This synthesis technique is useful to control the grain size with increased porosity to enhance the hydrophilic characteristics of the CaMgFe1.33Ti3O12 nanoceramic based sintered electro-ceramic nanocomposites. The highest porosity, lowest density and excellent surface-hydrophilicity properties were obtained at 1050 °C sintered ceramic. The performance of this impedance type humidity sensor was evaluated by electrical characterizations using alternating current (AC) in the 33%–95% relative humidity (RH) range at 25 °C. Compared with existing conventional resistive humidity sensors, the present sintered electro-ceramic nanocomposite based humidity sensor showed faster response time (20 s) and recovery time (40 s). This newly developed sensor showed extremely high sensitivity (%S) and small hysteresis of <3.4%. Long-term stability of the sensor had been determined by testing for 30 consecutive days. Therefore, the high performance sensing behavior of the present electro-ceramic nanocomposites would be suitable for a potential use in advanced humidity sensors.Since humidity sensors have been widely used in many sectors, a suitable humidity sensing material with improved sensitivity, faster response and recovery times, better stability and low hysteresis is necessary to be developed. Here, we fabricate a uniformly porous humidity sensor using Ca, Ti substituted Mg ferrites with chemical formula of CaMgFe A variety of techniques have been subsisted for detection of relative humidity (RH) in various systems or devices. RH is one of the most frequently influenced physical parameters in every field of life, e.g., domestic, pharmaceuticals, electronics, textile, biomedical field, and laboratories ,4,5,6,7.3) can show spontaneous high polarization and high magnetic Curie temperature [2) based flexible composite had shown good resistive sensitivity for humidity and gas sensor [In this context, lead-free ferroelectric ceramics  ferrites was found to be effective in order to minimize the magneto crystalline anisotropy and to enhance the electrical resistivity. The reduction in conductivity and dielectric loss of ferrites is very important for humidity sensor applications [Substitution of cations in magnetic and non-magnetic ferrites has ability to change their magnetic or electrical properties ,29,30. T ferrite  and infl ferrite  ferritesications .1.33Ti3O12) using inexpensive solid-state step-sintering from inexpensive iron oxide (Fe2O3), magnesium carbonate (MgCO3), calcium oxide (CaO) and titania (TiO2) powders and by optimizing the sintering conditions for humidity sensing application in the field of biomedical engineering. Since our previous report on capacitive sensor have shown successful effort [1.33Ti3O12 complex ceramic samples in the present study.The above survey indicates that the use of Ca, Ti doped magnesium ferrite nanocomposite might be potential candidate as an effective humidity sensor material. To the best of our knowledge, there is no existing report by any other group on the synthesis of Ca, Mg, and Ti oxide based complex ferrite materials. Therefore, for the first time, we aim to synthesize Ca, Mg, and Ti oxide based complex ferrite material  using commercial ceramic powders of CaO, MgCO3, Fe2O3 and TiO2  with proper molar ratio, as reported before [1.33Ti3O12 from the used molar ratio of the raw ingredients. All the ingredients powders were first, dry mixed thoroughly in normal grinder for 1 h and then, the mixed powders were ball-milled in aqueous medium of 70% alcohol in a ball mill  at 300 rpm for 72 h. The mixed slurry was kept at 250 °C for 5 h for self-combustion followed by drying at 220 °C in a programmable oven for 48 h. The cylindrical pellets (Φ10 mm × 1–2 mm) were prepared at a maximum uniaxial pressure of 450 MPa under a hydraulic press . The testing pellets were step-sintered in ambient gas atmosphere. Since, according to our previous studies, morphological, physical, and dielectric properties suggested that the material of sintering condition 1050 °C was best, only pellets sintered at 1050 °C were used as resistive or impedance type humidity sensors in the present investigation. In this particular sintering condition, mainly three steps were involved. First, the compact pellets were heated at 350 °C for 1 h at a ramp rate of 5 °C/min, then they were calcined at 550 °C for 3.5 h at a ramp of 10 °C/min, and finally, the pellets were sintered at 1050 °C for 1.3 h at a rate of 15 °C/min. After the final step, it was cooled down to 750 °C at 20 °C/min with a holding time of 3 h and then allowed to normal furnace cooling up to the room temperature . The solid-state step-sintering ensured the desired ranges of particle size, pore size, and porosity.The CaMgFed before . The emp1.33Ti3O12 was uniaxially pressed at high pressure of 450 MPa for 4 min to form the pellets of 10 mm in diameter and about 1.2 mm in thickness 2, NaCl, KCl and KNO3, which would produce the environments with different RHs of 33%, 55%, 75%, 85% and 95% RH, respectively at 25 °C.A measured amount of the dried milled powders of CaMgFehickness a. Then, hickness a. Finallhickness b. The RHhickness c were obX-ray diffraction (XRD) results were obtained by using X-ray diffractometer . The microstructure of the samples was analyzed by field emission-scanning electron microscopy . Pore size distribution (PSD) was computed by the ImageJ from the corresponding FESEM images. Density (ρ in g/cc), open porosity (%), and absorbed water or water absorption (%) of the porous materials were evaluated by modified Archimedes’ principle, as explored in our previous studies ,43,44 us1.33Ti3O12 nanoceramic derived sintered electro-ceramic nanocomposite humidity sensor was studied by impedance spectroscopy (IS)  using alternating current (AC) frequency, ranging from 100 Hz to 1 MHz at 25 °C. The sintered electro-ceramic nanocomposite used as sensing element and the schematic of humidity sensing measuring equipment are depicted in The impedance response to RH of CaMgFe2 (Powder diffraction file (PDF) No. 98-015-4609) and ferrite Fe2O3 (PDF No. 01-084-0308) are indicated in 3 have also been detected. A new peak of calcium carbonate  at 2θ = 29.59° indicates that the CaO and MgCO3 had reacted during mechano-chemical mixing. After sintering, the unsintered CaMgFe1.33Ti3O12 nanoceramic converted into mainly two new phases, which were resembled with the standard XRD patterns of orthorhombic armalcolite  and perovskite CaTiO3 (PDF No. 00-008-0092) at 1050 °C, are depicted in FWHM is the full width at half maxima of the XRD peak (in radian), κ is a constant (~0.9) that depends on the particle morphology, λ is the x-ray wavelength (λCuKα = 1.540546Å), ϵ is the crystal strain or elastic residual strain, and θ is the Braggs’ diffraction angle (in degree).X-ray diffraction (XRD) patterns of unsintered and sintered (at 1050 °C) ceramic materials are depicted in 2MgTi3O10 and 2θ = 33.32° for (440) plane of CaTiO3 are 20.7 nm and 5.9 nm, respectively. This result clearly indicates that the material sintered at 1050 °C contained mainly two phases, which were also observed in the scanning electron micrograph (see 3O4 (PDF No. 01-088-0315) for (311) plane clearly indicates the transformation of Fe3+ into Fe2+ from Fe2O3 after sintering at 1050 °C. According to the peak areas of the corresponding peaks, the amounts of Fe2MgTi3O10, CaTiO3 and Fe3O4 present in the sintered material are 85.80%, 12.16% and 2.04%, respectively  plane of Feraph see . Interes2MgTi3O10) and cubic perovskite (CaTiO3) can be calculated using Equations (2) and (3), respectively, and the values are illustrated to 3O4 at 2θ = 35.72°, the lattice parameters were not calculated in the present study:hkl is the inter-planer distance between the planes (hkl); and a, b and c are unit cell parameters. It was found that unit cell volume of both the crystal lower than that of standard unit cells of the corresponding crystals. It was affected by lattice strain. The interesting result obtained by the XRD was that the unit cell volume of the CaTiO3 was higher than that of Fe2MgTi3O10, which was reverse of the particle size obtained from the SEM micrograph of the sintered material . They were also confirmed by XRD study. The grain boundaries and grains were clearly revealed in the sintered materials at higher magnifications, as found at the large size armalcolite phase in A submicro-porous structure of the ceramic nanomaterials is depicted in the scanning electron micrographs of TiO3 see b. The sm2/g) of the unsintered sample could partially be the effect of large amount of closed pores in order to get detriment in open pores. The obtained changes in the both particle size and specific surface area suggest that the used step sintering technique, as a result of the formation of the different secondary or tertiary phases, inhibited the crystal growth and favored the increase in specific surface area. On the other hand, the sintered electro-ceramic showed the higher porosity (>40%), lowest density, the largest specific area (10–70 m2/g) and a wide PSD in comparison of unsintered CaMgFe1.33Ti3O12 nanoceramic. The large specific area enhanced active surface to adsorb or desorb water vapors and, thus, improved the resistive sensitivity with the variation of RH. Further, since large pores are necessary for a rapid response, this sintered electro-ceramic can easily improve the adsorption and condensation of water vapor [er vapor . Hence, The bulk density was conducted in order to measure the open porosity of the materials. The unsintered and sintered (1050 °C) materials showed bulk density of 1.989 ± 0.091 g/cc and 0.941 ± 0.035 g/cc, respectively; and the open porosity of unsintered and sintered (1050 °C) materials was 73.84% ± 1.24% and 40.26% ± 1.33%, respectively. The decreased value of density showed that the densification was significantly controlled by sintering condition. Densification was controlled using step-sintering conditions to enhance the porosity. The bulk density of the nanocomposite sintered at 1050 °C was significantly (50%) decreased due to lattice diffusion phenomenon during sintering with keeping substantial porosity . The por1.33Ti3O12 nanoceramic derived sintered electro-ceramic nanocomposite material based humidity sensor with different humidity conditions, i.e., 33%–95% RH, are depicted in The complex impedance spectra of the newly developed CaMgFeIt was found that the impedance value decreased with increasing of RH. This study describes the mechanism of impedance response with the function of humidity. From the SEM analysis it was confirmed that at 1050 °C sintering temperature, the porosity of the sintered nanocomposite was achieved to the desired level, which enhances the hydrophilic surface characteristics of the developed sensors material. When the porous sintered nanocomposite was placed in a humidity environment, the oxygen and water from surrounding atmosphere could be chemisorbed onto its hydrophilic surface, which played a vital role to alter the sensing characteristics of the sensor. In addition, due to increase of RH from 33% to 95%, more amount of water particles were absorbed on the composite’s surfaces. It mainly resulted to a decrease in impedance such a manner that the dielectric properties could be tailored. In 2 Hz–1 MHz at 25 °C. The RH dependent capacitance effect is depicted in + ions would be available in adsorbed water layers on the surface of sensor material, and, as a result, the capacitance value becomes higher [To analyze the effect of humidity on our present electro-ceramic nanocomposite device, we measured the impedance, capacitance and modulus at different humidity conditions in a frequency range of 10s higher . Impedan+ ions is increased due to the increasing of concentration of adsorbed water, which play a vital role in conduction mechanism and gradually the mobility of ions increase.The frequency dependent capacitive reactance of the sintered electro-ceramic nanocomposite, i.e., imaginary part of the impedance (Z″), for different RHs is depicted in 2, 103, 104, 105, and 106 Hz for sintered electro-ceramic nanocomposite sensors are depicted in 2 Hz, impedance of the nanocomposite humidity sensor device was 1.47 × 107 Ω at 33% RH and it decreased to 6 × 105 Ω at 95% RH. The impedance of this sensor decreased from 2.51 × 106 Ω at 33% RH to 4.8 × 105 Ω at 95% RH at 103 Hz. At a frequency of 104 Hz, the impedance changed from 2.02 × 105 Ω to 1.6 × 105 Ω. At a frequency of 105 Hz, impedance changed from 1.7 × 104 Ω to 1.6 × 104 Ω. At a frequency of 106 Hz, impedance changed from 2.2 × 103 Ω to 5.43 × 102 Ω. Therefore, it indicates that the humidity change affects the impedance considerably at intermediate and low frequencies, but the change is very small at higher frequencies. In order to evaluate the impedance characteristics of a nanocomposite sensor as a function of humidity, device sensitivity (SZ) can be estimated using Equation (4) [2, 103, 104 and 106 Hz, respectively. This result confirms that the highest sensitivity is observed at 102 Hz. The present device sensitivity was significantly higher compared to the other existing resistive type humidity sensors  [2 Hz frequency was adopted as the testing frequency in all the succeeding analyses of the present investigation.Humidity dependent impedance responses at 10tion (4) ,53:(4)S 100 Hz) ,55,56,572 Hz. In case of the present electro-ceramic nanocomposite sensing device, response time was 20 s when humidity was changed from 33% RH to 95% RH and recovery time was 40 s when humidity was changed from 95% RH to 33% RH, as depicted in 4-doped SnO2 ~60 s and ~100 s, Ba0.5Ni0.5SnO3 ~3 min and ~4 min, Li-doped mesoporous silica A-SBA-15 ~60 s and ~180 s, LiCl-doped mesoporous silica MCM-41 ~100 s and ~150 s, mesoporous ZnO–SiO2 composite ~50 s and ~100 s respectively, and so on) [In order to evaluate the performance of a humidity sensor, response and recovery behaviors are widely checked. The response time was measured by the time elapsed to reach 90% of the final signal at a given RH. On the other hand, the recovery time was measured by the time elapsed to come back within 10% of initial signal value. Here, response and recovery times of a sensor were measured by alternately exposing to 33% RH–95% RH. Both these times are normally very sensitive to structure and must be shortest for a practical application. In order to determine the response and recovery times, a graph of impedance versus time was plotted at AC voltage of 1 V at test frequency of 10d so on) ,59,60,614-doped SnO2 ~6%, CdTiO3 ~7%, BaTiO3 nanofibers ~5%, BaTiO3/polystyrene sulfonic sodium ~8%, and so on) [The humidity hysteresis response, which is maximum difference between the humidification and desiccation curve, is another crucial characteristic of humidity sensor. The humidity hysteresis of the present electro-ceramic nanocomposite sensor is shown in d so on) ,53,62,632 Hz with 1 V AC power supply at 25 °C. Very little fluctuation or almost no variation in impedance of the present device was found over the 30 days of measurements. As a matter of fact, the impedance or resistance of the sensor slightly increased in ambient atmosphere due to aging operation. It evidently confirms that the present resistive sensor has significantly good stability. The increased response of conductivity with RH of the present electro-ceramic nanocomposite could be explicated by the Grotthuss mechanism [+) in between the water molecules through tunneling [In addition to the aforementioned electrical parameters, stability is another one of the most important measuring properties for determining the advantages of a humidity sensor. In order to obtain the long-term stability, the present electro-ceramic nanocomposite based humidity sensor was studied in air at 25 °C for 30 days, while impedances were measured repeatedly at two-day intervals at different RH conditions, as depicted in echanism , which c1.33Ti3O12 nanoceramic derived sintered electro-ceramic nanocomposite via double hydrogen bonding and it is depicted in 1.33Ti3O12 nanoceramic derived sintered electro-ceramic nanocomposite was found via single hydrogen bonding (3O+) might be available for conduction [At lower RH condition, the water molecules were chemically adsorbed (chemisorption) onto the available active sites of the surface of oxide based CaMgFe bonding . Due to nduction .0Z′), M″ is an imaginary part of modulus (M″ = jωC0Z″), ε* is permittivity, Z* is complex impedance, ω is the angular frequency, i.e., ω = 2πf, f is linear frequency, and Z′ and Z″ are the real part and imaginary parts of impedance, respectively. C0 is the geometrical capacitance, i.e., C0 = ϵoA/t, where A is area of the electrode, t is thickness and ϵo is permittivity of air. To suppress the electrode polarization and space charge injection phenomena, the complex modulus analysis is an effective method. The frequency dependent M′ and M″ responses at different RH are shown in f) vs. M″ plot, as shown in maxf, where maxf is peak frequency. Since the relaxation frequency is increased with increased RH, the relaxation time decreases with the increase in humidity.The complex electric modulus (M*) can be expressed using Equation (5) :(5)M*(ω1.33Ti3O12 nanoceramic derived sintered electro-ceramic nanocomposites based humidity sensing device decreases with the increase in relative humidity.The complex modulus spectra (M′ vs. M″) of the present electro-ceramic nanocomposite sensor device at different relative humidity are depicted in 1.33Ti3O12 nanoceramic derived porous sintered electro-ceramic nanocomposites by using solid-state step-sintering. The typical structures and morphology were found by XRD and SEM. The desired grain size, high porosity and good surface-hydrophilicity properties were obtained for the nanocomposites sintered at 1050 °C. The sintered electro-ceramic exhibited excellent humidity sensing properties, including high sensitivity, faster response (20 s) and recovery (40 s) times, narrow hysteresis (<3.4%), and good stability over a long period. These advanced improved characteristics were attributed to suitable stable grain size and porous nature of the nanocomposite. The obtained electrical results confirm that our newly developed CaMgFe1.33Ti3O12 nanoceramic derived sintered electro-ceramic nanocomposite material based humidity sensor is better than many currently existing conventional metal oxide based humidity sensors [This investigation successfully fabricated impedance or resistive type humidity sensor based on CaMgFe sensors ,59,60,61 sensors , this ma sensors , particu


The elliptical array structure can be applied to tunable spectral detectors, filters and sensors at far-infrared and terahertz wavelengths.In this paper, we come up with a wavelength tunable absorber which is made up of periodically elliptical graphene arrays in the far-infrared and terahertz regions. Through simulation, we find that we can increase the length of long axis of the ellipse, raise the incidence angles of TM- and TE-polarization  within certain limits, and increase the chemical potential of graphene, so as to enhance the absorption of light in the elliptical graphene arrays. We also compare the absorption spectra of the original structure and the complementary structure, and find that the absorption of the original structure is higher than that of the complementary structure. In the end, we study the changes in the absorption rate of the double layer structure of the elliptical array with the increase in the thickness of SiO Since the first introduction of its synthesis via a ‘Scotch tape’ method in 2004, graphene has aroused great interest in the scientific community . As a siAlthough monolayer graphene absorbs only 2.3% of the incident energy in the visible and near-infrared ranges, in the mid-infrared and terahertz ranges, graphene is able to concentrate very large amounts of light energy. This is precisely because graphene supports the surface plasmon resonance (SPR) like metals. The graphene, which supports surface plasmon resonance, has many excellent properties that can supplement the defects of the metal. This is because the graphene, which supports SPR, is beneficial to the absorption of light energy . RecentlIn this work, we propose the study of optical absorption enhancement based on elliptical graphene. On the basis of silicon, we add a layer of silica with elliptical graphene. Due to the unique asymmetry of the ellipse, the proposed model possesses the advantage of having more free geometric parameters compared with the above-mentioned symmetric structures, and offers good flexibility for the manipulation of light. We not only discuss the influence of structural parameters and chemical potential, but also discuss the complementary structure and the double layer structure. We also study the changes in the resonant wavelength and absorption peak caused by different directions of the incidence of light in the polarization of TE and TM. Thus, the elliptical array structure can be applied to tunable spectral detectors, filters and sensors at far-infrared and terahertz wavelengths.nsi = 3.4, and the relative permittivity of silicon dioxide is εd = 3.9 [x-z plane is taken as the plane of incident. The Kubo formula, consisting of interband and intraband transitions, provides the surface conductivity of graphene σ [μc >> KBTm, the interband contribution can accordingly be neglected because of the Pauli exclusion principle. Thus, the conductivity of graphene is close to the Drude-like modelμc, relaxation time τ and photon frequency ω. The geometric schematic diagram of our elliptical graphene array is shown in εd = 3.9 ,17,18. Haphene σ ,20. In tconstant . Equatioμc = 0.4 eV and τ = 0.5 ps, respectively. The influence of chemical potential will be analyzed later. Through the finite-difference time-domain  method, we simulated the model and calculated the absorption performance. In the x and y directions, we used symmetric and anti-symmetric boundary conditions, respectively. In the propagation of the incident plane waves, perfectly matched layers are utilized in the z direction. The reflection, transmission, and absorption can be expressed as follows:S-parameters. In this work, we first set the chemical potential and relaxation time to At first, we studied the geometrical effects of the absorption of monolayer elliptical graphene array at normal incidence. When the long axis is changed and the short axis is constant, we can see clearly from L = 950 nm, a = 2500 nm, d = 300 nm and W = 750 nm, the absorption spectra for TM- and TE-polarization are displayed in x and y. Then we can clearly see that regardless of the mode, when the angle is between a minimum of 0° and a maximum of 70°, the absorption is increased by about 32%, which is two times greater than the original one. The cause of this phenomenon is that, as the incidence angle increases, the reflection of light becomes weaker. In At c plays a decisive role in the optical properties of graphene [L = 950 nm, a = 2500 nm, d = 300 nm and W = 750 nm, the absorption spectra at normal incidence are displayed in c. The results show that the chemical potential of graphene increases the blue shift of the spectrum, the resonance wavelength is shortened, and the absorption rate is increased. This is because when the chemical potential of graphene increases, the wavelength of the plasmons, such as the corresponding graphene, will increase. At the same time, with the increase of chemical potential, the conductivity of the surface of the graphene will increase as well. Therefore, the absorption of light is enhanced [μc = 0.2 and 0.4 eV. With the increase in the chemical potential of graphene, the SPR of graphene is enhanced. The enhancement of the local electric field leads to an increase in absorption. Therefore, we are able to control the chemical potential of graphene in order to achieve control of the absorption. The chemical potential μgraphene ,23. At Lenhanced ,22. ThisL = 950 nm, a = 2500 nm, d = 300 nm and W = 750 nm, We also studied the absorption spectra of the complementary structure under the same conditions. L = 950 nm, a = 2500 nm, d = 300 nm and W = 750 nm, the absorption spectra for TM- and TE-polarization are displayed in For the complementary structure, we also studied the absorption of the TM and TE modes at different incidence angles. With L = 950 nm, a = 2500 nm, d = 300 nm and W = 750 nm, h of SiO2. It is obvious that when h is increased, the resonance wavelength undergoes a red shift, and the absorption peak at the resonance wavelength moves slightly up and down. This is due to the change in the distance between the two graphene arrays, which results in a change in the coupling between them.Finally, we studied the double layer structure of the elliptical graphene array. With 2. Thus, the elliptical array structure we propose can be applied to tunable spectral detectors, filters and sensors at far-infrared and terahertz wavelengths. To conclude, we propose a wavelength-tunable absorber that is made up of periodic elliptical graphene arrays. By increasing the length of the major axis of the ellipse, increasing the incidence of x polarization within a certain range of angles, or increasing the graphene chemical potential, we can achieve tuning of the graphene resonant wavelength and raise the absorption rate of graphene. Among these adjustments, the change of the angle of incident light can increase the absorption rate to 56%. This is attributed to the SPR of graphene. We also found that the absorption of the structure is greater than that of its complementary structure. We compare the absorption changes caused by the incidence angle changes in the TM and TE modes. A larger angle of incidence causes higher absorption. In the end, we study the increase in the absorption of the double layer structure of the elliptical array with the increase in the thickness h of SiO


A315T mice), patient derived fibroblasts, and human cells expressing mutant forms of TDP-43.Mitochondrial dysfunction has been linked to the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Functional studies of mitochondrial bioenergetics have focused mostly on superoxide dismutase 1 (SOD1) mutants, and showed that mutant human SOD1 impairs mitochondrial oxidative phosphorylation, calcium homeostasis, and dynamics. However, recent reports have indicated that alterations in transactivation response element DNA-binding protein 43 (TDP-43) can also lead to defects of mitochondrial morphology and dynamics. Furthermore, it was proposed that TDP-43 mutations cause oxidative phosphorylation impairment associated with respiratory chain defects and that these effects were caused by mitochondrial localization of the mutant protein. Here, we investigated the presence of bioenergetic defects in the brain of transgenic mice expressing human mutant TDP-43 (TDP-43A315T mice, TDP-43 mutant fibroblasts, and cells expressing mutant TDP-43, we tested several bioenergetics parameters, including mitochondrial respiration, ATP synthesis, and calcium handling. Differences between mutant and control samples were evaluated by student t-test or by ANOVA, followed by Bonferroni correction, when more than two groups were compared. Mitochondrial localization of TDP-43 was investigated by immunocytochemistry in fibroblasts and by subcellular fractionation and western blot of mitochondrial fractions in mouse brain.In the brain of TDP-43A315T peripherally associated with brain mitochondria. However, there was no correlation between TDP-43 associated with mitochondria and respiratory chain dysfunction. In addition, we observed increased calcium uptake in mitochondria from TDP-43A315T mouse brain and cells expressing A315T mutant TDP-43.We did not observe defects in any of the mitochondrial bioenergetic functions that were tested in TDP-43 mutants. We detected a small amount of TDP-43While alterations of mitochondrial morphology and dynamics in TDP-43 mutant neurons are well established, the present study did not demonstrate oxidative phosphorylation defects in TDP-43 mutants, in vitro and in vivo. On the other hand, the increase in mitochondrial calcium uptake in A315T TDP-43 mutants was an intriguing finding, which needs to be investigated further to understand its mechanisms and potential pathogenic implications. Mutations in transactivation response element DNA-binding protein 43 (TDP-43) cause rare forms of familial amyotrophic lateral sclerosis (fALS) and frontotemporal lobe dementia (FTLD) , 2. The TDP-43 is a nuclear riboprotein involved in regulation of RNA splicing [In ALS-FTLD, mutant TDP-43 is depleted from the nucleus and accumulates in the cytosol, where it forms aggregates of phosphorylated protein , 6, and Mitochondria have been suggested to be one of the multiple potential targets of TDP-43 extra-nuclear mislocalization and aggregation, since mitochondrial dynamics and distribution has been described in cellular and animal models of the disease. In particular, it was shown that both TDP-43 overexpression and suppression impair mitochondrial movement in cultured neurons and that mutant TDP-43 co-localizes with mitochondria . FurtherMitochondrial axonal transport defects were identified in various TDP-43 animal models, from flies  to mice Recently, it was proposed that mutant cytosolic TDP-43 gains access to the mitochondrial matrix through a dysregulated import mechanism, and that TDP-43 in the matrix impairs mitochondrial respiratory chain activity by downregulation of complex I biosynthesis .Based on the growing body of evidence suggesting that mitochondria are targeted by mutant TDP-43, we decided to perform a thorough investigation of mitochondrial bioenergetics in multiple model systems, ranging from mutant TDP-43 transgenic mice to patient-derived cells harboring TDP-43 mutations, to asses the presence of mitochondrial dysfunction.All reagents were from Sigma-Aldrich  unless otherwise stated.A315T transgenic mice . In all experiments, N-Tg littermates were used as controls.We used the strain B6;CB-Tg(Prnp-TARDBP*A315T)95Balo/J of TDP43The criterion for determining disease onset was the development of abnormal hind-limb extension (clasping). The criterion for survival was the inability of the mouse to right itself, when placed on its side (loss of righting reflex).A315T transgenic mice and age and sex matched N-Tg littermates by differential centrifugation of homogenates on a discontinuous Percoll™ gradient as previously described [Forebrain mitochondrial fractions were freshly prepared from TDP43escribed . MitochoATP synthesis was measured in purified brain mitochondria using a luciferin-luciferase approach, as previously described . Glutama2O2 as described [2PO4, 200 μg/mL BSA, 1 μM Amplex Red, 4 U horseradish peroxidase, pH 7.2). Standard curves were used to calculate H2O2 emission rates after sequential addition of substrate , 1 μM rotenone, and 1.8 μM antimycin A.ROS emission was measured as Amplex Red  fluorescence (555 nm excitation and 581 nm emission wavelengths) in the presence of exogenous horseradish peroxidase and mitochondrial Hescribed , 19. Bri2+ uptake was estimated fluorimetrically with Fura 6 (340/380 nm excitation and 510 nm emission wavelengths) (Thermo Fisher Scientific) with sequential additions of 25 nmoles of Ca2+ to the incubation medium .Mitochondrial Ca2, 2 mM KH2PO4, 0.2 mM ATP, 200 μg/mL BSA, 5 mM glutamate, 2 mM malate, 2 μM safranin O, pH 7.2). Mitochondrial membrane potential decay curves were obtained by repetitive additions of 25 nmol Ca2+ or 2–16 nM of the respiratory chain uncoupler SF6847.Mitochondrial membrane potential was estimated using safranin O , as previously described . The inc2HPO4, 0.1 mg/ml BSA, pH 7.2 and 1 mM ADP. Glutamate (5 mM) and malate (2 mM) were used as oxidative substrates. Oxygen consumption was recorded with an oxygraph equipped with a Clark electrode , as described [For mitochondrial respiration, 100 μg of brain mitochondria were resuspended in 0.5 ml of respiration buffer containing 125 mM KCl, 20 mM Hepes, 4 mM KRespiratory chain complex I and complex IV enzymatic activities were measured spectrophotometrically, as previously described .Skin fibroblasts  were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 25 mM glucose, 4 mM glutamine, 1 mM pyruvate, and 10% fetal bovine serum. All fibroblast lines were coded to protect patients’ identity.HEK293T , were grown in DMEM supplemented with 25 mM glucose, 4 mM glutamine, 1 mM pyruvate, and 5% fetal bovine serum.All cells tested negative for mycoplasma contamination by PCR assays of the culture medium using previously described primer sets and amplification protocols .4 cells/well in replicates of eights in 96-well tissue culture plates in growth medium incubated at 37 °C in 5% CO2. The following day, all cells were washed with cultured medium and loaded with 50 nM tetramethylrhodamine methyl ester  and 450 nM Mito Tracker Green  for 30 min at 37 °C in phenol-free DMEM containing 5 mM glucose, 4 mM glutamine, and 1 mM pyruvate, half of the wells additionally contained the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone  to completely depolarize mitochondria and obtain background TMRM and MTG fluorescence, which were subtracted from total fluorescence levels. After washing with DMEM, MTG and TMRM fluorescence were simultaneously recorded in a plate reader equipped with a polychromator . MTG and TMRM fluorescence values were expressed as relative fluorescence units per milligram of total cellular proteins .For mitochondrial membrane potential and mitochondrial content measurements skin fibroblasts were seeded at the density of 1.5 × 104 cells/well in replicates of nines in 96-well tissue culture plates in growth medium incubated at 37 °C in 5% CO2. On the following day, triplicates of wells were incubated with either DMEM containing 5 mM glucose, 4 mM glutamine, and 1 mM pyruvate (ATP baseline), DMEM containing 5 mM 2-Deoxy-D-glucose (2DG), 4 mM glutamine, and 1 mM pyruvate to block glycolysis (ATP 2DG) or DMEM containing 5 mM glucose, 4 mM glutamine, 1 mM pyruvate, and 1 μM oligomycin to block the mitochondrial ATPase (ATP Oligo) for 90 min. Cells were washed with PBS and lysed in 30 μl of trichloroacetic acid (2.5% W/V) on ice for 30 min. Following lysis, 20 μl aliquots were pipetted into a separate plate for protein determination for data normalization, and 45 μl of Tris-acetate  buffer was added to each well of the remaining lysates. Bioluminescence was measured promptly after adding and mixing 20 μl of luciferase reagent. Luminescence values were normalized against an ATP standard and normalized by protein content.Total ATP content in fibroblasts was measured by luciferase reactions in a luminometry plate reader, according to the manufacturer’s guidelines . Cells were seeded at the density of 1.5 × 104 cells/well (fibroblasts) and 2 × 104 cells/well (HEK293T cells) in 200 μL of growth medium and incubated for 24 h at 37 °C in 5% CO2. After replacing the growth medium with 200 μL of XF Assay Medium supplemented with 5 mM glucose, 1 mM pyruvate and 4 mM glutamine, pre-warmed at 37 °C, cells were degassed for 1 h before starting the assay procedure, in a non-CO2 incubator. OCR and ECAR were recorded at baseline followed by sequential additions of 1 μM oligomycin, 2 μM FCCP, and 0.5 μM Antimycin A (AA) plus 0.5 μM Rotenone (Rot). Non-mitochondrial oxygen consumption (in the presence of AA + Rot) was subtracted from all OCR values, and outliers technical replicates outside the 2 standard deviation of the mean were discarded for both ECAR and OCR. In fibroblasts, values were normalized by the mean protein value of each line measured by modified Lowry protein assay. In HEK293T cells, values were normalized by total DNA content measured by fluorescence of DAPI stain, using DNA standards for quantification.Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured in human fibroblasts or TDP-43 transfected HEK293T cells with a Seahorse XF96 Flux Analyzer . Cell lines were seeded in 12 wells of a XF 96-well cell culture microplate at a density of 1 × 10HEK293T or HeLa cells were transiently transfected with plasmids expressing recombinant WT or mutant human TDP-43-myc or empty vector, using FuGENE-HD (Promega), according to the manufacturer’s protocol. For ER and mitochondrial contact analyses, HeLa cells were co-transfected with ddGFP plasmids targeted to mitochondria and ER  together with DSred2-Mito. Cells were imaged 48 h later on the Leica TCS SP5 confocal microscope on a live imaging stage, controlled at 37 °C.4, 1.25 mM KH2PO4, 10 mM glucose, 1 mM EGTA and 10 mM HEPES, pH 7.35), followed by replenishment of ER calcium with 2 mM calcium to induce store operated calcium entry, as described previously [Live cell calcium imaging was performed with HeLa cells co-transfected with WT or mutant TDP-43 and mitochondrially targeted GCamp6. 48 h after transfection, cells were perfused with 20 μM ATP in imaging medium containing  DMSO, freezing in liquid nitrogen, and storing them at −80 °C until the experiment. For mitochondria isolation, ~100 mg of frozen brain tissue was slowly (1 h) thawed on ice, then the cryopreservation solution was replaced with homogenization medium (HM) comprising 225 mM sucrose, 75 mM mannitol, 1 mM EGTA, 20 mM HEPES (pH 7.4), and 1 mg/ml bovine serum albumin essentially fatty acid free (BSA). Brain tissue was homogenized in 10 ml of HM with a Dounce tissue grinder  manually by 40 strokes, in ice (step 1). All further procedures were performed at 4 °C. The homogenate was centrifuged at 2000 g × 5 min; the supernatant was further centrifuged at 12000 g ×10 min (step 2). The pellet from this step was collected, resuspended in 1 ml of HM, loaded on top of 9 ml of 23% Percoll™ separation medium, and centrifuged at 31,000 g × 10 min. The Percoll™ separation medium was prepared by dissolving 225 mM sucrose, 75 mM mannitol, 1 mM EGTA, 20 mM HEPES in 100% Percoll™ and adjusting pH to 7.4; this medium was diluted with HM to 23% Percoll™. The pellet from this step was collected and washed 2 times by centrifuging at 12000 g × 10 min in HM. The final pellet was dissolved in 200 μl of HM devoid of BSA (HM-BSA). For digitonin treatment, this pellet was thoroughly resuspended in 10 ml of HM-BSA and 20 μl of 10% digitonin DMSO solution  was added to the suspension. After incubating on ice for 5 min, the suspension was centrifuged at 12000 g × 10 min, the pellet was resuspended in digitonin-free HM-BSA and centrifuged again at 12000 g × 10 min. The final solid pellet was resuspended in 100 μl of HM-BSA and stored frozen at −20 °C until the experiment.Brains from TDP43oC until the experiment.For assaying combined synaptic + nonsynaptic mitochondria, the isolation procedure was modified as follows. The supernatant from step 2 above was collected, and treated with 0.02% digitonin for 5 min in ice, then centrifuged at 12000 g × 10 min. The pellet was resuspended in 10 ml of HM and centrifuged 12,000 g × 10 min. To remove proteins electrostatically attached to mitochondria membranes, the pellet from this step (contains synaptic + non-synaptic mitochondria) was resuspended in 2 ml of medium comprising 6 M KCl, 20 mM HEPES (pH 7.4) and incubated in ice for 5 min with occasional gentle agitation. The suspension was diluted with HM-BSA to 10 ml and centrifuged 12,000 g × 10 min, and the pellet was collected. To prepare digitonin -treated mitochondria from this fraction, mitochondria pellet was diluted to 10 ml with HM-BSA and treated with 0.01% digitonin for 7 min in ice, with occasional gentle agitation. The suspension was centrifuged 12,000 g × 10 min, the pellet was collected and washed in 10 ml of HM-BSA and centrifuging 12,000 g × 10 min; the procedure was repeated once. The final solid mitochondria pellet was resuspended in 100 μl of HM-BSA and stored frozen at −20 Samples were lysed in RIPA buffer (Thermo Fisher Scientific) and centrifuged at 14,000 rpm at 4 °C for 20 min. Supernatants were diluted in 2X Laemmli sample buffer (Bio-Rad). Proteins were separated on 10 or 4–20% gradient Mini-PROTEAN TGX gels (Bio-Rad) and transferred to nitrocellulose blotting membranes (Bio-Rad). Membranes were probed with primary antibodies against FLAG tag , MCU (Sigma), MICU1 , cytochrome C , Cyclophilin D , Tim23 , VDAC1 (Abcam), COX1 (Abcam), GAPDH (Thermo Fisher Scientific), citrate synthase (Abcam), and an oxidative phosphorylation (OXPHOS) cocktail (Abcam) overnight at 4 °C. Blots were then probed with horseradish peroxidase-conjugated anti-mouse  or anti-rabbit (Thermo Fisher Scientific) secondary antibodies and detected using enhanced chemiluminescence (Bio-Rad).p value <0.05 was considered significant.All data are presented as mean ± standard deviation in all experiments, except for the results of Figs. A315T mice) expresses human A315T mutant TDP-43 with a N-terminal FLAG tag in the nervous system, under the control of the prion promoter. The expression of the transgene results in a 3-fold increase in the levels of TDP-43 in the brain. These transgenic mice develop a neurodegenerative disease characterized by abnormal gait starting at 2 moths of age, TDP-43 and ubiquitin pathology in brain and spinal cord, and motor neuron degeneration [A315T mice, CNS degeneration is accompanied by degeneration of the myoenteric plexus, resulting in premature death due to intestinal paralysis [A315T mice obtained from The Jackson Laboratories was 103.25 days , consistent with previously reported survival data [The B6.Cg-Tg(Prnp-TARDBP*A315T)95Balo/J mouse strain , further suggesting lack of bioenergetic differences.Since no defects in mitochondrial respiration and ATP synthesis were detected at presymptomatic (45 days) and at symptomatic (90 days) ages, in order to avoid potentially confounding effects due to major changes in brain cyto-architecture associated with neurodegeneration, we opted to continue the measurements of mitochondrial function at disease onset (60 days). The enzymatic activities (Vmax) of respiratory chain complexes I and IV (cytochrome oxidase) measured in isolated brain mitochondria Fig. , b did ning Fig.  were vir2O2 emission in all conditions did not differ in 60 days old TDP-43A315T and N-Tg littermate brain mitochondria , cyclophilin D (CypD), the MCU regulator MICU1, and the voltage dependent anion channel VDAC1, were unchanged in TDP-43ATaken together, these results indicated that brain mitochondria of TDP-43 mutant mice had well preserved calcium handling mechanisms, and also suggested that these mitochondria were in fact more resistant to calcium-induced MPTP than N-Tg controls.In order to test cellular energy metabolism in patient-derived cells expressing physiological levels of mutant TDP-43, we studied primary skin fibroblasts from 3 individuals harboring the N352S point mutation associated with ALS-FTLD  and 8 heFibroblasts were subjected to flux analyses of oxygen consumption (OCR) and extracellular acidification (ECAR) rates using the Seahorse XF96 platform. Average OCR rates at baseline respiration were not significantly different in TDP-43 and control fibroblasts Fig. . SimilarWe then assessed the ability of mitochondria to accumulate the potentiometric fluorescent dye TMRM in TDP-43 mutant fibroblasts and controls. This approach allows for evaluating differences in mitochondrial membrane potential. There were no significant differences in TMRM accumulation between TDP-43 mutant and control cells Fig. .Fig. 5MiNext, to examine the total mitochondrial content we utilized the fluorescent dye MTG, which accumulates in mitochondria in a virtually non-membrane potential dependent manner. We did not detect differences in the mitochondrial content between TDP-43 mutant and control cells Fig. . FurtherLastly, we analyzed steady state ATP content using a luciferase based assay, which did not show differences between TDP-43 mutant and control fibroblasts under baseline conditions Fig. . InhibitTaken together, these results indicated that energy metabolism in both TDP-43 mutant and control fibroblasts was highly reliant on glycolysis and that there was no significant difference in the ability to run glycolysis or OXPHOS between mutants and controls, thereby excluding that mutant cells had bioenergetic defects.To extend the analyses of cellular bioenergetics to a larger number of TDP-43 mutations, we expressed recombinant human TDP-43 with the Q331K, Q343K, M337 V, A315T, as well as wild type (WT) TDP-43, in HEK293T cells. Cells were transiently transfected by lipofection with plasmids encoding for various TDP-43 constructs, resulting in similar levels of protein expression for each construct, as determined by western blot analyses of cell homogenates and quantification Fig. , b. OCR TDP-43 was shown to alter the physical interactions between mitochondria and ER , leadingA315T mice  TDP-43 were transfected with the calcium reporter mtGCaMP6 targeted selectively to the mitochondrial matrix  Fig. 6f6f. ER caA315T mice [It was proposed that TDP-43, not only is associated with mitochondria, but is also imported inside the organelles due to an internal targeting amino acid signal, and that the mitochondrial import of TDP-43 is increased by the pathogenic mutations both in cells and in the TDP-4315T mice . To testA315T with mitochondria in vivo, in mouse brain. Since the transgenic construct contains a FLAG epitope, mitochondrial fraction was analyzed by western blot, using anti-FLAG antibodies. Importantly, we compared the mitochondrial fraction treated with KCl washes, with and without the addition of a low dose of the detergent digitonin to eliminate any potential contamination from non-mitochondrial membranes, such as ER and peroxisomes. Figure A315T-FLAG with brain mitochondria was clearly detected in the fractions not subjected to KCl washes or digitonin treatment (Fig. A315T-FLAG were completely undetectable after KCl washes, irrespective of digitonin treatment.Next, we used a biochemical approach to examine the association of TDP-43ent Fig. . HoweverNeither the matrix protein citrate synthase, nor the inter membrane space protein cytochrome c were significantly affected by KCl washes, with and without digitonin treatment, indicating that the dose of digitonin used did not compromise the integrity of the mitochondrial outer and inner membranes. Taken together, these results suggested that a portion of the protein was peripherally associated with brain mitochondria, presumably through electrostatic interactions.A315T, using a specific antibody cocktail. The levels of individual subunits of OXPHOS complexes I-V were unchanged in TDP-43A315T mitochondria, relative to N-Tg controls (Fig. Because it was proposed that TDP-43 in mitochondria results in downregulation of respiratory chain proteins, specifically complex I , we asseols Fig. , suggestA315T transgenic mice and patient-derived cells.Mitochondrial dysfunction is one of the known pathogenic events associated with ALS. In particular, bioenergetic impairment has been amply described in cellular and mouse models of fALS caused by SOD1 mutations  and, morA315T transgenic mice, patient fibroblasts, and transfected cell expressing TDP-43. Overall, we did not confirm previous findings of mitochondrial bioenergetics defects in any of these models. In particular, mitochondria isolated from the brain of TDP-43A315T transgenic mice did not show impairment of mitochondrial respiration, ATP generation, and calcium handling. Similarly, there was no impairment of bioenergetic functions in skin fibroblasts harboring a pathogenic TDP-43 mutation or in HEK293T cells overexpressing various mutant forms of TDP-43.In this study, we sought to validate the findings of bioenergetic dysfunction in TDP-43We found that a portion of TDP-43 was peripherally associated with the surface of intact mouse brain mitochondria. This association, despite not interfering with mitochondrial bioenergetics, could affect inter-organellar communication. Indeed, TDP-43 was shown to interfere with the structures that link mitochondria and ER, also known as mitochondria associated membranes or MAMs . Our ddGEarlier studies had shown that TDP-43 overexpression alters mitochondrial dynamics in neurons, resulting in decreased motility accompanied by abnormal clustering and fragmentation , 12, 38.A315T transgenic mice relative to controls. This result suggests that TDP-43 A315T mitochondria were less prone to undergo calcium induced permeability transition than controls, a further indication that they did not suffer from bioenergetic impairment.Interestingly, brain mitochondrial calcium uptake capacity was increased in TDP-43Like the mouse model, cultured cells expressing mutant TDP-43 did not show bioenergetic defects, both when endogenous levels of the protein were present, in patient-derived fibroblasts, and when the mutant proteins were overexpressed, in transfected cells. The results in fibroblasts were in line with a recent report, which compared C9Orf72 mutant and TDP-43 mutant fibroblasts, and detected respiratory chain defects in the former, but not the latter . FurtherA315T mouse model, since we utilized mice from the same transgenic line and at similar ages as in the aforementioned report. It is possible that differences resulted from the protocols of mitochondrial purification, which were partly dissimilar in the two studies. It could be hypothesized that mitochondria from mutant TDP-43 brains are more fragile and could be damaged by the isolation procedure, which was not the case in our study, because soluble proteins of the intermembrane space were retained in the mitochondrial preparation. On the other hand, membrane fragility could not explain a decline in the activity of individual respiratory chain complexes, such as complex I, whose activities are measured on fractionated mitochondrial membranes and do not depend on mitochondrial integrity. Specifically, we did not find evidence of a decrease in either complex I-driven respiration and ATP synthesis or complex I oxidoreductase activity in brain mitochondria from TDP-43A315T transgenic mice, suggesting that OXPHOS is preserved in these mitochondria. Furthermore, the levels of respiratory chain subunits that we analyzed were unchanged in TDP-43A315T transgenic mouse brain mitochondria, suggesting that their synthesis was not impaired.The reasons for the discrepancy between the present study and a recent report in which mitochondrial respiratory chain defects were associated with mitochondrial import of mutant TDP-43  is not iUnlike previous reports , 36, we Multiple lines of evidence support a role for mutant TDP-43 in causing alterations of mitochondrial motility, morphology, and distribution in neurons, which could result in failure to provide energy to critical sites of utilization. However, our present studies did not confirm reports of mutant TDP-43 causing impaired mitochondrial bioenergetics in vivo and in cultured cells, suggesting that, at least in the systems we investigated, mitochondrial OXPHOS dysfunction does not directly participate in disease pathogenesis. On the other hand, we report the intriguing finding of increased mitochondrial calcium uptake in mouse brain mitochondria and patient derived fibroblasts harboring A315T TDP-43. The pathological significance and especially the mechanisms underlying this observation remain to be determined, but they do not appear to involve changes in mitochondrial bioenergetics.


With the development of microelectronic devices, the insufficient heat dissipation ability becomes one of the major bottlenecks for further miniaturization. Although graphene-assisted epoxy resin (ER) display promising potential to enhance the thermal performances, some limitations of the reduced graphene oxide (RGO) nanosheets and three-dimensional graphene networks (3DGNs) hinder the further improvement of the resulting thermal interface materials (TIMs). In this study, both the RGO nanosheets and 3DGNs are adopted as co-modifiers to improve the thermal conductivity of the ER. The 3DGNs provide a fast transport network for phonon, while the presence of RGO nanosheets enhances the heat transport at the interface between the graphene basal plane and the ER. The synergy of these two modifiers is achieved by selecting a proper proportion and an optimized reduction degree of the RGO nanosheets. Moreover, both the high stability of the thermal conductivity and well mechanical properties of the resulting TIM indicate the potential application prospect in the practical field.The online version of this article (10.1186/s11671-017-2298-z) contains supplementary material, which is available to authorized users. Graphene-assisted thermal interface materials (TIMs) have attracted increasing attention because of their high thermal and mechanical performances –5. Kim e4 flow and a strict cooling rate of the substrate are needed during the CVD procedure [In order to give full play to the high thermal conductivity of the adopted graphene, high-quality three-dimensional graphene networks (3DGNs) prepared by chemical vapor deposition method have been adopted to hybridize with ER by our group . The betrocedure . TherefoIn this study, the RGO nanosheets and 3DGNs are adopted as fillers to enhance the thermal performances of the resulting ER. The specific functions of these two modifiers are discussed and proven. On the one hand, the 3DGNs provide a fast transport network, increasing the average mean path of phonons. On the other hand, the RGO nanosheets on the 3DGN surface improve the contact at the interface of the graphene basal plane and ER remarkably, which depresses the interface scattering of phonons. The further improvement of the resulting thermal performance resulting from the synergy of the RGO nanosheets and 3DGNs indicates that utilizing graphene with an optimizing manner is a useful strategy to prepare the high-performance TIMs.−2 in areal density and 12 mm in thickness was purchased from Haobo Co., Ltd.  and used as a template to fabricate the 3DGNs. Ethanol, HCl, FeCl3, and poly(methyl methacrylate)  were obtained commercially from the Beijing chemical reagent plant . Ethyl lactate, natural graphite, poly(methyl methacrylate), and acetone were received from Aladdin Co., Ltd. Polytetrafluoroethylene (PTFE) and sodium dodecyl benzene sulfonate were purchased from the Huangjiang Co., Ltd. . ER and curing agent were purchased from Sanmu Co. Ltd. . Deionized water (resistivity 18 MΩcm) was utilized to prepare all aqueous solutions.Nickel foam with 300 gmThe preparation of the RGO nanosheets and 3DGNs has been reported by our group –14, and −1 according to ASTM1640 and analyzed in the tensile mode. The dimensions of the samples were 2 × 4 cm. Laser flash analysis and differential scanning calorimetry were used to analyze the thermal transport performance of the fabricated composites.Morphology of the TIMs was obtained by a scanning electron microscope  and transmission electron microscope . Atomic force microscopy (AFM) results were recorded by Nanoscope IIIa  and E-Sweep  in tapping mode. Scanning Raman spectra were recorded by LabRam-1B Raman microspectrometer at 532 nm . X-ray photoelectron spectroscopy (XPS) measurements were performed on a RBD upgraded PHI-5000C ESCA system (Perkin Elmer). Fourier transform infrared spectroscopy (FTIR) curves were measured on IR Prestige-21 system (PerkinElmer). Mechanical properties of these composites were recorded by a Triton DMTA  instrument. The Tg and storage modulus were measured at a frequency of 1 Hz and a heating rate of 5 °C minAFM and SEM images of the prepared RGO nanosheets, 3DGNs, RGO-3DGNs, and RGO-3DGNs-ER are shown in Fig. 2g phonon at Brillouin zone center. Moreover, the defect density and average size of the RGO nanosheets can be calculated by the integrated intensity ratio of IG/ID [−1 and 3000–3700 cm−1 are induced from the skeletal vibration of the graphene basal plane and the O–H stretching vibration of adsorbed water [−1 arising from the O=C–OH can be seen only for the RGO nanosheets resulting from the surface functional groups [Raman curves of the adopted RGO nanosheets and 3DGNs are shown in Fig.  IG/ID . Accordi IG/ID ,1\documeaterials , 8. The ed water –20. A rel groups . After cThe corresponding thermal performances of various samples are shown in Fig. p represents the volume percentage of the graphene filler and K, Kg, and Ke are thermal conductivities of the resulting composite, graphene, and ER, respectively. H and δ are the thickness of the graphene and the thermal boundary resistance between the graphene and ER. After calculation, the similar δ values of the RGO-ER and RGO-3DGNs-ER samples prove that the added RGO nanosheets are loaded on the surface of the 3DGNs  rather than dispersing in the ER matrix. The presence of the 3DGNs not only provides a fast transport network for phonons but also acts as a scaffold for the RGO nanosheets. On the other hand, the surface functional groups of the RGO nanosheets enhance the close contact between the graphene basal plane and ER at their interface, which offsets the poor wettability of the 3DGNs. Therefore, the thermal performance of the resulting TIM is enhanced significantly , and a well stability of the thermal performance of the resulting TIM is revealed under high temperature . Moreover, the excellent mechanical properties including high ultimate strength and stretch limits indicate the potential promising prospect of the presented TIM.


Moreover, immune cell PD-L1 expression was an independent favorable prognostic factor for overall survival . In MSI-H gastric cancer, PD-L1 expression was observed to be independently associated with a longer survival.Programmed death-ligand 1 (PD-L1) is expressed in a subgroup of gastric cancers that may benefit from immunotherapy. Microsatellite instability-high (MSI-H) is a potential predictive factor for response to immunotherapy targeting the PD-1 or its ligand PD-L1. The relationship between PD-L1 expression and MSI-H status remains poorly understood. In this study, we investigated PD-L1 expression in patients with MSI-H gastric cancer. We analyzed PD-L1 expression in 78 MSI-H gastric cancer tissue samples using immunohistochemistry. PD-L1 expression was classified as expression on tumor cells or on immune cells. We observed PD-L1 expression in 48 gastric cancer samples (61.5%), consisting of 7 (9.0%) cases with tumor PD-L1 expression and 47 (60.3%) cases with immune cell PD-L1 expression. Immune cell PD-L1 expression was frequently associated with intestinal type cancer by the Lauren classification ( Gastric cancer is the fourth most commonly diagnosed cancer and the second leading cause of cancer-related mortality worldwide . The mosThe Cancer Genome Atlas (TCGA) research network proposed a molecular classification dividing gastric carcinoma into four subtypes. One of them, microsatellite instability-high (MSI-H) tumors, is associated with elevated mutation rates including mutations in genes encoding targetable oncogenic signaling proteins . The ratAlthough the relationship between MSI-H gastric carcinoma and PD-L1 status has not been fully explored, we hypothesized that a therapeutic strategy targeting the PD-1/PD-L1 interactions may be more effective in patients with MSI-H gastric carcinoma than in those with MSS gastric carcinoma. Accordingly, the connection between MSI-H status and PD-L1 expression in patients with gastric carcinoma requires investigation. In the present study, we investigated the clinicopathological characteristics of PD-L1 expression in patients with MSI-H gastric carcinoma and found that PD-L1 expression was an independent prognostic factor for the survival of such patients.The mean age of the patients with gastric carcinoma was 65.9 . The male-to-female ratio was 1.6:1. The mean follow-up period was 56.9 ± 26.1 months. Eighteen (23.1%) patients experienced a recurrence, and 16 (20.5%) patients died of the disease. Of the 78 gastric cancer cases, 15 (19.2%) were diagnosed as stage I, 30 (38.5%) as stage II, 23 (29.5%) as stage III, and 10 (12.8%) as stage IV. All patients were Epstein-Barr virus (EBV)-negative.TC+) and in immune cells (PD-L1IC+), respectively. Six of 7 patients with PD-L1TC+ status were also positive for PD-L1IC+ status. Table IC+ status was more frequently observed in intestinal type tumors (p = 0.015), as well as in those with a lower risk of lymph node metastasis (p = 0.027) and lower tumor, node, and metastasis (TNM) classification stages compared to PD- L1IC− group (p = 0.029). However, there was no significant association between PD-L1TC+ status and histological type of the tumor by the Lauren classification, pN status, or the TNM stage classification. Age, sex, location, and pT status demonstrated no significant correlation with PD-L1 IHC in either tumor cells or immune cells.Prominent staining of PD-L1 was observed at the periphery of the tumor, i.e., the infiltrating front . PD-L1IC+ status was significantly associated with a longer OS (log rank p = 0.011) but not with a longer DFS . Two of them were TNM stage II and five of them had TNM stage III disease. Of nine TNM stage IV patients, six patients died after recurrence and they were all PD-L1 negative. Other three patients survived without disease progression were unexceptionally PD-L1IC+ and their DFS were 63.8, 68.4, and 69.6 months, respectively. In multivariate analysis, the PD-L1IC− group showed a significantly shorter OS  when compared with the PD-L1IC+ group  and overall survival (OS) of patients with PD-L1Recently, anti-PD-1/PD-L1 antibodies have shown remarkable therapeutic effects in advanced solid cancers. However, the interaction between PD-L1 expression and MSI remains poorly understood. We investigated PD-L1 expression in patients with MSI-H gastric carcinoma and observed that PD-L1 was expressed in 61.5% of tumors and that it was an independent favorable prognostic factor for survival.TC+ in 9.0% of cases and PD-L1IC+ in 60.3% of cases. The positive rate of PD-L1 in tumor cells (9.0%) is generally lower than previous studies on gastric cancers (5.1% to 65.0%) [To establish an anti-PD-1/PD-L1 therapeutic strategy, it is important to explore the relationship between MSI-H gastric carcinoma and PD-L1 expression. Previous studies revealed an adverse prognostic effect of PD-L1 expression in various cancers –35. Howeo 65.0%) –26. It cTC+ status survived and none experienced recurrence during their long (mean: 55.7 months) follow-up period, which was interrupted to achieve statistical significance. Moreover, PD-L1 expression in TILs was observed in 85.7% of patients with a PD-L1TC+ status. This finding suggested that the expression of PD-L1 in tumor cells and intra- and peritumoral immune cells is closely related. Moreover, the release of interferon gamma (IFN-γ) by TILs can directly induce PD-L1 expression in tumor cells and immune cells [Unexpectedly, intra- or peritumoral immune cells with PD-L1 expression was associated with a longer OS. All 7 patients with PD-L1ne cells –42.Patients with gastric carcinoma and high TIL levels have been reported to have a favorable prognosis , 13, 43.Recent studies with a few MSI-H gastric carcinoma cases (indicated via IHC of mismatch repair proteins) demonstrated significantly higher rates of PD-L1 expression compared to MSS gastric carcinoma , 48, 49.In a phase 1b clinical trial (KEYNOTE-012 study) conducted to assess pembrolizumab, 8 out of 36 patients (22.2%) with PD-L1-positive gastric carcinoma or gastroesophageal junction cancer were judged to have a partial response. Among 4 patients with MSI-H gastric carcinoma, 2 had a partial response and the other 2 had progressive disease . One of In summary, PD-L1 expression, especially in intra- and peritumoral immune cells, is related to longer survival in patients with MSI-H gastric carcinoma. Further studies are necessary to elucidate the underlying immunological pathways and to predict which gastric carcinoma subclass may respond most favorably to anti-PD-1/PD-L1 immunotherapy.Patients who underwent surgery for primary gastric carcinoma from September 2004 to September 2011 at Samsung Medical Center were eligible for this study. Among them, 78 patients with MSI-H tumors were selected from our previous study cohort of 620 patients including 58 (85%) cases from 68 MSI-H gastric cancers , 6  . No patiFor MSI analysis, we performed multiplex polymerase chain reaction (PCR) with five quasi-monomorphic mononucleotide repeat markers as previously described . Brieflyin situ hybridization with BOND-MAX with an EBV-encoded RNA probe . Only sections that showed a strong signal within almost all tumor cell nuclei were considered positive.Three-micrometer-thick sections were cut from each tissue block and mounted on Superfrost-plus slides . We performed TC+. For peritumoral immune cells a score of 0, 1, 2, or 3 was given when < 1%; ≥ 1% but < 5%; ≥ 5% but < 10%; or ≥ 10% of cells per area were PD-L1-positive, respectively, as previously described [IC+. To reduce interobserver variation, all the cases were reviewed by two pathologists, and in cases with disagreement, the final interpretation was determined by consensus using the multi-head microscope.We performed IHC on all 78 MSI-H gastric carcinoma samples. Staining for PD-L1 in FFPE tissue sections was conducted using a rabbit anti-human PD-L1 monoclonal antibody . The percentages of tumor cells and peritumoral immune cells that stained positive for PD-L1 were analyzed independently by two pathologists (J.C. and K.M.K.). A tumor was considered positive for PD-L1 if there was histological evidence of cytoplasmic and/or membranous staining. Percentages of PD-L1 positive tumor cells were quantified as 1%, 2%, 3 to 5%, 6 to 10%, and then in 10% increments up to 100% and the samples were given a score of 0, 1+, 2+, or 3+ based on the intensity of staining , DFS, and OS of patients. For statistical analyses, we used SPSS 18.0 statistical software program . We compared PD-L1 expression and clinicopathological variables with Pearson's chi-square test and the chi-square test using linear-by-linear association. We used the Kaplan-Meier method to estimate DFS and OS. To evaluate the association between clinicopathological factors and survival, the Cox proportional hazard model was used.


Fluorescent organic dyes photobleach under intense light. Graphene has been shown to improve the photo-stability of organic dyes. In this paper, we investigated the Raman spectroscopy and photo-bleaching kinetics of dyes in the absence/presence of chemical vapor deposition (CVD)-grown graphene. We show that graphene enhances the Raman signal of a wide range of dyes. The photo-bleaching of the dyes was reduced when the dyes were in contact with graphene. In contrast, monolayer hexagonal boron nitride (h-BN) was much less effective in reducing the photo-bleaching rate of the dyes. We attribute the suppression of photo-bleaching to the energy or electron transfer from dye to graphene. The results highlight the potential of CVD graphene as a substrate for protecting and enhancing Raman response of organic dyes. It is generally known that organic dyes degrade upon intense light exposure. During trace analysis by Raman spectroscopy, dyes need to be exposed to an intense laser for several to several tens of minutes. Improving the photo-stability of dye will significantly improve the sensitivity of Raman-based sensing methods. Graphene, with a zero energy gap, can participate in both energy and electron transfer with organic dyes. Many studies reported the interaction between graphene and organic dyes, focusing on the impact of graphene on the photophysics of the dye. A number of studies have investigated the fluorescence quenching of organic dyes in the presence of exfoliated graphene and suggested that the quenching mechanism was energy or electron transfer between the dye and graphene . Swathi The fluorescence quenching increases the ratio of Raman intensity to that of fluorescence. Thus graphene can be used as a substrate for Raman Spectroscopy. Early studies showed that exfoliated graphene enhances the Raman intensity of some fluorescence dyes  and protoporphyrin (PPP)) . The degA much less appreciated benefit of using graphene substrate is its enhancement of the photochemical stability of organic dyes. The mechanism of such an effect was generally attributed to the barrier property of graphene. Zhao et al. studied the surface-enhanced Raman spectroscopy of R6G dyes sandwiched between CVD-grown graphene and the magnetron sputtered Ag film. They observed that the photo-stability of the dye was increased in the presence of graphene. They suggest that the graphene acts as a barrier film preventing the reaction between the oxygen in the air and the dye underneath graphene . Xu et aHere we showed that CVD-grown graphene enhances the Raman signals of a wide range of dyes including fluorescein isothiocyanate (FITC), pyrromethene, coumarin 540A, fluorescein 548, disodium fluorescein, and kiton red 620, suggesting a broad application of CVD-grown graphene in Raman enhancement. We also show that the fluorescence was quenched and the photo-bleaching rate was significantly slowed down by 46.3% in the presence of graphene. 2SO4:35% H2O2). Warning: Piranha is highly reactive. Avoid contact with large amount of organic materials. Work in a chemical hood and use full personal protective equipment.Silicon wafer with 300 nm of oxide layer was purchased from Universitywafer , Pyrromethene, coumarin 540A, fluorescein 548, disodium fluorescein, and kiton red 620 were purchased from Exciton . FITC and (3-Aminopropyl)triethoxysilane (APTES) were purchased from Sigma-Aldrich . The silicon substrate was cleaned with hot piranha solution . The water bath was kept at 100 °C for 15 min before the furnace was turned off and cooled from 1000 °C to room temperature under Ar/H2 flow. After the synthesis, the h-BN-copper substrate was coated with a thin layer of PMMA and the copper was etched away using a FeCl3 solution. The PMMA/h-BN film was transferred onto a silicon wafer with or without the dye absorbed on the surface; the PMMA polymer was removed with an acetone wash to leave the h-BN on the silicon wafer. The single layer h-BN used in this study was prepared by copper-catalyzed CVD using Borane-ammonia complex as the h-BN source ,18. The Binding of APTES was carried out by immersion of the silicon substrate into a 10 mM solution of the APTES in toluene (spectroscopic grade) . After 2Covalent of FITC on the APTES-functionalized silicon substrate was carried out by immersion of the substrate in a 0.2 mM solution of FITC in ethanol for 3 h then washed in ultrasonic baths of ethanol for 10 min and acetone for 20 min–27 h. The FITC is covalently linked to the APTES through the formation of a thiourea linkage .Absorption of pyrromethene, coumarin 540A, fluorescein 548, disodium fluorescein, and kiton red 620 on the APTES-functionalized silicon substrate was carried out by immersion of the substrate in a 0.2 mM solution of the corresponding dye in ethanol for 3 h then washed in ultrasonic baths of ethanol for 10 min and acetone for 90 min–6 h. The washing steps are intended to remove dyes in the state of agglomerated particles or multilayer adsorption, which may not form intimate contact with the graphene and complicates the data analysis. Absorption of FITC on the silicon substrate with oxide layer was carried out by immersion of the substrate in a 0.2 mM solution of FITC in ethanol for 3 h then washed in ultrasonic baths of ethanol for 10 min and acetone for 20 min. For the CVD-grown graphene substrate, 0.1 mM FITC dye solution in ethanol was spin-coated on the substrate at 3250 rpm.3 solution. The PMMA/graphene(h-BN) film was washed with D.I. water and kept floating on the water surface with graphene side facing downward. Then, the substrate with the dye layer facing upward was dipped into the water and the PMMA/graphene(h-BN) film was picked up. After drying in air for 30 min, the PMMA polymer was removed in acetone for 20 min~12 h to leave the graphene or h-BN on the surface of the dye layer, forming a Gr(or h-BN)/dye/SiO2/Si structure (Gr: graphene).FITC, pyrromethene, coumarin 540A, fluorescein 548, disodium fluorescein, orkiton red 620 was first absorbed on the silicon substrate or PTES-functionalized silicon substratewith the method mentioned above. Then, the CVD-grown graphene or h-BN was transferred onto the substrate with the help of PMMA and water . DetailsFluorescence measurement: Fluorescent spectra were collected on a custom-built set-up that consisted of a 532 nm single-longitudinal-mode solid-state laser, a 532 nm clean-up filter , an inverted microscope , a 532 nm long-pass edge filter (Semrock), and a single-stage spectrograph  with an attached back-illuminated charge-coupled device (CCD) camera thermoelectrically cooled to −55 °C . A 40× objective (NA: 0.60) was used in all of the micro-Raman experiments. Laser power was kept below 2 mW at the sample to avoid laser-induced heating and damage to the dye and graphene. The laser power was measured by using a power meter .−1) and FITC  were clearly observed on Gr/FITC sample (Gr: graphene), while the FITC sample in the absence of graphene barely showed any peak except the Si-Si overtone (969 cm−1) from the Si substrate [We use Raman spectroscopy to characterize the fluorescence and chemical stability of the dye. The single layer graphene sample was synthesized using a copper-catalyzed CVD method and was extensively characterized in our previous study . The atoubstrate ,21. Simi2 via APTES, ensuring that no more than a monolayer of FITC is present. For this reason, we chose FITC in our kinetic studies of photobleaching (see below). Note that in 2/Si (FITC was adsorbed on aminopropyltriethoxysilane (APTES) modified SiO2 surface) and Gr/FITC/APTES/SiO2/Si (FITC was sandwiched between graphene and the APTES modified SiO2/Si substrate), to compare the photochemical stability of FITC in the absence and presence of graphene. We attached FITC through APTES to the SiO2/Si substrate to achieve a high surface coverage of the dye through the reaction between the isothiocyanate group of the FITC and the amine group of the APTES, which forms a covalent thiourea linkage [We prepared two sets of samples with different structures: FITC/APTES/SiO linkage . By usin2/Si was 0.60 nm. The RMS roughness of Gr/FITC/APTES/SiO2/Si at location free of PMMA residues (white particles) was 0.48 nm. The smooth surface in both cases indicates minimal aggregation of the dye molecule. In 0 is attributed to the dark current of the Raman CCD (charge-coupled device) camera, and α and k are the pre-exponential factors and photo-bleaching rate, respectively. The double exponential behavior suggests that two parallel pathways are contributing the photo-bleaching of the dye and/or the presence of two populations of dyes in different photochemical environments. The reason behind the double exponential behavior will be studied in a future work. Here, the offset I−1 and 0.010±0.002 s−1 in the absence of graphene and are 0.044 ± 0.007 s−1 and 0.0060 ± 0.0006 s−1 in the presence of graphene (2/(α1 + α2) increased from ca. 0.4–0.5 in the absence of graphene to ca. 0.7 in the presence of graphene. At the same time, the rate constant for the slow decay decreased by 40% (from 0.01 s−1 to 0.006 s−1). Overall, the average photo-bleaching rate of dye in the presence of graphene is decreased by 46.3% (from 0.011 s−1 to 0.0063 s−1) compared to that in the absence of graphene. The average photo-bleaching rate The fast and slow components of the biexponential decay are 0.049 ± 0.009 sgraphene . Thus, tequation .(2)k¯=α2/Si without graphene  and is an efficient quencher for a number of fluorescence dyes [3O2 leads to the formation of semi-oxidized (X) radical form of the dye and HO2 or O2−. By adding a barrier layer between oxygen and dye, the D-O reaction will be effectively prevented, thus reducing the subsequent photo-bleaching and photodecomposition process. If D-O reaction is the predominant mechanism, similar observation will be found when the graphene was substituted by other oxygen diffusion barrier layers, such as h-BN. However, this mechanism is contradicted by the results from h-BN/FITC/SiO2/Si sample, as shown in The photo-bleaching of organic dye is commonly attributed to oxygen, which exists in a ground state triplet (nce dyes . The dyence dyes . QuenchiQuenching of fluorescence of dye could occur through either charge transfer or energy transfer and, in the latter case, two pathways are known: the Förster resonance energy transfer and the surface energy transfer ,27. The 2/Si. It is likely that the dyes already experience significant quenching by the Ag substrate, and therefore the photochemical stability can no longer be enhanced further by the quenching provided by graphene. In this case the effect of barrier film becomes important. In contrast, the dyes in our experiments rely on graphene to reduce their lifetime at the excited state. In this case, the difference between graphene and h-BN becomes significant. Although both are good gas barriers, the unquenched dyes may still be oxidized by the oxygen or other impurities trapped between graphene or h-BN and the SiO2. In this case, the better quenching efficiency of graphene, as can be seen by comparing the fluorescence background of the dye in the presence and absence of graphene (It is worth comparing our result with that of Zhao et al., who reported that the photochemical stability of dyes increases when they are adsorbed underneath graphene compared to those adsorbed above grapheme . In theigraphene b,c, play2 surface via APTES, ensuring that no more than a monolayer amount of dye is present.For other dyes, we observed inconsistent fluorescence quenching behavior (Finally, we comment on the limitations of our study. We chose to use FITC in our kinetic study because this dye can be covalently attached to the SiObehavior , which w2 and graphene.In summary, we have showed that CVD-grown graphene can be used as a substrate to enhance the Raman signal of a wide range of fluorescence dyes. By comparing graphene with h-BN, we showed thatalthough both are good barrier films for oxygen, graphene is much more effective in improving the photostability of FITC. Therefore, the energy/charge transfer plays an important role in improving the photochemical stability of the FITC dye sandwiched between SiO


Various MLG electrodes with different numbers of graphene layers were prepared with environmentally friendly poly:poly(styrene-sulfonate) (PEDOT:PSS) to produce ECD cells. Tests demonstrated the reproducibility and uniformity in optical performance, as well as the flexibility of the ECD fabrication. With the optimized MLG electrode, the ECD cells exhibited a very fast switching response for optical changes from transparent to dark states of a few hundred msec.For environmental reason, buildings increasingly install smart windows, which can dim incoming daylight based on active electrochromic devices (ECDs). In this work, multi-layered graphene (MLG) was investigated as an ECD window electrode, to minimize carbon dioxide (CO Among the global efforts being taken to preserve the earth, the energy losses due to windows in most types of construction, especially in buildings, have to be addressed in order to lower energy consumption, i.e. the energy used for space cooling and heating4. The functions of windows can generally be divided into technical and esthetic aspects. When the term “control” is added as a technical feature to the window’s typical function, then they are usually called “smart windows”. Smart windows have more active ability to control light and heat transmittance, either manually and/or automatically6. Among smart window technologies, including photochromic7, thermochromic10 and electrochromic approaches11, electrochromic devices can provide better performance in terms of ease operation and they are actively switchable to respond to environmental changes as well as personal demand.To address the increasing concerns of global warming, sustainable and renewable resources must be developed. However in addition, new technologies that can reduce the consumption of the generated energy are also highly necessary, in order to minimize carbon dioxide (CO12.The electrochromic (EC) phenomenon is generally defined as the ability to make reversible changes in optical properties, such as transmission, absorption and/or reflection. EC devices (ECDs) typically have structures of containing two conductors sandwiched with electrolytic cells that include an electrolyte and EC materials15, polythiophene17 have been widely investigated as potential materials due to their easy processability and effective cost.These EC materials have the intrinsic ability to reversibly change their visible color(s) when oxidized or reduced in response to an applied electrical potential, which makes them useful and controllable in EC devices. Among various EC materials, such as organic/inorganic materials, metal-complexes and organic polymers, polymers are the most attractive and promising materials, due to their simple processability, which enables large area fabrication, making them practical and cost effective for smart window applications. Consequently, conducting polymers including polyaniline12. The results were promising, in that it was demonstrated that the optical performance of PEDOT:PSS-based ECDs can be significantly improved by structure and/or material development. Besides, environmental-friendly PEDOT:PSS, which is a commercially available product, remains relatively stable under the electrochemical reaction with acceptable reproducibility. Therefore, PEDOT:PSS, with the trade name Clevious P, was also employed in this work. After EC material is selected, the conductors chosen as the electrodes are also very important for electrochemical processes.In our previous work, one of the well-known conducting polymer materials, poly:poly(styrene-sulfonate) (PEDOT:PSS), was investigated in order to optimize EC performance, especially to maximize optical contrast, by adjusting the ion diffusion length, which is affected by PEDOT:PSS layer thickness19, light-emitting diodes21 and also for ECDs23 because of its outstanding properties such as high conductivity and high optical transparency. However, the ITO surface has to be modified in order to reduce cathodic and anodic corrosion at the ITO surfaces, which affects the durability of the device28. Moreover, indium is becoming a scarce and expensive resource, which is affecting production schedules and cost. In addition, when poly: poly(styrene-sulfonate) (PEDOT:PSS) is coated onto an ITO surface, the acidic conditioned PEDOT:PSS leads to an unstable interface between the PEDOT:PSS and indium in the ITO, which then has a tendency to diffuse into neighboring layers, causing the degradation of the device performance and durability over time29. Consequently, it is mandatory to introduce electrochemically stable electrodes for electrochromic-based smart window and also for chameleon or camouflage applications.Among conductors, tin-doped indium oxide (ITO) is the most commonly used materials in various research and application fields, including photovoltaics31. However, compared to the performance of CNTs as electrodes, graphene is a promising material because graphene has lower surface roughness than CNTs, which affects electrical conductivity. Graphene also has outstanding characteristics, including a similar work function level (Φ ~ 4.5 eV) to ITO33. Although there have been a few reports of graphene assisted ECDs, there has been a lack of systematic investigation, for instance, by varying graphene amount and comparison of graphene flake dispersed solutions and stacked multi-layered graphene with less than 10 layers34. Therefore, in this paper the number of graphene layers grown on Cu will be optimized in order to maximize the PEDOT:PSS polymer based ECDs and the electrochemical and electrical characteristics of the different numbers of graphene layers used as electrodes will be investigated.Generally, carbon materials such as carbon nanotubes (CNTs) and graphene are employed as alternatives to ITO for electrochemical devices due to their intrinsic electrochemical stability and good electrical conductivityA schematic of the entire process flow for the graphene electrode-based ECD preparation is illustrated in Fig. In order to investigate ECD performance while using the graphene electrode, the optical and electrical characteristics of the prepared different numbers of graphene layers applied as electrodes was investigated. When the graphene layer was stacked in order to increase the number of graphene layers, the graphene surface was first UV/ozone treated to remove any adhesive residue on top of the transferred graphene surface. As shown in the optical transmittance spectra Fig.  after UV−1, the G-peak at ~1600 cm−1 and the 2D peak at ~2700 cm−1. No peaks in the D-peak position at ~1300 cm−1 indicates very high qualified single and stacked graphene layers have been obtained without defects. Moreover, the randomly placed hexagonal lattice among the stacked graphene layers, indicated by the intensity ratio of the G- and 2D-peaks (IG and I2D), was not changed significantly while the IG and I2D increased together  of the graphene films appears to have decreased with increasing numbers of transferred stacked graphene layers, from G1L to G6L Fig. . The ove+) between the electrolyte and PEDOT:PSS EC layer with various scan rates of 5, 10, 20, 30, 40 and 50 mV s−1 in the potential range of −3.0 to 1.5 V  behaviors were measured by potentiostatic cyclic voltammetry (CV). The graphene electrodes from G1L to G6L showed redox waves due to the insertion and extraction of lithium ions  for G4L is close to 1, promising a better reversible electrochemical reaction, while the G5L electrode shows much lower values of −Ipc/Ipa37. Therefore, following the comparison of the G4L and G5L electrodes, the G4L was selected and used as the electrode for further ECD performance investigations, due to its enhanced electrochemical activity38.In order to realize the optimum number of graphene layers for the electrode of the PEDOT:PSS-based EC reaction, the redox peak current densities for both G4L and G5L were observed in the CVs Fig. , Both thTt) and −0.5 to −3.5 V for the dark state (Td) with an offset voltage of −0.5 V during 40 s  electrode-based ECD cells prepared with PEDOT:PSS, the 4-layered graphene (G4L) electrode showed the best electrochemical behaviors in terms of electrochemical stability and reactivity, which enables the replacement of ITO as the electrode. Moreover, the optical switching time, which is one of the important parameters of ECD performance, was significantly enhanced and showed a fast optical change response of less than 1 sec from the dark to transparent state and only 500 msec from the transparent to dart states. Furthermore, the maximum contrast ratio was reached at ±2.5 V, which is low enough to improve device stability. Although there are still remaining issues, such as the mass production and reproducibility of graphene electrodes, this work can provide positive insights for the realization of graphene based devices employing redox-based processes.6, 99.0%) and lithium trifluoromethane-sulfonate  were acquired from Sigma-Aldrich (USA) and used without further purification.Single-layered graphene on thermal release film (TRF) was provided by Hanwha Techwin Co. Kr. (Korea). PEDOT:PSS was purchased from Heraeus Precious Metals GmbH & Co. (Germany) and used as received. Also, 1,2-dichlorobenzene , propylene carbonate , tetrabutylammonium hexafluorophosphate (TBAPF4)2S2O8 and then the released graphene layers are transferred onto the TRF using the method described in the literature39. Finally, the single-layered graphene  is detached from the TRF and laminated to form multi-layered graphene . MLGs are prepared by stacking SLG on the desired substrates using the lamination technique at 160 °C40.A graphene layer with polymer supports, grown on a copper foil, is released by etching the copper foil using aq. 0.1 M ammonium persulphate solution (NH3SO3 (10 mM) and TBAPF6 (100 mM) dissolved in a co-solvent system of DCB and PC (v:v = 3:1) was prepared as the liquid electrolyte (L-El) and injected into the ECD cells with the final structure of SLG or MLG/PEDOT:PSS//L-El//SLG or MLG. The active area was defined by the SLG or MLG area which was 1.0 × 1.0 cm.As the electrochromic material, the thickness-optimized PEDOT:PSS was spin-coated on the prepared SLG or MLG, then PEDOT:PSS coated SLG or MLG was covered with the same-layered graphene with a 100 μm-gap fixed by thermal adhesive tape to prevent infiltration by oxygen and moisture. Then, a mixture of LiCFThe film thickness of the spin-coated PEDOT:PSS was measured using an Alpha-step 500 profilometer. The optical transmittance measurement of the laminated graphene layers was performed using UV-visible-NIR spectroscopy . Raman spectra of the laminated graphene layers were obtained by Raman spectroscopy . The beam size of the microscope equipped with a 532-nm laser and a 50X objective was just 1 μm and the applied maximum power was 10 mW. Electrochemistry measurements were performed with a potentiostat . Cyclic voltammetry was conducted with a three-electrode cell in which ITO (with an active area for both the ITO and PEDOT:PSS of about 1 × 1 cm) was used as a working electrode. A platinum wire and saturated Ag/AgCl (SCE) were used as an auxiliary electrode and a reference electrode, respectively. The optical contrast ratio and response times were measured via the converted luminous transmittance (resulting in absorption and transmission changes) after applying a reverse bias voltage ranging from −3.5 to +3.5 V for both bleached and colored states, respectively, using an LCD electro-optical measurement system  in transmission mode with a standard illuminant D65  as a light source.


The right and responsibility of communities to participate in health service delivery was enshrined in the 1978 Alma Ata declaration , 3 and tIn working with communities, we note that there is a tension between the two extremes of over-stating and under-stating community capabilities. At the over-stating extreme, communities can be seen as having enormous capacity to solve their own problems. This extreme is problematic because it neglects to account for the role of broader contextual factors, such as state failures to provide adequate health services and infrastructure (e.g. roads and sanitation systems) that facilitate health, and broader structural and social determinants driven by colonial history, trade policies, legal systems and political conflicts that deny marginalized groups their rights. Focusing exclusively on community capabilities without considering these contextual factors can lead to solutions that may burden already poor people with unpaid work or financial costs that should be shouldered by the state.At the under-stating extreme, communities can also be seen as disenfranchised, powerless groups of people or clients, who lack valuable skills or assets and must wait passively for outside help. It fails to recognize that communities have expertise and local resources. They have the right to influence, direct and engage in programs that address their health needs. It also can suggest that communities have little role to play in demanding or supporting improvements in government services—contrary to evidence that social accountability can be an important mechanism for service improvement. Advancing health systems partnerships with communities that are equitable and effective in reaching health goals therefore requires a measured understanding of community capabilities. One that recognizes, harnesses and augments community assets, while also addressing the structural and social determinants of health, whose locus of interventions may lie outside of communities.As part of its thematic work on ‘Unlocking Community Capabilities’, the Future Health Systems research consortium examines how communities can be active participants in the planning, delivery, monitoring, and evaluation of their health systems, by identifying individual and collective capabilities in diverse social, political, and institutional environments to improve people’s health. This supplement builds on prior work undertaken by the consortium , 9 and iwhat communities have; how communities act, and for whom do communities act. These domains encompass material assets and resources, including information, skills and key external linkages that communities must have to support collective endeavours. In addition, it takes into account the governance processes and characteristics that support how assets and resources are shared and controlled; how communities function collectively; and the interests served by the community’s collective action and social processes. Each of these domains is necessary but not sufficient on its own to ensure that communities are empowered to improve their health and well-being. The review found that to date an unfortunate feature of health systems research involving community participation is the limited reporting and low quality of information related to community capability. The authors suggest that having a simpler framework explaining community capability may support better documentation and advance future understanding.In the first paper, George et al.  describeHaving started by seeking conceptual clarity about the domains that constitute community capability and their relation to the related concepts of social capital and community competence –24, the Anganwadi Centres) were not related to children’s receipt of minimal acceptable diets [Future research needs to balance ensuring local relevance with comparability across contexts, as well as teasing out more precisely which elements of community capability matter and why. For instance, Barman et al. were surprised to find that membership in credit groups by heads of households and children’s registration at government nutrition and early education centres (le diets . Moreovele diets  found thHealth systems research focussed on unlocking community capability entails working with marginalised communities . A key pAcross the papers, relationship building is a recurrent theme, also used to ensure inclusiveness within communities , 14–16. Ensuring that community interests are placed at the center of health initiatives is one of the reasons why community engagement can be sustained, as has been the case for over 20 years in Bangladesh . HoweverThe strategic relationship between communities and external actors requires careful mediation . In UganIn conclusion, unlocking community capabilities entails identifying local resources, building capacities, and brokering partnerships. Relationships feature centrally in this. Advancing these capabilities in service delivery models has the potential to advance social equity and strengthen health systems. But there are important nuances and tensions that underpin building and sustaining community capability. Community needs for tangible, material improvements must be met, while also fostering intangible yet powerful elements such as trust. While relationships across diverse actors and levels of the health system need to be brokered, inclusion of the most marginalized people within communities cannot be overlooked. Change needs to be pursued in two directions. Building communities’ internal capabilities needs to be supported, while also strengthening their linkages to external actors, mobilizing resources and supportive mechanisms across health system levels, and the foundations for broader, structural changes.As much as health systems initiatives seek to work with communities and strengthen their capabilities, communities are diverse and human agency can be ingeniously autonomous. Unlocking community capabilities enables communities to make use of their unique social systems and resources in ways that may not always align with outsider expectations . PolicymEngaging with these tensions to strengthen how health systems work with communities demands a careful consideration of how participatory health system interventions at the community level are supported. Interventions must navigate pre-existing power dynamics and the additional tensions created by introducing new resources and expectations. Questioning assumptions about the social processes that underpin how communities participate in health systems interventions is critical to developing more realistic expectations, adequate resources, and supportive principles of collaboration to facilitate community empowerment and broader social development. While there are no magic bullets , the pap