10.Remarkably, despite their classical ion channel structure, KARs can also signal via a non-canonical G-protein coupled metabotropic cascade . In contOur aim here is to provide an overview of recent advances in understanding the cellular regulation of KARs and their interacting proteins, and how these processes influence KAR-mediated synaptic transmission and plasticity. We highlight what is known, what remains to be established, and outline the future perspectives for KAR research and how it will impact on our understanding of brain function and dysfunction in disease.GRIK1-5 [Since their initial cloning , 19 the GRIK1-5 .KARs are heteromeric assemblies containing four subunits. Each subunit has a large extracellular N-terminal domain (NTD), helical transmembrane domains (TMD) including three membrane spanning domains  and a membrane re-entrant domain (M2), and an intracellular C-terminal domain (CTD). The latter part of the NTD  together with the extracellular loops between M3 and M4 (S2) form the ligand-binding domain (LBD) . RecentlFollowing protein synthesis, the NTDs initiate receptor assembly in the endoplasmic reticulum (ER) by facilitating dimer formation, and the dimerization of two dimers then leads to the formation of tetrameric receptors. Based on affinity for their ligand, KAR subunits have been grouped into low affinity (GluK1-3) and high affinity (GluK4-5) receptors. Studies on recombinant systems have shown that low affinity GluK1-3 subunits can form ion channels as both homomers and heteromers but high affinity GluK4 and GluK5 subunits can only form heteromeric functional ion channels when complexed with the low-affinity subunits , 26, 27.1 and GluK12 [Regions within the N- and C- terminal domains of KAR subunits can undergo alternative RNA splicing. For instance, the extracellular N-terminal domain of GluK1 can produce two variants, GluK1d GluK12 , while td GluK12 , 31. Spld GluK12 –34. C-ted GluK12 –37.In addition to splicing, further diversity arises from varying degrees of RNA editing in GluK1 and GluK2 subunits , 39. For2+ permeability [It is well established that GluK1/GluK2 editing is developmentally controlled through regulation of the enzyme that also catalyses GluK1/2 RNA editing, ADAR2 –44. For eability . Furthereability . Suppreseability . Therefoeability , 50, plaeability  and patheability .The accurate and timely delivery of KARs to specific pre-, post- and extrasynaptic locations is fundamental to many aspects of neuronal function. Most research efforts have focused on the processes of transcription, endocytosis, recycling and degradation . HoBRs, can be translated using both somatic and dendritically localised ribosome patterned rough ER. They then traffic from the ER using dendritic ER exit sites and utilise the somatic Golgi or dendritic Golgi outposts for mature glycosylation [In neurons, mRNAs can be trafficked to distant sites in axons and dendrites for local translation and processing –57. A rasylation , 58–62. sylation . For exasylation .Recently, using the RUSH system, which allows the synchronous release and visualization of cargo proteins trafficking through the secretory pathway , 66, it Although there is a strong base of knowledge about the activity-dependent regulation of KAR endocytosis and recycling , 67–71, As well as interactions with the GluK2 subunit, interactions with GluK5 also determine flux through the secretory pathway. ER retention sequences in the C-terminus of GluK5 facilitate its interaction with the COPI coat complex, driving retrograde Golgi to ER trafficking of KARs, and acting as an ER retrieval mechanism . This inFurther tight control of the ER exit and secretory pathway trafficking of KARs is also provided by post-translational modifications, most notably PKC phosphorylation of GluK2 at residues S846 and S868 , 75, whiThus, far from being a passive process, ER exit of KARs and flux through the secretory pathway is emerging as a major point of regulation in determining the surface expression of KARs under basal and activity-dependent conditions  including phosphorylation , SUMOyla2+ influx-, and CaMKII-dependent plasticity mechanism, which is absent in slices from GluK5−/− mice [−/− slices restored KAR-LTD, expression of a non-phosphorylatable GluK5 did not, demonstrating that direct phosphorylation of GluK5 by CaMKII is required for this form of KAR LTD [LTD of KARs at MF-CA3 synapses is induced by a spike timing-, Ca−/− mice . CaMKII  KAR LTD . Thus, tPKC phosphorylation of GluK2 at S846 and S868 regulates the surface expression of GluK2-containing KARs at several levels. It affects both GluK2 transit through the secretory pathway , 75 and SUMOylation is a post-translational modification that results from the conjugation of a member of the ~ 11kD SUMO family to lysine residues in substrate proteins . GluK2 iOne major functions of protein ubiquitination is to target proteins for lysosomal or proteasomal degradation . A recenKARs are involved in both excitatory and inhibitory neurotransmission, controlling both short and longer-term plasticity. These properties have been extensively reviewed , 11 and 2+–calmodulin complexes and the activation adenylate cyclase and PKA [Presynaptic KARs decrease glutamate release at CA3-CA1 pyramidal cell synapses –112. Int and PKA –115. ThuPostsynaptic KARs at MF-CA3 synapses undergo plastic changes, and exhibit several forms of LTD that can be induced by different stimulation protocols , 87. ChaFurthermore, in cultured neurons, activity-dependent up- or down-regulation of surface KARs can be induced by differential agonist application protocols , 68. KARAMPAR [Recently, a novel form of AMPAR-LTP was discovered at CA3-CA1 synapses that is mediated by activation of postsynaptic KARs (KAR-LTPAMPAR ). DespitAMPAR , 116, acAMPAR , 118, suAMPAR ).−/− mice lack this novel form of plasticity, indicating an absolute requirement for this subunit in induction of KAR-LTPAMPAR  .ARs while at the same time potentiating extrasynaptic GABAARs, in a process that decreases synaptic inhibitory drive to facilitate synaptic plasticity, while simultaneously protecting against neuronal over-excitation by promoting extrasynaptic inhibition [Moreover, postsynaptic KAR metabotropic signalling depresses synaptic GABAhibition .AR-mediated transmission, KARs separately regulate the potassium-chloride cotransporter KCC2. KCC2 is crucial because it establishes the electrochemical chloride gradient for postsynaptic inhibition through GABAARs [AR-mediated transmission [In addition to this direct effect on GABA GABAARs , 122 and GABAARs ) and epi GABAARs . KCC2 in GABAARs  and GluK GABAARs , which i GABAARs . KCC2 ansmission . In addismission . These fDysregulation of common molecular pathways underlies multiple neurological and neurodegenerative diseases and there is mounting evidence that KAR dysfunction could be one such feature .−/− mice exhibit significantly fewer seizures than wild-type counterparts [−/− mice [KAR dysfunction is particularly closely associated with temporal lobe epilepsy (TLE) , and Gluterparts . TLE is terparts –132 and −/− mice . Consist−/− mice  and gene−/− mice . TogetheIn addition to TLE, both gain  and lossMost recently it has been reported that complete ablation of all KAR subunits results in disrupted corticostriatal function and dramatic obsessive-compulsive-like behaviour with severe self-grooming . TogetheAlthough historically much less studied than AMPARs or NMDARs, there is a currently a renaissance in KAR research fuelled by the realisation that they are multifunctional neuronal modulators that have roles in health and disease far beyond those previously appreciated.To understand how KARs exert these regulatory effects, it is necessary to define how KARs themselves are controlled and how they interact with, and regulate, other proteins and systems. Furthermore, while there is a growing appreciation of the multifaceted roles played by KARs, a number of fundamental questions remain unanswered. For example, while it is widely-accepted that KARs can signal both ionotropically and metabotropically through G-proteins, how this dual signaling occurs, and what determines which ‘mode’ of signaling an individual KAR complex utilizes remains almost entirely unexplored. In addition, while we are beginning to understand the molecular events that control the specific distribution and synaptic localization of KARs, how these mechanisms cooperate to orchestrate the highly selective distribution of KARs in the brain remains an important unanswered question. Finally, although KARs can induce plasticity of AMPARs, exactly how this occurs, how widespread this form of plasticity is in the brain, and whether postsynaptic KARs can direct other forms of bidirectional synaptic plasticity remain unknown.We expect that further studies examining how KAR localisation and signalling at pre- and postsynaptic sites impacts on neuronal function will answer many of these remaining questions, and will transform our understanding of the roles KARs play in development, plasticity and disease.


The anomalous reflection angle covers the range of 10° to 90° with the efficiency higher than 80%, over a broad spectral range from 7 μm–40 μm of infrared spectrum. It reaches above 92% at the center wavelength in the spectral response. By increasing the periodicity of phase gradient, we can expand this frequency band even further without losing efficiency. The compact design of metasurface affords the adjustability of the electrochemical potential level of graphene by means of gating. Additionally, the impact of the number of graphene sheets for the optimum efficiency of the proposed structure is investigated. By adding the secondary graphene metasurface with opposite direction of phase gradient, we demonstrated the tunability of the reflection angle from  Anomalous refraction and reflection of light with these extraordinary surfaces has been utilized in several newly developed devices such as polarimetry, flat lenses, holography, surface plasmon couplers, solar sails, etc8, as the prominent applications of metasurfaces13. As controlling the light propagation in an embedded structure becomes a vital aim, one significant breakthrough for many viable application is to actively manipulate the direction of light, i.e. reflection or refraction by means of external voltage. The rapid progress of improvement and innovative design has become possible for various metasurfaces with the optically exotic graphene20. Graphene and other 2D materials might be one of the best candidates for designing externally controllable metasurfaces. By changing their electrochemical potential level, it is possible to change the optical characteristics (i.e. the surface conductivity) in an extraordinary fashion22.Due to their exotic manner of manipulation of electromagnetic waves, metasurfaces have recently become an essential research topic in the optics and photonics fields26. In this regard, the structures with low quality factors are the center of interests30. Broadband operation and higher efficiency of anomalous reflection are significant factors in realizing flexible metamirrors32. In parallel, trilayer metal-insulator-metal (MIM) metasurfaces with noble metals provide the efficient reflection in the desired direction33. Recently, triangularly shaped plasmonic metallic metasurfaces demonstrated continuous phase gradient configuration35. In an alternative design, magnetically polarized particles have shown an excellent plasmonic metasurface-like behavior with P-polarized electromagnetic waves36.Plasmonic metasurfaces with a phase gradient have the ability to alter and tilt the wavefront of electromagnetic waves27. Besides, cross-polarized metasurfaces do not offer high efficiencies useful for many applications like the solar sails4. Due to the overlapping of Fabry-Perot and the antenna resonances in the spectra of reflection, some metasurfaces act like an absorber metamaterial rather than a mirror, which results in a decrease of the anomalous reflection efficiency39. Efficiency of metamirrors can be easily defined by the power ratio of an anomalously reflected (i.e. reflected in predicted and desired direction) electromagnetic wave to the incident wave. The frequency band of operation is mainly determined by the generalized Snell’s law and the optical properties of materials. An additional influential factor contributing to the anomalous reflection is the near-field interaction of metasurface with the incident electromagnetic wave.In numerous studies, however, metasurfaces that operate at fixed frequencies or in extremely narrow bands suffer low efficiencies in the other frequency bands41. The structure studied here is sensitive to linearly polarized electromagnetic waves. Each graphene ribbon has a specific width, chosen to have a certain plasmon resonance frequency. This provides the strong near-field interaction, and therefore, highly efficient anomalous reflections. Both the periodicity of the phase gradient direction (x-direction) and the periodicity of the polarization direction (y-direction) are considered to design a structure and to investigate the behavior of its reflection accordingly with the assistance of the Finite-difference time-domain (FDTD) method. It is worth mentioning that similar structure with the trapezoid shaped silver plasmonic antennas array operating in the visble light spectrum has been reported by other research group42. However, unlike silver plasmon reflector, the reflection efficiency of our graphene based reflector can have more than 80% for wide wavelength band. Moreover, by leveraging the tunability of the Fermi level of the graphene sheets47, we propose uniquely tunable graphen plasmonic reflector structure in this work.In this paper, infrared multi-layered graphene metasurfaces controllable with bias voltages are designed to achieve exceptional high efficiencies2 with the refractive index of 1.38 is a dielectric layer sandwiched between a graphene multilayer and a metal ground plane. The imaginary part of the refractive index of the MgF2 is negligible in the discussed wavelength band48. The main objective of the dielectric material in our theoretical analysis is to provide a cavity for the metasurface, and the considered material could be replaced with a material with a similar refractive index. The thickness of the dielectric layer is 2.5 μm.The proposed metasurface reflector consists of a multi-sheet graphene layer on top of the dielectric material, followed by the Au ground plane as illustrated in Fig. Px = 20.1 μm and Py = 2.2 μm, with an etched triangle base of We = 2 μm and a length of Le = 20 μm. It is worthy to mention that size of the unit cell is set slightly larger (about 0.1–0.2 um) than the etched triangular hole, to make graphene layers be connected. This design is proposed since the etching of graphene can be more practical than producing triangular graphene islands in the fabrication process. However, the designs based on triangular islands, with specific electrochemical potential level could yield the similar results. In this paper the dimensions of the unit cell and etching dimensions are chosen in order to produce ultra-high efficiency metasurfaces and to make it practical one might need to manipulate the proposed dimensions, due to the fabrication process restrictions.Triangular holes etched through the graphene layer produce the phase gradients required for anomalous reflection. The dimensions of a unit-cell of the metasurfaces reflector are W, and the periodicity, Py of the triangular shape of graphenes (TSGs) in our model has a significant impact on tuning the supported plasmon resonance frequencies. It also affects its Lorentz like phase changes34. Since the width, W of TSG increases in x-direction, the resonance frequencies shift to lower frequencies through the phase gradient.It is expected the width, W is given by39,N, P, and μ represent the number of graphene sheets in each ribbon, the width and periodicity related factor, and the electrochemical potential level, respectively.The plasmon resonance frequency of graphene nano-ribbons (GNRs) with fixed 38. In the denominator, the W represents the width of GNRs, which applied to TSG in our model, varies in the y-direction. The effective permittivity of the graphene environment can be calculated from the permittivity of Air (εa) and substrate (εs). We can consider the average value of P is equal to 0.65 for our TSGs, since P = qW/π, where q is the first eigenvalue produced from the mode solution of GNRs37. An additional effective factor of the resonance and phase gradient is μ, where it could be tuned by gating or doping. By increasing the carrier density of the graphene layer, we can blue-shift the resonance frequency. This provides an important degree of freedom in adjusting the phase gradient to a specific frequency band as well as enhancing the reflection efficiency for a specific spectrum. The value of μ is considered to be 250 meV for each graphene sheet. Changing the value of chemical potential does not have an effect in changing the reflection angle. However, by manipulation of μ, we can intensify or diminish the anomalous reflection efficiency and even obscure this behavior. The number of graphene sheets, N, included in Eq.  and the free space wavelength of light are important in determining the angle of reflection. On the other hand, the periodicity in the y-direction (Py) and W determine the operating band where the phase gradient starts and ends.Since ∇Px. As W increases, the wavelength of resonance increases proportionally. Furthermore, increasing the Fermi energy level of the graphene layer blue-shifts the resonance wavelength. Therefore, as the number of graphene sheets increases, the Lorentz like phase change shifts to the longer W side. In the case of having only one graphene layer as shown in the Fig. Px is restricted to shorter W side, so that the phase shift over the unit-cell becomes almost zero, ∇ϕx ≈ 0. In addition, increasing N helps to provide a stronger near-field interaction of light. By increasing the number of the graphene sheets (N) the number of the conducting channels and the total number of the carriers will be increased, which intensifies the plasmonic response of the material, and the plasmon field intensity (i.e. the plasmonic near-field intensity)40. Additionally, it is experimentally proved that multilayer graphene structures provide broader tunability of the Fermi level, comparing to the single layer graphene structure41. In this regard, we purposely chose 20 graphene layers. We examined our proposed structure with different simulation methodologies (1) scaling the conductivity of a single layer graphene up to 20, compared with (2) considering 20 graphene sheets with a spacing of 5 nm to verify the consistency of our results. Figure N on the Lorentz like phase shift along the metasurface.The Fig. λ0 = Px, where we can observe the conversion of propagating waves to surface waves. Figure λ0 = 12 μm and λ0 = 17 μm with reflecting angles of θr = 37°, and θr = 58°, respectively. It shows that the reflection angle increases with wavelength, while the efficiency stays almost the same. The anomalous reflection efficiency for these two cases stays over 85%, which will be discussed in the next section.From the expression of Px, and Py on the overall operating wavelength bandwidth of the TSG metasurface reflector. The cut-off wavelength of anomalous reflection can be analytically predicted by both the surface conductivity of graphene and the generalized Snell’s law, simultaneously. According to Eq.  is determined by the intra-band term of the imaginary part of the conductivity function (The minimum wavelengths limit (Px. The reflection angle covers the broad wavelength band of 7 μm to 20 μm for unit-cell dimensions of Px = 20.1 μm and Py = 2.2 μm. According to Eq. (λL) should red-shift. For instance, the cut-off wavelength for the above unit-cell dimensions is λL = 20.1 μm, and this device is practically operable up to the predicted cut-off wavelength. Additionally, the metasurface with unit-cell of Px = 30.1 μm and Py = 2.2 μm is predicted to have the λL = 30.1 μm, which effectively supports up to λL = 27 μm. The metasurface with the periodicities of Px = 40.1 μm and Py = 2.2 μm supports effectively up to λL = 29 μm, while the predicted maximum cut-off wavelength is 40.1 μm.Figure λ = 14 μm. In other words, this unit-cell can reflect with the angles of 30° to 65° with an efficiency of higher than 80%. In the same regard, the metasurface with longer phase gradient periodicity Px = 30.1 μm supports the wavelength band from 7 μm to 27 μm, while its efficiency stays higher than 80% from 10 μm to 21 μm. The efficiency of this metasurface exceeds 90% at the middle of the operational band. Accordingly, the metasurface with the Px = 40.1 μm, supports the widest wavelength band from 7 μm up to 29 μm, with an efficiency higher than 80% over the wide band of 10 μm to 22 μm. These results exhibit high efficient anomalously reflecting metasurface reflector with flexible choice of wavelengths by choosing the proper physical dimensions of the phase gradient periodicity (Px).As it is shown in the Fig. Py, which is periodicity in y-direction is equal to 2.2 μm. Since the value of W in Eq.  is red-shifted. However, this value could not exceed the predicted cut-off wavelength of Snell’s law . Our simulation results disclosed that by increasing the thickness of the cavity we can reach the higher efficiency in the longer wavelengths at the same time of losing the efficiency in the shorter wavelengths.As it is discussed extensively, the reduction of the efficiency in the longer wavelengths is due to the approaching to the plasmonic cut-off wavelength or the cut-off wavelength supported by the Snell’s law. However, there is a general reduction of the efficiency in the longer wavelength, which is due to the cavity thickness  results in wider spanning range of the reflection angle. For instance, the total spatial range supported by the metasurface with periodicities of μ of graphene sheets, we add a second reflecting metasurface with phase gradient in the opposite direction. Figure h) comparing to the thickness of cavity. Each graphene metasurface is formed from 20 sheets of graphene layer and these two metasurfaces are supported by the same cavity and ground metal plane as shown in the Fig. h is considered to be 50 nm, which is very small comparing to the cavity thickness of 2.5 μm. By implementing the gates inside of the dielectric media or outside of the metasurface or changing the doping level of the graphene sheets, one can change the Fermi level of the graphene phase gradients. By reducing the μ1 to 100 meV, the first phase gradient metasurface loses its functionality and becomes transparent. But due to the second phase gradient metasurface, with μ2 = 250 meV and opposite direction of phase gradients, the reflection angle changes to −θr. This kind of functionality would bring flexibility in controlling the direction of reflectors at any frequency.As it is feasible to manipulate the anomalous reflection by means of changing the μ = 100 meV and also amplified absorption of two metasurfaces with 20 graphene sheets21. In spite of this absorption, 50% efficiency of anomalous reflection still provides better performance than several other metasurface configurations which do not have tunability with external voltage4.However, adding another phase gradient metasurface reduces the efficiency near to 50%. This reduction is due to the strong absorption of the transparent graphene layer with The results also reveal that the polarization of the electromagnetic wave must be perpendicular to the direction of the phase gradient. One can use several phase gradients in different directions in order to have a design supporting anomalous reflection for different polarization directions of the TE waves. This device could be actively controlled with the method illustrated in Fig. λL) of the metasurface is determined by the phase gradient periodicity (Px). In contrast, the minimum cut-off wavelength (λS) is due to the surface conductivity and the surface plasma frequency of the graphene layer. Graphene is an excellent candidate for a tunable metasurface reflector, as the surface conductivity of graphene can be controlled by means of external gating voltage. We found that adjustability of the Fermi level of graphene was not able to change the anomalous reflection direction from a phase gradient metasurface, but can possibly either disable or activate the anomalous reflection operation. Finally, we demonstrated tunability of the reflection angle from θr to −θr by separately controlling the electrochemical potential levels of double phase gradient layers embedded in opposite directions. In this case, one of the phase gradients is disabled by reducing the Fermi level to 100 meV, and the other layer with reverse phase gradient direction is activated by increasing its Fermi level to 250 meV. This novel configuration of the metasurface could be used as a highly efficient, ultra-wideband, electrically tunable metamirror in IR and THz frequency bands in several optical devices and systems.We designed an anomalously reflecting infrared metasurface with exceptional high efficiency and a wide frequency band of operation via triangularly etched multi-sheet graphene layers. Our design achieved the anomalous reflection angle, covering a range of 10° to 90° with an efficiency higher than 80% over a broad spectral range from 7 μm–40 μm. The efficiency of the anomalous reflection exceeds 92% at the center wavelength. In this design, the effective phase gradient is generated from the triangular shape on the graphene layers. This phase gradient and its gradual changes determined the anomalous reflection angles and reflection efficiency in the range of infrared incident wavelength. The maximum cut-off wavelength (In this paper we utilized the Finite-difference time-domain (FDTD) method in order to numerically analyze the anomalous reflection of the electromagnetic waves from the surface of the designed metasurface. Surface conductivity of the graphene sheets for various Fermi-levels in the mid-IR and THz frequency bands are imported to the simulation. Two different simulation approaches were pursued to simulate the plasmonic response of the 20 sheets of the graphene layer realistically; (1) Scaling the conductivity of a single graphene layer to 20, (2) Considering 20 separate sheets of graphene with the 5 nm separation distance between them. The results from both approaches were compared and verified no differences. Both simulation methods confirmed the efficiency and angle of the anomalously reflected light from the metasurface. The complex permittivity function of the gold material for the ground plane reflector is imported from the Johnson and Christy data. The smallest mesh sizes used in the simulation are 100 nm, 80 nm and 2 nm in cubic shape to obtain consistent data. We also verified our simulation results analytically by the numerical method using the equations presented in the context.Supplementary Information


We investigated the potential protective role of MOMAST(®) HY100 and MOMAST(®) HP30 on isolated rat colon, liver, heart, and prefrontal cortex specimens treated with Escherichia coli lipopolysaccharide (LPS), a validated ex vivo model of inflammation, by measuring the production of prostaglandin (PG)E2, 8-iso-PGF2α, lactate dehydrogenase (LDH), as well as cyclooxygenase (COX)-2, tumor necrosis factor α (TNFα), and inducible nitric oxide synthase (iNOS) mRNA levels. MOMAST(®) HY100 decreased LPS-stimulated PGE2 and LDH levels in all tested tissues. Following treatment with MOMAST(®) HY100, we found a significant reduction in iNOS levels in prefrontal cortex and heart specimens, COX-2 and TNFα mRNA levels in heart specimens, and 8-iso-PGF2α levels in liver specimens. On the other hand, MOMAST(®) HP30 was found to blunt COX-2, TNFα, and iNOS mRNA levels, as well as 8-iso-PGF2α in cortex, liver, and colon specimens. MOMAST(®) HP30 was also found to decrease PGE2 levels in liver specimens, while it decreased iNOS mRNA, LDH, and 8-iso-PGF2α levels in heart specimens. Both MOMAST(®) HY100 and MOMAST(®) HP30 exhibited protective effects on multiple inflammatory and oxidative stress pathways.MOMAST Olea europaea) polyphenols have healthy beneficial effects, including the prevention of several chronic diseases, such as cancer and aging-associated degenerative diseases [It has been well established that olive tree  HY100 and MOMAST(®) HP30  are polyphenolic liquid complexes from olive  pressing juice with a total polyphenolic content of 100 g/kg (at least 50% as HT) and 36 g/kg (at least 30% as HT), respectively ((®) HY100 and MOMAST(®) HP30 are also characterized by the presence of tyrosol and oleuropein.MOMASTectively . In addi(®) HY100 and MOMAST(®) HP30, both including HT and oleuropein, on the burden of oxidative stress/inflammation occurring on various isolated rat tissue  specimens exposed to Escherichia coli lipopolysaccharide (LPS), a well-established inflammatory stimulus. Specifically, we studied the effects of MOMAST(®) HY100 and MOMAST(®) HP30 on multiple inflammatory and oxidative stress pathways, by measuring the production of prostaglandin (PG)E2, 8-iso-PGF2α, lactate dehydrogenase (LDH), as well as cyclooxygenase (COX)-2, tumor necrosis factor α (TNFα), and inducible nitric oxide synthase (iNOS) mRNA levels. The results support a rational use of these polyphenolic complexes in the prevention of tissue damage occurring during inflammation.Considering the antioxidant effects displayed by both HT and oleuropein, the aim of the present study was to investigate the putative protective effects of MOMAST(®) HY100  and MOMAST(®) HP30  were tested in vitro to evaluate their effects on cell viability. We observed that both polyphenolic liquid complexes were well tolerated by Hypo-E22 and C2C12 cell lines (MOMASTll lines . Particu(®) HY100  and MOMAST(®) HP30  supplementation on oxidative stress and multiple inflammatory pathways in colon, liver, heart, and prefrontal cortex specimens challenged with LPS. As previously reported [2α, an isomer of prostaglandins produced by free radical-catalyzed peroxidation of membrane arachidonic acid, is a stable marker of lipid peroxidation and oxidative stress [(®) HP30 (110 and 220 µg/mL) was able to decrease 8-iso-PGF2α levels on rat prefrontal cortex, colon, liver, and heart tissues, challenged with LPS inflammatory stimulus  HY100  was effective in inhibiting LPS-induced 8-iso-PGF2α in rat liver specimens ((®) HY100 and MOMAST(®) HP30 . HT was also shown to decrease low density lipoproteins oxidation [(®) HY100 and MOMAST(®) HP30 on LDH level in inflamed tissues. LDH is a cytosolic enzyme, which can be considered a marker of tissue destruction [(®) HY100  treatment, we found a significant inhibition of LPS-induced LDH level in all tested tissues ((®) HP30  was also able to decrease LDH level induced by LPS in heart specimens . Actually, the reduction of LDH level could be related to the presence of HT [(®) HY100 and MOMAST(®) HP30. Finally, we evaluated the modulatory effects of MOMAST(®) HY100 and MOMAST(®) HP30 on pro-inflammatory markers, including PGE2, COX-2, TNFα, and iNOS. LPS was found to induce macrophage production of inflammatory cytokines such as TNFα, interleukin-1β (IL-1β), and IL-6, along with inflammatory mediators including nitric oxide (NO) and PGE2 [2 in both neoplastic and inflamed tissues [(®) HP30 was able to reduce COX-2, TNFα, and iNOS mRNA levels in prefrontal cortex, colon, and liver specimens  HY100 in heart tissue specimens ((®) HY100 in prefrontal cortex specimens ((®) HP30 in heart tissue specimens  HY100 was effective in reducing LPS-induced PGE2 levels in all tested tissues ((®) HP30 was able to decrease PGE2 levels only in liver tissue ((®) HY100 and MOMAST(®) HP30 on PGE2 levels, along with COX-2, TNFα, and iNOS mRNAs, support the protective effects of both polyphenolic liquid complexes in prefrontal cortex, colon, liver, and heart specimens. Accordingly, HT was shown to exert anti-inflammatory effects in LPS-stimulated RAW264.7 mouse macrophages by suppressing nuclear factor-kB (NF-κB) signaling and downregulating gene expression of iNOS, COX-2, TNFα, and IL-1β, and production of NO and PGE2 [(®) HY100 and MOMAST(®) HP30 could also be related to the presence of other phenolic compounds, including tyrosol and OE. In this context, tyrosol was found to significantly inhibit COX-2 gene and protein expression, as well as PGE2 secretion in human glioblastoma cells [®) HY100 and MOMAST(®) HP30 could display different effects in tissues. On one hand, Takeda et al. [As regards PGE tissues , MOMAST HY100 and MOMAST(®) HP30 exhibited protective effects as indicated by the blunting effect on the tested pro-inflammatory mediators. On the basis of these results, HT seems to be the main extract component involved in the pharmacological effects. Nevertheless, considering the inherent limitations of the ex vivo experimental model, further investigations including oxidative stress and inflammation biomarkers in in vivo studies are needed for a more accurate evaluation of MOMAST(®) HY100 and MOMAST(®) HP30 efficacy.In conclusion, both MOMAST® CRL-1772™) were cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal bovine serum and 1.2% (v/v) penicillin G/streptomycin in 75 cm2 tissue culture flask . The cultured cells were maintained in a humidified incubator with 5% CO2 at 37 °C. For cell differentiation, Hypo-E22 and C2C12 cell suspensions at a density of 1 × 106 cells/mL were treated with various concentrations  of phorbol myristate acetate  for 24 h or 48 h (induction phase). Thereafter, the PMA-treated cells were washed twice with ice-cold pH 7.4 phosphate buffer solution (PBS) to remove PMA and non-adherent cells, whereas the adherent cells were further maintained for 48 h (recovery phase). The morphology of the cells was examined under an inverted phase-contrast microscope. To assess the basal cytotoxicity of MOMAST(®) HY100 and MOMAST(®) HP30, a viability test was performed on 96-microwell plates, using a 3--2,5-diphenyltetrazolium bromide (MTT) test. Cells were incubated with MOMAST(®) HY100  and MOMAST(®) HP30 , corresponding to HT , for 24 h. Ten microliters of MTT (5 mg/mL) were added to each well and incubated for 3 h. The formazan dye formed was extracted with dimethyl sulfoxide and the absorbance was recorded as previously described [Rat Hypo-E22 cells (Cedarlane Cellution Biosystem) and mouse myoblast C2C12 cell lines  were housed in Plexiglass cages (40 cm × 25 cm × 15 cm), two rats per cage, in climatized colony rooms , on a 12 h/12 h light/dark cycle (light phase: 07:00–19:00 h), with free access to tap water and food, 24 h/day throughout the study, with no fasting periods. Rats were fed a standard laboratory diet .Housing conditions and experimentation procedures were strictly in accordance with the European Union ethical regulations on the care of animals for scientific research.2 inhalation (100% CO2 at a flow rate of 20% of the chamber volume per min) and colon, liver, heart, and prefrontal cortex specimens were immediately collected and maintained in a humidified incubator with 5% CO2 at 37 °C for 4 h, in DMEM buffer with added bacterial LPS (10 μg/mL) (incubation period).According to the recognized ethical principles of “Replacement, Refinement, and Reduction of Animals in Research”, colon, liver, heart, and prefrontal cortex specimens were obtained as residual material from vehicle-treated rats randomized in our previous experiments approved by the Local Ethical Committee of University “G. d’Annunzio” and the Italian Health Ministry . Rats were sacrificed by CO®) HY100  and MOMAST(®) HP30 . Tissue supernatants were collected, and the PGE2 and 8-iso-PGF2α levels (ng/mg wet tissue) were measured by radioimmunoassay (RIA), as previously reported [2α and anti-PGE2 were developed in the rabbit; the cross-reactivity against other prostanoids is <0.3%. One hundred microliters of prostaglandin standard or sample were incubated overnight at 4 °C with the 3H-prostaglandin  and antibody , in a volume of 1.5 mL of 0.025 M phosphate buffer. Free and antibody-bound prostaglandins were separated by the addition of 100 μL 5% bovine serum albumin and 100 μL 3% charcoal suspension, followed by centrifuging for 10 min at 4,000× g at 5 °C and decanting off the supernatants into scintillation fluid  for β emission counting. The detection limit of the assay method was 0.6 pg/mL. Additionally, tissue supernatants were assayed for lactate dehydrogenase (LDH) activity [−ΔΔCt method was used to quantify the relative abundance of mRNA and then determine the relative changes in individual gene expression (relative quantification) [During the incubation period, tissues were treated with scalar concentrations of MOMAST .p < 0.05. The number of animals randomized for each experimental group was calculated on the basis of the “Resource Equation” N = (E + T)/T (10 ≤ E ≤ 20) [https://www.nc3rs.org.uk/experimental-designstatistics.Statistical analysis was performed using GraphPad Prism version 5.01 for Windows . Means ± S.E.M. were determined for each experimental group and analyzed by one-way analysis of variance (ANOVA), followed by Newman–Keuls comparison multiple test. As for gene expression analysis, 1.00  was considered the theoretical mean for the comparison . Statist E ≤ 20) ,59,60, a


Graphene, having a perfect two-dimensional crystal structure, has many excellent features such as a high specific surface area, and extraordinary electrical, thermal and mechanical properties. However, during the production process, lattice defects will inevitably be produced. Therefore, the performance of graphene with various defects is much lower than its theoretical value. We summarize the major advances of research into graphene defects in engineering in this paper. Firstly, the main types and causes of defects in graphene are introduced. Secondly, the influence of different defects in graphene on the chemical, electronic, magnetic and mechanical properties is discussed. Also, the control methods of graphene defects are reviewed. Finally, we propose the future challenges and prospects for the study of the defects of graphene and other nano-carbon materials. According to the Mermin-Wagner theory, the long-range order of two-dimensional crystal structure could be destroyed by long wavelength fluctuation ,2. In ad2 hybrid orbital carbon atoms. Its thickness is about 0.335 nm, corresponding to the thickness of one carbon atom. Three-dimensional graphite, one dimensional nanotubes 95], Pt ,97, Ir , Cu 92]92], Ni  and so oID (the peak intensity of the defective structure) to IG (the peak intensity of the regular graphene structure) does not change or even increase. A recent study solves this problem by placing the graphene oxide into plasma methane [Although the above methods can reduce the defects of oxygen extrinsic atoms, the removal of external defects is often accompanied by the loss of carbon atoms. This will result in intrinsic defects. In Raman spectroscopy, the ratio of  methane . This isDue to its excellent physicochemical properties, graphene plays a significant role in today’s nanoscience. A variety of defects, including vacancies, interstitials, topological defects and line defects are unavoidable in experiments. The defects of graphene have a direct impact on the properties of the graphene. Although a large number of experiments or theoretical simulations have been carried out for the defects of graphene, based on the wide application prospect of graphene materials, the study of its defect characteristics needs further enrichment.


A new temperature-controllable humidifier for X-ray diffraction has been developed. It is shown that the humidifier can successfully maintain protein crystal growth at a temperature lower than room temperature. The room-temperature experiment has been revisited for macromolecular crystallography. Despite being limited by radiation damage, such experiments reveal structural differences depending on temperature, and it is expected that they will be able to probe structures that are physiologically alive. For such experiments, the humid-air and glue-coating (HAG) method for humidity-controlled experiments is proposed. The HAG method improves the stability of most crystals in capillary-free experiments and is applicable at both cryogenic and ambient temperatures. To expand the thermal versatility of the HAG method, a new humidifier and a protein-crystal-handling workbench have been developed. The devices provide temperatures down to 4°C and successfully maintain growth at that temperature of bovine cytochrome c oxidase crystals, which are highly sensitive to temperature variation. Hence, the humidifier and protein-crystal-handling workbench have proved useful for temperature-sensitive samples and will help reveal temperature-dependent variations in protein structures. Here, protein crystals are coated with a polymer hydrogel , which are grown at 4°C and are immediately damaged by rising temperature. These devices have already been applied to TR structural analysis of CcO with the SPring-8 Angstrom Compact Free Electron Laser (SACLA) ].To produce humid air from ambient temperature down to 4°C, our original humidity-control device , and bubbled in a water bottle equipped with a rubber heater kept at 28°C. The humid gas is transported and then cooled to room temperature (RT) during transportation before it reaches the air-mixing area. Mixing of the dry and humid gases is regulated using type SEC-N digital mass-flow controllers . The temperature t1 (°C) and humidity h1 (%RH) of the mixed air from the humidifier are measured using a type HYT271 humidity and temperature sensor . t1 is not controlled but is about RT.The humidifier works at ambient temperature and supplies wet gas to the air cooler. The wet gas is adjusted to a vapour pressure corresponding to the relative humidity (RH) at the desired temperature by mixing dry and saturated humid gases. The saturated humid gas is produced from the dry gas, which is supplied by an NThe air cooler comprises a heat exchanger and a double-pipe transfer tube. The heat exchanger was assembled with a tube that is passed through once in the shell to even out the coolant’s cooling. The transfer tube is lagged with an insulator and connects the heat exchanger to a humid-air nozzle. The coolant for the heat exchanger is chilled using an F32HE or F50HE refrigerated–heating circulator  and is flowed continuously through an outer side passage of the air cooler. The temperature of the outlet is adjusted using the temperature setting in the circulator.b) and 1c), respectively]. The outlet temperature, t2 (°C), is measured inside the humid-air nozzle using a T-type thermocouple, which is made of copper–constantan and is used for low-temperature applications such as cryogenics in crystallography.The humid-air nozzle was fabricated from a polyacetal tube . To prevent the influence of ambient air, the measurement sample is inserted into the nozzle tube 10 mm from its edge. In addition, two holes were created in the nozzle wall at the sample position. A 5 mm-diameter hole is used as a path for incident X-rays and to monitor the sample using a co-axial CCD camera. A second, 7 mm-diameter hole at the opposite side acts as a path for diffraction up to 1.67 Å  and 1.32 Å  resolution at a wavelength of 1 Å . The airstream is supplied by an N2 gas generator (KOFLOC Corporation Ltd) and is cooled using a coolant chilled by the F50HE refrigerated–heating circulator (JULABO GmbH) with an HE-25-025T2 multi-tubular heat exchanger . The dry N2 gas is charged into the chamber of the workbench to create a positive pressure inside it. The chamber has a large inner volume of 0.36 m3  and leaks because of two holes which allow insertion of bare hands into the chamber. Therefore, the airstream was supplied at a greater flow rate of 15 l min−1, and the chamber itself was also cooled by the coolant flowing in the wall piping of the chamber’s insulation panel. However, the workbench does not control humidity.The experimental environment around protein crystals should be maintained at the desired temperature while they are being collected. Gloveboxes are commonly used to maintain the experimental environment; however, most gloveboxes are not temperature controllable and are unsuitable for delicate work with bare hands. For such experiments, we developed a temperature-controlled airflow workbench, TeC-W . The beam size was 200 µm (height) × 100 µm (width) at the sample position with a photon flux of 9.5 × 1010 photons s−1 at a wavelength of 1 Å.To evaluate CcO crystal quality during the diffraction experiment, the mosaicity and unit-cell parameters for each diffraction image were processed using the 3.c)], was successful. Meanwhile, the low-temperature TeC-W workbench worked well to maintain the temperature within 4.0 ± 0.2°C. During CcO crystal-handing with bare hands, the temperature of the TeC-W workbench increased by 0.5°C.The TeCH-1 humidifier with the F50HE refrigerated–heating circulator successfully provided humid gas at a wide range of temperatures and humidity levels: 4–20°C and 20–99.5%RH, respectively. When using the F32HE refrigerated–heating circulator, the humidity SD tended to increase with higher humidity at 4°C, and the PID control did not work stably. The results suggested that improving the heat-insulating performance and the heat-exchange efficiency of the air cooler would allow more precise temperature and humidity control. The newly designed humid-air nozzle was also important for maintaining samples. When using the previous humid-air nozzle, which was similar to those of conventional cryostreams, the CcO crystal quality was not maintained, even at low temperatures. To avoid interfusion of ambient air into outlet air, insertion of CcO crystals into the humid-air nozzle, as described before . We previously reported a similar relaxation time and long-term crystal stability in tetragonal lysozyme crystals at RT (Baba et al., 2013To determine the optimum humidity for CcO crystals, we performed three trials at 4°C. First, we investigated temporal changes of lattice constants of the CcO crystals to evaluate the equilibration time in response to environmental changes in the CcO crystals under the almost saturated humidity of >98%RH by manual flow operation without PID control. This humidity value was the maximum without dew condensation inside the humidifier. Diffraction images were collected every 10 min for 60 min. It took ∼10 min for the lattice constants to reach equilibrium, and the lattices were then stably maintained for over 1 h Fig. 2. During Next, we decreased the humidity from 98%RH to determine the optimum humidity without PID control. A new CcO crystal was mounted, and we waited for 5 min after relaxation. Then we gradually decreased the humidity in 0.5–1% steps after each time delay of 2–3 min. The CcO crystal diffraction quality evaluated by crystal mosaicity and the resolution of the observed Bragg reflections remained intact at 96%RH or higher, but at 95%RH, the CcO crystal diffraction degraded in 5 min. The damage was not recovered with either rehydration to 96%RH or exposure to >99%RH.Finally, to improve the reproducibility of the humidity-control experiments, we examined PID control by comparison with lattice constants of three CcO crystals after relaxation under an optimum humidity of 98%RH. Under these conditions Fig. 3, the lat4.The TeCH-1 humidifier and TeC-W workbench successfully controlled temperature between 4 and 20°C. Using the combined device, we were able to perform a diffraction experiment on CcO crystals which were temperature controlled for crystal-handling operations and humidity controlled under the crystallization temperature. Because the HAG method has proved adaptable for protein crystals under various crystallization conditions, we intend to attempt to improve the performance of the TeCH-1 humidifier over a wider temperature range in future. In this study, we expanded the HAG method to low-temperature experiments.10.1107/S1600576719006435/ei5041sup1.pdfTo explain crystal damage at room temperature. DOI:


In this paper, the advances in radio-frequency (RF)/microwave biosensors based on graphene nanomaterials including graphene, graphene oxide (GO), and reduced graphene oxide (rGO) are reviewed. From a few frontier studies, recently developed graphene nanomaterials-based RF/microwave biosensors are examined in-depth and discussed. Finally, the prospects and challenges of the next-generation RF/microwave biosensors for wireless biomedical applications are proposed. Graphene is a carbon allotrope consisting of an atomically thin two-dimensional 2D) hexagonal lattice . This thD hexagon2·V−1·s−1) [−1·K−1) [In terms of physical properties, a pristine graphene without defects and impurities has a number of superior qualities such as high electronic mobility  , high op[−1·K−1) . Pristin[−1·K−1) , as show[−1·K−1) . In addi[−1·K−1) , graphen[−1·K−1) , microst[−1·K−1) , and gra[−1·K−1) .Meanwhile, graphene has excellent merits for biomedical applications, e.g., drug delivery and tissue engineering, because of its large surface area , chemicaIn particular, graphene oxide (GO) and reduced graphene oxide (rGO) have received much attention as excellent nanomaterials for drug delivery due to their structural and surface properties that are well suited for biomedical applications ,22. In pThis review outlines and highlights the recent trends and advances of RF/microwave biosensors based on graphene nanomaterials with their particular focus on the following: (1) graphene nanomaterials as biosensing materials, (2) the basic concept of RF/microwave biosensors, (3) RF/microwave devices and circuits based on graphene nanomaterials, (4) RF/microwave biosensors based on graphene nanomaterials, (5) the recent advancements and trends, and finally (6) the conclusions and prospects.In biomedical applications, all graphene derivatives are useful and extensively applied as components or as ingredients for biosensors, real-time bioimaging, cancer diagnosis and treatment, catalysis, and water purification ,33,34,352 hybridized carbon atoms organized in the form of a honeycomb lattice. Preparation or synthesis methods have been well reported such as mechanical exfoliation from graphite, chemical vapor deposition (CVD), and molecular beam epitaxy (MBE). For GO, its synthesis procedures introduce a wide variety of oxygen-containing functional groups, such as carboxyl, hydroxyl, carbonyl, ethoxy, and epoxy, on both planes of the GO sheets, thereby increasing its stability in aqueous solutions. This also provides easy functionalization and derivatization of these materials through both covalent and non-covalent modifications with different biocompatible polymers such as polyethylene glycol and chitosan, and conjugation with targeting moieties, including peptides and antibodies, to develop bio-functionalized nanocomposite systems with improved biological properties [As previously mentioned, graphene is a very thin nanomaterial with a single-layer of spoperties ,37. rGO For this reason, the excellent physicochemical and biocompatible properties of these graphene nanomaterials have created a wide variety of applications in the field of biomedicine. These are mostly in cancer research, ranging from nanocarriers and photo agents for drug/gene delivery to cancer cells as well as for photothermal and photodynamic therapies of cancer, both in vitro and in vivo, to its use as bioimaging agents for developing multifunctional theranostic platforms for carrying out more efficient detection and treatment of cancers . As showGenerally, a biosensor is an analytical device that combines a biological sensing element with a transducer . As showIn the previous session, the biological effects of graphene nanomaterials using diverse synthesis methods have been exploited for biomedical applications because of a large surface area that is capable of being immobilized on its surface and has easy-functionalization. Indeed, there are many possible approaches in the biomedical applications such as bioelectronics , tissue In particular, as pristine graphene has a thin film of infinite size with no imperfections which provides a large number of chemically active sites for charge-biomolecular interactions due to the large surface area, however these also lead to an enhanced sensitivity to the target biomolecules of very low concentrations and improved selectivity . For insOver the past decade, the feasibility of graphene in RF/microwave electronics was investigated owing to its excellent physical properties. As shown in 11 (or S22) is the reflection coefficient of the input (or output) port, and S21 (or S12) is the transmission coefficient of the output port to the input port (or output port to the input port), respectively. The measured S-parameters can be converted to many other parameters such as admittance (Y), impedance (Z), hybrid (H), transfer (T), and ABCD matrix [In RF/microwave measurement system, the measured data are primarily related to the S-parameters (or S-matrix), which is defined as the ratio of the output power (voltage) to the input power (voltage) in the frequency domain. In particular, the S-parameters for the two-part network are expressed asD matrix . Owing tGO has received considerable interests as a sensitive material for biosensing because electron transfer from the biomolecular binding mainly occurs at the defects or edges of GO structure. In addition, owing to the opposite properties of a hydrophobic and hydrophilic functional group on GO, this material exhibits good biocompatibility, high affinity for specific biomolecules. Hence, these properties of GO can provide many opportunities for the various approaches of biosensing platforms, including biosensors based on fluorescence resonance energy transfer, laser desorption/ionization mass spectrometry, surface-enhanced Raman spectroscopy, and electrochemical detection . R component revealed the sensing parameter for effectively detecting the glucose level with stable linearity and small fluctuation [ctuation .One of the excellent physical properties in graphene is a transparent and conducting material with low cost and low environmental impact. This is an ideal material for the integrant of biosensing devices in a range of transduction modes, from electrochemical transduction to optical transduction .Odorrana grahami, H. pylori, E. coli, and S. aureus [L)-capacitor (C) circuit, i.e., a resonant circuit for selecting a specific frequency. Also, this study demonstrated integration onto a tooth for specific bacteria detection in saliva via wireless circuitry [. aureus . These bircuitry .Recent advances in integrated biosensing platforms associated with remote sensing via RF/microwave wireless systems have focused on design and architecture of point-of-care (POC) diagnosis, attracting considerable interest in the biomedical applications. In particular, POC has significant diagnosis possibilities for use in the continuous and real-time monitoring of human metabolites as well as cancer biomarkers . In addiS. aureus, E. coli and so on, via bifunctional peptide. Recent advances in graphene nanomaterials such as synthesis techniques, electrical, thermal and mechanical analysis, surface treatment and device design have accelerated the development and application of graphene nanomaterials-based nanoelectronics as well as bioelectronics. In this review, we have examined the emerging advances of graphene nanomaterials-integrated biosensors including structures and merits of graphene nanomaterials and their biological functionalization in RF/microwave biomedical applications. From the developed RF/microwave biosensors, these biosensing schemes could be classified with passive RF/microwave devices and RF/microwave systems with graphene nanomaterials. Firstly, it was used as a biosensing scheme utilizing simple RF/microwave devices such as resonators and capacitors, with graphene nanomaterials like GO or rGO. In the case of latter, it was used as a biosensing scheme utilizing RF/microwave systems with graphene nanomaterials, e.g., graphene. These RF/microwave biosensors could be detectable of biomolecules, e.g., glucose, DNA, as well as bacteria, e.g., However, the research and development of these materials-based biosensing systems are in their infancy in the RF/microwave biomedical applications. This is because it is not only difficult to find the optimized frequency for biosensing, but devices and circuits also are dependent on the frequency. However, since there are great merits such as real-time, non-invasive, non-contact function, as a graphene nanomaterials-based RF/microwave biosensor, the biosensing scheme still needs to develop the robust biosensing platform integrated with wireless and flexible devices and circuits. In this case, there are also remains challenges how to find effective integration methods and how to secure stability for good performance of RF/microwave devices and systems with graphene nanomaterials. Before this challenge, the optimization of material fabrication and modification techniques to obtain large area, high quality, and uniform arrays will be essential for the highly sensitive and reproducible RF/microwave biosensors. Furthermore, the integration of graphene nanomaterials-based RF/microwave device needs to be optimized to minimize the entire device volume for portable, disposable and POC diagnosis and healthcare in the future.


The ‘leaky pipeline’ in academia is a clearly described phenomenon, but has not been examined in palliative care. We analysed the gender balance of speakers at the 9th World Research Congress of the European Association of Palliative Care (EAPC) to test the null hypothesis that there is no difference in the proportion of women and men with senior academic visibility in palliative care conference programmes.The final programme of the 2016 EAPC World Congress was examined, and the gender of each speaker was recorded. Presentations were assessed using a three-tier hierarchy of senior academic visibility: Free Communication sessions, Themed sessions and invited Plenaries (low to high). As there was only one Invited Plenary at EAPC 2016, we examined the gender balance at EAPC Plenaries from 2012 to 2016.2=25.4, p<0.001).Overall, the majority of speakers at EAPC 2016  were women. The proportion of women was highest in the Free Communication sessions . In the Themed sessions, women made up just over half of speakers . In 2016, there was 1 invited Plenary speaker, a man. From 2012 to 2016, just 6 of 23 invited Plenary speakers at EAPC conferences have been women (26.1%)  and unconscious gender bias need further exploration, as well as actions to address the imbalance. The gender balance in academia is the subject of considerable debate.Visibility, for example through conference talks and plenaries, is an important dimension of gender equality. A paucity of women conference speakers has been demonstrated in several academic disciplines.In June 2016, 1200 delegates attended the 9th World Research Congress of the European Association of Palliative Care (EAPC) in Dublin, making it one of the largest research conferences in palliative care globally. The programme featured 17 Free Communication sessions where researchers were chosen on the quality of anonymous (and gender-blind) abstracts to present their work in 8 min talks. In addition, there were seven Themed sessions in which speakers were invited on the basis of their level of authority on a specific subject to give 20 min talks. The most prestigious speaking opportunity is the invited Plenary where high-profile academics are invited to give longer talks (30 min) on the main conference stage.We aimed to analyse the gender balance of speakers at the 2016 EAPC World Congress to test the null hypothesis that there is no attrition of women in senior academic positions in palliative care, using the three-tiered hierarchy of Free Communication sessions, Themed sessions and Plenaries as a proxy for seniority.2 tests were used to assess differences. We did not analyse Meet the Expert sessions or Poster Discussion sessions.The final programme of the 2016 EAPC World Congress was examined, and the gender of each speaker was recorded. Speaker gender was determined either by direct observation of their talk or by inference from their first name. In eight cases, Google was used for more information. Where there was a substitution of speaker at the last minute, the gender of the speaker originally planned was used. At the 2016 EAPC, five top scoring abstracts were presented as 15 min Plenaries on the main stage. Since the decision to feature these presentations was made on the basis of an anonymous abstract, these were analysed with the Free Communication sessions. Since there was only one invited Plenary speaker at 2016 EAPC, we examined the gender balance of invited EAPC Plenary speakers over 5 years (2012–2016) by searching through relevant scientific programmes online. χAt EAPC 2016, there were a total of 17 Free Communication sessions comprising 102 talks. An additional five top scoring abstracts were presented on the main stage. There were seven Themed sessions which included 22 speakers. There was a single invited Plenary, the Ventafridda Lecture.Overall, the majority of speakers at EAPC 2016 were women. The proportion of women was highest in the Free Communication sessions. In the Themed sessions, women made up just over half of speakers. The Ventafridda Lecture was given by a man .2=25.4, p<0.001).Since there was only one invited Plenary speaker at EAPC 2016, we examined previous EAPC conference programmes to obtain a more representative view of gender balance of invited Plenary speakers for a 5-year period . At EAPCPalliative care is a profession where women make up the majority of the workforce,Given the large number of women working in palliative care, it is of concern that the most prestigious invited Plenaries are so infrequently given by women, and it is noteworthy that two EAPC conferences over the past 5 years have included no female Plenary speakers at all (2014 and 2016). These data suggest that there is attrition of women along the ‘academic pipeline’ in palliative care. However, the relative contribution of other potential factors such as self-selection  and unconscious gender bias  is unclear. In addition, palliative care is multiprofessional, with gender unequally represented within each of the professions. The potential contribution of discipline should also be explored.Given that there was a single Plenary speaker in 2016, we examined the gender balance of Plenary speakers over 5 years. This was based on the assumption that variation from conference to conference is likely to outweigh any temporal trends. The proportion of female Plenary speakers was one-third or fewer for each of the 5 years examined. There was no indication that the proportion of female Plenary speakers improved over time. Indeed, the 2012 and 2013 conferences had the greatest proportion of female Plenary speakers (one-third).Speaking at academic conferences is important for career advancement. Such opportunities not only facilitate networking and collaboration, but are used as a marker of quality by promotions panels.Failure of promotion on merit is deleterious for academia and ultimately for patient care. Whether these data represent a leaky pipeline, gender bias, self-selection or all three, it is essential that as a specialty we take this issue seriously. We hope that the data presented here provide a starting point for more in-depth consideration of this issue and appropriate strategies to tackle it.


CA-170 is currently the only small-molecule modulator in clinical trials targeting PD-L1 and VISTA proteins – important negative checkpoint regulators of immune activation. The reported therapeutic results to some extent mimic those of FDA-approved monoclonal antibodies overcoming the limitations of the high production costs and adverse effects of the latter. However, no conclusive biophysical evidence proving the binding to hPD-L1 has ever been presented. Using well-known in vitro methods: NMR binding assay, HTRF and cell-based activation assays, we clearly show that there is no direct binding between CA-170 and PD-L1. To strengthen our reasoning, we performed control experiments on AUNP-12 – a 29-mer peptide, which is a precursor of CA-170. Positive controls consisted of the well-documented small-molecule PD-L1 inhibitors: BMS-1166 and peptide-57. Alongside chemo- and radiotherapy, surgery, and other “targeted treatments”, cancer immunotherapy  is now regarded as the fifth pillar of cancer treatment, mainly due to a rapid development of potent immune checkpoint-blocking (ICB) therapeutic inhibitors ,2,3,4. T2 and is maintained by polar and hydrophobic interactions. More polar interactions comprising, hydrogen bonds and salt bridges, are observed at the periphery of the hydrophobic core based mainly on alkyl-π and π-π interactions. Interestingly, only PD-1 undergoes significant structural rearrangement upon binding, most notably in the CC′ loop, which transits from an open conformation in the apo form to a closed one in C β strand ..10]. PD-Physiologically, the PD-1/PD-L1 complex negatively regulates the immune response and provides so called T cell homeostasis ,15. CancCA-170, a small-molecule inhibitor against PD-L1/L2 and VISTA, with low nanomolar potencies for the treatment of advanced solid tumors and lymphomas . CA-170 is also undergoing phase II clinical trials for lung cancer, head and neck/oral cavity cancer, MSI-H positive cancers and Hodgkin lymphoma in India . According to the preclinical in vitro and in vivo data reported by the company, CA-170 demonstrated dose-dependent enhancement in the proliferation of PD-L1, PD-L2, and VISTA-inhibited T lymphocytes, and exhibited antitumor effects similar to those of antibodies, including tumor shrinkages and prolonged stable disease without adverse effects [In the recent years, several attempts for the development of non-mAb PD-1/PD-L1 inhibitors, such as macrocyclic peptides, peptidomimetic molecules and nonpeptidic small molecules, were presented and disclosed in particular by Bristol-Myers Squibb (BMS) and Aurigene Discovery Technologies Limited  is routinely employed to characterize small proteins, protein-protein and ligand-protein interactions . PerformCA-170 and AUNP-12 to hPD-L1 were assessed with the 1H and 1H-15N HMQC NMR spectroscopy. In the first experiment, the CA-170 compound was titrated against the 15N-labeled hPD-1-binding single domain of hPD-L1 (amino acids 18-134) and against the entire extracellular domain of the hPD-L1 protein (residues 18-239). In both 1D and 2D NMR spectra, no changes were observed for signals of the protein of hPD-L1, even when the 10-fold excesses of compound was used  with the KD at the millimolar level indicated by slight chemical shift perturbations (AUNP-12 at the molar ratio of 1/5. The rest of the results of NMR experiments for AUNP-12 with hPD-L1 (18-239) and with hPD-1 showed no interaction of the peptide and the proteins , which is currently a standard methodology to determine potency of the inhibitors in numerous publications and patents, due to its robustness and absence of false-positive results [50 of well-known hPD-L1 inhibitors as the positive reference: BMS-1166 and peptide-57 to cross-validate the results with the literature  or hPD-1 either. Lack of the activity in the precursor peptide does not rule out the possibility that its fragments alone may be potent. Moreover, we do not exclude the possibility that this family of compounds may act downstream from the hPD-1 receptor or on any other T cell-activating pathway, since the compounds clearly present promising therapeutic results according to the data provided by Aurigene and Curis [Based on the above consideration we state that neither nd Curis .AUNP-12, is a hybrid of the peptide sequences extracted from hPD-1, that are supposed to be close to its interaction surface with hPD-L1. However, if we look at the locations of particular peptide fragments of AUNP-12  Cas: 1673534-76-3, AUNP-12 was purchased from SelleckChem  BMS-1166 was synthesized according to the protocol from described in [peptide-57 was synthesized as described in [ribed in , peptideribed in .Escherichia coli BL21 (DE3). Bacterial cells were cultured at 37 °C in LB or M9 minimal medium containing 15NH4Cl as the sole nitrogen source to achieve 15N isotope labeling. Proteins expression was induced with 1 mM Isopropyl β-d-1-thiogalactopyranosid (IPTG) at OD600 of 0.8 and the cells were cultured overnight. For hPD-1, hPD-L1 and mPD-L1 temperature was lowered to 28 °C, for hPD-L1(18-239) temperature was left at 37 °C. The inclusion bodies purification was carried out as described previously [l-Arginine hydrochloride, 2 mM EDTA, 5 mM cystamine and 0.5 mM cysteamine for hPD-1 and 0.1 M Tris pH 8.0, 1 M l-Arginine hydrochloride, 0.25 mM oxidized glutathione and 0.25 mM reduced glutathione for hPD-L1, human PD-L1(18-239) and mPD-L1. After refolding, proteins were dialyzed 3 times against solution containing 10 mM Tris pH 8.0 and 20 mM NaCl. Finally, proteins were purified by SEC (size-exclusion chromatography) on HiLoad 26/600 Superdex 75 column  in 25mM sodium phosphate pH 6.4 with 100 mM NaCl for hPD-1 or in PBS pH 7.4 for hPD-L1, hPD-L1(18-239) and mPD-L1.The gene encoding hPD-1  was cloned into pET-24d, the gene encoding hPD-L1  was cloned into pET-21b, the genes encoding hPD-L1  and mPD-L1 (amino acids 38-134) was cloned into pET-28a, respectively. Proteins were expressed in the v/v) of D2O was added to the samples to provide the lock signal. All spectra were recorded at 300 K using a Bruker Avance III 600 MHz spectrometer. Binding of the compounds was analyzed by titrating the 15N-labeled hPD-L1/hPD-1 and recording the 1H and 1H−15N HMQC spectra prior to and after the addition of the compounds.For NMR measurements, the buffer was exchanged by gel filtration to PBS pH 7.4. 10% (50) of tested compounds, measurements were performed on individual dilution series. After mixing all components according to Cis-Bio protocol, the plate was left for 2h incubation at room temperature followed by TR-FRET measurement on Tecan Spark 20M. Collected data was background subtracted on the negative control, normalized on the positive control, averaged and fitted with normalized Hill’s equation to determine the IC50 value using Mathematica 12.HTRF assay was performed using the certified Cis-Bio assay kit at 20 µL final volume using their standard protocol . To determine the half maximal inhibitory concentration   and Jurkat T cells overexpressing hPD-1 and carrying a luciferase reporter gene under the control of Nuclear Factor of Activated T-cells Response Element (NFAT-RE)  were cultured in RPMI-1640 medium  supplemented with 10% Fetal Bovine Serum  and 200 mM L- Glutamine (Biowest) in the presence of G418  and Hygromycin B Gold  as selection antibiotics. The overexpression of hPD-L1 and TCR ligand in aAPCs and PD-1 in ECs were confirmed by flow cytometry and western blot analysis, respectively. PCR tests for 50 values) were calculated from the Hill’s curve fitting to the experimental data.The activity of the inhibitors of hPD-1/hPD-L1 immune checkpoint was examined using the hPD-1/hPD-L1 Blockade Bioassay (Promega), according to the manufacturer’s instructions. hPD-L1 aAPCs were seeded on 96-well (white) plates at the density 10 000 cells/well 17h prior to the experiment. The 2.5-fold dilutions of the small molecules or peptide-57 were first prepared in DMSO. On the day of the assay the compounds were diluted 1000-fold in the assay buffer  to maintain the constant concentration of DMSO . The 2.5-fold dilutions of nivolumab, a positive control anti-hPD-1 monoclonal antibody , were prepared in the assay buffer on the day of the assay. The culture medium was discarded from the wells and serial dilutions of either the small-molecule or antibody was added. Afterwards, Jurkat hPD-1 cells were seeded at the density of 20,000 cells per well in the assay’s plates. After 6h of the incubation in standard culture conditions, assay plates were equilibrated at ambient temperature for 10min, followed by a 20min incubation with the Bio-GloTM Assay reagent (Promega). The luminescence was detected using the Infinite M200 reader. Half maximal effective concentrations  containing protease inhibitor cocktail (Sigma Aldrich). Following the electrophoresis and transfer to PVDF membranes, the membranes were blocked with 4% BSA  in TBS-N buffer and incubated with primary antibody solution at 4 °C overnight. After that, four washes with TBS-N buffer, the incubation with secondary antibody for 1h at room temperature, and additional four washes were done. Then, the detection was performed with the use of Clarity Western ECL Substrate  and ChemiDoc MP system (BioRad).The following antibodies and dilutions were used: rabbit monoclonal anti-hPD-L1 , cat. 13684), rabbit monoclonal anti-α-Tubulin , goat peroxidase-conjugated anti-rabbit .For the analysis of the expression of human PD-L1 on the surface of aAPC cells flow cytometry analysis was performed. The cells were seeded on 6-well plates and cultured till sub-confluency. The cells were detached from the plates with TrypLe Select Enzyme , placed on ice, washed 2 times with Flow Cytometry Staining Buffer  and stained with mouse anti-human PD-L1 antibody . 17-4714 antibody was used as an isotype control of the staining. The cells were analyzed with the FACSCanto II cytometer.CA-170 is the first small-molecule inhibitor targeted at PD-L1 that was introduced to phase I and II clinical trials. We, however, unambiguously determined that there is in fact no direct binding between CA-170 and its peptidic precursor AUNP-12 to PD-L1. In the NMR binding assay, neither of these molecules showed any interactions with the one- or two-domain PD-L1 in the human and mice variants as well as human PD-1. CA-170 and AUNP-12 were also not able to dissociate complex formation in the isolated system of the HTRF assay and cell-based assay mimicking in vivo conditions. All measurements were cross-validated with positive controls: peptide-57 and small-molecule BMS-1166. Therefore, proposed mechanism of the direct blocking of hPD-1/hPD-L1 interactions with CA-170 has to be changed.